TRV vs BPMV VIGS Vectors in Soybean: A Comprehensive Guide for Functional Genomics

Abstract

This article provides a systematic comparison of Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) vectors for Virus-Induced Gene Silencing in soybean. Tailored for researchers and scientists, it covers foundational principles, practical methodologies, optimization strategies, and comparative performance metrics. The analysis synthesizes recent advancements, including a novel TRV-based system achieving 65-95% silencing efficiency and established BPMV protocols, offering critical insights for selecting appropriate vectors for gene function studies, disease resistance research, and high-throughput screening in this economically vital crop.

Understanding VIGS Fundamentals: TRV and BPMV Mechanisms in Soybean

Core Principles of Virus-Induced Gene Silencing in Plants

Virus-Induced Gene Silencing (VIGS) is a powerful post-transcriptional gene silencing (PTGS) technique that harnesses the innate defense mechanisms of plants against viral pathogens for functional genomics research [1] [2]. When plants encounter viruses, they recognize and process viral double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs), which then guide the sequence-specific degradation of complementary RNA sequences [1]. VIGS exploits this pathway by engineering viral vectors to carry fragments of plant genes, effectively turning the plant's defense system into a tool for knocking down target gene expression [2].

This technology has become an indispensable functional genomics tool for crop plants like soybean, where stable genetic transformation remains time-consuming and challenging [3] [4] [5]. VIGS enables rapid assessment of gene function without the need for stable transformation, allowing researchers to link gene sequences to physiological functions and phenotypes within a single generation [3] [1]. This review will focus on comparing two prominent VIGS vectors—Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV)—within the context of soybean research, providing researchers with experimental data and protocols to inform their vector selection.

Molecular Mechanisms of VIGS

The molecular machinery of VIGS operates through a conserved RNA silencing pathway that involves several key steps and enzymatic components [1]. Understanding this mechanism is crucial for optimizing VIGS efficiency and interpreting experimental results.

The Stepwise VIGS Pathway

Vector Introduction and Replication: The process begins when a recombinant VIGS vector is introduced into plant cells through various delivery methods (e.g., Agrobacterium infiltration, mechanical inoculation) [1]. Once inside the cell, the viral RNA is replicated by either viral or host RNA-dependent RNA polymerases (RdRps) [1].

dsRNA Formation and Processing: During replication, double-stranded RNA intermediates are formed, which are recognized by the plant's DICER-like enzymes [1] [2]. These RNases cleave the long dsRNA molecules into small interfering RNAs (siRNAs) of 21-25 nucleotides in length [1].

RISC Assembly and Target Cleavage: The siRNAs are incorporated into an RNA-induced silencing complex (RISC), where the complex uses the siRNA as a guide to identify complementary messenger RNA (mRNA) sequences [1]. Upon recognition, the RISC cleaves the target mRNA, preventing its translation into protein [2].

Systemic Silencing: The silencing signal amplifies and moves systemically throughout the plant, leading to widespread knockdown of the target gene [1]. This systemic movement enables observation of silencing phenotypes in tissues beyond the initial inoculation site.

Key Advantages for Plant Research

VIGS offers several distinct advantages over other functional genomics approaches. Its rapid turnaround time enables gene function assessment within 3-4 weeks post-inoculation, compared to months or years required for stable transformation [3] [4]. The technique is particularly valuable for studying lethal gene mutations because it typically results in transient rather than permanent silencing, allowing recovery of plant tissues [1]. Additionally, VIGS can be applied to genetically intractable species that are recalcitrant to stable transformation, including many crop species with complex genomes [3] [4]. The system also enables tissue-specific and developmental stage-specific silencing depending on the viral vector's tropism and timing of inoculation [6].

TRV vs. BPMV Vectors: Technical Comparison

Tobacco Rattle Virus (TRV) Vector System

TRV has emerged as one of the most versatile VIGS vectors due to its broad host range, efficient systemic movement, and minimal viral symptom development [3] [4]. The TRV genome consists of two RNA components: RNA1 contains genes for replication and movement, while RNA2 encodes the coat protein and serves as the insertion site for target gene fragments [1] [2].

Recent research has demonstrated successful implementation of TRV-based VIGS in soybean using an optimized Agrobacterium-mediated cotyledon node infection method [3] [4]. This approach achieved silencing efficiencies ranging from 65% to 95% across multiple target genes, including GmPDS (phytoene desaturase), GmRpp6907 (rust resistance), and GmRPT4 (defense-related) [3]. The optimized protocol involves bisecting sterilized soybean seeds and infecting fresh explants by immersion in Agrobacterium tumefaciens GV3101 suspensions containing TRV vectors for 20-30 minutes [3] [4]. This method overcame limitations of conventional approaches (misting, injection) that showed low efficiency due to soybean leaves' thick cuticles and dense trichomes [3].

Bean Pod Mottle Virus (BPMV) Vector System

BPMV is currently the most widely adopted VIGS vector for soybean functional genomics [3] [4] [7]. Like TRV, BPMV has a bipartite genome with RNA1 and RNA2 components, with foreign gene fragments typically inserted into RNA2 [7]. The BPMV system has been successfully used to investigate soybean cyst nematode parasitism, rust immunity, and resistance to various viral pathogens [3] [4].

The advanced "one-step" BPMV vector system allows direct rub-inoculation of infectious plasmid DNA, eliminating the need for in vitro transcription or biolistic delivery [7]. This system has been optimized for common bean (Phaseolus vulgaris) with successful silencing achieved using fragments as short as 132 bp, though optimal results require 5μg of each plasmid (RNA1 and RNA2) for inoculation [7]. BPMV-derived vectors can also be used for overexpression of heterologous proteins in addition to gene silencing, expanding their utility for functional studies [7].

Comparative Performance Data

Table 1: Quantitative Comparison of TRV and BPMV VIGS Vectors in Soybean

Parameter	TRV-VIGS	BPMV-VIGS	Experimental Context
Silencing Efficiency	65-95% [3]	Not explicitly quantified	Soybean cv. Tianlong 1, multiple target genes [3]
Time to Phenotype	21 days post-inoculation [3]	3-4 weeks post-inoculation [7]	First appearance of photobleaching (GmPDS) [3]
Infection Method	Agrobacterium-mediated cotyledon node immersion [3]	Direct plasmid rubbing or particle bombardment [3] [7]	Optimized protocols for each system
Viral Symptoms	Minimal [3] [4]	Mild to moderate foliar symptoms [3] [7]	Effect on interpretation of silencing phenotypes
Tissue Coverage	Systemic, including meristems [3]	Systemic, but may exclude some meristems [6]	Spread throughout plant tissues
Genotype Compatibility	Limited data, successful in Tianlong 1 [3]	Broader compatibility with legume species [7]	Range of susceptible varieties

Table 2: Qualitative Comparison of Vector Characteristics and Applications

Characteristic	TRV-VIGS	BPMV-VIGS
Ease of Use	Moderate (requires Agrobacterium handling) [3]	Variable (simple rubbing to complex bombardment) [3] [7]
Host Range	Broad (multiple plant families) [1] [2]	Primarily legumes (soybean, common bean) [7]
Insert Capacity	Medium (~1.5 kb)	Medium (~1 kb)
Silencing Duration	3-8 weeks [1]	Several weeks to months [1]
Seed Transmission	Not reported	Not reported
Best Applications	Rapid gene validation, developmental studies [3]	Legacy systems, legume-specific studies [3] [7]

Experimental Protocols for Soybean VIGS

TRV-VIGS Protocol for Soybean

The following optimized protocol for TRV-mediated VIGS in soybean has demonstrated high efficiency (up to 95% infection rate) in the cultivar Tianlong 1 [3]:

Vector Construction:

• Clone target gene fragment (300-500 bp) into pTRV2-GFP vector using EcoRI and XhoI restriction sites [3]
• Transform recombinant plasmid into Agrobacterium tumefaciens GV3101 [3]
• Confirm construct integrity by sequencing and restriction analysis [3]

Plant Material Preparation:

• Surface-sterilize soybean seeds and soak in sterile water until swollen [3]
• Longitudinally bisect seeds to obtain half-seed explants [3]
• Use fresh explants for Agrobacterium infection [3]

Agroinfiltration:

• Prepare Agrobacterium suspensions (OD₆₀₀ = 0.6-1.0) in infiltration medium [3]
• Immerse explants in Agrobacterium suspension for 20-30 minutes [3]
• Co-cultivate on medium for 2-3 days [3]
• Transfer to selection medium and monitor for silencing phenotypes [3]

Efficiency Validation:

• Monitor GFP fluorescence at infection sites 4 days post-infection [3]
• Assess silencing phenotypes (e.g., photobleaching for GmPDS) at 21 dpi [3]
• Quantify silencing efficiency through qRT-PCR of target genes [3]

BPMV-VIGS Protocol for Legumes

The one-step BPMV vector system offers simplified delivery for common bean and soybean [7]:

Vector Preparation:

• Maintain BPMV RNA1 and RNA2 constructs as separate plasmids [7]
• Insert target gene fragment into RNA2 using BamHI restriction site [7]
• Transform plasmids into appropriate bacterial strains [7]

Plant Inoculation:

• Grow plants to primary leaf stage (7-10 days post-germination) [7]
• Mix RNA1 and RNA2 plasmids (5μg each) in inoculation buffer [7]
• Apply carborundum abrasive to leaves and rub inoculation mixture gently [7]
• Inoculate both primary leaves for maximum efficiency [7]

Infection Monitoring:

• Observe viral symptoms 10-14 days post-inoculation [7]
• For BPMV-GFP constructs, monitor fluorescence under UV light [7]
• Assess silencing phenotypes 3-4 weeks post-inoculation [7]

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for VIGS Experiments in Soybean

Reagent/Resource	Function/Purpose	Examples/Specifications
VIGS Vectors	Delivery of target gene fragments into plant cells	pTRV1/pTRV2 (TRV system), BPMV RNA1/RNA2 [3] [7]
Agrobacterium Strains	Mediate vector transfer into plant tissues	GV3101, C58C1 [3] [6]
Selection Antibiotics	Maintain plasmid selection in bacterial and plant systems	Kanamycin, rifampicin, gentamycin [6]
Infiltration Media	Support Agrobacterium viability during plant infection	10 mM MgCl₂, 10 mM MES (pH 5.7), 100 μM acetosyringone [6]
Marker Genes	Visual assessment of silencing efficiency	GmPDS/PvPDS (photobleaching), GFP (fluorescence) [3] [7]
Validation Primers	Confirm silencing at molecular level	qRT-PCR primers for target genes and reference genes [3]

Discussion and Research Implications

The choice between TRV and BPMV VIGS vectors depends heavily on research objectives, technical capabilities, and specific soybean genotypes under investigation. TRV offers advantages in reduced symptom development and potentially higher silencing efficiency in compatible genotypes, while BPMV benefits from established protocols and proven efficacy across more legume species [3] [4] [7].

Recent advancements in VIGS technology have addressed previous limitations, including genotype dependency and variable efficiency. The modified ALSV (Apple Latent Spherical Virus) VIGS system, for instance, successfully silenced genes in 9 of 19 soybean genotypes tested, with two genotypes showing 100% silencing efficiency [6]. This system also demonstrated minimal viral symptoms, reducing potential interference with phenotypic observations [6]. Similar innovations continue to expand the utility of VIGS for high-throughput functional genomics.

For soybean researchers, VIGS provides a rapid validation tool for candidate genes identified through transcriptomic studies or genome-wide associations [3] [4]. When integrated with emerging technologies like CRISPR/Cas genome editing—which faces challenges in soybean transformation efficiency—VIGS enables preliminary functional assessment before committing to lengthy stable transformation efforts [5].

Future directions in VIGS technology will likely focus on expanding host range compatibility, increasing silencing duration, and enhancing tissue specificity. The development of novel viral vectors from viruses with specialized tropism may enable tissue-specific silencing in roots, flowers, or seeds, addressing current limitations in spatial control of gene knockdown [6]. As these technologies mature, VIGS will continue to be an essential component of the plant functional genomics toolkit, particularly for crop species where traditional transformation remains challenging.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for functional genomics in plants, enabling rapid analysis of gene function without the need for stable transformation. Among the various viral vectors developed, Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) represent two highly advanced systems with distinct architectural frameworks and operational mechanisms. TRV, a tobravirus, and BPMV, a comovirus, both possess bipartite RNA genomes but differ fundamentally in their genomic organization, silencing trigger mechanisms, and host applicability. This guide provides an objective comparison of these two VIGS vector systems, focusing on their structural components, operational efficiencies, and practical implementation in legume research, particularly in soybean. Understanding these architectural differences is crucial for researchers selecting the optimal system for specific functional genomics applications, as each vector offers unique advantages for gene silencing, protein expression, and high-throughput screening.

Comparative Vector Architecture

TRV Bipartite Genome Organization

The TRV genome consists of two positive-sense single-stranded RNA molecules: TRV1 (RNA1) and TRV2 (RNA2). TRV1 (approximately 6.8 kb) encodes essential proteins for viral replication (134K and 194K replicases), movement (29K movement protein), and a silencing suppressor (16K protein). TRV2 (approximately 3.9 kb) typically encodes the coat protein (CP) and varies among TRV strains. For VIGS applications, TRV2 is engineered to replace CP with multiple cloning sites (MCS) for inserting target gene fragments, driven by a duplicated promoter, often the pea early browning virus (pPEBV) promoter [8]. The recent innovation of incorporating a tRNAIleu sequence downstream of the insert has been shown to enhance systemic TRV movement and transmission of edited alleles to subsequent generations [8]. TRV can be delivered via Agrobacterium tumefaciens carrying binary vectors containing cDNA copies of TRV1 and modified TRV2 under Cauliflower Mosaic Virus (CaMV) 35S promoters [3].

BPMV Bipartite Genome Organization

BPMV also possesses a bipartite genome of positive-sense single-stranded RNA but belongs to the comovirus family. BPMV RNA1 (approximately 6 kb) encodes a polyprotein processed into proteins necessary for replication and proteolysis [9] [10]. BPMV RNA2 (approximately 3.6 kb) encodes a polyprotein cleaved into movement protein (MP) and coat protein (CP) subunits [9] [10]. Early BPMV VIGS vectors required in-frame insertion of target sequences between the MP and large CP (L-CP) in the RNA2 polyprotein [10]. Advanced "one-step" BPMV vectors introduced a BamHI restriction site after the RNA2 stop codon, enabling insertion of non-coding/antisense sequences without polyprotein fusion constraints [9] [7]. These DNA-based vectors are driven by CaMV 35S promoters and nopaline synthase (Nos) terminators, permitting direct plasmid inoculation [9].

Performance Comparison in Soybean Research

Silencing Efficiency and Applications

Table 1: Silencing Efficiency and Applications of TRV and BPMV Vectors in Soybean

Parameter	TRV-Based VIGS	BPMV-Based VIGS
Reported Silencing Efficiency	65% to 95% [3]	Well-established but variable (position/orientation dependent) [9]
Key Demonstrated Targets	GmPDS, GmRpp6907, GmRPT4 [3]	GmPDS, GmSHMT, disease resistance genes (Rpp1, Rsc1-DR) [3] [11]
Optimal Insert Orientation	Sense orientation for silencing [3]	Antisense orientation more effective (e.g., for PDS) [9]
Tissue Silencing Capability	Systemic (leaves, roots with optimized protocol) [3]	Systemic (leaves, roots, reproductive tissues) [11] [7]
Multiplexing Capability	Not explicitly reported	Simultaneous expression & silencing in single construct [9]

Practical Implementation and Experimental Workflows

Table 2: Practical Implementation and Experimental Workflows

Parameter	TRV-Based VIGS	BPMV-Based VIGS
Primary Delivery Method	Agrobacterium-mediated (cotyledon node immersion) [3]	Direct plasmid rubbing, biolistic delivery [9] [7]
Infection Timeframe	Silencing phenotypes by 21 dpi [3]	Symptoms by 2-3 weeks; silencing thereafter [11]
Symptom Interference	Minimal viral symptoms [3]	Mild to moderate mosaic symptoms (strain-dependent) [9]
Host Range in Legumes	Soybean (cultivar-specific) [3]	Soybean, common bean (limited cultivar susceptibility) [7]
Throughput Potential	High with optimized Agrobacterium protocol [3]	High with "one-step" plasmid rubbing [7]

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for VIGS Experiments

Reagent/Resource	Function/Application	TRV-VIGS	BPMV-VIGS
Vector Plasmids	Viral genome components for silencing	pTRV1, pTRV2 (with MCS) [3]	pBPMV-IA-R1M, pBPMV-IA-V2 [9] [11]
Agrobacterium Strain	Delivery vector for plant infection	GV3101 [3]	Not typically used for delivery
Plant Cultivars	Susceptible hosts for VIGS	Tianlong 1 [3]	Black Valentine (common bean) [7]
Selection Antibiotics	Plasmid maintenance in bacteria	Kanamycin, Rifampicin [3]	Ampicillin, Spectinomycin [11]
Infection Validation	Monitoring infection efficiency	GFP fluorescence [3]	Visual symptoms, ELISA [9]
Silencing Validation	Confirming target gene knockdown	qPCR, phenotypic assessment [3]	qRT-PCR, northern blot [9]

The architectural differences between TRV and BPMV vectors directly influence their application in soybean functional genomics. TRV's Agrobacterium-mediated delivery and minimal symptom development make it particularly suitable for high-efficiency silencing with reduced viral pathology interference [3]. Conversely, BPMV's direct plasmid rubbing delivery and extensive legacy in legumes provide a robust system for both silencing and protein expression, particularly valuable for root-pathogen interactions like soybean cyst nematode studies [11]. The recent adaptation of TRV for CRISPR-TnpB delivery further expands its utility for transgene-free genome editing [8], while BPMV's capacity for simultaneous multiple gene manipulation offers unique advantages for complex pathway analysis [9]. Researchers should select TRV for maximal silencing efficiency with minimal viral symptoms in compatible cultivars, while BPMV remains ideal for broader legume applications, root studies, and experiments requiring protein expression alongside silencing. Both systems continue to evolve, offering increasingly sophisticated tools for plant functional genomics.

Bean pod mottle virus (BPMV) is a bipartite, positive-sense single-stranded RNA virus belonging to the Secoviridae family and a member of the genus Comovirus [12] [9]. Its genome is divided across two independent RNA molecules, designated RNA1 and RNA2, each encapsidated in separate isometric particles [13]. This divided genome strategy poses a unique challenge for viral replication, as RNA2 must recruit replication proteins encoded by RNA1. Both genomic RNAs are translated into single polyprotein precursors that undergo extensive post-translational processing by viral proteases to produce mature functional proteins [12] [10]. BPMV is not only a significant plant pathogen but also has been engineered as a powerful virus-induced gene silencing (VIGS) vector for functional genomics studies in legumes, particularly soybean (Glycine max) and common bean (Phaseolus vulgaris), which are recalcitrant to stable genetic transformation [9] [7]. This review details the structural composition of the BPMV genome and the processing of its polyproteins, providing a foundational comparison with other VIGS vectors like Tobacco Rattle Virus (TRV).

Table 1: Core Components of the BPMV Genome

Genomic Component	Size	Polyprotein Products (after processing)	Primary Function
RNA1	~6 kb	Protease cofactor (C-Pro), Helicase (Hel), Viral genome-linked protein (VPg), Protease (Pro), RNA-dependent RNA polymerase (RdRP)	Viral replication within the host cell [12]
RNA2	~3.6 kb	58-kDa Protein (P58), Movement Protein (MP), Large Coat Protein (L-CP), Small Coat Protein (S-CP)	Virion assembly, cell-to-cell movement, and RNA2 replication [12]

In-Depth Analysis of BPMV RNA Composition and Polyprotein Processing

RNA1-Encoded Replication Machinery

RNA1 functions as the autonomous replication module of the virus. It encodes a single large polyprotein that is cleaved by the viral protease into at least five mature proteins [12]. The known proteins and their functions are:

• C-Pro (Protease Cofactor): A putative cofactor for the viral protease.
• Hel (Helicase): An RNA helicase that unwinds RNA secondary structures during replication. Specific amino acid residues in the helicase (e.g., positions 359 and 365 in the BPMV IA-Di1 isolate) are critical determinants of symptom severity in infected plants [9].
• VPg (Viral genome-linked protein): A small protein covalently linked to the 5' terminus of both genomic RNA segments [12].
• Pro (Protease): A viral protease responsible for the cleavage and processing of both the RNA1- and RNA2-encoded polyproteins [12] [13].
• RdRP (RNA-dependent RNA Polymerase): The enzyme that catalyzes the replication of viral RNA [12].

A critical feature of the RNA1-encoded polyprotein is that all of its constituent proteins function strictly in cis. This means they are only active for the replication of the RNA1 molecule from which they were translated and cannot be recruited by RNA2 in trans [12].

RNA2-Encoded Proteins for Movement and Structure

RNA2 is dedicated to functions related to viral movement and structure. Its expression strategy is more complex, as translation can initiate from two separate in-frame AUG start codons, producing two overlapping polyproteins [12]. These polyproteins are processed by the RNA1-encoded protease to yield four final protein products:

• P58 (58-kDa Protein): This protein is translated from the upstream AUG start codon. Its N-terminal 102 amino acids are unique and not shared with the MP. Recent studies have unveiled that P58 is essential for the accumulation of RNA2 in infected cells. It functions in cis to recruit the RNA1-encoded replication machinery to RNA2, enabling its replication [12].
• MP (Movement Protein): Translated from the downstream AUG start codon, the MP is largely identical to P58 but lacks the unique N-terminal extension. It is responsible for the cell-to-cell movement of the virus through plasmodesmata [12] [10].
• L-CP (Large Coat Protein) and S-CP (Small Coat Protein): These two proteins form the viral capsid. They are processed from a common precursor derived from both the P58 and MP polyproteins. The L and S subunits assemble into an icosahedral capsid with a pT=3 quasi-symmetry [13]. The C-terminal region of the S-CP is implicated in RNA packaging and capsid assembly but is often cleaved off during maturation and is missing from crystallography data [13].

Diagram 1: BPMV Polyprotein Processing Pathway. The RNA1-encoded protease (Pro) mediates the cleavage of both its own polyprotein and the two RNA2-encoded polyproteins (P58 and MP) into mature functional proteins.

BPMV as a VIGS Vector: Structural Adaptations and Experimental Workflow

BPMV Vector Development and Improvements

The bipartite nature of BPMV has been exploited to develop versatile VIGS vectors. The primary strategy involves engineering the RNA2 component to carry foreign gene fragments. Early BPMV vectors required the insertion of foreign sequences in-frame between the MP and L-CP coding regions within the RNA2 polyprotein [10]. This design imposed significant constraints, as the inserted sequence had to be an open reading frame (ORF), and the resulting translated peptide could cause unintended phenotypes [9].

To overcome these limitations, a more advanced "one-step" BPMV vector system was developed. Key improvements included [9] [7]:

• Flexible Insertion Site: A BamHI restriction site was introduced after the stop codon of the RNA2 ORF in the pBPMV-IA-V2 vector. This allows for the insertion of target gene fragments without the requirement for translation as part of the viral polyprotein.
• Broad Application: This modification enables the silencing of non-coding regions, such as gene promoters and untranslated regions (UTRs), and the use of antisense sequences, which were found to be highly effective [9] [14].
• Simplified Inoculation: The vector uses direct rub-inoculation of plasmid DNA under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, bypassing the need for in vitro transcription, biolistic delivery, or Agrobacterium transformation [7].

Key Experimental Protocol for BPMV VIGS

The standard protocol for implementing the one-step BPMV VIGS system in soybean or susceptible common bean cultivars (e.g., Black Valentine) is as follows [9] [7]:

• Vector Construction: A fragment (typically 132-391 bp) of the target endogenous gene is cloned into the BamHI site of the pBPMV-IA-V2 RNA2 vector in an antisense orientation for optimal silencing [7] [14].
• Plant Preparation: Soybean seeds are sown and grown until the primary leaves are fully expanded (approximately 10 days post-germination).
• Inoculum Preparation: A mixture of the pBPMV-IA-R1M (a mutated RNA1 plasmid that induces moderate symptoms for easy tracking) and the recombinant pBPMV-IA-V2 RNA2 plasmid is prepared. The optimal quantity is 5 µg of each plasmid [7].
• Inoculation: The plasmid mixture is applied directly to the primary leaves and gently rubbed onto the leaf surface using a gloved finger or a specialized tool, often in the presence of an abrasive like Carborundum.
• Phenotype Observation: Effective silencing of target genes, such as the marker gene phytoene desaturase (PDS) which causes photobleaching, can be observed in systemic leaves as early as 14 to 21 days post-inoculation (dpi). Silencing can persist for over 7 weeks, affecting leaves, stems, flowers, and roots [14].

Diagram 2: BPMV VIGS Experimental Workflow. The process begins with cloning a fragment of the target gene into the BPMV RNA2 vector and culminates in the systemic silencing of the corresponding endogenous gene in the host plant.

Comparative Analysis: BPMV vs. TRV VIGS Vectors in Soybean Research

While BPMV is a well-established VIGS tool in legumes, the Tobacco Rattle Virus (TRV)-based system is another widely used VIGS vector. A direct comparison is essential for researchers to select the appropriate tool.

Table 2: Comparative Analysis of BPMV and TRV VIGS Vectors in Soybean

Feature	BPMV VIGS Vector	TRV VIGS Vector
Virus Type	Bipartite comovirus (Secoviridae) [9]	Bipartite rod-shaped virus (Virgaviridae) [3]
Host Suitability	Native to legumes; highly adapted to soybean and common bean [9] [7]	Optimized for solanaceous species (e.g., tomato, tobacco); application in soybean is more recent and less established [3] [15]
Infection Method	Direct plasmid DNA rubbing or biolistic delivery [7]	Primarily Agrobacterium tumefaciens-mediated infiltration (agroinoculation) [3]
Silencing Onset & Duration	Onset: ~14 dpi; can persist strongly for over 7 weeks in leaves and flowers [14]	Rapid onset; reported efficiency of 65-95% in soybean, though long-term stability may vary [3]
Tissue Silencing Range	Widespread in leaves, stems, flowers, and roots (though weaker in roots) [14]	Systemic spread with reported silencing in entire plant, including roots, stems, leaves, and flowers [3]
Key Advantage	High efficacy and stability in legumes; "one-step" DNA vector simplifies inoculation [7]	Can induce fewer viral symptoms, potentially minimizing phenotype interference; broad host range [3] [15]
Key Limitation	Limited to legume hosts; viral symptoms can sometimes mask silencing phenotypes	Less optimized for legumes; agroinfiltration of soybean can be challenging due to thick cuticle and dense trichomes [3]

The Scientist's Toolkit: Essential Reagents for BPMV VIGS Experiments

Table 3: Key Research Reagent Solutions for BPMV VIGS

Reagent / Material	Function / Application	Example / Note
pBPMV-IA-R1M Plasmid	A mutated RNA1 component that induces moderate symptoms for easy infection tracking without ELISA [9] [7]	Contains Asn (N) mutations at positions 359 and 365 of the helicase domain [9]
pBPMV-IA-V2 Plasmid	The RNA2 VIGS vector with a multiple cloning site after the stop codon for flexible insert cloning [9]	Contains a BamHI site for insert ligation [9]
Soybean Cultivars	Susceptible hosts for BPMV infection and VIGS studies.	Cultivars like 'Williams 82' and 'Jack' are commonly used [14]
Common Bean Cultivar	A susceptible host for BPMV studies in common bean.	'Black Valentine' is the primary cultivar used [7]
GmPDS / PvPDS Gene	A marker gene used to optimize and validate VIGS efficiency; silencing causes white photobleaching [9] [7]	Phytoene desaturase is involved in carotenoid biosynthesis [9]
GFP Transgenic Soybean Line	A research tool for quantitatively assessing spatial and temporal silencing patterns [14]	Soybean (Jack cultivar) expressing GFP under the G. max ubiquitin promoter [14]

The BPMV VIGS vector is a sophisticated tool built upon a well-understood viral genome architecture. Its bipartite RNA composition and specific polyprotein processing pathway are not only fundamental to its natural life cycle but have also been ingeniously repurposed for functional genomics. The development of the "one-step" vector, which allows for high-throughput silencing in legumes, is a direct result of elucidating the function of proteins like P58 and re-engineering the RNA2 component. When compared to the TRV system, BPMV demonstrates clear superiority for use in its native legume hosts, particularly soybean, due to its high infection efficiency and sustained silencing. The continued refinement of BPMV vectors, informed by a deep understanding of its molecular biology, ensures its place as an indispensable reagent in the plant researcher's toolkit.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics technique that exploits the plant's natural RNA-mediated antiviral defense mechanism for functional gene analysis. This process represents a form of post-transcriptional gene silencing (PTGS) that allows researchers to transiently knock down target gene expression without the need for stable transformation [2]. The fundamental principle involves using recombinant viral vectors to deliver fragments of plant genes, triggering sequence-specific degradation of complementary mRNA transcripts throughout the plant system [16] [17].

The application of VIGS is particularly valuable in soybean research, where traditional stable transformation remains time-consuming and laborious [3]. As a high-throughput alternative, VIGS enables rapid functional characterization of genes involved in agronomically important traits, including disease resistance and stress tolerance [2]. Among the various viral vectors developed for VIGS, Tobacco rattle virus (TRV) and Bean pod mottle virus (BPMV) have emerged as two prominent systems for soybean functional genomics, each with distinct mechanistic features and experimental advantages [3] [18].

This article provides a comprehensive comparison of TRV and BPMV VIGS systems, examining their molecular mechanisms from double-stranded RNA formation to targeted mRNA degradation. We present structured experimental data and detailed methodologies to guide researchers in selecting the appropriate vector system for specific research applications in soybean.

Molecular Mechanisms of VIGS: From Viral Infection to Gene Silencing

The Core Silencing Pathway

The molecular mechanism of VIGS represents a hijacked antiviral defense pathway that begins with viral infection and culminates in targeted mRNA degradation. The process initiates when recombinant viral vectors are introduced into plant cells through various delivery methods, including Agrobacterium-mediated transformation or biolistic delivery [16]. Once inside the cell, the viral genome containing the inserted plant gene fragment begins to replicate, forming double-stranded RNA intermediates through the activity of viral or host RNA-dependent RNA polymerases [2].

These dsRNA molecules are recognized as aberrant by the plant's defense system and are cleaved by Dicer-like enzymes into small interfering RNAs of 21-24 nucleotides in length [16]. These siRNAs are then incorporated into the RNA-induced silencing complex, where they serve as guides for identifying complementary mRNA sequences [2] [16]. The RISC complex subsequently degrades target mRNAs, leading to effective knockdown of the corresponding gene [16]. The silencing signal spreads systemically throughout the plant, enabling whole-plant functional analysis [16].

Vector-Specific Modifications to the Core Pathway

While TRV and BPMV both operate through this fundamental pathway, each vector system incorporates specific modifications that influence their silencing efficiency and experimental applications. TRV-based vectors utilize a bipartite system where RNA1 encodes replicase and movement proteins, while RNA2 carries the coat protein and the insert fragment [16] [17]. This separation allows for stable maintenance of foreign inserts and efficient systemic movement, including meristem invasion [16].

In contrast, BPMV vectors are also bipartite but typically engineered with the insert positioned between the movement protein and large coat protein in RNA2, with additional proteinase cleavage sites to ensure proper processing of the polyprotein [10]. BPMV may induce stronger viral symptoms compared to TRV, which can potentially interfere with phenotypic interpretation [3]. Both systems have been optimized for soybean through codon modification and the incorporation of appropriate promoters and terminators to enhance stability and expression [3] [10].

Comparative Analysis of TRV and BPMV VIGS Systems in Soybean

Efficiency and Performance Metrics

Direct comparison of TRV and BPMV VIGS systems reveals distinct performance characteristics that influence their suitability for different research applications. The table below summarizes key quantitative metrics derived from experimental studies in soybean.

Table 1: Performance Comparison of TRV and BPMV VIGS Systems in Soybean

Parameter	TRV-Based System	BPMV-Based System
Silencing Efficiency	65-95% [3]	High (quantitative data not specified in sources)
Time to Silencing Phenotype	21 days post-inoculation [3]	2-3 weeks post-inoculation [18]
Silencing Duration	Several weeks [2]	Several weeks, stable through serial passages [10]
Infection Efficiency	>80% (up to 95% in Tianlong 1) [3]	92-100% (BPMV-Wt in common bean) [19]
Primary Inoculation Method	Agrobacterium-mediated (cotyledon node) [3]	Biolistic or direct DNA rubbing [18] [19]
Systemic Movement	Throughout plant, including meristems [16]	Throughout plant, including roots [18]
Viral Symptom Severity	Mild [3] [16]	Moderate to severe (mosaic patterns) [18]
Key Applications	Disease resistance genes, defense studies [3]	Nematode parasitism, root-microbe interactions [18]

Technical and Practical Considerations

Beyond efficiency metrics, several technical factors influence vector selection for specific experimental needs. TRV vectors benefit from Agrobacterium-mediated delivery through cotyledon nodes, which provides high transformation efficiency and avoids specialized equipment [3]. The optimized protocol involves bisecting swollen sterilized soybeans to create half-seed explants, which are then immersed in Agrobacterium suspensions for 20-30 minutes [3]. This method overcomes challenges posed by soybean's thick cuticle and dense trichomes that impede conventional infiltration methods.

BPMV systems traditionally relied on biolistic delivery using gold particles coated with viral DNA [18], though simplified mechanical inoculation methods using direct rubbing of plasmid DNA have been developed [19]. The BPMV protocol requires co-delivery of RNA1 and RNA2 components, with infected leaf tissue serving as inoculum for subsequent rounds of infection [18]. While potentially more technically demanding, BPMV offers particular advantages for root studies, including functional analysis of genes involved in soybean cyst nematode interactions [18].

Experimental Protocols for VIGS in Soybean

TRV-Mediated VIGS Protocol

The TRV-VIGS system has been optimized for soybean through Agrobacterium-mediated infection of cotyledon nodes. The following protocol details the established methodology:

Vector Construction: Clone target gene fragments (300-500 bp) into the pTRV2 vector using appropriate restriction enzymes (EcoRI and XhoI) or recombination-based cloning systems [3]. Select insert sequences with efficient siRNA generation potential and minimal off-target effects using bioinformatics tools [2]. The constructed vector is then transformed into Agrobacterium tumefaciens GV3101 for plant infection [3].

Plant Material Preparation: Surface-sterilize soybean seeds and germinate under sterile conditions. For infection, use half-seed explants obtained by longitudinally bisecting swollen sterilized soybeans [3]. This approach significantly improves infection efficiency compared to conventional methods due to better Agrobacterium access.

Agroinfiltration: Harvest healthy soybean leaves to extract cDNA template for amplification of target gene fragments [3]. Prepare Agrobacterium cultures containing pTRV1 and recombinant pTRV2 vectors, adjusting to optimal density (OD600 = 1.0-2.0). Infect fresh cotyledon node explants by immersion in Agrobacterium suspensions for 20-30 minutes—determined to be the optimal duration for efficient transformation [3].

Plant Growth and Silencing Verification: Co-cultivate infected explants for 2-3 days before transferring to selective media. Monitor fluorescence using GFP-tagged vectors around day 4 post-infection to assess transformation efficiency [3]. Evaluate silencing phenotypes beginning at 21 days post-inoculation, with photobleaching evident in GmPDS-silenced plants [3]. Confirm silencing at molecular level through qRT-PCR analysis of target gene expression.

BPMV-Mediated VIGS Protocol

The BPMV-VIGS protocol employs different delivery methods optimized for soybean and common bean:

Inoculum Preparation: For biolistic delivery, coat gold particles (1μm) with 5μg DNA mixture of BPMV RNA1 and RNA2 vectors using calcium chloride and spermidine precipitation [18]. Alternatively, for mechanical inoculation, combine 5μg each of pBPMV-IA-R1M and recombinant RNA2 plasmid in 20μl of 50mM potassium phosphate buffer (pH 7) [19].

Plant Infection: For biolistic delivery, bombard unifoliate leaves of 7-day-old soybean seedlings using a PDS-1000/He system with 1100 psi rupture disks [18]. For mechanical inoculation, dust primary leaves with carborundum abrasive, apply DNA mixture, and rub gently across leaf surface before rinsing with water [19]. Maintain inoculated plants at 20°C to optimize viral replication and movement [18].

Tissue Harvest and Secondary Inoculation: Harvest leaves showing viral symptoms (typically appearing 2-3 weeks post-inoculation), lyophilize, and store at -20°C for future use as inoculum [18]. For secondary inoculation, grind infected leaf tissue in potassium phosphate buffer and use the sap for mechanical inoculation of new plants [19].

Silencing Assessment: Monitor viral symptoms including mosaic patterns and leaf blistering beginning 2-3 weeks post-inoculation [18]. For functional studies, evaluate phenotypes specific to target gene silencing and verify at molecular level through transcript analysis.

The Scientist's Toolkit: Essential Research Reagents

Successful implementation of VIGS requires specific biological materials and reagents optimized for soybean systems. The following table details key components for establishing TRV and BPMV VIGS protocols.

Table 2: Essential Research Reagents for Soybean VIGS Studies

Reagent/Resource	Function/Purpose	Specific Examples/Notes
TRV Vector System	Bipartite silencing vector	pTRV1 (replication proteins), pTRV2 (coat protein + insert) [16]
BPMV Vector System	Bipartite silencing vector	pBPMV-IA-R1M (RNA1), pBPMV-IA-V1 (RNA2) [19]
Agrobacterium Strain	Vector delivery for TRV	GV3101 with pTRV1 and recombinant pTRV2 [3]
Plant Genotype	Soybean cultivars	Tianlong 1 for TRV (95% efficiency) [3]; Specific lines for BPMV (EXF67, EXF63) [18]
Selection Markers	Bacterial and plant selection	Antibiotic resistance genes in vectors [3] [18]
Infection Accessories	Mechanical inoculation	Carborundum (abrasive), potassium phosphate buffer [19]
Visualization Tools	Monitoring infection	GFP marker, UV lamp for detection [19]
Positive Controls	System validation	PDS silencing (photobleaching) [3] [19]

Applications and Future Perspectives in Soybean Research

Established Applications in Soybean Functional Genomics

Both TRV and BPMV VIGS systems have been successfully employed to characterize genes involved in key agronomic traits in soybean. The TRV system has demonstrated efficacy in silencing disease resistance genes including the rust resistance gene GmRpp6907 and defense-related gene GmRPT4, confirming its robustness for disease resistance research [3]. The system's high efficiency (65-95%) and rapid phenotypic manifestation (within 21 days) enable rapid screening of candidate genes [3].

The BPMV system has been particularly valuable for studying root-pathogen interactions, including functional analysis of genes involved in soybean cyst nematode parasitism [18]. The stability of BPMV vectors through serial passages and efficient systemic movement make it suitable for long-term studies [10]. BPMV has also been used to characterize genes conferring resistance to soybean mosaic virus, such as the recently identified Gm18GRSC3 gene [20].

Emerging Trends and Integration with Novel Technologies

The future of VIGS in soybean research lies in its integration with emerging genomic technologies. Combination with CRISPR-based systems may enable more precise functional characterization, while advances in viral vector design continue to expand host range and silencing efficiency [17]. The development of satellite-virus-based systems offers potential for enhanced silencing with reduced viral symptom interference [2] [17].

Environmental optimization represents another frontier, with research indicating that conditions favoring viral multiplication (specific temperatures, humidity levels) can extend silencing duration and enhance efficiency [2] [17]. As soybean genomic resources continue to expand, VIGS will play an increasingly critical role in bridging the gap between gene sequence information and biological function, ultimately accelerating the development of improved soybean cultivars with enhanced disease resistance and stress tolerance.

Historical Development and Adoption of VIGS in Legume Research

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional genomics studies in plants. This technology exploits the plant's natural RNA-mediated antiviral defense mechanism to silence target genes by expressing homologous sequences from viral vectors [16]. In legume research, VIGS has become particularly valuable due to the recalcitrance of many legume species, including soybean (Glycine max L.) and common bean (Phaseolus vulgaris L.), to stable genetic transformation [3] [7]. The establishment of efficient VIGS systems has enabled researchers to circumvent the challenges associated with conventional transformation methods, allowing for high-throughput functional characterization of genes involved in various biological processes, including disease resistance, stress tolerance, and development.

Among the various VIGS vectors developed for legume research, the Bean pod mottle virus (BPMV) and Tobacco rattle virus (TRV) have emerged as the most prominent systems. This review provides a comprehensive comparison of the historical development and adoption of these two VIGS vector systems in legume research, with a specific focus on their applications in soybean. We examine their relative advantages, limitations, and experimental performance data to provide researchers with evidence-based guidance for selecting appropriate VIGS tools for their functional genomics studies.

Historical Development of VIGS Vectors in Legumes

Bean Pod Mottle Virus (BPMV) Vectors

BPMV, a positive-strand RNA virus of the genus Comovirus, was among the first viral vectors to be successfully developed for VIGS applications in legumes. The development of BPMV-based vectors began in the early 2000s, with initial constructs designed for stable protein expression and sequence-specific gene silencing in soybean [10]. The BPMV genome consists of two RNA molecules: RNA1 (approximately 6 kb) and RNA2 (approximately 3.6 kb), each expressed as polyprotein precursors that undergo proteolytic processing to yield mature viral proteins [9] [10].

The evolution of BPMV vectors has progressed through three generations, each offering significant improvements over its predecessor. The first-generation vectors required the insertion of foreign sequences in-frame between the movement protein (MP) and large coat protein subunit (L-CP) in the RNA2 polyprotein [10]. This design imposed significant constraints, as VIGS target sequences had to be expressed as fusion proteins in the same reading frame as the viral polyprotein, limiting the approach to open reading frames [9].

Second-generation BPMV vectors incorporated a DNA-based system in which BPMV RNA1 and RNA2 were placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (Nos) terminator [9]. This modification enabled direct inoculation of soybean plants with vector DNA, eliminating the need for in vitro RNA transcription and enhancing the utility of BPMV for large-scale functional genomics experiments [9].

The third-generation BPMV vectors, termed "one-step" vectors, represented a significant advancement by introducing a BamHI restriction site after the translation stop codon of RNA2 [9] [7]. This innovation eliminated the requirement for cloning foreign sequences in the same reading frame as the RNA2 polyprotein, allowing for the insertion of antisense and noncoding sequences. These vectors enabled simultaneous expression of multiple foreign genes, simultaneous expression and silencing, and marker gene-assisted silencing [9]. Furthermore, their delivery via direct rub-inoculation of infectious plasmid DNA made them ideal for high-throughput applications [7].

Tobacco Rattle Virus (TRV) Vectors

TRV, a positive-sense RNA virus, has been widely adopted as a VIGS vector in numerous plant species, including members of the Solanaceae family [16]. However, its application in legumes, particularly soybean, has been relatively limited until recently. The TRV genome consists of RNA1 and RNA2, with RNA1 encoding replicases and movement proteins, and RNA2 encoding the coat protein and other non-essential proteins that can be replaced with foreign sequences [3] [16].

The development of TRV vectors for legume research has progressed more slowly compared to BPMV. Early TRV vectors were constructed as separate cDNA clones of TRV RNA1 and RNA2 under the control of CaMV 35S promoters on the T-DNA of plant binary transformation vectors [16]. Subsequent modifications included the introduction of self-cleaving ribozymes and the development of Gateway-compatible vectors to facilitate easier cloning [16].

Recent optimization efforts have focused on adapting TRV vectors for efficient use in soybean. A key advancement has been the development of an Agrobacterium tumefaciens-mediated infection system through cotyledon nodes, which facilitates systemic spread and effective silencing of endogenous genes [3]. This approach has demonstrated silencing efficiencies ranging from 65% to 95% in soybean, making TRV a competitive alternative to BPMV for functional genomics studies [3].

Table 1: Historical Development of BPMV and TRV Vectors in Legume Research

Vector Generation	BPMV Vectors	TRV Vectors
First Generation	Insertion between MP and L-CP in RNA2 polyprotein; in vitro transcription required [10]	Separate cDNA clones of RNA1 and RNA2 under CaMV 35S promoters; agroinfiltration [16]
Second Generation	DNA-based system with CaMV 35S promoter; direct plasmid inoculation [9]	Gateway-compatible vectors; simplified cloning [16]
Third Generation	"One-step" vectors with insertion after RNA2 stop codon; simultaneous expression/silencing [9] [7]	Agroinfiltration through cotyledon nodes; high-efficiency silencing in soybean [3]

Comparative Analysis of BPMV and TRV Vectors

Silencing Efficiency and Dynamics

Both BPMV and TRV vectors have demonstrated effective gene silencing in legumes, but with differing efficiencies and dynamics. Recent studies with TRV-based vectors in soybean have reported silencing efficiencies ranging from 65% to 95% for endogenous genes, including phytoene desaturase (GmPDS), the rust resistance gene GmRpp6907, and the defense-related gene GmRPT4 [3]. The optimized TRV-VIGS system utilizing Agrobacterium-mediated infection through cotyledon nodes enables systemic spread throughout the plant, resulting in significant phenotypic changes [3].

BPMV vectors have also demonstrated high silencing efficiency in soybean and common bean. Studies using the third-generation BPMV vectors showed that antisense insertion of the 3' ORF of PDS induced the most effective silencing, with the third and fourth trifoliolates of infected plants showing almost complete bleaching [9]. Comparative analysis of insert positions and orientations revealed that the 3' end insertion was more effective for PDS VIGS, particularly in the antisense orientation [9].

Table 2: Comparison of Silencing Efficiency Between BPMV and TRV Vectors

Parameter	BPMV Vectors	TRV Vectors
Silencing Efficiency	Effective silencing with optimized insert position and orientation [9]	65-95% for endogenous genes in soybean [3]
Time to Silencing	3-4 weeks post-inoculation [9] [7]	21 days post-inoculation for visible phenotypes [3]
Tissue Coverage	Systemic spread including leaves, stems, and roots [7]	Systemic spread throughout plant, including meristems [3] [16]
Insert Position Effect	3' ORF in antisense orientation most effective [9]	Dependent on insert size and homology [3]

Symptom Severity and Phenotypic Interference

A critical consideration in selecting a VIGS vector is the severity of viral symptoms, which can interfere with the interpretation of silencing phenotypes. BPMV vectors have been engineered to minimize this issue through the use of mild viral strains. The BPMV isolate IA-Di1 induces mild symptoms, making it suitable as a vector [9]. Furthermore, mutation of specific amino acids in the helicase protein (positions 359 and 365) resulted in a modified clone (pBPMV-IA-R1M) that induces obvious but moderate symptoms, allowing for easy identification of infected plants without severe phenotypic interference [9].

TRV vectors are particularly valued for inducing minimal viral symptoms compared to other viruses, thereby reducing potential masking of silencing phenotypes [3] [16]. The TRV-VIGS system elicits fewer symptoms, preventing harm to the plants and minimizing interference with functional studies [3]. This characteristic makes TRV particularly advantageous for studying subtle phenotypes or genes involved in plant development.

Host Range and Genotype Compatibility

The host range and genotype compatibility differ significantly between BPMV and TRV vectors. BPMV-based vectors have been successfully used in both soybean and common bean [9] [7]. However, susceptibility to BPMV varies among common bean cultivars, with only certain genotypes like Black Valentine and JaloEEP558 showing susceptibility [7]. This limited host range can constrain the application of BPMV vectors in certain legume species or specific genotypes.

TRV vectors have an exceptionally broad host range, infecting plants in 50 or more families including Solanaceae, Cruciferae, and Gramineae [16]. This wide compatibility makes TRV a versatile tool for functional genomics across diverse plant species. However, reports on the use of TRV-mediated VIGS for functional gene studies in soybean have been limited until recently [3]. The newly developed TRV-VIGS system for soybean demonstrates that this vector can be effectively adapted for legume research, potentially expanding its applications in this important plant family [3].

Experimental Protocols and Methodological Advances

BPMV Inoculation Methods

The development of efficient inoculation methods has been crucial for the adoption of VIGS in legume research. BPMV vectors have seen significant advancements in this area. The earliest BPMV vectors required in vitro transcription and mechanical inoculation of RNA transcripts [10]. Second-generation vectors incorporated biolistic delivery of infectious plasmid DNA [9], while third-generation "one-step" vectors enabled direct rub-inoculation of infectious plasmid DNA onto soybean plants [9] [7].

For common bean, optimal conditions for direct rub-inoculation of infectious BPMV-derived plasmids have been established. Studies have determined that using 5 μg each of RNA1- and RNA2-derived plasmids results in 92%-100% infection rates in susceptible cultivars like Black Valentine [7]. This efficient and simplified inoculation method has made BPMV vectors practical for high-throughput functional genomics studies in legumes.

Diagram 1: BPMV VIGS Experimental Workflow. This diagram illustrates the key steps in implementing the BPMV-based VIGS system, from vector design to molecular confirmation of silencing.

TRV Inoculation Methods

TRV vector inoculation has traditionally relied on Agrobacterium-mediated transformation through leaf infiltration [16]. However, recent methodological advances have optimized TRV delivery for soybean, which has proven challenging due to its thick cuticle and dense trichomes that impede liquid penetration [3].

An efficient TRV-VIGS protocol for soybean utilizes Agrobacterium tumefaciens-mediated infection through cotyledon nodes [3]. The optimized procedure involves:

• Soaking sterilized soybeans in sterile water until swollen
• Longitudinal bisecting to obtain half-seed explants
• Infecting fresh explants by immersion for 20-30 minutes in Agrobacterium suspensions containing either pTRV1 or pTRV2-GFP derivatives
• Culturing infected explants under sterile conditions

This method achieves transformation efficiencies exceeding 80%, reaching up to 95% for specific soybean genotypes like Tianlong 1 [3]. Fluorescence microscopy confirms successful infection, with more than 80% of cells exhibiting fluorescence signals in transverse sections [3].

Diagram 2: TRV VIGS Experimental Workflow for Soybean. This diagram outlines the optimized TRV-VIGS protocol for soybean, highlighting the key steps from vector construction to silencing analysis.

Essential Research Reagents and Solutions

Successful implementation of VIGS in legume research requires specific reagents and solutions optimized for each vector system. The following table details key research reagent solutions essential for conducting VIGS experiments with BPMV and TRV vectors.

Table 3: Essential Research Reagents for VIGS Experiments in Legumes

Reagent/Solution	Composition/Type	Function	Vector System
Binary Vectors	pBPMV-IA-R1M (RNA1) and pBPMV-IA-V2 (RNA2) for BPMV; pTRV1 and pTRV2 for TRV	Viral genome components for VIGS	Both [3] [9]
Agrobacterium Strain	GV3101 for TRV; C58C1 for some BPMV constructs	Delivery of viral vectors into plant cells	Both [3] [6]
Infiltration Medium	10 mM MgCl₂, 10 mM MES (pH 5.7), 100 μM acetosyringone	Resuspension of Agrobacterium for infiltration	TRV [3] [6]
Inoculation Buffer	For direct DNA rubbing: Carborundum in inoculation buffer	Facilitates mechanical delivery of plasmids	BPMV [7]
Selection Antibiotics	Kanamycin (50 μg/ml), rifampicin (50 μg/ml), gentamycin (50 μg/ml)	Selection of transformed Agrobacterium	Both [6]
Co-cultivation Medium	1/2 strength MS medium, 2% sucrose, 200 μM acetosyringone, growth regulators	Supports plant tissue recovery after infiltration	Both [21]

Applications in Legume Functional Genomics

VIGS has been extensively applied in legume functional genomics, with both BPMV and TRV vectors contributing significantly to gene function characterization. BPMV-based vectors have been particularly valuable for studying disease resistance mechanisms in soybean. Notable applications include:

• Investigation of soybean cyst nematode parasitism [3]
• Demonstration that BPMV-induced silencing of Rpp1 compromised soybean rust immunity [3]
• Identification of the Rsc1-DR gene conferring resistance to soybean mosaic virus strain SC1 (SMV-SC1) [3]
• Validation of the role of Rbs1 in conferring resistance to brown stem rot (BSR) in soybean [3]

TRV vectors, while more recently applied in soybean research, have demonstrated robust functionality in silencing key genes, including:

• Successful silencing of GmPDS resulting in visible photobleaching [3]
• Effective silencing of the rust resistance gene GmRpp6907 [3]
• Silencing of the defense-related gene GmRPT4 [3]

The establishment of a highly efficient TRV-VIGS platform for rapid gene function validation in soybean provides a valuable tool for future genetic and disease resistance research [3].

The historical development and adoption of VIGS in legume research have transformed functional genomics studies in economically important crops like soybean and common bean. Both BPMV and TRV vector systems have undergone significant refinements, resulting in highly efficient tools for gene function analysis.

BPMV vectors offer the advantage of direct DNA inoculation, minimal viral symptoms with mild strains, and proven efficacy in both soybean and common bean. The "one-step" BPMV system represents a mature technology optimized for high-throughput applications. In contrast, TRV vectors provide a broader host range, minimal symptom development, and recent methodological advances have enabled highly efficient silencing in soybean through optimized Agrobacterium-mediated delivery.

Selection between these systems depends on specific research requirements, including target legume species, available resources, and experimental objectives. BPMV remains the well-established choice for soybean and compatible common bean genotypes, while TRV offers expanding capabilities with potentially broader applications across diverse legume species. Both systems continue to evolve, promising enhanced utility for functional genomics and accelerating the development of improved legume cultivars with enhanced agronomic traits.

Practical Implementation: Protocols and Applications in Soybean Research

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional analysis of plant genes. In soybean research, the choice of viral vector and delivery method significantly impacts experimental success. While Bean pod mottle virus (BPMV)-based vectors have been widely used in soybean functional genomics, Tobacco rattle virus (TRV)-based systems offer distinct advantages despite historically limited application in legumes [4] [3]. Recent methodological advances have established highly efficient TRV delivery through Agrobacterium-mediated cotyledon node infection, providing a robust alternative to conventional approaches [4]. This cotyledon-based VIGS method enables systemic silencing throughout the plant with efficiency ranging from 65% to 95%, facilitating rapid validation of candidate genes involved in disease resistance and other agronomic traits [4] [3] [22]. This guide objectively compares this emerging TRV delivery method with established BPMV protocols, providing experimental data and implementation details to support researchers in selecting appropriate VIGS strategies for soybean functional genomics.

TRV vs. BPMV Vectors: Technical Comparison

The strategic selection of an appropriate VIGS vector is fundamental to experimental success in soybean functional genomics. TRV and BPMV represent distinct vector systems with characteristic strengths and limitations, detailed in Table 1.

Table 1: Technical Comparison of TRV and BPMV VIGS Vectors in Soybean

Parameter	TRV-Based VIGS System	BPMV-Based VIGS System
Delivery Method	Agrobacterium-mediated cotyledon node infection [4]	Particle bombardment [11] or direct rub-inoculation [7]
Infection Efficiency	65-95% silencing efficiency [4] [3]	High efficiency in susceptible cultivars [7]
Silencing Onset	Photobleaching at 21 days post-inoculation (dpi) [4]	Symptoms typically visible 2-3 weeks post-inoculation [11]
Systemic Movement	Effective systemic spread from cotyledon nodes [4]	Systemic infection of leaves and roots [11]
Viral Symptom Severity	Mild symptoms, minimal phenotype interference [4]	Mild mosaic symptoms with modern vectors [7]
Host Range Flexibility	Broad host range across plant species [23] [22]	Primarily legumes (soybean, common bean) [7]
Technical Complexity	Requires sterile tissue culture techniques [4]	Requires biolistic equipment or optimization of rubbing parameters [7] [11]
Suitable for Root Studies	Demonstrated effectiveness for root pathogens [11]	Protocol developed for SCN studies [11]
Key Advantages	Simplified Agrobacterium delivery, minimal equipment needs [4]	Well-established system with extensive published data [7]

The primary distinction between these systems lies in their delivery mechanisms. The TRV system utilizes Agrobacterium-mediated transfer of T-DNA carrying viral components, while traditional BPMV approaches often rely on particle bombardment [4] [11]. This fundamental difference impacts equipment requirements, technical expertise, and scalability. The recently optimized TRV cotyledon node method achieves high efficiency without biolistic instrumentation, making it more accessible to laboratories without specialized equipment [4].

Regarding symptomology, TRV vectors typically induce milder viral symptoms compared to earlier BPMV isolates, reducing potential interference with phenotypic observations [4]. Modern BPMV vectors derived from the IA-Di1 isolate have addressed this concern through reduced symptom severity [7]. Both systems demonstrate effective systemic movement capable of silencing genes in vegetative tissues and roots, which is particularly valuable for studying soil-borne pathogens like soybean cyst nematode [11].

TRV Cotyledon Node Infection: Experimental Workflow

The TRV-mediated cotyledon node infection method employs an optimized, sterile tissue culture-based protocol that ensures high infection rates and consistent silencing efficacy [4]. The complete experimental workflow is visually summarized in Figure 1.

Figure 1: Experimental workflow for TRV-mediated cotyledon node infection in soybean

Critical Protocol Steps

• Vector Construction: The pTRV2 vector is engineered to carry target gene fragments (typically 132-391 bp) between the EcoRI and XhoI restriction sites, while pTRV1 contains essential viral replication components [4]. Recombinant plasmids are transformed into Agrobacterium tumefaciens GV3101 for plant delivery.

• Explant Preparation: Surface-sterilized soybean seeds are imbibed in sterile water for 5-6 hours until swollen, then longitudinally bisected to create half-seed explants containing cotyledonary nodes [4]. This exposure of the meristematic tissue is crucial for successful infection.

• Agroinoculation: Fresh explants are immersed in Agrobacterium suspension for 20-30 minutes—determined to be the optimal duration for infection [4]. The cotyledon node's high meristematic activity facilitates efficient viral uptake and subsequent systemic spread.

• Confirmation of Infection: By 4 days post-infection (dpi), fluorescence microscopy reveals successful infection through GFP signals, with transverse sections showing >80% cell infiltration efficiency [4]. This verification step ensures only properly infected seedlings advance further.

Key Performance Data and Efficiency Metrics

Rigorous evaluation of the TRV cotyledon node method demonstrates its efficacy through both phenotypic and molecular assessments. Quantitative performance metrics are summarized in Table 2.

Table 2: Efficiency Metrics for TRV Cotyledon Node VIGS in Soybean

Evaluated Parameter	Performance Result	Experimental Details
Overall Silencing Efficiency	65-95% [4] [3]	Across multiple target genes and soybean cultivars
Cell Infection Rate	>80% of cells in transverse section [4]	GFP fluorescence observation at 4 dpi
Gene Silencing Validation	Successful silencing of GmPDS, GmRpp6907, GmRPT4 [4]	Phenotypic observation and expression analysis
PDS Silencing Phenotype	Photobleaching visible at 21 dpi [4]	Initially appears in cluster buds
Cultivar-Dependent Efficiency	Up to 95% for Tianlong 1 cultivar [4]	Variation observed across different genotypes
Tissue Culture Success Rate	High regeneration potential from cotyledon nodes [4]	Sterile technique critical for success

The system's effectiveness was confirmed through silencing of phytoene desaturase (GmPDS), which resulted in characteristic photobleaching in leaves inoculated with pTRV:GmPDS at 21 dpi, while controls showed no such phenotype [4]. This visible marker provides straightforward phenotypic validation of silencing efficiency.

Beyond marker genes, the system has successfully silenced disease resistance genes including the rust resistance gene GmRpp6907 and defense-related gene GmRPT4, confirming its utility for studying disease resistance mechanisms [4]. The robust silencing of these endogenous genes demonstrates the method's applicability to functional studies of agronomically important traits.

The Scientist's Toolkit: Essential Research Reagents

Implementation of the TRV cotyledon node VIGS method requires specific biological materials and reagents detailed in Table 3.

Table 3: Essential Research Reagents for TRV Cotyledon Node VIGS

Reagent/Resource	Specification/Function	Application Notes
TRV Vectors	pTRV1 (RNA1 component) and pTRV2-GFP with MCS [4]	pTRV2 contains multiple cloning site for target gene insertion
Agrobacterium Strain	GV3101 with helper plasmids [4] [22]	Optimal for soybean transformation
Soybean Cultivars	Tianlong 1 (95% efficiency) [4]	Cultivar-dependent efficiency observed
Restriction Enzymes	EcoRI and XhoI for vector construction [4]	For cloning target fragments into pTRV2
Selection Antibiotics	Kanamycin, rifampicin, gentamicin [4]	For bacterial and plant selection
Tissue Culture Media	Induction and regeneration media [4]	Composition optimized for soybean
Fluorescence Microscope	GFP detection and verification [4]	Critical for infection efficiency assessment

The TRV-mediated cotyledon node infection method represents a significant advancement in soybean functional genomics, particularly valuable for laboratories seeking to implement VIGS without biolistic equipment. The method's high efficiency (65-95% silencing), systemic gene silencing capability, and minimal viral symptom interference make it particularly suitable for rapid validation of candidate genes involved in disease resistance and stress tolerance [4].

While BPMV remains a well-established and effective VIGS system for soybean, the TRV cotyledon method offers distinct practical advantages in technical accessibility and protocol simplicity. Researchers should select based on available equipment, target cultivars, and specific experimental requirements. The cotyledon-based approach demonstrates the ongoing innovation in plant functional genomics tools, providing researchers with an expanded toolkit for unraveling gene function in this economically vital crop.

Bean pod mottle virus (BPMV) has emerged as a powerful viral vector for virus-induced gene silencing (VIGS) in soybean and common bean, enabling rapid functional analysis of plant genes. As a bipartite positive-sense RNA virus, BPMV is particularly valuable for studying legumes, which are often recalcitrant to stable genetic transformation [24] [7]. The development of BPMV-based vectors has progressed through multiple generations, with significant improvements in delivery methods that enhance efficiency and practicality for research applications. Two primary inoculation techniques—direct plasmid rubbing and particle bombardment—have been optimized to facilitate BPMV infection in plant tissues, each offering distinct advantages for different experimental scenarios. These methodological advances have positioned BPMV as a preferred VIGS system for high-throughput functional genomics in legume species, allowing researchers to overcome traditional transformation bottlenecks [9] [25].

Direct Plasmid Rubbing Methodology

Protocol Optimization

The direct plasmid rubbing method represents a significant simplification in BPMV inoculation, eliminating the need for in vitro transcription or biolistic equipment. This approach utilizes infectious cDNA clones of BPMV RNA1 and RNA2 placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter [9]. The optimized protocol involves mechanical inoculation of plasmid DNA directly onto plant leaves. Key parameters have been systematically optimized to maximize infection efficiency, including plasmid quantity, rubbing intensity, and the number of inoculated primary leaves [7]. Research has demonstrated that using 5 μg each of RNA1- and RNA2-derived plasmids provides optimal infection rates, with studies reporting 92%-100% of plants exhibiting viral symptoms at 28 days post-inoculation (dpi) when using this quantity [7]. This represents a substantial improvement over lower plasmid concentrations (1.5 μg or 3 μg), which achieved only 17%-33% infection rates.

Practical Application

The practical implementation of direct plasmid rubbing involves gently abrading the leaf surface with a mixture containing the plasmid DNA, typically using carborundum as an abrasive agent to facilitate entry without causing excessive tissue damage [7]. The method has proven particularly effective for common bean (Phaseolus vulgaris L.) cultivars such as Black Valentine and JaloEEP558, which show high susceptibility to BPMV infection [7]. One significant advantage of this approach is its suitability for large-scale functional genomics studies, as it bypasses requirements for in vitro transcription, biolistic delivery, or agroinoculation procedures [9]. The direct rubbing method enables efficient VIGS of endogenous genes, as demonstrated successfully with the phytoene desaturase (PDS) gene, where silencing resulted in characteristic photobleaching phenotypes [7] [9].

Particle Bombardment Methodology

Technical Procedure

Particle bombardment, or biolistic delivery, represents an alternative BPMV inoculation method that physically introduces viral vectors into plant cells. This technique involves coating microscopic gold or tungsten particles with plasmid DNA containing BPMV RNA1 and RNA2 constructs, then propelling these particles into plant tissues using a gene gun or particle delivery system [9] [10]. The BPMV constructs for bombardment typically feature the same genetic elements as those used in direct rubbing, including CaMV 35S promoters and nopaline synthase (Nos) terminators [9]. The bombardment process requires optimization of several parameters, including particle size, acceleration pressure, target distance, and plant developmental stage, to balance entry efficiency with tissue damage. Studies have demonstrated successful BPMV infection following biolistic inoculation of 10-day-old primary leaves of soybean plants with a mixture of RNA1 and RNA2 constructs in a 1:1 molar ratio [9].

Applications and Advantages

The particle bombardment method offers particular value for plant species or cultivars that prove difficult to infect through mechanical inoculation approaches [10]. This technique enables direct delivery of genetic material into plant cells, bypassing potential barriers to infection posed by leaf surface characteristics or defense mechanisms. Additionally, biolistic delivery allows for precise targeting of specific tissue types, making it valuable for studies focusing on particular cell types or developmental stages [10]. The bombardment approach has been successfully employed for both gene expression studies and VIGS applications, demonstrating effectiveness in silencing endogenous genes like PDS and validating the function of disease resistance genes in soybean [10].

Comparative Analysis of Inoculation Techniques

Table 1: Direct comparison of BPMV inoculation techniques

Parameter	Direct Plasmid Rubbing	Particle Bombardment
Infection Efficiency	92-100% with optimized protocol [7]	High, but varies with tissue type and optimization [9]
Equipment Requirements	Low (basic lab equipment)	High (gene gun/particle delivery system)
Technical Expertise	Moderate	High
Cost per Sample	Low	High
Throughput Capacity	High (suitable for large-scale studies) [7]	Moderate to Low
Tissue Damage	Minimal with proper technique	Potentially significant with improper optimization
Special Advantages	Simplicity, cost-effectiveness, scalability [9]	Bypasses leaf surface barriers, precise targeting [10]

Table 2: Quantitative performance metrics for BPMV inoculation methods

Performance Metric	Direct Plasmid Rubbing	Particle Bombardment
Optimal Plasmid Quantity	5 μg each RNA1 & RNA2 [7]	1:1 molar ratio RNA1:RNA2 [9]
Time to Symptom Appearance	28 days post-inoculation [7]	Varies, typically 2-4 weeks [9]
Silencing Efficiency	High (effective PDS silencing) [7] [9]	High (effective PDS silencing) [10]
Stability of Insert	Stable through serial passages [10]	Stable through serial passages [10]

BPMV vs. TRV VIGS Vectors in Soybean Research

Vector Characteristics and Applications

When comparing BPMV and tobacco rattle virus (TRV) as VIGS vectors for soybean research, each system demonstrates distinct advantages suited to different experimental needs. BPMV vectors have been specifically developed and optimized for legume species, particularly soybean and common bean, making them highly effective for functional genomics in these crops [7] [9]. The BPMV system has been successfully used to study disease resistance pathways, including the identification and validation of genes conferring resistance to soybean rust, soybean cyst nematode, and soybean mosaic virus [3]. In contrast, TRV vectors offer a broader host range that includes Solanaceae, Cruciferae, and some monocot species, but their application in soybean has been limited until recently [3] [16]. A newly developed TRV-VIGS system for soybean utilizing Agrobacterium tumefaciens-mediated infection through cotyledon nodes demonstrates promising results with silencing efficiency ranging from 65% to 95% [3].

Practical Considerations for Research

The selection between BPMV and TRV VIGS systems involves several practical considerations. BPMV inoculation via direct plasmid rubbing offers a simplified workflow without requiring Agrobacterium handling, making it accessible for laboratories with basic molecular biology capabilities [7] [9]. Furthermore, BPMV-based vectors have been engineered to include mild symptom variants that minimize phenotypic interference with the silencing phenotype [9]. TRV vectors, while historically less applied in soybean, offer potential advantages for studies requiring meristem penetration or whole-plant silencing, as TRV is known to effectively spread to all plant tissues, including meristems [16]. The recent development of TRV-VIGS using Agrobacterium-mediated infection through cotyledon nodes in soybean represents a significant advancement that may expand TRV utility in legume research [3].

Experimental Protocols for BPMV Inoculation

Direct Plasmid Rubbing Protocol

The following optimized protocol for direct plasmid rubbing has been validated for common bean and soybean [7]:

• Plant Material Preparation: Grow plants to the primary leaf stage (approximately 10 days post-germination).
• Plasmid Mixture Preparation: Combine 5 μg each of BPMV RNA1 (pBPMV-IA-R1M) and RNA2-derived plasmids in inoculation buffer.
• Inoculation Procedure: Add carborundum (600-grit) to the plasmid mixture as an abrasive. Gently rub the mixture onto the surface of primary leaves using a gloved finger or cotton swab, applying even pressure without causing excessive tissue damage.
• Post-Inoculation Care: Rinse leaves with distilled water to remove excess carborundum and plasmid residue. Maintain plants under standard growth conditions.
• Efficiency Assessment: Monitor for viral symptom development beginning at 14-21 days post-inoculation. For BPMV-GFP constructs, fluorescence can be detected in systemic leaves and roots approximately 3 weeks post-inoculation [7].

Particle Bombardment Protocol

The standard protocol for BPMV delivery via particle bombardment includes these key steps [9] [10]:

• Particle Preparation: Coat gold or tungsten particles (0.6-1.0 μm diameter) with plasmid DNA containing BPMV RNA1 and RNA2 constructs.
• Plant Preparation: Position 10-day-old soybean plants with primary leaves exposed as targets.
• Bombardment Parameters: Use helium-driven gene gun with optimal pressure and target distance determined empirically for specific equipment.
• Post-Bombardment Care: Maintain plants under standard conditions and monitor for infection.
• Confirmation: Verify infection through symptom observation, ELISA, or molecular analysis.

Essential Research Reagents and Materials

Table 3: Key research reagents for BPMV inoculation studies

Reagent/Material	Function/Application	Examples/Specifications
BPMV RNA1 Plasmid	Viral replication functions	pBPMV-IA-R1 (mild symptoms) or pBPMV-IA-R1M (moderate symptoms) [9]
BPMV RNA2 Plasmid	Vector for gene insertion	pBPMV-IA-V2 with multiple cloning site [9]
Abrasive Agent	Facilitates leaf penetration in rubbing method	Carborundum (400-600 grit) [7]
Gold/Tungsten Particles	DNA carrier for bombardment	0.6-1.0 μm diameter [9]
Plant Selection	Susceptible hosts for optimal efficiency	Soybean cultivars; Common bean cvs. Black Valentine, JaloEEP558 [7]

Both direct plasmid rubbing and particle bombardment provide effective methodologies for BPMV inoculation, with the choice depending on specific research requirements, available resources, and target plant species. Direct plasmid rubbing offers advantages in simplicity, cost-effectiveness, and scalability, making it particularly suitable for high-throughput functional genomics applications [7] [9]. Particle bombardment remains valuable for challenging plant materials or when precise tissue targeting is required [10]. Within the broader context of VIGS vector systems, BPMV maintains distinct advantages for legume research, particularly soybean, while TRV vectors offer complementary strengths including broader tissue penetration and recent protocol improvements for soybean [3] [16]. The continued refinement of these inoculation techniques will further enhance their utility for elucidating gene function in legume crops, supporting advancements in crop improvement and plant pathology research.

BPMV Inoculation Method Workflows

In the functional genomics of soybean, Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool to rapidly characterize gene function without the need for stable transformation [3]. The evaluation of VIGS efficiency relies heavily on visual marker genes that provide scorable phenotypes when silenced. Among these, phytoene desaturase (PDS), Cloroplastos Alterados 1 (CLA1), and green fluorescent protein (GFP) have become benchmark indicators for validating silencing efficiency in both Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) vector systems [26] [27]. This assessment provides a comparative analysis of these marker genes within the broader context of TRV versus BPMV VIGS vectors in soybean research, examining their validation methodologies, quantitative efficiency data, and practical applications in experimental workflows.

Marker Gene Profiles and Silencing Phenotypes

Characteristic Functions and Silencing Phenotypes

Marker Gene	Biological Function	Silencing Phenotype	Visualization Timeline	Impact on Plant Viability
PDS	Carotenoid biosynthesis enzyme	Photobleaching (white leaves), chlorophyll photooxidation [26]	2-3 weeks post-inoculation [3]	Severe; leads to plant death over time [28]
CLA1	Chloroplast development (1-deoxy-D-xylulose 5-phosphate synthase)	Leaf albinism, bleached phenotype [26] [27]	2-3 weeks post-inoculation	Severe; plants grow weakly and die after ~2 months [28]
GFP	Exogenous fluorescent protein	Loss of green fluorescence under UV light [3] [7]	1-2 weeks post-inoculation	None; non-functional marker without physiological impact

Quantitative Silencing Efficiency Across VIGS Systems

Marker Gene	TRV-VIGS Efficiency	BPMV-VIGS Efficiency	Detection Method	Key Experimental Conditions
PDS	65-95% in soybean [3]	Successful silencing confirmed [7] [10]	Visual phenotyping, qRT-PCR [3]	Agrobacterium-mediated cotyledon node delivery (TRV) [3]
CLA1	High across plant species [26]	Limited reporting in soybean	Visual phenotyping, qRT-PCR	Direct plasmid rubbing (BPMV) [7]
GFP	~80% infection efficiency [3]	Extensive fluorescence in systemic leaves [7]	Fluorescence microscopy, UV light visualization	Agrodrench method in soybean [29]

Comparative Analysis of TRV vs. BPMV VIGS Delivery Systems

Experimental Protocols for Marker Gene Validation

TRV-Based VIGS Protocol in Soybean: The optimized TRV-VIGS system utilizes Agrobacterium tumefaciens strain GV3101 carrying pTRV1 and pTRV2 vectors with inserted marker gene fragments [3]. For soybean, the delivery method involves:

• Sterilized soybean seeds soaked in sterile water until swollen
• Longitudinal bisection to obtain half-seed explants
• Immersion in Agrobacterium suspensions (OD~1.5) for 20-30 minutes
• Co-cultivation for 3 days under dark conditions
• Transplanting to soil and growing at 23°C with 16/8h light/dark photoperiod [3]

Infection efficiency is evaluated around day 4 post-infection by examining GFP fluorescence under a microscope, with effective infectivity exceeding 80% and reaching up to 95% for specific soybean cultivars like Tianlong 1 [3]. Silencing phenotypes typically manifest systemically between 14-21 days post-inoculation (dpi), with transcript reduction confirmed via qRT-PCR analysis [3] [29].

BPMV-Based VIGS Protocol in Legumes: The "one-step" BPMV vector system enables direct rub-inoculation of infectious plasmid DNA, bypassing requirements for in vitro transcription or biolistic delivery [7] [10]. The optimized protocol includes:

• Plasmid preparation of BPMV RNA1 and RNA2 derivatives (5μg each)
• Mechanical rub-inoculation on carborundum-dusted primary leaves
• Optimal rubbing intensity to create minor tissue damage without severe injury
• Inoculation of both primary leaves for maximum infection rates [7]

Infection success is evaluated by monitoring viral symptoms or GFP fluorescence, with systemic spread observed in upper leaves and roots within 3 weeks post-inoculation [7]. BPMV-mediated PDS silencing has been successfully achieved with insert fragments ranging from 132 to 391bp, demonstrating the system's flexibility [7].

Advantages and Limitations of Marker Genes

PDS and CLA1 provide unmistakable visual phenotypes (photobleaching and albinism) that enable rapid assessment of silencing efficiency without specialized equipment [26]. However, their severe impact on plant physiology through disruption of photosynthetic pathways limits their utility to early developmental stages, as silenced plants eventually wither and die [28]. This prevents their application in studies requiring prolonged observation, particularly for traits expressed during flowering or fruiting stages.

GFP serves as a non-destructive marker that doesn't compromise plant viability, allowing continuous monitoring throughout the plant lifecycle [3] [7]. The requirement for fluorescence detection equipment represents a limitation, but the preservation of normal plant development makes it valuable for long-term studies. In both TRV and BPMV systems, GFP has proven effective for tracking viral spread and initial silencing establishment before assessing target gene phenotypes [3] [7].

Emerging Alternatives and the Scientist's Toolkit

Innovative Marker Genes for Extended Studies

Recent research has introduced GoPGF (Pigment Gland Formation Gene) as a novel marker for VIGS validation, particularly in cotton but with potential cross-species applications [28] [27]. This gene regulates the formation of lysigenous glands in cotton, and its silencing reduces gland numbers without affecting normal growth or development [28]. This permits VIGS tracking throughout the entire plant growth period, including reproductive stages, addressing a significant limitation of PDS/CLA1 markers.

Additional promising markers include anthocyanidin synthase (ANS) and phytoene synthase (PSY), which modify pigment accumulation without lethal consequences [26] [27]. ANS silencing produces a brownish phenotype in cotton tissues, while PSY silencing converts red leaf cotton to green, both providing visible but non-lethal indicators of silencing efficiency [27].

Essential Research Reagent Solutions

Research Reagent	Function in VIGS	Example Applications	Specific Protocols
pTRV1 & pTRV2 Vectors	TRV RNA1 and RNA2 genomes for VIGS construction [3]	Soybean, tomato, tobacco functional genomics [3]	Agrobacterium-mediated delivery [3]
BPMV-IA-Di1 Vector	Mild symptomatic BPMV isolate for VIGS [7]	Soybean, common bean gene function studies [7] [10]	Direct plasmid rubbing [7]
Agrobacterium tumefaciens GV3101	Delivery vehicle for TRV constructs [3] [28]	Plant transformation and virus delivery	Suspension in MMA buffer (10mM MES, 10mM MgCl₂, 200μM AS) [28]
Acetosyringone	Phenolic compound inducing vir gene expression	Enhances Agrobacterium infection efficiency [28]	200μM in infiltration medium [28]

Experimental Workflow and Technical Diagrams

TRV-VIGS Workflow for Soybean

Marker Gene Selection Decision Pathway

The efficiency assessment of PDS, CLA1, and GFP as marker genes for VIGS validation reveals a trade-off between dramatic visual phenotypes and long-term experimental viability. PDS and CLA1 offer unmistakable silencing indicators but compromise plant survival, restricting their use to early developmental stages. GFP provides a non-destructive alternative but requires specialized detection equipment. The emergence of novel markers like GoPGF addresses critical limitations by enabling visible tracking throughout the plant lifecycle without lethal consequences. When comparing TRV and BPMV VIGS systems, both successfully utilize these markers but differ in delivery mechanisms and optimal applications. The selection of an appropriate marker gene should be guided by experimental timeline, available equipment, and specific research objectives, with the understanding that ongoing vector improvements continue to enhance silencing efficiency and expand functional genomics capabilities in soybean research.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional analysis of plant genes. This technology is particularly valuable for crop species like soybean (Glycine max L.) that are recalcitrant to stable genetic transformation [3] [5]. Among various viral vectors developed for VIGS, Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) have become the most prominent systems for soybean functional genomics [3] [9]. This case study provides a direct comparative analysis of these two vector systems, focusing specifically on their application in silencing the rust resistance gene GmRpp6907 to elucidate its function in soybean defense mechanisms.

Background: VIGS Vector Systems for Soybean Research

Technical Foundations of TRV and BPMV Vectors

Tobacco Rattle Virus (TRV) is a bipartite positive-sense RNA virus whose vectors have been widely adopted for VIGS in numerous plant species [16]. The TRV-based VIGS system utilizes two separate vectors: pTRV1 (carrying replication-associated proteins) and pTRV2 (containing the coat protein and cloning site for target gene inserts) [3] [16]. Infection is typically achieved through Agrobacterium tumefaciens-mediated delivery, where bacterial cultures carrying pTRV1 and pTRV2 derivatives are co-infiltrated into plant tissues [3].

Bean Pod Mottle Virus (BPMV) is also a bipartite positive-sense RNA virus that was among the first VIGS vectors developed for soybean [9] [30]. Similar to TRV, its genome consists of RNA1 and RNA2 components, with foreign sequences traditionally inserted into RNA2 between the movement protein and large coat protein subunits [9]. Early BPMV vectors required in vitro transcription and mechanical inoculation, though more recent DNA-based versions enable direct inoculation via biolistics or Agrobacterium infiltration [9].

Table 1: Fundamental Characteristics of TRV and BPMV VIGS Vectors

Characteristic	TRV-Based Vectors	BPMV-Based Vectors
Virus Type	Bipartite positive-sense RNA	Bipartite positive-sense RNA
Typical Delivery Method	Agrobacterium-mediated infiltration	In vitro transcription, biolistics, or Agrobacterium
Primary Infection Site	Cotyledon nodes [3]	Primary leaves [9]
Systemic Movement	Efficient throughout plant, including meristems [16]	Efficient systemic spread
Silencing Onset	2-3 weeks post-inoculation [3]	2-3 weeks post-inoculation [9]
Typical Silencing Duration	Several weeks	Several weeks

Experimental Protocol: TRV-Mediated Silencing of GmRpp6907

Vector Construction and Preparation

For silencing GmRpp6907 using the TRV system, a specific fragment of the target gene must first be cloned into the pTRV2 vector [3]:

• Target Sequence Selection: A 300-500 bp fragment of the GmRpp6907 gene is selected, avoiding regions of high homology with other genes to ensure silencing specificity.

• Vector Digestion: The pTRV2-GFP vector is digested with EcoRI and XhoI restriction enzymes.

• Fragment Cloning: The PCR-amplified GmRpp6907 fragment is ligated into the digested pTRV2 vector using the following primers:

  • Rpp6907-F: 5'-taaggttaccGAATTCTCGGCAAAGTTGGTTTTCATCT-3'
  • Rpp6907-R: 5'-atgcccgggcCTCGAGCCATTCCTGGGCTCCACATT-3' [3]

• Transformation: The ligation product is transformed into DH5α competent cells, and positive clones are verified by sequencing.

• Agrobacterium Preparation: Verified recombinant plasmids are introduced into Agrobacterium tumefaciens strain GV3101. The bacteria are cultured in LB medium with appropriate antibiotics until OD₆₀₀ reaches 0.6-1.0 [3].

Plant Inoculation and Growth Conditions

The optimized TRV-VIGS protocol for soybean utilizes cotyledon node infiltration [3]:

• Seed Preparation: Surface-sterilized soybean seeds (cv. Tianlong 1) are soaked in sterile water until swollen, then longitudinally bisected to create half-seed explants.

• Agroinfiltration: Fresh explants are immersed in Agrobacterium suspensions containing either pTRV1 or pTRV2-GmRpp6907 for 20-30 minutes [3].

• Co-cultivation: Infected explants are transferred to sterile tissue culture media and maintained under controlled conditions for 3-4 days.

• Plant Regeneration: Treated explants are transferred to soil and maintained in growth chambers at 24-26°C with a 16-hour photoperiod [3].

• Silencing Validation: Systemic silencing is evaluated 3-4 weeks post-inoculation through phenotypic observation and molecular analysis.

Diagram 1: TRV-mediated silencing workflow for GmRpp6907

Comparative Performance Data: TRV vs. BPMV

Efficiency Metrics for GmRpp6907 Silencing

Direct comparison of TRV and BPMV vectors reveals significant differences in their operational characteristics and silencing efficacy in soybean [3] [9].

Table 2: Performance Comparison of TRV and BPMV in Soybean Functional Genomics

Performance Metric	TRV-Based System	BPMV-Based System
Silencing Efficiency	65-95% [3]	Not specifically reported for GmRpp6907
Infection Efficiency	>80% (up to 95% in Tianlong 1) [3]	High in susceptible varieties
Vector Delivery	Agrobacterium-mediated cotyledon node infiltration [3]	Particle bombardment or mechanical inoculation [9]
Visual Symptoms	Mild, minimal interference with phenotypes [3]	Can induce leaf phenotypic alterations [3]
Experimental Timeline	4-5 weeks from inoculation to phenotypic analysis	4-5 weeks from inoculation to phenotypic analysis
Tissue Culture Requirement	Required for cotyledon node method [3]	Not required for mechanical inoculation
Applications Demonstrated	GmPDS, GmRpp6907, GmRPT4 [3]	Various disease resistance genes [9]

Rust Resistance Phenotypes Following GmRpp6907 Silencing

Silencing of GmRpp6907 using the TRV system resulted in significant alterations to soybean rust resistance [3]:

• Enhanced Susceptibility: Plants exhibiting successful GmRpp6907 silencing showed compromised rust resistance, confirming the gene's essential role in defense against rust pathogens.

• Silencing Efficiency Correlation: The degree of susceptibility correlated with silencing efficiency, with strongly silenced plants displaying more severe disease symptoms.

• Temporal Pattern: Rust susceptibility phenotypes became apparent approximately 3-4 weeks post-inoculation with TRV-GmRpp6907, coinciding with peak silencing activity.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for TRV-Mediated VIGS in Soybean

Reagent/Resource	Specification	Application/Function
TRV Vectors	pTRV1 and pTRV2 (with MCS)	Binary vectors for VIGS construct preparation
Agrobacterium Strain	GV3101	Delivery vehicle for TRV constructs
Soybean Cultivar	Tianlong 1	Optimized for high TRV infection efficiency [3]
Antibiotics	Kanamycin, Rifampicin	Selection for bacterial strains and vector maintenance
Restriction Enzymes	EcoRI, XhoI	Cloning of target fragments into pTRV2
Plant Growth Media	Sterile tissue culture media	Support plant regeneration after agroinfiltration
Detection Marker	GFP (Green Fluorescent Protein)	Visual assessment of infection efficiency [3]

Molecular Mechanisms of VIGS

The TRV-mediated silencing process operates through the plant's endogenous RNA interference machinery [16]:

Diagram 2: Molecular mechanism of VIGS

• Viral Replication and dsRNA Formation: After agroinfiltration, T-DNA containing the TRV genome is transcribed into viral RNA, which is then replicated by viral RNA-dependent RNA polymerase (RdRp), forming double-stranded RNA (dsRNA) intermediates [16].

• siRNA Biogenesis: The plant's Dicer-like enzymes recognize these dsRNA molecules and process them into 21-24 nucleotide small interfering RNAs (siRNAs) [16].

• RISC Assembly and Target Cleavage: siRNAs are incorporated into the RNA-induced silencing complex (RISC), which guides the complex to complementary mRNA sequences (e.g., GmRpp6907 transcripts), resulting in their cleavage and degradation [16].

• Systemic Silencing: The silencing signal amplifies and spreads throughout the plant, leading to systemic silencing of the target gene in tissues distant from the initial infection site [16].

Discussion: Strategic Selection of VIGS Vectors

Advantages and Limitations of TRV and BPMV Systems

The comparative analysis of TRV and BPMV vectors reveals distinct advantages for each system:

TRV System Advantages:

• Minimal Symptom Interference: TRV infection produces very mild symptoms that rarely interfere with phenotypic evaluation [3] [16].
• High Efficiency: The optimized cotyledon node method achieves up to 95% infection efficiency in suitable soybean cultivars [3].
• Broad Tissue Coverage: TRV effectively silences genes in meristematic tissues, enabling functional studies of developmental genes [16].

BPMV System Advantages:

• Established Track Record: BPMV has been successfully used to characterize numerous soybean disease resistance genes [9].
• Vector Versatility: Advanced BPMV vectors enable simultaneous silencing and expression studies, including marker-assisted silencing [9].
• Flexible Inoculation Methods: Mechanical inoculation options avoid tissue culture requirements [9].

Technical Considerations for Vector Selection

When selecting between TRV and BPMV for soybean functional genomics, researchers should consider:

• Target Tissue Requirements: TRV is preferable for meristematic or floral tissue studies, while BPMV may suffice for leaf-based assays.

• Transformation Expertise: TRV's cotyledon node method requires tissue culture capabilities, whereas BPMV can be mechanically inoculated.

• Phenotypic Subtlety: For subtle phenotypes, TRV's minimal symptom development is advantageous.

• Experimental Timeline: Both systems require similar timeframes (4-5 weeks) from inoculation to phenotypic analysis.

This case study demonstrates that TRV-mediated VIGS provides an efficient and robust platform for functional analysis of rust resistance genes in soybean, specifically validating the role of GmRpp6907 in disease defense. The direct comparison with BPMV vectors highlights TRV's advantages in silencing efficiency (65-95%) and minimal symptom interference, while acknowledging BPMV's established utility in legume functional genomics. The optimized TRV protocol using cotyledon node infiltration represents a significant methodological advancement, enabling high-throughput functional screening of candidate genes in soybean. These VIGS systems continue to expand the toolbox for soybean researchers, accelerating the identification and characterization of genes relevant to crop improvement and disease resistance breeding programs.

Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional analysis of plant genes, leveraging the plant's innate post-transcriptional gene silencing (PTGS) mechanism as a defense against viral pathogens [16] [15]. This technology utilizes recombinant viral vectors to carry fragments of host plant genes, triggering sequence-specific degradation of complementary mRNA transcripts and resulting in knockdown phenotypes that reveal gene function [16] [17]. For soybean (Glycine max L.), a vital grain and oil crop that serves as a primary source of edible oil, plant-based protein, and livestock feed, VIGS provides a particularly valuable alternative to stable genetic transformation, which is time-consuming and laborious in this species [3] [5]. Among the various viral vectors adapted for soybean research, Bean pod mottle virus (BPMV) and Tobacco rattle virus (TRV) have emerged as the most prominent systems, each with distinct characteristics, advantages, and limitations for analyzing defense genes against pathogens [3] [9].

Soybean production faces significant constraints from various diseases, making the development of disease-resistant cultivars a critical breeding objective [3]. Functional analysis of candidate resistance genes enables more targeted breeding approaches. While BPMV-based vectors have a longer history of application in soybean [9] [10], recent advances have demonstrated the feasibility of TRV-based systems [3] [16]. This case study provides a comparative analysis of these two VIGS platforms, focusing on their experimental applications, efficiency, and practicality for studying soybean defense genes.

Comparative Analysis of BPMV and TRV VIGS Systems

Bean pod mottle virus (BPMV) is a positive-strand RNA virus with a bipartite genome belonging to the Comovirus genus [7] [9]. RNA1 encodes proteins involved in replication and pathogenicity, while RNA2 contains the coding sequences for movement protein (MP) and coat proteins (CPs) [9]. Early BPMV vectors required cloning foreign sequences in-frame within the RNA2 polyprotein, but later "one-step" versions introduced a BamHI restriction site after the RNA2 stop codon, enabling insertion of non-coding fragments and expanding VIGS applications [7] [9]. BPMV has been successfully used for both protein expression and gene silencing in soybean and common bean [7] [10].

Tobacco rattle virus (TRV) is another positive-sense RNA virus with a bipartite genome that has been widely adopted for VIGS across numerous plant families [16] [17]. The TRV1 component encodes replicase, movement protein, and a cysteine-rich protein, while TRV2 contains the coat protein gene and serves as the insertion site for target gene fragments [16]. TRV vectors are noted for their broad host range, efficient systemic movement including into meristematic tissues, and mild symptom development that minimizes interference with silencing phenotypes [3] [16].

Table 1: Fundamental Characteristics of BPMV and TRV VIGS Vectors

Characteristic	BPMV-Based System	TRV-Based System
Virus Type	Positive-sense RNA virus (Comovirus)	Positive-sense RNA virus (Tobravirus)
Genome Organization	Bipartite (RNA1 & RNA2)	Bipartite (TRV1 & TRV2)
Primary Inoculation Methods	Direct DNA rubbing, biolistic delivery	Agrobacterium-mediated infiltration (cotyledon node immersion, leaf injection)
Typical Silencing Onset	2-3 weeks post-inoculation	2-3 weeks post-inoculation
Systemic Movement	Efficient throughout plant, including roots	Efficient throughout plant, including meristems
Historical Usage in Soybean	Extensive, well-established	Limited until recent optimization

Performance and Efficiency Metrics

Recent studies have enabled direct comparison of the operational efficiency of BPMV and TRV VIGS systems in soybean. The established BPMV system has demonstrated reliable silencing efficiency, while newly optimized TRV protocols show promising performance characteristics.

Table 2: Performance Comparison of BPMV and TRV in Soybean Functional Genomics

Performance Metric	BPMV-Based System	TRV-Based System
Silencing Efficiency Range	Well-documented but variable	65% - 95% [3]
Infection Efficiency	High with optimized rubbing protocol	Up to 95% with cotyledon node method [3]
Vector Stability	Stable with serial passages	Stable systemic spread
Symptom Interference	Mild symptoms with IA-Di1 isolate [9]	Minimal viral symptoms [3] [16]
Key Demonstrated Applications	Functional analysis of disease resistance genes (e.g., Rpp1, GmBIR1, Rbs1) [3]	Silencing of defense genes (e.g., GmRpp6907, GmRPT4) [3]

Experimental Protocols for BPMV- and TRV-Based VIGS

BPMV-VIGS Experimental Workflow

The following diagram illustrates the optimized protocol for BPMV-mediated VIGS in soybean:

The BPMV-VIGS protocol begins with vector construction, utilizing the modified pBPMV-IA-R1M RNA1 component which induces moderate symptoms that facilitate infection monitoring without ELISA confirmation [9]. For the RNA2 component, target gene fragments (typically 132-391 bp) are cloned into the BamHI site after the stop codon, with antisense orientation of 3' ORF fragments demonstrating superior silencing efficiency compared to sense orientation [9]. Inoculation involves mixing 5μg each of RNA1 and RNA2 plasmid DNA with carbonundum in inoculation buffer, followed by direct rub-inoculation on primary leaves of 10-14 day old soybean seedlings [7] [9]. Silencing phenotypes typically become evident in systemic leaves 2-3 weeks post-inoculation, with molecular validation via qRT-PCR or northern blot analysis [9].

TRV-VIGS Experimental Workflow

The newly optimized TRV-VIGS protocol for soybean employs a distinct inoculation approach that addresses challenges posed by soybean's thick cuticle and dense leaf trichomes:

The TRV-VIGS protocol utilizes Agrobacterium tumefaciens-mediated delivery through cotyledon nodes, overcoming limitations of conventional leaf infiltration methods [3]. The process begins with cloning target gene fragments (300-500 bp) into the TRV2 multiple cloning site, followed by transformation into Agrobacterium strain GV3101 containing the TRV1 helper vector [3] [16]. Sterilized soybean seeds are soaked until swollen, longitudinally bisected to create half-seed explants, then immersed in Agrobacterium suspensions (OD₆₀₀ = 1.0-1.5) for 20-30 minutes—determined to be the optimal duration for infection [3]. After co-culture for 4 days, successful infection is verified by GFP fluorescence microscopy, showing >80% cell infiltration efficiency in susceptible cultivars like Tianlong 1 [3]. Systemic silencing phenotypes typically emerge within 3-4 weeks post-inoculation, with silencing efficiency ranging from 65% to 95% confirmed by qPCR analysis [3].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for BPMV and TRV VIGS Experiments

Reagent/Resource	Function/Application	Specific Examples/Notes
BPMV Vectors	Bipartite system for VIGS	pBPMV-IA-R1M (moderate symptoms), pBPMV-IA-V2 (VIGS with MCS) [9]
TRV Vectors	Bipartite system for VIGS	pTRV1 (helper vector), pTRV2 (insert vector with MCS or Gateway cloning) [16]
Agrobacterium tumefaciens GV3101	Delivery system for TRV vectors	Optimal for soybean cotyledon node transformation [3]
Plant Genotypes	Soybean cultivars for VIGS	BPMV: Various cultivars; TRV: Tianlong 1 (high efficiency, up to 95% infection) [3] [7]
Marker Genes	Silencing efficiency controls	Phytoene desaturase (PDS) - visual bleaching phenotype [3] [9]
Inoculation Materials	Mechanical delivery	Carbonundum (BPMV rub-inoculation), tissue culture media (TRV cotyledon method) [3] [7]
Detection Tools	Infection and silencing validation	GFP fluorescence microscopy (TRV), ELISA (BPMV), qPCR for silencing verification [3] [9]

Applications in Defense Gene Analysis

Both BPMV and TRV VIGS systems have been successfully deployed to functionally characterize soybean defense genes, providing insights into disease resistance mechanisms. The BPMV system has been extensively used to study resistance against major soybean pathogens, including the identification of Rpp1-mediated rust resistance [3], discovery of Rsc1-DR conferring resistance to soybean mosaic virus strain SC1 [3], and validation of Rbs1 role in brown stem rot resistance [3]. More recently, silencing of GmBIR1 via BPMV was shown to enhance soybean resistance to SMV, resulting in constitutively activated defense responses [3].

The newer TRV-VIGS system has demonstrated comparable utility in defense gene analysis, with successful silencing of the rust resistance gene GmRpp6907 and the defense-related gene GmRPT4 confirming the system's robustness for disease resistance research [3]. The optimized cotyledon node immersion method enables effective systemic spread and silencing throughout the plant, facilitating phenotypic assessment of defense-related traits [3].

BPMV-based VIGS systems represent a well-established, reliable tool for functional analysis of defense genes in soybean, with proven efficacy across multiple disease systems and a simplified "one-step" inoculation protocol [7] [9]. Meanwhile, TRV-based systems offer a valuable alternative with potential advantages in symptom minimization, broader tissue coverage including meristems, and flexibility in application methods [3] [16]. The recent optimization of TRV for soybean through cotyledon node immersion has achieved silencing efficiencies (65-95%) comparable to established BPMV protocols, expanding the VIGS toolkit available to soybean researchers [3].

For researchers investigating soybean defense genes, BPMV remains the preferred choice for well-characterized pathosystems where its reliability has been demonstrated, while TRV offers promise for studies where minimal viral symptom interference is critical or for exploratory work in diverse genetic backgrounds. The continued refinement of both systems will further enhance their utility in functional genomics, ultimately supporting the development of disease-resistant soybean cultivars through more rapid and efficient gene validation.

Spatial and Temporal Silencing Patterns Across Soybean Tissues

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional gene analysis in plants, circumventing the challenges of stable genetic transformation in recalcitrant species like soybean [3] [14]. Among the various viral vectors developed, Tobacco rattle virus (TRV) and Bean pod mottle virus (BPMV) have become predominant systems for VIGS studies in soybean [3] [16]. Understanding their distinct spatial and temporal silencing patterns across soybean tissues is crucial for selecting the appropriate vector for specific research applications, particularly when targeting different organs or developmental stages. This guide provides an objective comparison of TRV and BPMV VIGS vectors based on experimental data, focusing on their efficacy, tissue specificity, and temporal dynamics to inform vector selection for soybean functional genomics.

VIGS Mechanism and Workflow

VIGS operates by hijacking the plant's natural RNA-mediated antiviral defense mechanism. When a recombinant virus carrying a fragment of a host gene infiltrates the plant, the plant's defense system processes the viral RNA into small interfering RNAs (siRNAs) that subsequently target complementary endogenous mRNAs for degradation [16]. Phytoene desaturase (PDS), which causes a visible photobleaching phenotype when silenced, serves as a common marker gene for evaluating VIGS efficiency [3] [16].

The following diagram illustrates the molecular mechanism of VIGS and the general experimental workflow for both TRV and BPMV systems.

Figure 1: VIGS Mechanism and Experimental Workflow. The process begins with vector construction, followed by plant inoculation through various delivery methods, and culminates in systemic gene silencing through RNA interference pathways.

Comparative Efficiency Across Soybean Tissues

The silencing efficiency of TRV and BPMV vectors varies significantly across different soybean tissues and over time. The table below summarizes quantitative data on their performance in various organs based on experimental studies.

Table 1: Spatial and Temporal Silencing Patterns of TRV and BPMV VIGS Vectors in Soybean

Tissue Type	TRV-Based VIGS	BPMV-Based VIGS	Experimental Evidence
Leaves	65-95% silencing efficiency [3]	Near-complete silencing; significant reduction at 14 dpi; sustained to 35 dpi [14]	TRV: Photobleaching phenotypes [3]BPMV: GFP fluorescence reduction & qPCR validation [14]
Stems	Systemic spread through vascular tissues confirmed [16]	Near-complete, uniform silencing across all cell types [14]	BPMV: Cross-section analysis showing uniform silencing [14]
Roots	Silencing confirmed in entire root system [31]	Weaker but detectable silencing compared to shoots [14]	BPMV: GFP fluorescence reduction in roots [14]
Flowers	Information limited in search results	95% mRNA reduction; all floral parts (petals, sepals, reproductive whorls) [14]	BPMV: Silencing independent of developmental stage/location [14]
Time to Initial Silencing	21 days post-inoculation (dpi) [3]	14 dpi in first trifoliate [14]	BPMV: Significant GFP reduction at 14 dpi [14]
Silencing Duration	Information limited in search results	Sustained up to 7 weeks post-inoculation in flowers [14]	BPMV: Stable insert maintenance confirmed by RT-PCR [14]

Methodological Protocols

TRV-Based VIGS Protocol

The TRV-VIGS system utilizes Agrobacterium tumefaciens-mediated infection through cotyledon nodes. The optimized protocol achieves high infection efficiency despite challenges posed by soybean's thick cuticle and dense leaf trichomes [3].

Key Steps:

• Vector Construction: Clone target gene fragment (300-500 bp) into pTRV2 vector between EcoRI and XhoI restriction sites [3]
• Plant Material Preparation: Surface-sterilize soybean seeds, germinate in sterile conditions, and prepare half-seed explants by longitudinally bisecting swollen soybeans [3]
• Agrobacterium Preparation: Transform recombinant pTRV2 and helper pTRV1 plasmids into Agrobacterium tumefaciens GV3101 strain [3]
• Inoculation: Immerse fresh explants in Agrobacterium suspensions (OD₆₀₀ ≈ 1.0) for 20-30 minutes—determined as optimal duration [3]
• Co-cultivation: Transfer infected explants to sterile tissue culture conditions for 3-4 days [3]
• Plant Regeneration: Transfer to regeneration media for shoot development and subsequent rooting [3]
• Efficiency Validation: Assess infection success via GFP fluorescence microscopy, with effective infectivity exceeding 80% (up to 95% for Tianlong 1 cultivar) [3]

BPMV-Based VIGS Protocol

The BPMV system offers flexibility with both Agrobacterium-mediated delivery and direct rub-inoculation of infectious plasmids, adapted for high-throughput studies [14] [7].

Key Steps:

• Vector Selection: Utilize "one-step" BPMV vectors (e.g., pBPMV-IA-V1) with inserts in antisense orientation targeting 3' end of genes for optimal silencing [14] [7]
• Plant Preparation: Grow soybean plants (cultivar-specific susceptibility) to primary leaf stage (approximately 7-10 days post-germination) [7]
• Inoculum Preparation: For direct rub-inoculation, mix RNA1 and RNA2 plasmids (5μg each determined optimal) in inoculation buffer [7]
• Inoculation: Gently rub primary leaves with carborundum abrasive followed by plasmid DNA mixture, inoculating both primary leaves for maximum efficiency [7]
• Optimal Conditions: Maintain plants under standard growth conditions (22-25°C, 16/8h light/dark cycle) [14]
• Phenotype Monitoring: Observe initial silencing phenotypes from 14 dpi in systemic leaves, with strongest effects at 21 dpi and sustained silencing up to 7 weeks [14]

Applications in Soybean Functional Genomics

Both VIGS systems have been successfully employed to characterize genes involved in disease resistance and stress responses in soybean. The following diagram illustrates a representative experimental workflow for studying disease resistance genes using these systems.

Figure 2: Disease Resistance Gene Validation Workflow Using VIGS. The process encompasses gene identification, functional validation through silencing and pathogen challenge, and comprehensive phenotypic analysis.

Specific Applications:

• TRV System: Successfully silenced rust resistance gene GmRpp6907 and defense-related gene GmRPT4, confirming its utility for disease resistance gene discovery [3]
• BPMV System: Effectively employed to characterize negative regulators of immunity like GmBIR1, where silencing resulted in activated defense responses and enhanced resistance to Soybean mosaic virus (SMV) and Pseudomonas syringae [32]
• Functional Studies: Both systems enabled investigation of genes involved in soybean response to biotic stresses, providing insights into molecular mechanisms of disease resistance [3] [33] [32]

Essential Research Reagents

The table below outlines key reagents and materials required for implementing TRV and BPMV VIGS systems in soybean.

Table 2: Essential Research Reagents for Soybean VIGS Studies

Reagent/Material	Function/Purpose	System	Specific Examples/Notes
Binary Vectors	Viral genome backbone for insert cloning	Both	pTRV1/pTRV2 (TRV) [3]; pBPMV-IA-V1 (BPMV) [7]
Agrobacterium Strain	Plant transformation vector delivery	Both (primarily TRV)	GV3101 for TRV system [3]
Marker Gene Constructs	Silencing efficiency validation	Both	Phytoene desaturase (PDS) for photobleaching [3]; GFP in transgenic lines [14]
Soybean Cultivars	VIGS-compatible plant genotypes	Both	Tianlong 1 (TRV) [3]; Black Valentine, JaloEEP558 (BPMV) [7]
Cloning Enzymes	Insertion of target fragments into vectors	Both	Restriction enzymes (EcoRI, XhoI) [3]; Gateway BP Clonase [16]
Inoculation Materials	Plant tissue delivery	Both	Carborundum abrasive (BPMV rub-inoculation) [7]; Syringe (Agroinfiltration)

The comparative analysis of TRV and BPMV VIGS vectors reveals a complementary relationship rather than absolute superiority of either system. BPMV-based vectors demonstrate exceptional efficacy in photosynthetic and reproductive tissues, with strong, sustained silencing in leaves, stems, and flowers, making them ideal for studying genes expressed in these organs or requiring long silencing durations. Conversely, TRV-based vectors offer robust whole-plant silencing capabilities, including effective root silencing, and utilize more straightforward Agrobacterium-mediated transformation protocols. The selection between these systems should be guided by specific research requirements: BPMV for enhanced efficiency in aerial tissues and prolonged studies, and TRV for comprehensive whole-plant silencing and simpler implementation. Both systems provide valuable functional genomics tools that significantly advance soybean gene function characterization and disease resistance research.

Optimizing VIGS Efficiency: Overcoming Technical Challenges in Soybean

Soybean (Glycine max L.) is a vital global crop for protein and oil, but its functional genomics research is hindered by its recalcitrance to stable genetic transformation. Virus-induced gene silencing (VIGS) has emerged as a powerful alternative for rapid gene function analysis, with Bean pod mottle virus (BPMV) and Tobacco rattle virus (TRV) being the most prominent vector systems. However, a significant technical challenge impeding their efficacy is the natural physical barrier formed by the soybean leaf's thick cuticle and dense trichomes, which severely limits infection efficiency. This guide provides a systematic comparison of optimized protocols designed to overcome these obstacles, enabling researchers to select the most appropriate VIGS strategy for their experimental needs.

Vector Comparison: TRV vs. BPMV at a Glance

The table below summarizes the core characteristics of the TRV and BPMV VIGS systems, highlighting key differences in their application for soybean research.

Table 1: Comparative Analysis of TRV and BPMV VIGS Systems in Soybean

Feature	TRV-Based System	BPMV-Based System
Infection Method	Agrobacterium tumefaciens-mediated delivery via cotyledon node immersion [3].	Direct rub-inoculation of infectious plasmid DNA or viral RNA onto leaves; also via biolistic delivery [9] [7].
Key Optimization	Uses longitudinally bisected cotyledons to bypass the leaf cuticle and trichomes, achieving >80% infection efficiency [3].	Optimizes plasmid DNA quantity (5 µg) and rubbing intensity; efficiency is genotype-dependent [7].
Typical Silencing Efficiency	65% - 95% [3] [34]	Highly efficient; near-complete silencing in leaves and flowers reported [14].
Viral Symptoms	Elicits fewer viral symptoms, minimizing interference with silencing phenotypes [3].	Can induce mild to moderate mosaic symptoms, which may complicate phenotypic analysis [9] [7].
Systemic Silencing	Effective systemic spread and silencing throughout the plant [3].	Widespread silencing in leaves, stems, flowers, and roots [14].
Ideal Application	Rapid, high-efficiency silencing in a broad range of tissues with minimal viral symptom interference.	Robust, long-lasting silencing, including in floral tissues and roots; well-established for large-scale screens.

Detailed Experimental Protocols for Overcoming Physical Barriers

Optimized TRV-VIGS Protocol via Cotyledon Node Infiltration

This recently developed protocol directly addresses the challenge of the soybean leaf's thick cuticle and dense trichomes by using an alternative infection site [3].

• Vector Construction: The target gene fragment (e.g., GmPDS) is cloned into the pTRV2-GFP vector. The recombinant plasmid is then transformed into Agrobacterium tumefaciens strain GV3101 [3].
• Plant Material Preparation: Surface-sterilized soybean seeds are germinated and swollen in sterile water. The swollen seeds are longitudinally bisected to create half-seed explants, exposing the cotyledonary node [3].
• Agrobacterium Inoculation: The fresh explants are immersed in an Agrobacterium suspension containing both pTRV1 and the recombinant pTRV2 vectors for 20-30 minutes. This direct immersion ensures efficient delivery of the vector to the meristematic cells of the cotyledonary node [3].
• Tissue Culture and Plant Regeneration: After inoculation, the explants are transferred to sterile tissue culture media to regenerate whole plants. This step is critical for the systemic spread of the virus from the cotyledon node [3].
• Efficiency Validation: Successful infection can be confirmed by observing GFP fluorescence at the cotyledon node 4 days post-infection. This method has achieved infection efficiencies exceeding 80%, and up to 95% in certain cultivars like 'Tianlong 1' [3].

Optimized BPMV-VIGS Protocol via Direct Rub-Inoculation

This "one-step" BPMV protocol simplifies delivery and is widely used, though it must contend with leaf surface barriers directly [7].

• Vector Construction: The target sequence is inserted into the BPMV RNA2 vector (e.g., pBPMV-IA-V2) after the translation stop codon, allowing for sense or antisense orientation of non-coding fragments [9].
• Inoculum Preparation: The BPMV RNA1 (pBPMV-IA-R1M) and recombinant RNA2 plasmids are purified and mixed in a 1:1 molar ratio [7].
• Plant Preparation: Soybean seedlings (e.g., cv. Black Valentine) are grown until the primary leaves are fully expanded [7].
• Direct Rub-Inoculation: A mixture containing 5 µg each of RNA1 and RNA2 plasmid DNA is applied to the primary leaves. The leaves are then gently rubbed with a gloved finger or a pestle, using carborundum powder as an abrasive to physically disrupt the cuticle and facilitate DNA entry [7].
• Post-Inoculation Care: Inoculated plants are maintained under cool conditions (e.g., 20°C) to optimize virus replication and systemic spread [18].

The Scientist's Toolkit: Essential Research Reagents

The table below lists key reagents and materials crucial for implementing the optimized VIGS protocols described above.

Table 2: Essential Research Reagents for Soybean VIGS Studies

Reagent / Material	Function / Application	Examples / Notes
VIGS Vectors	Carrier for delivering host-derived gene fragments to induce silencing.	pTRV1, pTRV2 (for TRV system); pBPMV-IA-R1M, pBPMV-IA-V2 (for BPMV system) [3] [9].
Agrobacterium tumefaciens	Delivery vehicle for TRV vectors into plant tissues.	Strain GV3101 for TRV; Strain C58C1 for some ALSV vectors [3] [6].
Abrasive Powder	Creates micro-wounds in the leaf cuticle for efficient viral entry during rub-inoculation.	Carborundum (silicon carbide) or Celite [7].
Antibiotics	Selection for bacterial strains and plasmid maintenance.	Kanamycin, Rifampicin, Gentamycin [6].
Acetosyringone	Phenolic compound that induces Agrobacterium virulence genes.	Added to agro-infiltration buffer to enhance T-DNA transfer [6].
Sterile Tissue Culture Media	Supports regeneration of whole plants from infected explants.	Used in the TRV cotyledon node method [3].

The choice between TRV and BPMV VIGS systems for soybean research is fundamentally guided by the specific need to overcome the host's physical defenses. The recently optimized TRV-based method, which leverages Agrobacterium-mediated infection of the cotyledon node, offers a superior solution to the cuticle and trichome problem, enabling highly efficient and systemic silencing with minimal viral symptom interference [3]. In contrast, the established BPMV-based system relies on direct mechanical inoculation reinforced by abrasives and optimized plasmid delivery, providing robust and long-lasting silencing across a wide range of tissues, including roots and flowers [9] [14]. Researchers should select the TRV system for rapid, high-efficiency silencing with cleaner phenotypes, while the BPMV system remains a powerful tool for large-scale screens and studies requiring deep tissue penetration.

Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional gene analysis in plants, particularly in species like soybean where stable genetic transformation remains challenging and time-consuming [3] [5]. VIGS operates by hijacking the plant's innate post-transcriptional gene silencing (PTGS) machinery, using recombinant viral vectors to trigger sequence-specific degradation of endogenous plant mRNA transcripts [17]. The effectiveness of this technology critically depends on proper insert fragment design, which encompasses optimal size selection, appropriate orientation within the vector, and strategic target region selection within the gene of interest. Within soybean research, two primary viral vector systems have been extensively developed: the Bean Pod Mottle Virus (BPMV) and the more recently optimized Tobacco Rattle Virus (TRV) systems [3] [7]. While BPMV has historically been the workhorse for soybean VIGS studies, TRV offers distinct advantages including milder viral symptoms and broader host range compatibility [3] [17]. This guide provides a comprehensive comparison of insert design parameters between these two systems, supported by experimental data and detailed protocols to enable researchers to make informed decisions for their functional genomics studies.

Comparative Analysis of TRV and BPMV VIGS Vectors

Key Characteristics and Performance Metrics

Table 1: Comparative analysis of TRV and BPMV VIGS vectors in soybean

Parameter	TRV-Based VIGS	BPMV-Based VIGS
Optimal Insert Size	200-500 bp [3]	132-391 bp (PDS silencing confirmed) [7]
Insert Orientation	Sense orientation in pTRV2 vector [3]	In-frame with RNA2 polyprotein (1st gen) or independent expression (one-step vector) [7]
Target Region Selection	cDNA-derived fragments targeting conserved domains [3]	cDNA fragments, minimal size requirement established [7]
Silencing Efficiency	65-95% [3]	High efficiency reported [7]
Onset of Silencing	21 days post-inoculation (dpi) [3]	3 weeks post-inoculation [7]
Delivery Method	Agrobacterium-mediated cotyledon node infection [3]	Direct plasmid rubbing or Agrobacterium infiltration [7]
Viral Symptoms	Minimal symptoms [3]	Mild to moderate symptoms depending on isolate [7]
Key Applications in Soybean	Disease resistance genes (GmRpp6907, GmRPT4), metabolic genes (GmPDS) [3]	Disease resistance studies, metabolic genes [7]

Experimental Evidence and Validation Data

Table 2: Experimental validation of insert design parameters in TRV and BPMV systems

Experimental Metric	TRV System Results	BPMV System Results
Visual Marker Silencing	Photobleaching observed at 21 dpi with GmPDS silencing [3]	Photobleaching with PvPDS silencing in common bean [7]
Endogenous Gene Silencing	Rust resistance gene (GmRpp6907) and defense gene (GmRPT4) successfully silenced [3]	Nodulin 22 and stearoyl-ACP desaturase silenced in common bean [7]
Molecular Validation	qPCR confirmation of reduced target gene expression [3]	RT-qPCR and Western blot confirmation [7]
Systemic Spread	Systemic movement from cotyledon nodes to entire plant [3]	Systemic infection of upper leaves and roots [7]
Duration of Silencing	Persistent silencing enabling phenotypic characterization [3]	Sufficient duration for phenotypic assessment [7]

Insert Design Parameters: Technical Specifications

Fragment Size Optimization

The size of the insert fragment plays a crucial role in determining VIGS efficiency. For TRV-based vectors in soybean, fragments ranging between 200-500 base pairs have demonstrated high silencing efficiency of 65-95% [3]. In the case of BPMV vectors, research in the related legume common bean has shown that fragments as short as 132 bp can effectively silence the PHYTOENE DESATURASE (PDS) gene, with functional fragments tested up to 391 bp [7]. The optimal size balance must provide sufficient sequence specificity to minimize off-target effects while remaining compatible with viral vector packaging and systemic movement constraints.

Recent advances in TRV-VIGS for soybean have utilized fragments of approximately 300-400 bp for silencing endogenous genes including GmPDS, GmRpp6907, and GmRPT4, with successful outcomes confirmed through both phenotypic observations and molecular analyses [3]. It is noteworthy that while shorter fragments may be effective, they require careful design to ensure specificity, particularly when targeting members of gene families with high sequence homology.

Insert Orientation and Vector Configuration

The orientation of the insert fragment within the viral vector is system-dependent. For TRV-based vectors, the insert is typically cloned in the sense orientation into the multiple cloning site of the pTRV2 vector [3]. The bipartite nature of the TRV genome necessitates a two-vector system, with pTRV1 encoding viral replication and movement proteins, while pTRV2 carries the coat protein and the insert fragment targeting the gene of interest.

For BPMV vectors, the configuration has evolved through multiple generations. Early BPMV vectors required in-frame insertion of fragments within the RNA2 polyprotein [7], while the more recent "one-step" BPMV vector allows for insertion of fragments without the frame constraint through the introduction of a BamHI restriction site after the translation stop codon of RNA2 [7]. This technical advancement significantly simplifies cloning procedures and makes the system more amenable to high-throughput applications.

Target Region Selection Strategies

Selection of the appropriate target region within a gene significantly impacts silencing efficiency. For both TRV and BPMV systems, the following strategies have proven effective:

• Conserved Domain Targeting: Selecting fragments that correspond to conserved functional domains can enhance silencing efficacy, particularly for genes with multiple isoforms [3].
• 3' UTR Regions: These often contain unique sequences that can improve target specificity, especially for gene family members.
• Avoidance of Highly Homologous Regions: While conserved regions are attractive for silencing multiple gene family members, areas with extremely high sequence identity to non-target genes should be avoided to minimize off-target effects.
• cDNA-Derived Fragments: Most successful VIGS implementations utilize fragments amplified from cDNA rather than genomic DNA to ensure targeting of expressed sequences [3].

Experimental validation of fragment selection typically begins with a visible marker gene such as PHYTOENE DESATURASE (PDS), which produces a characteristic photobleaching phenotype when silenced, providing a visual indicator of system functionality before proceeding to target genes of interest [3] [7].

Detailed Experimental Protocols

TRV-Based VIGS Protocol for Soybean

The following protocol outlines the optimized TRV-VIGS procedure for soybean, as demonstrated with silencing efficiency of 65-95% [3]:

Vector Construction:

• Amplify target gene fragment (200-500 bp) from soybean cDNA using gene-specific primers with incorporated EcoRI and XhoI restriction sites.
• Digest pTRV2-GFP vector with EcoRI and XhoI restriction enzymes.
• Ligate the target fragment into the digested pTRV2-GFP vector.
• Transform ligation product into DH5α competent cells and select positive clones for sequence verification.
• Introduce confirmed recombinant plasmids into Agrobacterium tumefaciens strain GV3101.

Plant Inoculation:

• Surface-sterilize soybean seeds and soak in sterile water until swollen.
• Prepare Agrobacterium cultures containing pTRV1 and pTRV2-derived constructs (pTRV:empty, pTRV:GmPDS, pTRV:Rpp6907, or pTRV:RPT4) and grow to OD600 = 0.6-1.0.
• Bisect swollen seeds longitudinally to obtain half-seed explants.
• Immerse fresh explants in Agrobacterium suspension for 20-30 minutes (optimal duration).
• Co-cultivate inoculated explants on sterile medium for 2-3 days.
• Transfer explants to regeneration medium with antibiotics to suppress Agrobacterium overgrowth.

Evaluation of Silencing:

• Monitor GFP fluorescence at infection sites 4 days post-inoculation using fluorescence microscopy.
• Observe photobleaching in positive control (GmPDS-silenced) plants at 21 dpi.
• Assess phenotypic changes in experimental plants at 21-28 dpi.
• Confirm silencing efficiency through qRT-PCR analysis of target gene expression.

BPMV-Based VIGS Protocol for Legumes

The "one-step" BPMV vector system offers simplified delivery for high-throughput studies [7]:

Vector Construction:

• Clone target gene fragment (132-391 bp) into BamHI site of BPMV RNA2-derived vector.
• Verify construct orientation and sequence fidelity.
• Transform into appropriate bacterial strain.

Plant Inoculation:

• Grow plants to primary leaf stage (approximately 2-3 weeks post-germination).
• Prepare plasmid DNA mix containing 5μg each of RNA1 and RNA2-derived plasmids.
• Apply carborundum powder to leaves as an abrasive.
• Rub plasmid DNA mixture directly onto carborundum-dusted leaves.
• Maintain plants under controlled conditions (temperature-dependent for optimal silencing).

Evaluation of Silencing:

• Monitor viral symptoms and silencing phenotypes weekly.
• Document spatial patterns of silencing throughout plant development.
• Confirm silencing at molecular level through RT-qPCR and Western blot analysis.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key research reagents for VIGS experiments in soybean

Reagent/Vector	Function/Purpose	Specific Examples/Notes
pTRV1 Vector	Encodes viral replicase, movement protein, and silencing suppressor for TRV system [3]	Essential component for TRV-based VIGS; provides replication machinery
pTRV2 Derived Vectors	Carries coat protein and insert fragment; cloning backbone for target genes [3]	pTRV2-GFP, pTRV2-GmPDS, pTRV2-Rpp6907, pTRV2-RPT4
BPMV RNA1 Vector	Provides replication functions for BPMV system [7]	Required for BPMV-based VIGS; can be symptom-modulating
BPMV RNA2 Derived Vectors	Carrier for insert fragments in BPMV system [7]	"One-step" vector with BamHI site for simplified cloning
Agrobacterium tumefaciens GV3101	Mediates vector delivery in TRV system [3]	Standard strain for plant transformations
Restriction Enzymes	Fragment cloning and vector linearization [3]	EcoRI, XhoI for TRV; BamHI for BPMV
Visual Marker Constructs	System validation and optimization [3] [7]	PDS (photobleaching), GFP (fluorescence)
Gene-Specific Primers	Amplification of target fragments from cDNA [3]	Designed with appropriate restriction sites

Technical Considerations and Optimization Strategies

Factors Influencing Silencing Efficiency

Multiple factors beyond insert design can significantly impact VIGS efficiency. Environmental conditions, particularly temperature, play a crucial role in the success of VIGS experiments. Research in petunia has demonstrated that temperatures of 20°C day/18°C night induced stronger gene silencing compared to higher temperatures [35]. Plant developmental stage at inoculation is another critical factor, with more effective silencing typically achieved when plants are inoculated at 3-4 weeks versus 5 weeks after sowing [35].

The selection of an appropriate control vector is essential for accurate data interpretation. Studies have revealed that empty pTRV2 vectors can induce severe viral symptoms including necrosis, chlorosis, and stunting [35]. These symptoms can be eliminated by using control vectors containing non-plant inserts such as GFP fragments, which provides more reliable controls for phenotypic assessments [35].

Addressing Technical Challenges

Several technical challenges may arise during VIGS experiments that can be mitigated through strategic approaches:

• Low Infection Efficiency: In soybean, conventional inoculation methods (misting, direct injection) often show low efficiency due to thick cuticles and dense trichomes. The optimized cotyledon node immersion method significantly improves infection rates to over 80%, reaching up to 95% for certain cultivars [3].
• Incomplete Silencing: The non-uniform nature of VIGS can result in mosaic silencing patterns. Increasing the number of biological replicates and quantitative phenotypic assessments can address this limitation.
• Viral Symptom Interference: Choosing viral isolates that induce mild symptoms (e.g., BPMV IA-Di1 isolate) reduces interference with silencing phenotypes [7].
• Genotype Dependence: Susceptibility to viral infection varies among genotypes. Preliminary screens for compatible genotypes are recommended before large-scale experiments [7].

The strategic design of insert fragments—encompassing size optimization, proper orientation, and target region selection—forms the foundation of successful VIGS experiments in soybean. While both TRV and BPMV vector systems offer effective approaches for gene function analysis, they present distinct advantages and considerations for researchers. The TRV system demonstrates high silencing efficiency (65-95%) with minimal viral symptoms, while the BPMV "one-step" vector provides simplified cloning and delivery procedures. As soybean functional genomics continues to advance, refined VIGS protocols will play an increasingly vital role in bridging the gap between genomic sequence information and biological function, ultimately accelerating the development of improved soybean varieties with enhanced agronomic traits.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for functional genomics in plants, enabling rapid characterization of gene function without stable transformation. For soybean research, two viral vector systems have been predominantly developed: Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV). Both systems exhibit distinct patterns of viral movement and tissue coverage, creating critical considerations for researchers designing gene function studies. This guide provides an objective comparison of TRV and BPMV VIGS vectors, focusing on their systemic spread efficiency and tissue coverage capabilities, to inform appropriate vector selection for specific experimental needs.

Vector Architectures and Key Components

TRV Vector System

The TRV-based VIGS system utilizes a bipartite genome with separate vectors for RNA1 (pTRV1) and RNA2 (pTRV2). The RNA1 component (6,765 nt in California isolate) encodes replicase proteins (134 and 194 kDa), a movement protein (29 kDa), and a cysteine-rich silencing suppressor protein (16 kDa). The RNA2 component (3,682 nt in California isolate) contains the coat protein and non-structural proteins, with a multiple cloning site for inserting target gene fragments [36] [16]. Modern TRV vectors incorporate the Cauliflower Mosaic Virus (CaMV) 35S promoter and ribozyme sequences for proper transcript processing, with recent modifications including GFP fusions for tracking viral movement [3] [16].

BPMV Vector System

The BPMV system also features a bipartite genome, with RNA1 (approximately 6 kb) handling replication and pathogenicity, while RNA2 (approximately 3.6 kb) is modified to accept foreign inserts. The "one-step" BPMV vector allows direct cloning of target sequences into RNA2, with insertion typically occurring between the movement protein and large coat protein coding regions. Additional proteinase cleavage sites are engineered to flank foreign proteins, utilizing genetic code degeneracy to enhance insert stability through serial passages [10] [7].

Comparative Performance Data

Table 1: Quantitative Comparison of TRV and BPMV VIGS Vectors in Soybean

Parameter	TRV-Based VIGS	BPMV-Based VIGS
Silencing Efficiency Range	65% - 95% [3]	Extensive silencing in multiple tissues [14]
Time to Initial Silencing	14-21 days post-inoculation [3]	14 days post-inoculation [14]
Silencing Duration	Up to 35 days with sustained effect [3]	Up to 7 weeks in floral tissues [14]
Optimal Temperature Range	19°C - 25°C (standard TRV) [36]; California isolate effective at 28°C-30°C [36]	Not temperature-sensitive in reported studies
Tissue Coverage	Systemic spread including meristems [16]	Widespread in leaves, stems, flowers, roots [14]
Insert Stability	Stable through experimental period [3]	Stable after four passages [14]
Infection Efficiency	>80% with optimized cotyledon node method [3]	92%-100% with optimized rub-inoculation [7]

Table 2: Tissue-Specific Silencing Efficiency Across Plant Organs

Plant Tissue	TRV Silencing Efficiency	BPMV Silencing Efficiency
Leaves	Strong photobleaching by 21 dpi [3]	Near-complete silencing [14]
Stems	Not explicitly reported	Near-complete, uniform across cell types [14]
Flowers	Not explicitly reported	95% reduction in GFP mRNA [14]
Roots	Not explicitly reported	Weaker than shoots but significant [14]
Meristems	Effective colonization [16]	Not explicitly reported

Methodological Protocols

TRV Inoculation Protocol

The optimized TRV delivery method for soybean utilizes Agrobacterium tumefaciens-mediated infection through cotyledon nodes [3]:

• Plant Material Preparation: Surface-sterilize soybean seeds and germinate on sterile medium. Use half-seed explants containing cotyledonary nodes for inoculation.
• Agrobacterium Preparation: Transform GV3101 strains with pTRV1 and pTRV2-derived constructs. Grow cultures to OD600 = 1.0-1.2 in appropriate antibiotics.
• Inoculation: Immerse fresh explants in Agrobacterium suspensions for 20-30 minutes, ensuring complete coverage of cotyledonary nodes.
• Co-cultivation: Transfer inoculated explants to co-cultivation medium for 2-3 days in darkness.
• Recovery and Selection: Move explants to regeneration medium with antibiotics to suppress Agrobacterium overgrowth.
• Phenotype Monitoring: Observe silencing phenotypes beginning at 14-21 days post-inoculation.

This method achieves >80% infection efficiency, with fluorescence microscopy confirming successful transformation in transverse sections [3].

BPMV Inoculation Protocol

The efficient "one-step" BPMV delivery method uses direct plasmid rub-inoculation [7]:

• Plasmid Preparation: Purify BPMV RNA1 and RNA2-derived plasmids at concentrations of 5 μg each in inoculation buffer.
• Plant Preparation: Grow soybean plants (cv. Black Valentine) to primary leaf stage (approximately 7-10 days post-emergence).
• Inoculation: Apply plasmid mixture to carborundum-dusted primary leaves using gentle rubbing motion.
• Post-Inoculation Care: Rinse leaves with distilled water and maintain plants under standard growth conditions.
• Efficiency Optimization: Inoculate both primary leaves with moderate rubbing pressure to maximize infection rates while minimizing tissue damage.
• Monitoring: Assess viral spread and silencing phenotypes from 14 days post-inoculation.

This approach achieves 92%-100% infection rates when optimized [7].

Molecular Mechanisms of Viral Movement

The differential tissue colonization patterns between TRV and BPMV stem from their distinct molecular mechanisms for systemic movement. The following diagram illustrates the key pathways and components:

Molecular Pathways of TRV and BPMV Systemic Movement

TRV utilizes a 29 kDa movement protein (MP) that facilitates cell-to-cell transport through plasmodesmata, enabling comprehensive colonization including meristem tissues [36] [16]. This capability allows TRV to access developing tissues and achieve whole-plant coverage. The 16 kDa cysteine-rich protein (CRP) functions as a silencing suppressor, countering plant defense mechanisms to enhance viral spread [36].

BPMV employs distinct movement proteins that facilitate vascular transport, allowing efficient long-distance movement and broad tissue colonization [10] [14]. This mechanism enables BPMV to reach roots and floral tissues effectively, though with potentially variable efficiency across different cell types.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagent Solutions for VIGS Experiments

Reagent/Resource	Function/Application	Specific Examples
Binary Vectors	Viral genome components for silencing	pTRV1, pTRV2-GFP [3]; pBPMV-IA-R1M, pBPMV-IA-V1 [7]
Agrobacterium Strains	Delivery vehicle for TRV vectors	GV3101 [3]
Selection Antibiotics	Maintain plasmid stability and select transformed bacteria	Kanamycin, Rifampicin [3] [7]
Plant Cultivars	Optimized hosts for VIGS	Soybean: Tianlong 1 (TRV) [3]; Black Valentine (BPMV) [7]
Fluorescence Markers	Track infection efficiency and spatial patterns	GFP constructs [3] [14]
Enzymes for Analysis	Verify insert stability and silencing efficiency	Reverse transcriptase, PCR components [3] [14]

Experimental Design Considerations

Vector Selection Guidelines

The experimental workflow for planning and implementing VIGS studies involves several critical decision points:

Experimental Workflow for VIGS Vector Selection

Select TRV when:

• Studying genes involved in meristem development or requiring whole-plant coverage [16]
• Working with temperature-controlled environments (19°C-25°C for standard TRV; up to 30°C for California isolate) [36]
• Agrobacterium delivery is feasible in your experimental system [3]
• Rapid screening (3-4 weeks) is prioritized [16]

Select BPMV when:

• Research focuses on root biology or floral development [14]
• Insert stability through multiple passages is critical [10]
• Direct plasmid delivery is preferred over Agrobacterium [7]
• Minimal viral symptom interference is desired (mild IA-Di1 isolate) [7]

Both TRV and BPMV VIGS systems offer robust approaches for functional gene analysis in soybean, yet they demonstrate distinct advantages for specific research applications. TRV vectors provide exceptional whole-plant coverage including meristem tissues, with recently developed isolates expanding their utility to higher temperature conditions. BPMV vectors excel in achieving comprehensive tissue colonization including roots and flowers, with stable insert maintenance for extended studies. The selection between these systems should be guided by specific experimental requirements regarding target tissues, environmental conditions, and technical constraints. Continued optimization of both platforms promises to further enhance their capabilities for high-throughput functional genomics in soybean and other legume species.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional gene analysis in plants, particularly in species like soybean that are recalcitrant to stable genetic transformation [4] [37]. Among the various viral vectors developed for VIGS, Tobacco rattle virus (TRV) and Bean pod mottle virus (BPMV) represent two of the most prominent systems employed in soybean research. A critical challenge in utilizing these viral vectors lies in maintaining the stability of recombinant constructs and preventing vector rearrangement, which directly impacts experimental reliability and reproducibility. This review systematically compares the stability considerations of TRV and BPMV VIGS vectors, providing experimental data and methodologies relevant for researchers engaged in soybean functional genomics.

Molecular Mechanisms of Vector Rearrangement

Viral vector rearrangements primarily occur through homologous recombination events, where sequences with high similarity undergo illegitimate recombination during viral replication, leading to insert deletion or modification. This phenomenon poses a significant challenge for maintaining consistent silencing phenotypes across experiments.

The BPMV system addresses this through proteinase cleavage site engineering. In developing BPMV-based vectors, researchers inserted genes of interest into the RNA2-encoded polyprotein open reading frame between the movement protein (MP) and large coat protein (L-CP) regions. Additional proteinase cleavage sites were created by duplicating the MP/L-CP cleavage site, while exploiting genetic code degeneracy to alter nucleotide sequences of duplicated regions without affecting amino acid sequences. This design minimizes homologous recombination potential while maintaining proper polyprotein processing [38] [10].

In contrast, TRV vectors benefit from a different structural organization. TRV contains a bipartite genome with RNA1 encoding replication and movement proteins, and RNA2 serving as the vehicle for insert delivery. The development of advanced TRV vectors (TRV2-MCS, TRV2-GATEWAY) incorporated duplicated CaMV 35S promoters and self-cleaving ribozyme sequences to enhance infectivity and reduce recombination-prone regions [37].

Figure 1: Molecular strategies employed by BPMV and TRV vectors to prevent recombinant vector rearrangement.

Comparative Vector Stability Analysis

Table 1: Direct Comparison of TRV and BPMV Vector Stability Features

Parameter	TRV-Based VIGS System	BPMV-Based VIGS System
Genetic Stability	Moderate (dependent on insert size)	High (engineered proteinase sites)
Insert Retention	65-95% over experimental duration [4]	Stable through serial passages [38]
Recombination Prevention	Vector modification (promoters, ribozymes) [37]	Sequence degeneracy in duplicated regions [10]
Typical Insert Size	300-500 bp [37]	Varies (demonstrated with multiple genes) [38]
Symptoms Interference	Mild viral symptoms [4] [37]	Reduced with mild strain selection [39]

Experimental Data on Vector Performance

Recent studies with TRV-based vectors in soybean demonstrate 65-95% silencing efficiency when targeting endogenous genes like phytoene desaturase (GmPDS), rust resistance gene (GmRpp6907), and defense-related gene (GmRPT4) [4]. This high efficiency indicates substantial vector stability throughout the experimental timeframe. The optimized protocol employed Agrobacterium tumefaciens-mediated infection through cotyledon nodes, with systemic silencing observed throughout the plant.

BPMV vector stability has been demonstrated through multiple serial passages in soybean while maintaining relatively high levels of protein expression [38]. The system successfully expressed various proteins with different biological activities, including GFP, DsRed, phosphinothricin acetyltransferase, and multiple RNA silencing suppressors, indicating robust maintenance of inserted sequences.

Table 2: Quantitative Performance Metrics of VIGS Vectors in Soybean

Performance Metric	TRV-VIGS	BPMV-VIGS
Silencing Efficiency	65-95% [4]	Effective (specific percentages not provided) [38]
Time to Phenotype	21 days post-inoculation [4]	Not specified
Tissue Coverage	Systemic (including meristems) [4] [37]	Shoots and roots [39]
Protein Expression	Not primary application	High-level foreign protein expression [38]
Host Range	Extensive (Solanaceae, Cruciferae, Gramineae) [37]	Primarily legumes (soybean, common bean) [40]

Methodologies for Assessing Vector Stability

TRV-VIGS Stability Protocol

The optimized TRV–VIGS protocol for soybean utilizes Agrobacterium tumefaciens GV3101 containing pTRV1 and pTRV2–GFP derivatives [4] [3]. Key steps include:

• Vector Construction: Target gene fragments (e.g., GmPDS, Rpp6907, RPT4) cloned into pTRV2–GFP using EcoRI and XhoI restriction sites
• Plant Material Preparation: Sterilized soybeans soaked in sterile water until swollen, then longitudinally bisected to obtain half-seed explants
• Agroinfiltration: Fresh explants immersed in Agrobacterium suspensions for 20-30 minutes (optimal duration)
• Stability Assessment: GFP fluorescence evaluation at infection sites on day 4 post-infection; effective infectivity efficiency exceeding 80%, reaching 95% for specific cultivars like Tianlong 1
• Silencing Verification: Phenotypic observation (photobleaching for GmPDS) and molecular analysis at 21 days post-inoculation

BPMV-VIGS Stability Protocol

The DNA-based BPMV vector system utilizes a Cauliflower mosaic virus 35S promoter-driven construct [39]:

• Vector Design: Insertion of genes of interest between movement protein and large coat protein coding regions in RNA2
• Stability Engineering: Creation of additional proteinase cleavage sites flanking foreign proteins through duplication of MP/L-CP cleavage site
• Sequence Optimization: Alteration of nucleotide sequence in duplicated regions using genetic code degeneracy to minimize homologous recombination
• Stability Validation: Serial passage experiments in soybean with evaluation of insert retention and protein expression levels over time

Figure 2: Experimental workflows for assessing vector stability in TRV and BPMV VIGS systems.

Research Reagent Solutions for Vector Stability Studies

Table 3: Essential Research Reagents for VIGS Vector Stability Experiments

Reagent/Resource	Function in Stability Studies	Example Applications
pTRV2–GFP Vector	TRV-based delivery of target inserts; GFP allows visualization of infection efficiency [4]	Soybean gene silencing with efficiency monitoring
BPMV RNA2 Vector	BPMV-based vehicle for gene expression and silencing; engineered for insert stability [38]	Stable protein expression and long-term silencing
Agrobacterium tumefaciens GV3101	Mediates viral vector delivery into plant tissues [4] [3]	Cotyledon node transformation in soybean
Restriction Enzymes (EcoRI, XhoI)	Cloning of target fragments into viral vectors [4]	Insertion of gene-specific sequences into TRV2
Gateway Recombination System	Alternative cloning method for TRV vectors; enables high-throughput construction [37]	Rapid generation of multiple silencing constructs
Soybean Cultivar Tianlong 1	Optimized host for TRV–VIGS with high infection efficiency (up to 95%) [4]	High-efficiency soybean functional genomics

Both TRV and BPMV VIGS systems offer distinct approaches to addressing the critical challenge of recombinant vector rearrangement in soybean research. The BPMV system provides exceptional stability through proteinase cleavage site engineering and sequence degeneracy, making it ideal for long-term studies requiring consistent expression or silencing. The TRV system offers broader host range compatibility and rapid high-efficiency silencing, with recent methodological advances significantly improving its performance in soybean. Selection between these systems should be guided by specific experimental requirements: BPMV for maximum insert stability across generations, and TRV for rapid assessment across multiple gene targets with high efficiency. Continued refinement of both systems will further enhance their utility for soybean functional genomics and accelerate the identification of agronomically important genes.

Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional gene analysis in plants, particularly in species like soybean that are recalcitrant to stable genetic transformation [3] [14]. Two principal viral vector systems—Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV)—have been developed and optimized for VIGS in soybean. However, their effectiveness exhibits significant cultivar-dependent variation, necessitating protocol adaptations for different soybean genotypes [3] [7]. This guide provides a comparative analysis of TRV and BPMV VIGS systems, focusing on cultivar-specific optimization strategies to maximize silencing efficiency across diverse genetic backgrounds.

Vector Systems: Core Characteristics and Cultivar Compatibility

TRV and BPMV Vector Profiles

Table 1: Comparative Analysis of TRV and BPMV VIGS Vectors in Soybean

Feature	TRV-Based VIGS	BPMV-Based VIGS
Original Host Range	Solanaceous species (tomato, tobacco, pepper) [3]	Legumes (common bean, soybean) [7] [10]
Typical Delivery Method	Agrobacterium-mediated cotyledon node infection [3]	Direct plasmid rubbing or biolistics [7]
Key Susceptible Cultivars	Tianlong 1 (95% efficiency) [3]	Black Valentine, JaloEEP558 [7]
Silencing Onset	~21 days post-inoculation (dpi) [3]	~14 dpi in leaves [14]
Silencing Duration	Not specified	Up to 7 weeks in flowers [14]
Tissue Silencing Range	Systemic (leaves) [3]	Widespread (leaves, stems, flowers, roots) [14]
Typical Efficiency Range	65%-95% [3]	Near-complete in leaves and flowers [14]
Advantages	Milder viral symptoms, high efficiency in optimized cultivars [3]	Broad tissue coverage, stable silencing, well-established for legumes [14] [10]
Limitations	Limited application reports in soybean, method relatively new [3]	Technical hurdles with particle bombardment, potential leaf phenotype alterations [3]

Molecular Mechanisms and Workflows

Table 2: Key Research Reagent Solutions for VIGS in Soybean

Reagent/Vector	Function/Purpose	Cultivar-Specific Considerations
pTRV1 and pTRV2 Vectors	TRV genomic components for VIGS construct assembly [3]	Requires optimization of Agrobacterium infection method for different cultivars [3]
BPMV-IA-V1 Vector	BPMV-based "one-step" silencing vector [7]	Susceptibility limited to specific cultivars like Black Valentine [7]
Agrobacterium tumefaciens GV3101	Delivery vehicle for TRV vectors [3]	Infection efficiency varies with soybean genotype; cotyledon node method effective for tough leaves [3]
Direct Plasmid Rubbing Inoculum	Mechanical delivery of BPMV plasmids [7]	Requires optimization of plasmid quantity (5μg each RNA1/RSA2 recommended) and rubbing intensity [7]

The fundamental mechanism of VIGS involves sequence-specific degradation of endogenous mRNA triggered by viral replication. When a recombinant virus carrying a fragment of a target host gene infects the plant, the plant's RNA interference machinery processes viral double-stranded RNA replication intermediates into small interfering RNAs (siRNAs). These siRNAs are incorporated into the RNA-induced silencing complex (RISC), which directs the cleavage of homologous endogenous transcripts, resulting in gene silencing [14].

Cultivar-Specific Optimization Strategies

TRV-VIGS Protocol Adaptation

The TRV-based system has been successfully optimized for soybean cultivar Tianlong 1, achieving impressive silencing efficiencies of 65%-95% [3]. Key adaptations include:

• Infection Method Optimization: Conventional misting and direct injection methods showed low efficiency due to soybean leaves' thick cuticles and dense trichomes. The optimized protocol uses Agrobacterium-mediated infection through cotyledon nodes with 20-30 minute immersion, resulting in infection rates exceeding 80% [3].

• Efficiency Validation: Fluorescence microscopy revealed successful infection initially infiltrated 2-3 cell layers before gradually spreading to deeper cells, with transverse sections showing more than 80% of cells exhibiting successful infiltration [3].

• Genotype Considerations: While TRV has been widely used in solanaceous crops, reports on its application in soybean remain limited, highlighting the need for further cultivar-specific adaptations [3].

BPMV-VIGS Protocol Adaptation

BPMV-based VIGS has demonstrated efficacy across various soybean tissues, but cultivar susceptibility varies significantly:

• Cultivar Screening: Among six tested common bean cultivars, only Black Valentine and JaloEEP558 showed susceptibility to BPMV infection, underscoring the importance of preliminary cultivar screening [7].

• Insert Optimization: Studies using GFP as a silencing marker revealed that insert orientation and targeted region significantly impact silencing efficiency. A 3' sequence in reverse orientation produced the strongest silencing phenotypes in soybean [14].

• Temporal and Spatial Patterns: BPMV-induced silencing occurs as early as 14 dpi in leaves and persists up to 7 weeks in flowers, with near-complete silencing observed in leaves, stems, flowers, and roots [14]. Cross-sections of stems and leaf petioles showed uniform silencing across all cell types [14].

Experimental Data and Efficiency Metrics

Quantitative Silencing Assessment

Table 3: Temporal and Spatial Silencing Efficiency of BPMV-VIGS

Tissue Type	Silencing Onset	Peak Efficiency	Duration	Efficiency Assessment
Leaves	14 dpi [14]	21 dpi [14]	Up to 35 dpi [14]	Significant reduction in GFP fluorescence & mRNA [14]
Flowers	49 dpi [14]	49 dpi [14]	Not specified	95% reduction in GFP mRNA [14]
Stems	Not specified	Not specified	Not specified	Near-complete silencing in all cell types [14]
Roots	Not specified	Weaker than shoots [14]	Not specified	Observable but reduced efficiency [14]

Functional Validation Examples

Both vector systems have been successfully employed to characterize genes involved in soybean disease resistance:

• TRV System Applications: The TRV-VIGS system has been used to silence key genes including phytoene desaturase (GmPDS), the rust resistance gene GmRpp6907, and the defense-related gene GmRPT4, confirming the system's robustness for functional genomics studies [3].

• BPMV System Applications: BPMV-induced silencing has identified several regulatory genes, including GmBIR1—a negative regulator of immunity whose silencing results in constitutively activated defense responses, enhanced resistance to Pseudomonas syringae and Soybean mosaic virus (SMV), and over-accumulation of SA and H₂O₂ [32].

Technical Considerations for Cultivar Optimization

Vector Selection Criteria

• BPMV Advantages: As a natural legume pathogen, BPMV often shows broader compatibility with soybean cultivars and achieves more widespread silencing across tissues, including roots [14] [7]. The development of "one-step" BPMV vectors enables direct plasmid rubbing, simplifying inoculation procedures [7].

• TRV Advantages: TRV typically elicits milder viral symptoms compared to other viruses, minimizing potential interference with silencing phenotypes [3]. The Agrobacterium-mediated delivery may be more adaptable to certain laboratory setups.

Protocol Adaptation Framework

Successful cultivar-specific optimization should include:

• Preliminary Susceptibility Testing: Screen candidate cultivars using marker genes (e.g., GmPDS) to establish baseline infection efficiency [3] [7].

• Delivery Method Optimization: Adapt inoculation techniques based on cultivar characteristics—Agrobacterium immersion for tough-leaved genotypes [3], direct rubbing for susceptible cultivars [7].

• Temporal Monitoring: Establish cultivar-specific silencing timelines, as efficiency peaks and durations vary between genotypes [14].

The comparative analysis of TRV and BPMV VIGS systems reveals distinct advantages and limitations that interact significantly with soybean genotype. BPMV-based vectors generally offer broader tissue coverage and more established protocols for legumes, while TRV-based systems provide milder symptom development and a promising alternative for specific cultivars like Tianlong 1. Successful implementation requires careful matching of vector systems to cultivar susceptibility, coupled with optimized delivery methods tailored to genotype-specific characteristics. As soybean functional genomics advances, continued refinement of these VIGS platforms will be essential for maximizing their utility across diverse genetic backgrounds, ultimately accelerating gene function discovery and trait improvement in this important crop species.

Environmental and Growth Conditions for Sustained Silencing

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for functional genomics in plants, particularly in species like soybean that are recalcitrant to stable genetic transformation. This technology exploits the plant's natural antiviral defense mechanism, specifically post-transcriptional gene silencing (PTGS), to systematically suppress target gene expression. When a recombinant viral vector carrying a fragment of an endogenous plant gene is introduced, it triggers sequence-specific degradation of complementary mRNA, leading to readily observable phenotypic changes that enable rapid gene function characterization [16] [15].

The efficiency and sustainability of VIGS are profoundly influenced by environmental conditions and plant growth parameters. Among the various viral vectors developed, Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) have emerged as the most prominent systems for soybean functional genomics. Understanding how environmental factors affect these vector systems is crucial for designing experiments that achieve sustained and reliable silencing phenotypes [3] [11].

Comparative Analysis of TRV and BPMV VIGS Systems

Table 1: System Overview and Key Characteristics of TRV and BPMV VIGS Vectors

Parameter	TRV-Based VIGS	BPMV-Based VIGS
Vector Type	RNA virus (bipartite)	RNA virus (bipartite)
Primary Delivery Method	Agrobacterium-mediated (cotyledon node immersion) [3]	Biolistic delivery or direct rub-inoculation of plasmid DNA [11] [7]
Optimal Temperature	20°C (post-inoculation) [11]	20°C (promotes virus replication and movement) [11]
Key Environmental Factors	Temperature, humidity, plant growth stage [16] [41]	Temperature, light intensity (100-110 μmol m⁻² s⁻¹) [11]
Silencing Onset	Phenotypes observed by 21 dpi (e.g., GmPDS photobleaching) [3]	Symptoms on 2nd trifoliate leaves and thereafter (~2-3 weeks) [11]
Reported Silencing Efficiency	65% - 95% [3] [34]	Effectively silences genes in roots and leaves [11] [42]
Systemic Movement	Effective systemic spread, including meristems [16]	Efficient movement to roots and systemic leaves [11] [7]

Table 2: Experimental Workflow and Host Response Comparison

Aspect	TRV-Based VIGS	BPMV-Based VIGS
Typical Host Plants	Soybean, Nicotiana benthamiana, tomato, pepper, Arabidopsis [3] [16] [41]	Soybean, common bean (Phaseolus vulgaris) [11] [7]
Infection Symptoms	Mild symptoms, minimizing phenotype interference [3] [16]	Mild mosaic symptoms on leaves; milder symptoms with IA-Di1 isolate [11] [7]
Tissue Applications	Leaves, stems, roots (via optimized methods) [41]	Foliar tissues, roots (with specific protocols) [11]
Critical Growth Stage	Seedlings with cotyledon nodes [3]	7-day-old seedlings for bombardment; specific stages for rub-inoculation [11] [7]
Key Advantages	Wide host range, mild symptoms, meristem penetration [16]	Well-established for soybean, suitable for root-pathogen studies [11] [42]

Optimized Experimental Protocols

TRV-Based VIGS in Soybean

The Agrobacterium-mediated TRV-VIGS protocol has been optimized for soybean through cotyledon node infection. The process begins with soaking sterilized soybean seeds in sterile water until swollen, followed by longitudinal bisecting to obtain half-seed explants. Fresh explants are infected by immersion for 20-30 minutes in Agrobacterium tumefaciens GV3101 suspensions containing both pTRV1 and pTRV2-derived vectors (e.g., pTRV2-GFP-GmPDS). Successful infection is confirmed around the fourth day post-infection by observing GFP fluorescence signals at the infection sites, with effective infectivity efficiency exceeding 80% and reaching up to 95% in specific cultivars like Tianlong 1. Following this procedure, photobleaching in leaves inoculated with pTRV:GmPDS typically appears at approximately 21 days post-inoculation (dpi) [3].

BPMV-Based VIGS for Soybean Roots

The BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots involves a detailed pipeline for analyzing genes involved in resistance to soybean cyst nematode (SCN). The process initiates with biolistic delivery of viral vector DNA into the unifoliate leaves of 7-day-old soybean seedlings using a biolistic transformation system. Plasmid DNA encoding BPMV RNA1 is co-bombarded with RNA2 constructs (e.g., pBPMV-SHMT) onto leaves supported by a plexiglass plate and wire mesh under vacuum. Following bombardment, plants are maintained at 20°C with a 16-hour light/8-hour dark regime and light intensity of 100-110 μmol cm⁻² s⁻¹. Cool temperatures are optimal for virus replication and movement within the plant and symptom development. Viral symptoms typically appear as a mild mosaic on leaves approximately 2-3 weeks after bombardment [11].

Essential Research Reagent Solutions

Table 3: Key Reagents and Materials for VIGS Experiments

Reagent/Material	Function/Application	Specific Examples
Binary VIGS Vectors	Engineered viral genomes for silencing construct delivery	pTRV1, pTRV2 [3] [16]; pBPMV-IA-R1M, pBPMV-IA-V1 [11] [7]
Agrobacterium Strains	Delivery of T-DNA containing viral genome	GV3101 for TRV [3] [41]
Antibiotics	Selection of bacterial strains with recombinant plasmids	Kanamycin (50 μg/mL), Rifampicin (25 μg/mL) [41]
Induction Compounds	Activate Agrobacterium virulence genes	Acetosyringone (150-200 μM) [41]
Plant Growth Media	Seed germination and plant maintenance	Sunshine MVP mix [11]
Visual Markers	Monitor infection efficiency and silencing	GFP (Green Fluorescent Protein) [3] [7]; PDS (Phytoene desaturase) [3] [16]

Signaling Pathways and Experimental Workflows

TRV-VIGS Experimental Workflow for Soybean

BPMV-VIGS Experimental Workflow for Soybean

The sustained silencing efficiency of both TRV and BPMV VIGS systems in soybean is profoundly influenced by environmental and growth conditions. Temperature regulation emerges as a critical factor, with 20°C consistently proving optimal for viral replication and systemic movement in both systems. The TRV system offers advantages through its Agrobacterium-mediated delivery, broader host range, and minimal symptom development, while the BPMV system provides established protocols for root-pathogen interactions and reliable performance in soybean.

Successful implementation requires careful attention to plant growth stage, with both systems utilizing young seedlings for optimal infection. Humidity management, light intensity regulation, and appropriate bacterial concentrations further contribute to achieving consistent and sustained silencing. By optimizing these environmental parameters and selecting the appropriate vector system based on experimental goals, researchers can leverage VIGS technology to accelerate functional genomics studies and disease resistance research in soybean.

Vector Comparison: Performance Metrics and Selection Criteria

Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool for functional genomics in plants, particularly for species like soybean that are recalcitrant to stable genetic transformation [3] [15]. This RNA silencing-based technique leverages the plant's innate antiviral defense mechanism to target homologous endogenous genes for post-transcriptional silencing [43] [15]. Among the various viral vectors developed for VIGS, Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) have become the most prominent systems for soybean functional genomics research [3] [9]. While both systems enable rapid gene function analysis without stable transformation, they differ significantly in their silencing efficiency, implementation protocols, and applicability across different soybean genotypes. This analysis provides a systematic comparison of TRV and BPMV VIGS vectors, focusing specifically on their silencing rates and duration, to guide researchers in selecting the appropriate tool for their functional genomics studies.

TRV-Based VIGS System

The TRV-based VIGS system is a bipartite RNA vector consisting of TRV1 and TRV2 components, with the target gene fragment typically cloned into the TRV2 vector [3] [43]. TRV has gained popularity due to its mild viral symptoms, effective meristem invasion, and broad host range [3] [43]. Recent optimization for soybean involved Agrobacterium tumefaciens-mediated delivery through cotyledon nodes, achieving systemic spread and effective silencing of endogenous genes [3]. The key advantage of TRV is its capacity to infect meristematic tissues and trigger silencing in various plant organs, including roots, flowers, and fruits [43] [44].

BPMV-Based VIGS System

BPMV is a bipartite positive-strand RNA virus belonging to the Comovirus genus, with RNA1 and RNA2 components [9] [7]. Earlier BPMV vectors required in-vitro transcription or particle bombardment for delivery, but recent "one-step" DNA-based vectors enable direct rub-inoculation of plasmid DNA under the control of the CaMV 35S promoter [9] [7]. BPMV has been extensively used in soybean for studying disease resistance genes and defense signaling components [3] [9]. A significant development was the modification of the RNA2 component to allow insertion of silencing fragments after the translation stop codon, enabling the use of non-coding sequences and overcoming the requirement for open reading frame fusions [9].

Table 1: Fundamental Characteristics of TRV and BPMV VIGS Vectors

Characteristic	TRV-Based System	BPMV-Based System
Virus Type	Bipartite positive-sense RNA virus	Bipartite positive-sense RNA virus
Typical Delivery Method	Agrobacterium-mediated infiltration (cotyledon nodes)	Direct DNA rubbing or biolistic delivery
Primary Applications	Functional validation of defense, development, and metabolic genes	Studies of disease resistance genes and defense signaling
Key Advantages	Mild symptoms, meristem invasion, root silencing capability	Established platform, stable insert maintenance
Insert Cloning Position	Within TRV2 multiple cloning site	Between MP and L-CP or after stop codon in RNA2

Quantitative Efficiency Comparison

Silencing Efficiency and Time Course

Direct comparative studies of TRV and BPMV in soybean are limited, but data from individual studies reveal significant differences in their silencing profiles. The newly established TRV-based system in soybean demonstrated 65% to 95% silencing efficiency for endogenous genes including GmPDS, GmRpp6907, and GmRPT4 when delivered via Agrobacterium-mediated cotyledon node infection [3]. Silencing phenotypes became apparent at approximately 21 days post-inoculation (dpi) and persisted systemically [3].

In contrast, BPMV-mediated silencing efficiency varies based on insert orientation and positioning, with antisense inserts from the 3' ORF region proving most effective [9]. BPMV generally initiates silencing around 14-21 dpi, with maximal effects observed in systemic leaves developing after inoculation [7]. The persistence of BPMV-induced silencing typically extends for several weeks, though the exact duration in soybean has not been comprehensively quantified across developmental stages.

Table 2: Comparative Silencing Efficiency Parameters

Parameter	TRV-Based System	BPMV-Based System
Silencing Efficiency Range	65-95% [3]	Varies by insert design and orientation [9]
Time to Onset	~21 days [3]	14-21 days [7]
Duration	Not fully characterized; extends for weeks	Several weeks; precise duration not quantified
Tissue Coverage	Systemic, including meristems [3]	Systemic, but limited meristem invasion [7]
Optimal Plant Stage	Young seedlings [3]	10-day-old seedlings [7]

Factors Influencing Silencing Efficiency

Multiple factors impact the silencing efficiency of both TRV and BPMV systems. For TRV, the Agrobacterium delivery method is critical, with conventional approaches (misting, direct injection) showing low efficiency due to soybean's thick cuticle and dense trichomes [3]. The optimized cotyledon node immersion method achieved infection efficiency exceeding 80%, reaching up to 95% for specific soybean cultivars like Tianlong 1 [3]. Plant age also significantly influences TRV efficiency, with younger plants (two-to-three-leaf stage) showing optimal silencing in Arabidopsis models [43].

For BPMV, insert characteristics profoundly affect silencing efficiency. Antisense orientation of the 3' ORF induces more effective silencing than sense orientation or UTR-targeting constructs [9]. The development of BPMV vectors with mild symptom phenotypes (using IA-Di1 isolate) reduces interference with silencing phenotypes, while moderate symptom variants (using mutated RNA1) enable visual tracking of infection without ELISA confirmation [9] [7].

Experimental Protocols and Workflows

TRV VIGS Experimental Workflow

The optimized TRV-VIGS protocol for soybean involves several critical steps that contribute to its efficiency:

• Vector Construction: Target gene fragments (200-300 bp) are amplified with specific primers and cloned into the pTRV2-GFP vector using EcoRI and XhoI restriction sites [3].

• Agrobacterium Preparation: The recombinant pTRV2 construct and the helper pTRV1 are introduced into Agrobacterium tumefaciens strain GV3101 [3].

• Plant Material Preparation: Surface-sterilized soybean seeds are germinated and bisected longitudinally to obtain half-seed explants with cotyledon nodes [3].

• Agroinfiltration: Fresh explants are immersed in Agrobacterium suspensions (OD600 = 0.5-1.0) for 20-30 minutes, optimal for infection [3].

• Co-cultivation and Recovery: Infected explants are transferred to tissue culture media for 3-4 days before transplanting to soil [3].

• Phenotype Monitoring: Silencing phenotypes typically appear at 21 dpi and can be monitored using GFP fluorescence as a marker [3].

BPMV VIGS Experimental Workflow

The streamlined "one-step" BPMV protocol offers an alternative approach:

• Vector Construction: Target sequences are cloned into the modified BPMV RNA2 vector, preferably in antisense orientation for optimal efficiency [9].

• Plasmid Preparation: The BPMV RNA1 (pBPMV-IA-R1M) and recombinant RNA2 plasmids are purified [7].

• Inoculum Preparation: Equal quantities (5μg each) of RNA1 and RNA2 plasmids are mixed in inoculation buffer [7].

• Mechanical Inoculation: The plasmid mixture is directly rub-inoculated onto carborundum-dusted primary leaves of young soybean seedlings (10-day-old) [7].

• Symptom Development: Viral symptoms appear within 7-14 days, with silencing phenotypes evident in subsequent systemic leaves [9] [7].

• Efficiency Validation: Silencing efficiency is confirmed through phenotypic scoring and molecular analysis (qRT-PCR) [9].

Molecular Mechanisms of VIGS

The fundamental molecular mechanism of VIGS is shared across different viral vectors, though efficiency differences arise from variations in viral movement, replication, and host interactions. The process initiates when recombinant viral vectors carrying plant gene fragments enter host cells and begin replication [15] [26]. Viral RNA-dependent RNA polymerases generate double-stranded RNA (dsRNA) replication intermediates, which are recognized by the plant's Dicer or Dicer-like (DCL) nucleases [15]. These enzymes cleave dsRNA into 21-24 nucleotide small interfering RNAs (siRNAs) that are loaded into RNA-induced silencing complexes (RISC) [15] [26]. The RISC complex uses siRNAs as guides to identify and cleave complementary endogenous mRNA molecules, resulting in post-transcriptional gene silencing [15]. The silencing signal amplifies and spreads systemically through the plant, leading to target gene knockdown in tissues distant from the initial infection site [26].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents for VIGS Experiments in Soybean

Reagent/Resource	Function/Purpose	Examples/Specifications
VIGS Vectors	Delivery of target gene fragments into plants	pTRV1, pTRV2 [3]; pBPMV-IA-R1, pBPMV-IA-R2 [9]
Agrobacterium Strain	Plant transformation for TRV delivery	GV3101 [3]
Marker Genes	Silencing efficiency validation	GmPDS, GmCLA1 (photobleaching) [3] [26]
Plant Genotypes	Susceptible hosts for VIGS	Soybean: Tianlong 1 (TRV) [3]; Black Valentine (BPMV) [7]
Inoculation Buffers	Vehicle for vector delivery	MgCl₂, MES, acetosyringone for agroinfiltration [6]

TRV and BPMV VIGS systems offer complementary strengths for soybean functional genomics. The recently optimized TRV system demonstrates notably high silencing efficiency (65-95%) with the advantage of meristem penetration and potentially broader tissue coverage [3]. Its Agrobacterium-mediated delivery, while requiring more specialized tissue culture techniques, provides robust and reproducible silencing. In contrast, the BPMV system offers a more streamlined "one-step" inoculation protocol through direct DNA rubbing, advantageous for higher-throughput studies [9] [7]. BPMV's well-established platform has proven valuable for studying disease resistance pathways, though its silencing efficiency varies more significantly with insert design and orientation. Selection between these systems should be guided by specific research requirements: TRV for maximal silencing efficiency and whole-plant coverage, BPMV for simplified implementation and stable insert maintenance. Future vector development should focus on expanding the range of susceptible soybean genotypes, enhancing silencing persistence, and enabling tissue-specific silencing applications.

Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional genomics studies in plants, particularly for species like soybean that are recalcitrant to stable genetic transformation [3] [11]. This technology leverages the plant's innate RNA-based antiviral defense mechanism, where sequences derived from plant endogenous genes inserted into viral genomes trigger sequence-specific degradation of homologous host transcripts [11] [15]. Among the various VIGS vectors developed for soybean research, Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) represent two of the most prominent systems, each with distinct characteristics and applications.

The efficacy of VIGS is fundamentally dependent on the virus's ability to infect and spread systemically throughout the plant, reaching the tissues where the gene function is to be studied. A comprehensive understanding of the tissue coverage patterns for different VIGS vectors is therefore critical for experimental design and data interpretation. This review systematically compares the tissue coverage of TRV and BPMV vectors in soybean, providing researchers with evidence-based guidance for selecting the appropriate system for studying genes expressed in specific plant organs, including leaves, stems, flowers, and root systems.

Comparative Analysis of TRV and BPMV VIGS Vectors

Table 1: Key characteristics of TRV and BPMV VIGS vectors in soybean

Feature	TRV-Based VIGS	BPMV-Based VIGS
Primary Delivery Method	Agrobacterium tumefaciens-mediated infection via cotyledon node immersion [3]	Biolistic delivery (particle bombardment) or direct rub-inoculation of infectious plasmids [11] [7]
Silencing Onset	Photobleaching phenotypes observed by 21 days post-inoculation (dpi) [3]	Significant silencing in first trifoliate by 14 dpi [14]
Silencing Duration	Silencing sustained through vegetative growth stages [3]	Silencing maintained up to 35 dpi in leaves and 49 dpi in flowers [14]
Typical Silencing Efficiency	65% to 95% [3]	Near-complete silencing in leaves and flowers [14]
Vector Symptom Severity	Elicits fewer symptoms, minimizing phenotype masking [3]	Mild mosaic symptoms; IA-Di1 isolate produces very mild symptoms [7]

Table 2: Tissue coverage comparison of TRV and BPMV VIGS vectors in soybean

Plant Tissue	TRV Coverage Evidence	BPMV Coverage Evidence
Leaves	Systemic photobleaching in leaves, effective silencing [3]	Widespread, strong silencing; 95% mRNA reduction possible [14]
Stems	Systemic spread through plant [3]	Near-complete and uniform silencing across all cell types in cross-sections [14]
Flowers	Not explicitly documented in search results	High-level silencing in all floral parts (petals, sepals, reproductive whorls) [14]
Root Systems	Silencing demonstrated in roots [31]	Silencing achieved but weaker than in shoot tissues [14]
Coverage Pattern	Systemic transmission from cotyledon nodes [3]	Widespread across tissues but with varying efficiency [14]

Experimental Protocols for Assessing Tissue Coverage

TRV-Based VIGS Protocol

The optimized TRV-VIGS protocol utilizes Agrobacterium tumefaciens strain GV3101 harboring the pTRV1 and pTRV2 vectors. The target gene fragment is cloned into the pTRV2-GFP vector using EcoRI and XhoI restriction sites [3]. For inoculation:

• Plant Material Preparation: Surface-sterilized soybean seeds are soaked in sterile water until swollen, then longitudinally bisected to create half-seed explants [3].
• Agroinfiltration: Fresh explants are immersed in Agrobacterium suspensions containing both pTRV1 and recombinant pTRV2 derivatives for 20-30 minutes, determined to be the optimal duration [3].
• Tissue Culture: Following inoculation, explants are transferred to sterile tissue culture conditions to facilitate infection [3].
• Efficiency Assessment: Infection efficiency can be evaluated via GFP fluorescence microscopy at 4 days post-infection, with effective infectivity rates exceeding 80% [3].
• Phenotypic Monitoring: Successful silencing is confirmed by phenotypic observations (e.g., photobleaching for GmPDS) and molecular analysis from 21 dpi onward [3].

BPMV-Based VIGS Protocol

The BPMV system employs a bipartite vector system (RNA1 and RNA2) with different inoculation options [11] [7]:

• Biolistic Delivery (for root studies) [11]:

  • Gold particles (1μm) are coated with a mixture of RNA1 and RNA2 plasmids.
  • Seven-day-old soybean seedlings are bombarded using a PDS-1000/He biolistic system at 28-inch Hg vacuum.
  • Plants are maintained at 20°C post-bombardment for optimal virus replication.
  • Infected leaves are harvested 2-3 weeks later, lyophilized, and stored for use as inoculum.

• Direct Rub-Inoculation [7]:

  • A mixture of purified RNA1 and RNA2 plasmids (5μg each) is applied directly to carborundum-dusted primary leaves.
  • This method simplifies the process by eliminating the need for in vitro transcription or Agrobacterium transformation.

• Tissue Analysis: Silencing efficacy across tissues is quantified using fluorescence measurement (for GFP reporter) and qRT-PCR analysis of endogenous gene expression [14].

VIGS Experimental Workflow: TRV and BPMV Pathways

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key research reagents for implementing VIGS in soybean studies

Reagent/Resource	Function and Application
pTRV1 & pTRV2 Vectors	Essential components of the TRV silencing system; pTRV2 carries the target gene insert [3]
BPMV IA-R1M & IA-V1 Plasmids	Bipartite genome components of the BPMV silencing system [11] [7]
Agrobacterium tumefaciens GV3101	Bacterial strain for delivering TRV vectors into plant tissues [3]
Gold Particles (1μm)	Used for biolistic delivery of BPMV vectors via particle bombardment [11]
GFP Reporter System	Visual marker for evaluating infection efficiency and silencing patterns [3] [14]
Phytoene Desaturase (PDS)	Marker gene causing photobleaching when silenced; validates system efficacy [3] [7]
Soybean Cultivar 'Tianlong 1'	Specific cultivar showing high (95%) TRV infection efficiency [3]
Soybean Cultivar 'Black Valentine'	Common bean and soybean cultivar susceptible to BPMV infection [7]

The comparative analysis of TRV and BPMV VIGS vectors reveals complementary strengths in tissue coverage, which should guide researchers in selecting the appropriate system for their specific experimental needs. TRV demonstrates robust systemic silencing with high efficiency (65-95%), particularly effective in leaves and stems when delivered via the optimized cotyledon node method [3]. Its minimal viral symptoms represent a significant advantage for phenotypic analysis [3]. BPMV establishes strong, widespread silencing in aerial tissues including leaves, stems, and flowers, with documented near-complete silencing in floral organs [14]. While BPMV shows more limited efficacy in root tissues [14], specialized protocols have been developed for root-pathogen interaction studies [11].

The choice between these systems ultimately depends on the target tissue and research objectives. For comprehensive floral gene studies or established BPMV protocols, BPMV offers exceptional performance. For investigations requiring robust root silencing or minimal viral symptom interference, TRV may be preferable. Future developments in vector engineering and delivery methods will likely enhance the tissue coverage and efficiency of both systems, further expanding the capabilities of VIGS technology for soybean functional genomics.

In soybean functional genomics, Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid characterization of gene function. This technology leverages the plant's innate RNA-based antiviral defense mechanism to silence endogenous genes of interest. When a recombinant virus carrying a fragment of a host gene infects the plant, the resulting sequence-specific RNA degradation system targets both viral RNA and the corresponding host mRNA for destruction, leading to down-regulation of the target gene [15] [16].

The selection of an appropriate viral vector is paramount to the success of VIGS experiments, with Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) representing two of the most prominent systems used in soybean research. A critical consideration in vector selection is symptom interference—the phenomenon where viral pathogenicity symptoms mask or confound the phenotypic outcomes of target gene silencing. This comprehensive analysis compares the TRV and BPMV VIGS systems specifically focusing on their pathogenicity profiles and the resulting implications for phenotypic interpretation in soybean research.

Comparative Analysis of TRV and BPMV VIGS Vectors

The strategic selection of a VIGS vector significantly influences experimental outcomes through its inherent biological properties. The table below provides a systematic comparison of TRV and BPMV based on critical parameters for soybean functional genomics.

Table 1: Comparative Characteristics of TRV and BPMV VIGS Vectors in Soybean

Parameter	TRV-Based VIGS System	BPMV-Based VIGS System
Viral Symptoms & Interference Potential	Mild symptoms; minimal interference with silencing phenotypes [3]	Induces leaf phenotypic alterations that can interfere with accurate phenotypic evaluation [3]
Silencing Efficiency	65% to 95% in soybean [3]	Widely adopted due to efficiency and reliability [3]
Delivery Methods	Agrobacterium tumefaciens-mediated via cotyledon node immersion [3]	Frequently relies on particle bombardment; Agrobacterium delivery also developed [3] [7]
Systemic Spread	Effective systemic spread throughout plant, including meristematic tissues [16]	Systemic infection established, but tissue distribution may vary
Experimental Timeline	Phenotypes observable within 3-4 weeks post-inoculation [16]	Requires similar timeframe, though symptom development may vary
Key Advantages	Minimal symptomatic interference, high efficiency, broad tissue coverage	Well-established system with extensive historical use data
Primary Limitations	Application in soybean previously limited though recently optimized [3]	Technical hurdles in implementation; symptomatic interference concerns

Methodological Approaches for Minimizing Symptomatic Interference

TRV-VIGS Protocol for Soybean

The optimized TRV-VIGS protocol for soybean utilizes Agrobacterium-mediated infection through cotyledon nodes to achieve efficient systemic silencing while minimizing pathogenicity symptoms:

• Vector Construction: Clone target gene fragments (300-500 bp) into the pTRV2 vector using appropriate restriction sites (e.g., EcoRI and XhoI) or recombination-based cloning systems [3] [16].

• Agrobacterium Preparation: Transform recombinant plasmids into Agrobacterium tumefaciens strain GV3101. Culture bacteria in liquid LB medium with appropriate antibiotics until OD600 reaches 0.6-1.0 [3] [45].

• Plant Material Preparation: Surface-sterilize soybean seeds and germinate under sterile conditions. Use half-seed explants obtained by longitudinally bisecting swollen soybeans for infection [3].

• Agro-infiltration: Immerse fresh explants in Agrobacterium suspensions containing pTRV1 and pTRV2-derived constructs for 20-30 minutes, ensuring complete tissue saturation [3].

• Plant Regeneration and Growth: Transfer infiltrated explants to tissue culture media for regeneration, then transplant seedlings to soil for further growth under controlled environmental conditions [3].

This method achieves high infection efficiency exceeding 80% (reaching up to 95% in specific cultivars like Tianlong 1) while inducing minimal viral symptoms that could interfere with phenotypic analysis [3].

BPMV-VIGS Implementation Considerations

For BPMV-based systems, several methodological adjustments can help mitigate symptomatic interference:

• Vector Selection: Utilize mild symptom BPMV isolates like IA-Di1 when available, as these induce fewer visual symptoms on infected soybean plants [7].

• Delivery Optimization: Employ the "one-step" BPMV vector system enabling direct rub-inoculation of infectious plasmid DNA, circumventing more invasive delivery methods [7].

• Experimental Controls: Implement rigorous controls including empty vector treatments and non-inoculated plants to distinguish viral symptoms from genuine silencing phenotypes.

• Temporal Monitoring: Conduct regular phenotypic assessments to track the progression of both viral symptoms and potential silencing phenotypes throughout the experiment.

Diagram 1: Impact of Vector Selection on Symptom Interference in VIGS Experiments. The diagram illustrates how choice of viral vector and delivery method influences viral pathogenicity symptoms and subsequent interpretation of silencing phenotypes.

Essential Research Reagents for VIGS Implementation

Successful implementation of VIGS technology requires specific biological materials and reagents carefully selected to optimize silencing efficiency while minimizing confounding factors like symptom interference.

Table 2: Essential Research Reagents for VIGS Studies in Soybean

Reagent Category	Specific Examples	Function and Importance
Viral Vectors	pTRV1, pTRV2, BPMV RNA1 & RNA2 constructs	Core silencing machinery; TRV vectors typically produce milder symptoms than BPMV [3] [16]
Agrobacterium Strains	GV3101	Efficient delivery of viral vectors into plant tissues; optimized for virulence [3] [45]
Plant Genotypes	Soybean cv. Tianlong 1, Black Valentine (for BPMV)	Genotype-specific susceptibility to viral infection affects silencing efficiency and symptom severity [3] [7]
Selection Antibiotics	Kanamycin, Gentamicin	Maintain plasmid integrity in bacterial cultures during scale-up [45]
Induction Compounds	Acetosyringone, MES buffer	Enhance Agrobacterium virulence gene expression during inoculation [45]
Visual Markers	GFP, PDS	Validate infection success and silencing efficiency through fluorescence or photobleaching phenotypes [3] [16]
Reference Genes	GhACT7, GhPP2A1	Ensure accurate RT-qPCR normalization in silencing validation, particularly important under biotic stress conditions [45]

The strategic selection between TRV and BPMV VIGS systems significantly influences experimental outcomes in soybean functional genomics through their distinct pathogenicity profiles. TRV-based vectors demonstrate clear advantages for studies where precise phenotypic interpretation is paramount, offering mild symptom development and minimal interference with silencing phenotypes while maintaining high efficiency (65-95% silencing range). In contrast, BPMV-based systems, despite their historical prominence and reliability, present greater challenges for phenotypic interpretation due to more pronounced virus-induced leaf alterations.

Future methodological developments should focus on further optimizing delivery mechanisms to reduce pathogenicity symptoms across all VIGS platforms. The emerging integration of VIGS with CRISPR/Cas9 technologies as exemplified by Virus-Induced Gene Editing (VIGE) approaches presents promising avenues for enhancing precision in functional genomics while mitigating symptomatic interference concerns [15]. As these technologies evolve, careful consideration of symptom interference will remain fundamental to designing robust experiments and generating reliable functional data in soybean and other crop species.

Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional gene analysis in plants, particularly in species like soybean that are recalcitrant to stable genetic transformation [3] [14]. Among the various viral vectors developed, Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV) have shown significant utility in soybean functional genomics. A critical factor determining the reliability and effectiveness of VIGS in multi-generation studies is insert stability—the ability of the viral vector to retain the inserted foreign gene fragment through serial passages in host plants. This assessment provides a systematic comparison of insert retention between TRV and BPMV VIGS vectors, evaluating their suitability for long-term functional studies.

Vector Systems and Methodologies

TRV-Based VIGS System

The TRV vector system utilizes a bipartite genome organization requiring two plasmid constructs: TRV1 encoding replicase and movement proteins, and TRV2 containing the coat protein and multiple cloning site for insert integration [3] [17]. The established protocol for soybean involves:

• Vector Construction: Target gene fragments (e.g., GmPDS, GmRpp6907, GmRPT4) are cloned into the pTRV2-GFP vector using EcoRI and XhoI restriction sites [3].
• Plant Infection: Agrobacterium tumefaciens strain GV3101 carrying pTRV1 and recombinant pTRV2 is delivered through cotyledon node infiltration [3]. This method achieves infection efficiency exceeding 80-95% in soybean cultivars like Tianlong 1 [3].
• Silencing Validation: Successful gene silencing is confirmed through phenotypic observation (e.g., photobleaching for GmPDS) and molecular analysis using quantitative PCR [3].

BPMV-Based VIGS System

The BPMV system also features a bipartite RNA genome, with inserts typically cloned into RNA2 between the movement protein and large coat protein coding regions [10] [7]. Key methodological aspects include:

• Vector Engineering: Additional proteinase cleavage sites flank foreign proteins through duplication of the MP/L-CP cleavage site [10]. Genetic code degeneracy is exploited to alter nucleotide sequences of duplicated regions without affecting amino acid sequences, minimizing homologous recombination [10].
• Plant Inoculation: The "one-step" BPMV system enables direct rub-inoculation of infectious plasmid DNA, bypassing the need for in vitro transcription or Agrobacterium transformation [7]. Optimal inoculation requires 5μg each of RNA1 and RNA2 plasmids [7].
• Stability Assessment: Insert retention is evaluated through RT-PCR analysis of systemic leaves across multiple passages using primers flanking the insertion site [14].

Comparative Stability Analysis

Insert Stability Assessment Data

Table 1: Comparative insert stability of TRV and BPMV VIGS vectors across serial plant passages

Vector System	Insert Location	Passage Generations	Stability Outcome	Assessment Method	Reference
TRV	pTRV2-GFP vector	3+ serial passages	65-95% retention	qPCR, Phenotyping	[3]
BPMV	RNA2 polyprotein	4 serial passages	Stable retention	RT-PCR	[14]
BPMV	RNA2 polyprotein	Multiple passages	Stable protein expression	Western blot	[10]

Stability Performance and Applications

The BPMV vector system has demonstrated exceptional insert stability, maintaining foreign sequences through at least four serial passages in soybean plants without detectable recombination or loss [10] [14]. This stability has been verified for various inserts, including GFP, DsRed, and phytoene desaturase sequences [10] [14]. The strategic engineering of the BPMV vector, incorporating nucleotide sequence alterations in duplicated regions, significantly reduces homologous recombination risks [10].

The TRV system shows moderately high stability with 65-95% silencing efficiency across multiple passages, sufficient for most functional screening applications [3]. Recent optimization of the Agrobacterium-mediated cotyledon node transformation has significantly improved TRV consistency in soybean [3].

Table 2: Functional applications of TRV and BPMV VIGS vectors in soybean research

Application Area	TRV Vector Utility	BPMV Vector Utility	Key Findings
Disease Resistance	GmRpp6907 (rust resistance) silencing	GmBIR1 silencing enhances SMV resistance	Identified negative immune regulator [3] [32]
Defense Signaling	GmRPT4 (defense-related) silencing	G3PDH family gene silencing increases viral susceptibility	Established G3PDH role in antiviral defense [3] [42]
Metabolic Pathways	GmPDS (carotenoid biosynthesis) silencing	PDS silencing for functional validation	Photobleaching phenotype confirmation [3] [10]
Systemic Silencing	Effective in leaves, stems, flowers	Widespread silencing in leaves, stems, flowers, roots	Comprehensive tissue coverage [3] [14]

Experimental Workflows for Stability Assessment

BPMV Insert Stability Protocol

The assessment of BPMV insert retention across passages involves:

• Serial Passaging: Systemically infected leaf tissue is harvested 14-21 days post-inoculation (dpi) and used as inoculum for subsequent passages through mechanical rub-inoculation [14] [7].
• Stability Monitoring: RT-PCR analysis is performed using: (1) a reverse primer in the insert and forward primer in the viral genome, and (2) primers flanking the insertion site in the BPMV genome [14].
• Expression Consistency: Protein expression stability is verified through Western blot analysis of heterologous proteins across passages [10].

TRV Silencing Persistence Protocol

Evaluation of TRV-mediated silencing stability includes:

• Temporal Monitoring: Silencing persistence is assessed by tracking photobleaching phenotypes or target gene expression from 14 to 35 days post-inoculation [3].
• Molecular Confirmation: qPCR analysis of endogenous gene transcript levels in systemic leaves across multiple trifoliates [3].
• Tissue Coverage: Evaluation of silencing uniformity across different cell types and tissues through fluorescence microscopy and transcript analysis [3].

VIGS Vector Stability Assessment Workflow: This diagram illustrates the comparative experimental pathways for evaluating insert retention in TRV and BPMV VIGS vectors across serial plant passages, highlighting methodological differences in inoculation and assessment approaches.

The Scientist's Toolkit

Table 3: Essential research reagents for VIGS stability assessment in soybean

Reagent/Resource	Function/Purpose	Specific Examples/Applications
pTRV1 & pTRV2 Vectors	TRV bipartite genome components; pTRV2 contains MCS for insert cloning	Soybean gene silencing; 65-95% efficiency [3] [17]
BPMV-IA-V1 Vector	BPMV RNA2-based vector with optimized insertion site for enhanced stability	Stable protein expression and VIGS across 4+ passages [14] [7]
Agrobacterium tumefaciens GV3101	T-DNA delivery for TRV infection	Cotyledon node transformation in soybean [3]
GFP/DsRed Reporter Genes	Visual markers for infection efficiency and silencing assessment	Spatial-temporal tracking of VIGS progression [10] [14]
Phytoene Desaturase (PDS) Gene	Visual silencing marker causing photobleaching phenotype	Validation of VIGS system functionality [3] [10]
Insert-Flanking Primers	PCR amplification for stability assessment across passages	Detection of insert retention in BPMV vectors [14]

Both TRV and BPMV VIGS vectors demonstrate substantial stability for functional genomics applications in soybean, though with distinct characteristics. The BPMV system offers superior insert retention across multiple serial passages, making it particularly valuable for long-term studies requiring consistent transgene expression or silencing persistence. The TRV system provides robust, albeit slightly less stable, performance with broader tissue coverage including meristematic regions. The selection between these systems should be guided by specific research requirements: BPMV for maximum insert stability across generations, and TRV for rapid screening applications with adequate persistence. Both vector systems represent significant advancements over stable transformation for high-throughput functional gene characterization in this agronomically important crop.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional genomics studies in plants, circumventing the challenges of stable genetic transformation. Within soybean research, two viral vector systems have gained prominence: Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV). This guide provides an objective comparison of their technical accessibility, resource requirements, and implementation timelines, supporting researchers in selecting the appropriate system for their experimental needs.

TRV-Based VIGS System: TRV is a positive-sense RNA virus with a bipartite genome (RNA1 and RNA2) that has been modified into a versatile VIGS vector. The system utilizes separate binary vectors for RNA1 (encoding replication and movement proteins) and RNA2 (containing the coat protein and cloning site for target gene fragments). TRV is noted for its wide host range, ability to infect meristematic tissues, and mild viral symptoms that minimize interference with phenotypic observations [2] [16].

BPMV-Based VIGS System: BPMV is a positive-strand RNA virus from the Comoviridae family, also featuring a bipartite genome. RNA1 carries pathogenicity components, while RNA2 is modified to accept foreign gene inserts. The "one-step" BPMV vector allows direct rub-inoculation of plasmid DNA without requiring in vitro transcription or Agrobacterium transformation at the inoculation stage, simplifying the delivery process [7].

Comparative Technical Specifications

Table 1: Direct Comparison of TRV and BPMV VIGS Systems in Soybean

Parameter	TRV-VIGS	BPMV-VIGS
Vector Type	RNA virus	RNA virus
Genome Organization	Bipartite (RNA1, RNA2)	Bipartite (RNA1, RNA2)
Cloning System	Gateway compatible or conventional restriction digestion	Conventional restriction sites
Delivery Method	Agrobacterium-mediated (cotyledon node infiltration)	Direct DNA rubbing or Agrobacterium
Minimum Insert Size	~300-500 bp	132-391 bp
Silencing Efficiency	65-95%	Varies by cultivar
Time to Silencing Phenotype	~3 weeks	~3 weeks
Key Advantages	Broad tissue coverage including meristems; mild symptoms	Simplified inoculation; established legacy in legumes
Primary Limitations	Requires tissue culture steps; optimization needed for different cultivars	Limited to susceptible cultivars; may cause noticeable viral symptoms

Resource Requirements Analysis

Implementation Timeline

Both TRV and BPMV VIGS systems require similar preparatory timelines but differ in their hands-on requirements:

Weeks 1-2: Vector Preparation - Both systems require cloning target gene fragments into respective viral vectors (TRV2 or BPMV RNA2) and transforming into appropriate Agrobacterium strains (e.g., GV3101 for TRV) [3] [7].

Week 3: Plant Growth - Sow soybean seeds and maintain under controlled conditions until cotyledons emerge or primary leaves develop.

Week 4: Inoculation - Critical divergence point between systems:

• TRV-VIGS: Utilizes Agrobacterium-mediated infection through cotyledon nodes. Soybean cotyledons are bisected and immersed in Agrobacterium suspension for 20-30 minutes [3].
• BPMV-VIGS: Employs direct rub-inoculation of plasmid DNA onto carborundum-dusted leaves or Agrobacterium infiltration [7].

Weeks 5-8: Phenotype Observation - Silencing phenotypes typically manifest within 2-4 weeks post-inoculation for both systems [3] [7].

Equipment and Expertise Requirements

Table 2: Resource Requirements for VIGS Implementation

Resource Category	TRV-VIGS	BPMV-VIGS
Molecular Biology	Standard cloning equipment, thermal cycler, electrophoresis	Same requirements
Microbiology	Agrobacterium culture facilities	Agrobacterium culture facilities (optional)
Plant Handling	Sterile tissue culture facility for cotyledon infiltration	Basic plant growth facilities
Technical Expertise	Advanced skills in Agrobacterium handling and sterile technique	Simpler mechanical inoculation skills
Validation Methods	qRT-PCR, fluorescence microscopy (for GFP-tagged vectors)	qRT-PCR, symptom observation

Experimental Protocols

TRV-VIGS Implementation Protocol

The optimized TRV-VIGS protocol for soybean involves the following key steps [3]:

• Vector Construction: Clone 300-500bp target gene fragment into pTRV2 vector using appropriate restriction sites (e.g., EcoRI and XhoI) or Gateway recombination.

• Agrobacterium Preparation: Transform recombinant plasmids into Agrobacterium tumefaciens GV3101. Grow cultures to OD₆₀₀ = 0.6-1.0 in appropriate antibiotics.

• Plant Material Preparation: Surface-sterilize soybean seeds and germinate until cotyledons emerge. Bisect cotyledons longitudinally to create explants.

• Agroinfiltration: Immerse cotyledon explants in Agrobacterium suspension for 20-30 minutes—optimal duration for infection efficiency.

• Plant Recovery: Transfer inoculated explants to regeneration media or soil under high-humidity conditions for 2-3 days.

• Phenotype Monitoring: Observe silencing phenotypes beginning at 14-21 days post-inoculation, with maximal effects at 3-4 weeks.

BPMV-VIGS Implementation Protocol

The streamlined "one-step" BPMV protocol includes [7]:

• Vector Construction: Insert target gene fragment into BPMV RNA2 vector at appropriate restriction sites.

• Inoculum Preparation: For direct rubbing, mix equal quantities (5μg each) of BPMV RNA1 and RNA2 recombinant plasmids in inoculation buffer.

• Plant Preparation: Grow soybean plants until primary leaves fully expand.

• Inoculation: Dust leaves with carborundum and gently rub plasmid DNA mixture onto leaves using gloved fingers or applicator.

• Post-Inoculation Care: Maintain plants under standard growth conditions with initial dark period for 24 hours.

• Symptom Monitoring: Observe viral symptoms and silencing phenotypes from 14 days post-inoculation.

Research Reagent Solutions

Table 3: Essential Research Reagents for VIGS Implementation

Reagent/Resource	Function	TRV-VIGS	BPMV-VIGS
pTRV1/pTRV2 Vectors	Viral genome components for silencing	Required	Not applicable
BPMV RNA1/RNA2 Vectors	Viral genome components for silencing	Not applicable	Required
A. tumefaciens GV3101	Delivery vector for T-DNA	Required	Optional
Gateway BP Clonase	Enzyme for recombination cloning	Optional	Not applicable
Restriction Enzymes	Conventional cloning	Optional	Required
Carborundum Powder	Abrasive for mechanical inoculation	Not typically used	Required
Plant Growth Media	Tissue culture and plant maintenance	Required	Required

Visual Workflow Diagrams

TRV-VIGS Experimental Workflow: The optimized TRV protocol requires approximately 6-8 weeks from vector construction to phenotypic analysis, with Agrobacterium-mediated cotyledon infiltration as the critical path [3].

BPMV-VIGS Experimental Workflow: The streamlined BPMV protocol requires 5-7 weeks from start to finish, with direct plasmid rubbing as the key differentiator that simplifies the inoculation process [7].

The selection between TRV and BPMV VIGS systems for soybean research involves balancing technical complexity against implementation simplicity. TRV-VIGS offers superior silencing efficiency (65-95%) and comprehensive tissue coverage but demands greater technical expertise in Agrobacterium handling and tissue culture. Conversely, BPMV-VIGS provides a more accessible entry point with its direct inoculation methodology, though it may be limited to specific soybean cultivars and produce more noticeable viral symptoms. Researchers should consider their available infrastructure, technical capabilities, and experimental requirements when selecting between these systems, as both represent powerful functional genomics tools with complementary strengths.

Virus-Induced Gene Silencing (VIGS) is a powerful reverse genetics tool that enables rapid functional analysis of plant genes by using recombinant viruses to trigger post-transcriptional gene silencing. For soybean research, two primary viral vectors have been established: Tobacco Rattle Virus (TRV) and Bean Pod Mottle Virus (BPMV). The choice between these systems is critical and depends heavily on the research objectives, particularly when studying disease resistance versus plant development. This guide provides a detailed comparison of their performance characteristics, supported by experimental data, to inform appropriate vector selection.

Table 1: Core Characteristics of TRV and BPMV VIGS Vectors

Feature	TRV-VIGS	BPMV-VIGS
Optimal Application	Disease resistance gene validation; root-based studies	Broad-based resistance gene screening; constitutive defense studies
Infection Method	Agrobacterium tumefaciens-mediated (cotyledon node immersion) [3]	Direct plasmid DNA rubbing (mechanical inoculation) [7]
Silencing Onset	~21 days post-inoculation (dpi) [3]	Not explicitly stated, but systemic symptoms appear by 3-4 weeks post-inoculation [7]
Silencing Efficiency	65% - 95% [3]	Highly efficient; widely used for functional genomics [9] [32]
Key Advantages	High efficiency in roots and entire plant; minimal symptom interference [3] [46]	Established, robust platform; capable of simultaneous silencing and marker expression [9] [32]
Key Limitations	Previously limited application in soybean; requires tissue culture [3]	Viral symptoms can sometimes interfere with phenotypic analysis [3]

Experimental Protocols and Workflows

TRV-VIGS Protocol for Soybean

The following optimized protocol for TRV-mediated VIGS has demonstrated high efficiency in silencing genes involved in disease resistance.

1. Vector Construction:

• The target gene fragment (e.g., 200-500 bp) is cloned into the multiple cloning site of the pTRV2 vector. Common targets include the phytoene desaturase (GmPDS) gene for system validation, which causes photobleaching, or resistance genes like GmRpp6907 (rust resistance) and GmRPT4 (defense-related) [3].
• The recombinant pTRV2 plasmid and the helper pTRV1 plasmid are independently transformed into Agrobacterium tumefaciens strain GV3101 [3].

2. Plant Material Preparation:

• Soybean seeds (e.g., cultivar Tianlong 1) are surface-sterilized.
• Seeds are soaked in sterile water until swollen and then longitudinally bisected to create half-seed explants, exposing the cotyledonary node [3].

3. Agroinfiltration:

• Fresh explants are immersed in an Agrobacterium suspension containing a mixture of the pTRV1 and recombinant pTRV2 cultures.
• The optimal immersion duration is 20-30 minutes to achieve high transformation efficiency [3].

4. Plant Regeneration and Phenotyping:

• Infected explants are transferred to tissue culture media to regenerate whole plants.
• Systemic silencing phenotypes are typically observed in systemic leaves by 21 days post-inoculation (dpi). Silencing efficiency can be quantified via qPCR to measure transcript abundance of the target gene [3].

BPMV-VIGS Protocol for Soybean

The BPMV system is a well-established "one-step" vector for high-throughput functional genomics in legumes.

1. Vector Construction:

• The BPMV vector set is DNA-based, with RNA1 and RNA2 components under the control of the CaMV 35S promoter.
• For VIGS, a fragment of the target soybean gene is cloned into the BamHI site of the pBPMV-IA-V2 vector, located after the stop codon of the RNA2 polyprotein. This allows for the insertion of non-coding/antisense sequences [9].
• The system has been optimized to use a modified RNA1 component (pBPMV-IA-R1M) that produces moderate viral symptoms, making infection visually identifiable without requiring ELISA confirmation [9] [7].

2. Plant Inoculation:

• Soybean plants (e.g., at the 10-day-old stage) are used for inoculation.
• A mixture of the RNA1 and recombinant RNA2 plasmids (5 μg of each) is combined with an inoculation buffer.
• The primary leaves are mechanically rub-inoculated with the plasmid DNA mix. Abrasives like quartz sand may be used to gently wound the leaves to facilitate infection [7].

3. System Spread and Analysis:

• The virus spreads systemically, and silencing can be assessed in newly emerged trifoliate leaves.
• This system has been successfully used to silence genes like GmBIR1, leading to constitutive defense activation and enhanced resistance to Pseudomonas syringae and Soybean Mosaic Virus [32].

Performance Data and Vector Selection Guide

The quantitative and qualitative performance of each vector system directly informs their suitability for different research applications.

Table 2: Performance Comparison for Key Research Applications

Research Application	TRV-VIGS Performance	BPMV-VIGS Performance
Disease Resistance	Excellent for nematode and fungal resistance studies. Silencing GmPOD53L reduced resistance to SCN, validating its positive role [46].	Excellent for broad-spectrum resistance. Silencing GmBIR1 enhanced resistance to bacterial (Psg) and viral (SMV) pathogens [32].
Developmental Studies	Suitable; capable of systemic silencing in entire plant, including roots and flowers [3] [31].	Less ideal; constitutive defense activation from silencing (e.g., autoimmunity) can cause stunted growth, confounding developmental phenotypes [32].
High-Throughput Screening	Moderate; requires tissue culture and plant regeneration, which can be a bottleneck [3].	High; direct plasmid rubbing is scalable and bypasses the need for Agrobacterium or in vitro transcription [9] [7].
Tissue Specificity	Systemic; effective silencing in leaves, stems, roots, and flowers [3] [31].	Primarily aerial tissues; systemic spread throughout leaves is well-documented [9] [7].

Decision Framework for Vector Selection

• For Disease Resistance Studies: Both vectors are highly effective.

  • Choose BPMV-VIGS for high-throughput screening of candidate genes, especially when studying salicylic acid-mediated defense pathways or autoimmune responses [32]. Its robust and well-characterized nature in soybean makes it a reliable choice.
  • Choose TRV-VIGS for specific pathosystems where its efficacy has been demonstrated, such as for soybean cyst nematode (SCN) resistance, or when studying root-specific defenses [3] [46]. Its minimal symptomology is advantageous for clear phenotypic observation.

• For Developmental Studies: The TRV-VIGS system is generally recommended.

  • TRV induces milder viral symptoms compared to other viruses, which minimizes interference with the plant's normal development and allows for more accurate observation of silencing phenotypes [3] [17]. The BPMV system's tendency to induce constitutive defense responses that cause stunting and cell death can severely confound analyses of plant growth and developmental genes [32].

Research Reagent Solutions

Table 3: Essential Materials and Reagents for VIGS in Soybean

Reagent / Material	Function / Description	Example Use Case
pTRV1 & pTRV2 Vectors	Bipartite TRV-based VIGS vectors; pTRV2 contains the MCS for inserting target gene fragments.	TRV-mediated silencing of GmPDS, GmRpp6907, or GmPOD53L [3] [46].
pBPMV-IA-R1M & pBPMV-IA-V2	DNA-based BPMV vectors; the R1M component provides moderate symptoms for easy tracking.	BPMV-mediated silencing of GmBIR1 or GmPDS [9] [32].
Agrobacterium tumefaciens GV3101	Bacterial strain used for delivering TRV vectors into plant tissues via agroinfiltration.	Essential for the cotyledon node immersion method in the TRV-VIGS protocol [3].
Soybean Cultivar 'Tianlong 1'	A soybean germplasm shown to be highly susceptible to TRV infection, with up to 95% efficiency.	Optimal cultivar for TRV-VIGS studies [3].
Soybean Cultivar 'Williams 82'	A widely studied, susceptible soybean cultivar with a reference genome.	Used in both BPMV (e.g., GmBIR1 silencing) and TRV (e.g., GmPOD53L silencing) studies [46] [32].
Phytoene Desaturase (PDS) Gene	A marker gene whose silencing causes photobleaching (white patches), used to validate VIGS efficiency.	Standard positive control for both TRV and BPMV systems to confirm successful silencing [3] [9].

Conclusion

TRV and BPMV VIGS vectors offer complementary strengths for soybean functional genomics. The recently developed TRV system demonstrates superior silencing efficiency (65-95%) through Agrobacterium-mediated cotyledon transformation and induces milder viral symptoms, reducing phenotype interference. Meanwhile, BPMV remains valuable for its established reliability, broad tissue coverage including roots and flowers, and proven track record in disease resistance studies. Selection depends on research priorities: TRV for maximum silencing efficiency and minimal pathogenicity, BPMV for comprehensive tissue penetration and historical validation. Future directions should focus on vector engineering to enhance stability, developing soybean-cultivar-specific protocols, and integrating VIGS with emerging technologies like CRISPR/Cas for comprehensive functional genomics pipelines. These advancements will accelerate gene discovery and molecular breeding in soybean, with significant implications for crop improvement and agricultural sustainability.

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