TRV-VIGS Protocol: A High-Throughput Reverse Genetics Tool for Functional Genomics

Abstract

This article provides a comprehensive guide to the Tobacco Rattle Virus-induced Gene Silencing (TRV-VIGS) protocol, a powerful reverse genetics tool for rapid functional gene analysis in plants. Tailored for researchers and scientists, the content covers the foundational mechanism of VIGS, detailed methodological steps for vector construction and Agrobacterium-mediated inoculation, troubleshooting for common optimization challenges, and validation techniques to confirm silencing efficiency. By synthesizing recent methodological advances and applications across diverse species, this resource serves as an essential reference for employing TRV-VIGS in high-throughput gene screening and functional genomics studies.

Understanding TRV-VIGS: Principles, Mechanisms, and Vector Systems

Virus-Induced Gene Silencing (VIGS) is a powerful reverse genetics technique that harnesses the innate antiviral defense mechanisms of plants to silence target genes of interest. Among the various viral vectors developed for VIGS, the Tobacco Rattle Virus (TRV) has emerged as the most widely adopted and effective system across numerous plant species [1] [2]. The TRV-VIGS system represents a breakthrough in functional genomics because it enables researchers to investigate gene function without the need for stable genetic transformation, which is often time-consuming, labor-intensive, and unavailable for many plant species [3] [4]. This protocol has proven particularly valuable for perennial crops, woody species, and plants with complex genetics where traditional transformation methods remain challenging.

The fundamental principle underlying TRV-VIGS involves the plant's natural RNA silencing machinery, which recognizes and degrades viral double-stranded RNA (dsRNA) during infection. When recombinant TRV vectors carry fragments of host genes, this defense system is tricked into targeting corresponding endogenous mRNA transcripts for degradation, resulting in post-transcriptional gene silencing (PTGS) [2]. This process allows for rapid functional characterization of candidate genes through loss-of-function phenotypes, typically within weeks rather than the months or years required for conventional transformation approaches. The efficiency and speed of TRV-VIGS make it an indispensable tool for high-throughput functional genomics, especially with the expanding availability of genomic resources for non-model plant species.

The Molecular Mechanism of TRV-Mediated Gene Silencing

Key Stages in the TRV-VIGS Process

The molecular mechanism by which TRV-VIGS triggers PTGS involves a precisely coordinated sequence of events that hijacks the plant's antiviral defense system. This process can be divided into several distinct stages, beginning with vector delivery and culminating in targeted gene silencing, each governed by specific molecular components and interactions.

Vector Delivery and Viral Replication: The process initiates with the delivery of TRV-based vectors into plant cells, typically mediated by Agrobacterium tumefaciens carrying the modified viral genome [3] [5]. The TRV genome consists of two positive-sense, single-stranded RNA components: RNA1, which encodes proteins essential for viral replication and movement, and RNA2, which can be engineered to carry fragments of host target genes [3]. Following delivery, the viral RNA is uncoated and serves as a template for replication, producing complementary RNA strands that form double-stranded RNA (dsRNA) intermediates during viral replication.

Recognition and Dicing of dsRNA: These dsRNA molecules are recognized by the plant's innate antiviral surveillance system as foreign invaders. A key enzyme, Dicer-like (DCL), specifically DCL2 and DCL4 in Arabidopsis, cleaves the long dsRNA molecules into small interfering RNAs (siRNAs) of 21-24 nucleotides in length [2]. These viral-derived siRNAs (vsiRNAs) represent the core silencing signals that guide the sequence-specific degradation of homologous RNA transcripts.

RISC Assembly and Target Gene Silencing: The vsiRNAs are incorporated into an RNA-induced silencing complex (RISC), where they serve as guides to identify complementary RNA sequences for degradation. The catalytic component of RISC, Argonaute protein (AGO), typically AGO1 in Arabidopsis, cleaves the target mRNA strands that exhibit perfect or near-perfect complementarity to the guide vsiRNA [2]. When the TRV vector carries a fragment of a host gene, this process leads to the degradation of corresponding endogenous mRNAs, resulting in effective silencing of the target gene and the appearance of loss-of-function phenotypes.

Systemic Silencing Spread: A crucial feature of TRV that makes it particularly effective for VIGS is its ability to spread systemically throughout the plant, including meristematic tissues [2]. The silencing signal is amplified and transported cell-to-cell through plasmodesmata and via the phloem vasculature, enabling whole-plant silencing rather than being restricted to the initial infection sites.

Visualizing the TRV-VIGS Mechanism

The following diagram illustrates the key molecular stages of the TRV-VIGS mechanism, from initial infection to systemic gene silencing:

Diagram 1: Molecular mechanism of TRV-VIGS showing key stages from vector delivery to systemic gene silencing.

Critical Experimental Parameters for Optimizing TRV-VIGS Efficiency

Quantitative Optimization Data Across Plant Species

Successful implementation of TRV-VIGS depends on numerous experimental parameters that must be optimized for each plant species. The table below summarizes key optimization data from recent studies across diverse plant systems:

Table 1: Optimization parameters for TRV-VIGS across different plant species

Plant Species	Optimal Infiltration Method	Best Plant Stage	Optimal Agrobacterium OD₆₀₀	Key Marker Gene	Silencing Efficiency	Citation
Soybean (Glycine max)	Cotyledon node immersion (20-30 min)	Half-seed explants	0.6-0.8	GmPDS	65-95%	[5]
Atriplex canescens	Vacuum infiltration (0.5 kPa, 10 min)	Germinated seeds (1-3 cm radicle)	0.8	AcPDS	~16.4% (phenotypic), 40-80% (transcript)	[4]
Walnut (Juglans regia)	Seedling spray (5-10 true leaves)	Seedlings with 5-10 true leaves	1.0-1.5	JrPDS	Up to 48%	[3]
Arabidopsis thaliana	Agroinfiltration (two- to three-leaf stage)	Two- to three-leaf stage	1.5	AtPDS	90-100%	[2]
Lycoris chinensis	Leaf tip needle injection	Young leaves emerging from bulb	0.6-0.8	LcCLA1	Higher than LcPDS	[1]

Protocol Variations and Methodological Considerations

The data presented in Table 1 reveals significant variation in optimal parameters across species, highlighting the necessity for protocol optimization when establishing TRV-VIGS in new plant systems. Agrobacterium concentration (OD₆₀₀) typically ranges from 0.6 to 1.5, with higher concentrations sometimes necessary for species with physical barriers like thick cuticles or dense trichomes [5]. The developmental stage of plant material proves critical, with younger tissues generally showing higher silencing efficiency, as demonstrated by the 90-100% success rate in two- to three-leaf stage Arabidopsis compared to dramatically reduced efficiency in older plants [2].

Infiltration methodology must be adapted to plant morphology and tissue structure. For instance, Lycoris leaves with a waxy surface required a specialized leaf tip needle injection method, which used only 1-2 mL of bacterial solution and took 15-20 seconds per leaf compared to conventional methods requiring at least 5 mL and 1-2 minutes per leaf [1]. Similarly, soybean's thick cuticle and dense trichomes necessitated the development of a cotyledon node immersion protocol to achieve satisfactory infection rates [5]. These examples underscore the importance of customizing delivery methods based on specific anatomical features of target species.

Marker gene selection also influences protocol assessment, with Phytoene desaturase (PDS) serving as the most common visual marker due to the photobleaching phenotype resulting from disrupted carotenoid biosynthesis [3] [4]. However, alternative markers like Cloroplastos Alterados 1 (CLA1) may provide superior visual phenotypes in some species, as demonstrated in Lycoris where LcCLA1 silencing produced more pronounced chlorosis compared to LcPDS [1].

Detailed TRV-VIGS Protocol for Plant Functional Genomics

Vector Construction and Agrobacterium Preparation

The foundational step in establishing a TRV-VIGS system involves proper vector construction and preparation of Agrobacterium strains carrying the viral components:

Vector Design and Cloning:

• Select a 300-500 bp fragment from the target gene sequence with high specificity to minimize off-target silencing [4]. Online tools like the SGN-VIGS predictor can assist in identifying optimal target regions.
• Clone the fragment into the pTRV2 vector using appropriate restriction sites (commonly EcoRI and XhoI) or Gateway recombination cloning [5].
• For visual tracking of infection, consider using pTRV2-GFP vectors that incorporate a green fluorescent protein reporter, enabling monitoring of viral spread through fluorescence microscopy [5] [4].

Agrobacterium Preparation:

• Introduce the recombinant pTRV2 and pTRV1 vectors into Agrobacterium tumefaciens strain GV3101 through freeze-thaw transformation or electroporation [4].
• Plate transformed bacteria on YEP agar containing appropriate antibiotics (50 mg/L kanamycin and 50 mg/L rifampicin) and incubate at 28°C for 48 hours [4].
• Inoculate single colonies into YEP liquid medium with antibiotics and culture at 28°C with shaking at 200 rpm until reaching mid-logarithmic growth phase (OD₆₀₀ = 0.6-0.8) [5] [4].
• Centrifuge bacterial cultures at 6000 rpm for 8 minutes and resuspend in infiltration buffer (10 mM MES, 200 μM acetosyringone, 10 mM MgCl₂, 0.03% Silwet-77) adjusted to the optimal OD₆₀₀ for the target species [4].
• Combine equal volumes of TRV1 and TRV2-derived Agrobacterium suspensions and incubate at room temperature in darkness for 3 hours to induce virulence gene expression before inoculation [4].

Plant Inoculation and Post-Infection Management

Inoculation Methodology: The appropriate inoculation method depends on the target species and plant developmental stage:

• Vacuum Infiltration: Suitable for germinated seeds and small seedlings. Submerge materials in Agrobacterium suspension and apply vacuum (0.5 kPa for 5-10 minutes) to facilitate bacterial entry [4].
• Cotyledon Node Immersion: Particularly effective for soybean. Bisect sterilized, swollen seeds to create half-seed explants, then immerse in Agrobacterium suspension for 20-30 minutes [5].
• Leaf Infiltration: Use needleless syringe for species with accessible leaf mesophyll, such as Nicotiana benthamiana and Arabidopsis [2].
• Spray Inoculation: Effective for walnut seedlings with 5-10 true leaves, though may result in lower efficiency than other methods [3].

Post-Inoculation Management:

• Maintain inoculated plants in high-humidity conditions for 24-48 hours to facilitate infection.
• Grow plants under controlled environmental conditions appropriate for the species, typically 16h light/8h dark photoperiod at 22-25°C [4].
• Monitor for silencing phenotypes beginning at 10-15 days post-inoculation (dpi), with full phenotypes typically visible by 21-28 dpi [5].
• For soil-grown plants, irrigate with appropriate nutrient solutions (e.g., ½-strength Hoagland solution) to maintain plant health throughout the silencing period [4].

Efficiency Assessment and Validation

Phenotypic Assessment:

• For marker genes like PDS, monitor photobleaching symptoms in newly emerged leaves beginning at 10-15 dpi [4].
• Document phenotypic progression through photography, noting the distribution and intensity of silencing symptoms.

Molecular Validation:

• Extract RNA from silenced tissues and perform quantitative RT-PCR to measure transcript abundance of target genes [5] [4].
• Calculate silencing efficiency as the percentage reduction in target gene expression compared to empty vector controls.
• For systems incorporating GFP reporters, visualize infection efficiency and distribution using fluorescence microscopy [5].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Essential research reagents and materials for TRV-VIGS experiments

Reagent/Material	Specification/Example	Function/Purpose	Application Notes
TRV Vectors	pTRV1, pTRV2, pTRV2-GFP	Viral genome components for silencing	pTRV1 encodes replication/movement proteins; pTRV2 carries target gene fragment [3]
Agrobacterium Strain	GV3101	Vector delivery into plant cells	Optimized for plant transformation; requires appropriate antibiotic resistance [5] [4]
Infiltration Buffer	10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂, 0.03% Silwet-77	Facilitates Agrobacterium infection	Acetosyringone induces virulence genes; Silwet-77 enhances tissue penetration [4]
Marker Genes	PDS, CLA1	Visual assessment of silencing efficiency	PDS silencing causes photobleaching; CLA1 results in chlorosis [1] [4]
Antibiotics	Kanamycin (50 mg/L), Rifampicin (50 mg/L)	Selection of transformed Agrobacterium	Concentration may require optimization for different strains [4]
Plant Growth Media	YEP (Agrobacterium), ½-strength Hoagland solution (plants)	Support microbial and plant growth	Specific plant species may require customized nutrient formulations

Applications and Advances in TRV-VIGS Technology

Research Applications Across Plant Species

TRV-VIGS has enabled functional gene characterization across diverse plant species, facilitating research in areas that were previously genetically intractable. In soybean, this system has been successfully deployed to validate disease resistance genes, including the rust resistance gene GmRpp6907 and the defense-related gene GmRpt4, achieving 65-95% silencing efficiency through cotyledon node transformation [5]. In cotton, TRV-VIGS has elucidated regulatory networks controlling pigment gland development and secondary metabolite biosynthesis, revealing how the VQ domain-containing protein JAVL negatively regulates gland size while the MYC2-like transcription factor GoPGF exerts opposing effects [6].

For halophytic species like Atriplex canescens, TRV-VIGS has overcome the limitations of stable transformation systems, enabling functional studies of stress-responsive genes such as aquaporins (AcTIP2;1 and AcPIP2;5) with 60.3-69.5% knockdown efficiency [4]. In ornamental species including Lycoris, established VIGS systems using leaf tip needle injection have opened avenues for investigating genes controlling flowering time and pigment biosynthesis, traits with significant horticultural importance [1].

Troubleshooting and Technical Considerations

Despite its broad utility, several technical challenges can impact TRV-VIGS efficiency. Incomplete silencing may result from suboptimal fragment selection, insufficient viral spread, or target gene redundancy. This can be addressed by testing multiple target regions (5', central, and 3' gene fragments) and ensuring proper plant growth conditions to support viral movement [4]. Limited systemic spread occasionally occurs in certain species or tissues, which may be improved by adjusting inoculation methods or using younger plant materials [2].

Viral symptom interference can sometimes mask silencing phenotypes, though TRV generally produces mild symptoms compared to other viral vectors [5]. Silencing persistence varies across species, typically maintaining effectiveness for several weeks to months, but may require reinfection for long-term studies. Recent advances include the development of fluorescent protein markers to track silencing progression and the optimization of tissue culture-based infection methods for challenging species [5].

The TRV-VIGS system represents a sophisticated application of plant-virus interactions that leverages the natural RNA silencing machinery to create a powerful functional genomics tool. The core mechanism—from viral delivery and replication to dsRNA recognition, siRNA biogenesis, RISC assembly, and systemic silencing spread—provides researchers with an efficient method for post-transcriptional gene silencing across an expanding range of plant species. As protocols continue to be refined and adapted for new species, TRV-VIGS will play an increasingly important role in characterizing gene functions, validating candidate genes from omics studies, and accelerating crop improvement programs, particularly for species resistant to conventional transformation approaches. The ongoing optimization of delivery methods, vector design, and efficiency assessment protocols ensures that TRV-VIGS remains at the forefront of plant functional genomics methodologies.

TRV Genome Organization and the Roles of RNA1 and RNA2

Genomic Architecture of Tobacco Rattle Virus

Tobacco Rattle Virus (TRV), a member of the genus Tobravirus (family Virgaviridae), possesses a bipartite, positive-sense, single-stranded RNA genome encapsidated in rod-shaped particles [7] [8]. This divided genome is a hallmark of the genus and is central to its functionality and experimental versatility [8]. The two genomic components, designated RNA1 and RNA2, are encapsidated separately in viral particles of different lengths [7]. The L (long) particles contain RNA1, which is approximately 6.8 kilobases (kb) in size, while the S (short) particles contain RNA2, which exhibits more size variation, ranging from 1.8 to about 4.5 kb depending on the isolate [8]. Both RNAs are 5'-capped and possess a 3' end that can adopt a tRNA-like structure, though it cannot be aminoacylated [9] [8].

Table 1: Core Components of the TRV Genome

Component	Size	Encapsidated In	Key Characteristics
RNA1	~6.8 kb	Long particles (180-215 nm)	Highly conserved among isolates; capable of independent replication and cell-to-cell movement [9] [8].
RNA2	1.8-4.5 kb	Short particles (46-115 nm)	Genetically variable and prone to recombination; length depends on the viral isolate [9] [8].

The following diagram illustrates the general organization of the TRV genome and its relationship with the Virus-Induced Gene Silencing (VIGS) vector system:

Detailed Functional Analysis of RNA1 and RNA2

RNA1: The Replication and Movement Module

RNA1 functions as an autonomous unit, encoding proteins essential for viral replication and intra-plant movement [8]. It contains four primary open reading frames (ORFs):

• The 134-141 kDa and 194-201 kDa Replication Proteins: The 194-201 kDa protein is produced by the readthrough of an opal termination codon that ends the 134-141 kDa ORF. Together, these proteins form the viral RNA-dependent RNA polymerase (RdRp) responsible for replicating both genomic RNAs [9] [8].
• The 29-30 kDa Movement Protein (MP): This "30K"-like protein is crucial for the cell-to-cell movement of the virus through plasmodesmata [8]. It is expressed via a subgenomic RNA and is a key determinant for the systemic spread of the virus within the host plant [9].
• The 12-16 kDa Cysteine-Rich Protein (CRP): This protein acts as a potent suppressor of the plant's RNA silencing defense machinery, a key antiviral response [9] [8]. For Pea early-browning virus (PEBV), a closely related tobravirus, this protein has also been identified as a determinant of seed transmission [8].

Notably, infection with RNA1 alone (an "nm-type" isolate) is sufficient to cause systemic infection, albeit without the production of viral particles. Such infections often result in more severe necrotic symptoms [8].

RNA2: The Transmission and Structural Module

RNA2 is more variable in sequence and structure than RNA1 and is not required for the fundamental processes of replication and movement [9] [7]. Its primary ORFs include:

• The Coat Protein (CP): This 22-24 kDa protein is the sole structural component of the viral capsid, forming the characteristic rod-shaped particles [7] [8]. The CP is translated from a subgenomic RNA [8].
• The Non-Structural Proteins 2b and 2c: These proteins are essential for the transmission of the virus by its natural vectors, soil-inhabiting nematodes of the genera Trichodorus and Paratrichodorus [9] [8]. The 2b protein is absolutely required for transmission, while the requirement for 2c varies by strain [8]. In laboratory strains maintained by mechanical inoculation, the ORFs for 2b and 2c are often partially or completely deleted [8].

A key feature of RNA2 is its "rule-breaking" architecture found in some isolates, where one or several ORFs are located upstream of the CP gene. The biological role of these CP-preceding proteins, such as the 35 kDa protein in German potato isolates or the hypothetical proteins in the SYM isolate, remains an area of active investigation and may be associated with host-specific infection abilities [9].

Table 2: Protein Functions Encoded by TRV RNA1 and RNA2

Genomic RNA	Encoded Protein	Size	Function
RNA1	134-141 kDa / 194-201 kDa	~130-201 kDa	Viral RNA replication (RdRp) [9] [8].
	29-30 kDa (Movement Protein)	~30 kDa	Cell-to-cell movement through plasmodesmata [8].
	12-16 kDa (Cysteine-Rich Protein)	~12-16 kDa	Suppression of host RNA silencing; role in virion formation [9] [8].
RNA2	Coat Protein (CP)	22-24 kDa	Forms the protective rod-shaped capsid [7] [8].
	2b	27-40 kDa	Required for nematode transmission [8].
	2c	18-33 kDa	Affects nematode transmission in some strains [8].

The Scientist's Toolkit: Essential Reagents for TRV-VIGS Research

The TRV-based Virus-Induced Gene Silencing system is a powerful tool for functional genomics. Its implementation relies on a standardized set of biological reagents and vectors.

Table 3: Key Research Reagent Solutions for TRV-VIGS

Reagent / Solution	Function / Description	Example Use in Protocol
pTRV1 Vector	A binary plasmid containing cDNA of TRV RNA1, providing replication and movement functions [4] [10].	Co-infiltrated with pTRV2-derived vectors into plant tissues.
pTRV2 Vector	A binary plasmid containing cDNA of TRV RNA2, modified to include a Multiple Cloning Site (MCS) for inserting gene fragments [4] [10].	The backbone for constructing VIGS vectors (e.g., TRV2:PDS).
Agrobacterium tumefaciens GV3101	A disarmed strain used to deliver the pTRV1 and pTRV2 vectors into plant cells via T-DNA transfer [4] [10].	Host for plasmid vectors; resuspended in infiltration buffer for inoculation.
Infiltration Buffer	A solution facilitating Agrobacterium delivery into plant tissues, typically containing MES, MgCl₂, acetosyringone, and a surfactant like Silwet-77 [4].	Used to resuscent Agrobacterium cells to an OD600 of 0.8-1.0 for inoculation.
Marker Gene Vectors (e.g., TRV2:PDS)	Control vectors containing a fragment of a plant gene (e.g., Phytoene Desaturase) that produces a visible photobleaching phenotype when silenced, used to validate VIGS efficiency [4] [3] [10].	Essential positive control for optimizing and assessing silencing protocols.

Established Protocols for TRV-Mediated VIGS

The application of TRV-VIGS has been successfully optimized for a wide range of plant species. Below are detailed methodologies for two distinct systems, demonstrating the protocol's adaptability.

Protocol: VIGS in Halophyte ModelAtriplex canescens

This protocol establishes an efficient TRV-VIGS system for a non-model plant, Atriplex canescens, valuable for studying abiotic stress tolerance genes [4].

• Vector Construction: Clone a 300-400 base pair (bp) fragment of the target gene (e.g., AcPDS) into the pTRV2 vector using standard molecular biology techniques (e.g., restriction digestion with EcoRI/BamHI and ligation) [4].
• Agrobacterium Preparation:
  • Transform the recombinant pTRV2 vector and the pTRV1 vector separately into Agrobacterium tumefaciens strain GV3101.
  • Culture single colonies in YEP liquid medium with appropriate antibiotics (50 mg/L kanamycin, 50 mg/L rifampicin) at 28°C with shaking until the OD600 reaches 0.6-0.8.
  • Pellet the bacterial cells by centrifugation (6000 rpm, 8 min) and resuspend in infiltration buffer (10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂, 0.03% Silwet-77) to a final OD600 of 0.8 [4].
• Plant Inoculation:
  • Mix equal volumes of the Agrobacterium suspensions carrying pTRV1 and the recombinant pTRV2. Incubate the mixture at room temperature in darkness for 3 hours.
  • Use pre-germinated seeds with radicles of 1-3 cm in length as inoculation material.
  • Subject the germinated seeds to vacuum-assisted agroinfiltration: submerge them in the Agrobacterium mixture and apply a vacuum of 0.5 kPa for 10 minutes (e.g., two cycles of 5 minutes each) [4].
• Post-Inoculation Care:
  • Transplant the inoculated materials into pots with vermiculite.
  • Maintain plants in a greenhouse at 22°C with a 16/8 hour light/dark cycle.
  • Systemic silencing phenotypes, such as photobleaching for AcPDS, typically appear in newly emerged leaves around 15 days post-inoculation (dpi) [4].
• Validation: Confirm silencing efficiency by quantifying the reduction in target gene transcript abundance using quantitative reverse transcription PCR (qRT-PCR). This protocol achieved a 40-80% reduction in AcPDS expression [4].

Protocol: VIGS in Woody Plant Walnut (Juglans regiaL.)

This protocol demonstrates the adaptation of TRV-VIGS for a woody tree species, overcoming challenges associated with its genetic transformation [3].

• Vector Construction: Identify and clone a ~255 bp fragment of the target gene (e.g., JrPDS) into the pTRV2 vector. A fragment of this length was found to be optimal for silencing efficiency in walnut [3].
• Agrobacterium Preparation:
  • Introduce the pTRV1 and recombinant pTRV2 plasmids into Agrobacterium tumefaciens GV3101.
  • Grow the bacteria to an OD600 of 0.6-0.8 and resuspend in infiltration buffer to a final OD600 of 0.5, which was identified as the optimal density for walnut [3].
• Plant Inoculation via Cotyledonary Nodes:
  • Soak walnut seeds until swollen and bisect them longitudinally to create half-seed explants, exposing the cotyledonary node.
  • Immerse the fresh explants in the mixed Agrobacterium suspension for 20-30 minutes [3]. The cotyledonary node method proved more effective than spraying or leaf injection in walnut.
• Post-Inoculation Care:
  • Co-culture the explants and then transfer them to growing medium.
  • Maintain plants in a light incubator at 24°C with a 16/8 hour light/dark cycle.
  • The photobleaching phenotype for JrPDS is observable at 21 dpi [3].
• Efficiency: This optimized protocol for walnut achieved a silencing efficiency of up to 48%, providing a valuable tool for functional gene validation in this species [3].

The bipartite nature of the TRV genome, cleanly separating replication and movement functions (RNA1) from transmission and structural functions (RNA2), is the foundation of its success as both a pathogen and a biotechnological tool. The modularity of RNA2 allows for its extensive engineering as a VIGS vector without compromising viral replication. The well-established protocols for species ranging from model plants to recalcitrant woody crops underscore the robustness and versatility of the TRV-VIGS system. A deep understanding of the distinct roles played by RNA1 and RNA2 enables researchers to better troubleshoot experiments, optimize vectors for specific hosts, and continue to push the boundaries of functional genomics.

Advantages of TRV-VIGS Over Stable Transformation and Other Silencing Methods

In the field of plant functional genomics, the ability to rapidly characterize gene function is fundamental. While stable genetic transformation has been a cornerstone technique, it presents significant limitations in terms of time, labor, and applicability across diverse plant species. Virus-Induced Gene Silencing (VIGS), particularly systems based on Tobacco rattle virus (TRV), has emerged as a powerful alternative that circumvents many of these constraints [11]. This protocol review examines the distinct advantages of TRV-VIGS over stable transformation and other silencing methodologies, providing researchers with a clear rationale for its adoption in functional genomic studies.

TRV-VIGS operates by harnessing the plant's innate post-transcriptional gene silencing (PTGS) machinery [12] [11]. When a recombinant TRV vector carrying a fragment of a plant gene of interest infects the host, it triggers a sequence-specific RNA degradation mechanism that ultimately leads to the knockdown of the corresponding endogenous mRNA [12]. This process enables researchers to observe loss-of-function phenotypes without the need for stable genetic modification.

Key Advantages of TRV-VIGS

The following sections detail the specific benefits of using TRV-VIGS, with quantitative comparisons provided to underscore its efficiency.

Speed and Time Efficiency

TRV-VIGS dramatically accelerates the process from gene identification to phenotypic analysis. The table below compares the typical timelines between stable transformation and TRV-VIGS.

Table 1: Time Efficiency Comparison Between Stable Transformation and TRV-VIGS

Experimental Stage	Stable Transformation	TRV-VIGS
Vector Construction	2-4 weeks	2-3 weeks
Plant Transformation/Inoculation	2-3 months	1 day
Selection & Regeneration	3-6 months	Not Applicable
Phenotype Observation	T1 generation (≥ 3 months)	2-4 weeks post-inoculation [12]
Total Time	6+ months	3-5 weeks

As evidenced, TRV-VIGS can reduce the experimental timeline from over six months to just several weeks, enabling high-throughput functional screening [12] [11].

Technical Simplicity and Cost-Effectiveness

The TRV-VIGS system bypasses the most technically challenging and genotype-dependent stages of stable transformation.

• No Tissue Culture Required: Stable transformation relies on efficient in vitro regeneration systems, which are unavailable for many plant species or are highly genotype-specific [5] [4]. TRV-VIGS utilizes simple inoculation methods like agroinfiltration or vacuum infiltration on seeds or seedlings [5] [4].
• Lower Operational Costs: By eliminating the need for sterile tissue culture facilities, specialized media, and prolonged maintenance of transformants, TRV-VIGS significantly reduces the cost per experiment.

Applicability to Recalcitrant Species

TRV-VIGS is particularly valuable for studying non-model organisms and recalcitrant species where stable transformation is inefficient or non-existent. Successful implementations have been demonstrated across a wide taxonomic range:

• Woody Plants: Camellia drupifera (tea oil camellia) [13] and Atriplex canescens (a halophytic shrub) [4].
• Ornamentals: Iris japonica [14] and Petunia hybrida [15].
• Major Crops: Soybean [5] [10], cotton [16], tomato [17], and pepper [11].

High Silencing Efficiency and Robust Systemic Spread

A key strength of the TRV vector is its ability to spread systemically throughout the plant, including meristematic tissues, inducing strong and widespread silencing phenotypes [12].

Table 2: Documented Silencing Efficiency of TRV-VIGS in Various Plant Species

Plant Species	Target Gene	Silencing Efficiency	Citation
Soybean (Glycine max)	GmPDS	65% - 95%	[5] [10]
	GmRpp6907, GmRPT4	65% - 95%	[5] [10]
Camellia drupifera	CdCRY1	~69.8%	[13]
	CdLAC15	~90.9%	[13]
Atriplex canescens	AcPDS	40% - 80% (transcript reduction)	[4]
	AcTIP2;1, AcPIP2;5	60.3% - 69.5% (transcript reduction)	[4]
Iris japonica	IjPDS	36.67% (optimized in 1-yr seedlings)	[14]

Comparison with Other VIGS Vectors

While other viral vectors like Bean Pod Mottle Virus (BPMV) are used in soybean, TRV-based systems often present distinct benefits [5] [10]. A major advantage is that TRV typically elicits milder viral symptoms compared to other viruses, which prevents the masking of the true silencing phenotype and allows for more accurate phenotypic evaluation [5] [11]. Furthermore, TRV has a very broad host range, making it a versatile tool for establishing VIGS in new species [12] [11].

The TRV-VIGS Mechanism: A Visual Workflow

The following diagram illustrates the core mechanism of the TRV-VIGS system, from Agrobacterium delivery to the generation of a visible phenotype.

Critical Experimental Protocol and Optimization

A successful TRV-VIGS experiment depends on a carefully optimized protocol. The workflow below outlines the key steps from preparation to analysis.

Target Gene Fragment Selection and Vector Construction

• Fragment Design: Amplify a 200-500 bp fragment from the target gene's cDNA [12]. The fragment should be specific to the target gene to avoid off-target silencing. Tools like the SGN VIGS Tool (https://vigs.solgenomics.net/) can help select unique regions and predict silencing efficiency [13] [16] [4].
• Vector Assembly: Clone the selected fragment into a pTRV2 vector (e.g., pTRV2-GFP) using appropriate restriction enzymes (e.g., EcoRI and XhoI) or recombination-based cloning [5] [12]. The recombinant pTRV2 and the helper pTRV1 vector are then transformed into Agrobacterium tumefaciens strain GV3101 [5] [4].

Plant Inoculation Methods

The choice of inoculation method is critical and depends on the plant species. The table below compares effective techniques.

Table 3: Optimized Inoculation Methods for Different Plant Species

Plant Species	Optimal Inoculation Method	Key Details	Efficiency/Result
Soybean [5] [10]	Cotyledon Node Immersion	Bisect swollen seeds, immerse explants in Agrobacterium suspension for 20-30 min.	Infection efficiency >80%, up to 95%.
Atriplex canescens [4]	Vacuum Infiltration of Germinated Seeds	0.5 kPa for 10 min, using decorticated seeds.	~16.4% silencing efficiency (phenotypic).
Camellia drupifera [13]	Pericarp Cutting Immersion	Used for firmly lignified capsules.	Infiltration efficiency ~93.94%.
Tomato [17]	Agroinfiltration + Environmental Control	Combine inoculation with low temp (15°C) & low humidity (30%).	Enhanced silencing maintained in fruits.
Petunia [15]	Apical Meristem Inoculation	Inoculation of mechanically wounded shoot apical meristems.	Induced most effective and consistent silencing.

Key Factors for Optimization

• Plant Developmental Stage: Inoculating younger seedlings generally yields higher silencing efficiency. In petunia, plants inoculated at 3-4 weeks after sowing showed stronger silencing than those at 5 weeks [15]. In iris, one-year-old seedlings provided the best efficiency [14].
• Environmental Conditions: Temperature is a major factor. Research in petunia and tomato showed that lower growth temperatures (e.g., 15-20°C) after inoculation significantly enhance the silencing effect and its persistence [17] [15].
• Agroinoculum Concentration: An optical density at OD600 of 0.8 to 1.0 for the Agrobacterium suspension is commonly used for optimal infection without causing excessive stress [4] [15].

The Scientist's Toolkit: Essential Research Reagents

Table 4: Key Reagents for TRV-VIGS Experiments

Reagent / Material	Function / Description	Example Use Case
pTRV1 & pTRV2 Vectors	Binary T-DNA vectors containing TRV genome components; pTRV2 has MCS for target insert.	Essential for all TRV-VIGS experiments [5] [12].
Agrobacterium tumefaciens GV3101	Disarmed bacterial strain for delivering T-DNA vectors into plant cells.	Standard strain for agroinfiltration [5] [4].
Infiltration Buffer (MES, AS, MgCl₂)	Buffer to prepare Agrobacterium suspension, induces virulence genes.	10 mM MES, 200 µM AS, 10 mM MgCl₂ [4].
Silwet L-77	Surfactant that reduces surface tension, improving infiltration efficiency.	Added at ~0.03% to infiltration buffer [4].
Marker Genes (PDS, CHS)	Endogenous reporter genes whose silencing causes visual phenotypes (photobleaching, white flowers).	Used to validate and optimize the system in new species [5] [14] [15].
Control Vector (e.g., pTRV2-sGFP)	Contains a non-plant insert (e.g., GFP) to minimize viral symptoms in control plants.	Crucial for proper phenotyping, eliminates severe necrosis from empty vector [15].

The TRV-VIGS system represents a paradigm shift in plant functional genomics, offering a rapid, efficient, and versatile alternative to stable transformation. Its primary advantages—significant time savings, applicability to genetically recalcitrant species, high silencing efficiency, and systemic spread—make it an indispensable tool for modern plant biologists. By following the optimized protocols and critical factors outlined in this application note, researchers can robustly implement this powerful technology to accelerate the discovery of gene functions in a wide array of plant species, thereby advancing both basic science and crop improvement efforts.

Virus-Induced Gene Silencing (VIGS) is a powerful reverse genetics technique that harnesses the plant's innate RNA-based antiviral defense mechanism to silence endogenous genes [12]. When a recombinant virus carrying a fragment of a plant gene infects the host, it triggers sequence-specific degradation of complementary mRNA, leading to knock-down of the target gene's expression [2]. Among the various viral vectors developed for VIGS, the Tobacco Rattle Virus (TRV) has emerged as one of the most widely used and versatile systems, primarily due to its exceptionally broad host range [12]. TRV-based VIGS can effectively silence genes in plants across numerous families, including Solanaceae, Cruciferae, Gramineae, and many others in both dicots and monocots [12]. This extensive compatibility, combined with its ability to infect meristematic tissues and induce mild viral symptoms, makes TRV an indispensable tool for functional genomics in model plants and recalcitrant crops alike [12].

The molecular characteristics of TRV contribute significantly to its wide applicability. TRV is a positive-sense RNA virus with a bipartite genome. RNA1 encodes proteins essential for replication and movement, including the 134K and 194K replicases, a movement protein (MP), and a 16K cysteine-rich protein. RNA2 typically encodes the coat protein (CP) and other non-structural proteins, which can be replaced with plant gene fragments for VIGS applications without compromising viral infectivity [12]. This genetic flexibility, coupled with the virus's efficient systemic movement, enables researchers to rapidly assess gene function without the need for stable transformation [3].

TRV-VIGS Mechanism and Workflow

Molecular Mechanism of TRV-Induced Silencing

The TRV-VIGS process exploits the plant's post-transcriptional gene silencing (PTGS) pathway, which naturally functions as an antiviral defense mechanism [12]. The process begins when a recombinant TRV vector containing a fragment of the target plant gene is introduced into plant cells, typically via Agrobacterium-mediated delivery. Once inside the cell, the viral RNA genome is released and replicated by viral RNA-dependent RNA polymerase (RdRp), generating double-stranded RNA (dsRNA) intermediates [12]. These dsRNA molecules are recognized as aberrant by the plant's defense system and are cleaved by Dicer-like (DCL) enzymes into small interfering RNAs (siRNAs) of 21-24 nucleotides [12]. These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), which uses the siRNA as a guide to identify and cleave complementary mRNA sequences, including both viral RNAs and endogenous plant mRNAs that share sequence homology with the inserted fragment [12]. This results in targeted degradation of the host gene's transcripts and a corresponding reduction in gene expression, enabling functional characterization through phenotypic analysis.

General TRV-VIGS Experimental Workflow

The implementation of TRV-VIGS follows a systematic workflow that can be adapted to various plant species. The process begins with the selection of a target gene fragment, typically 200-500 base pairs in length, which is cloned into the TRV2 vector [12]. Simultaneously, the TRV1 vector containing genes for viral replication and movement is prepared. Both constructs are transformed into Agrobacterium tumefaciens strains such as GV3101 or GV1301 [18]. The bacterial cultures are grown to optimal density (OD600 typically 0.8-2.0, depending on the plant species), induced with acetosyringone to enhance T-DNA transfer, and mixed in equal ratios [18]. The inoculation method varies based on the target plant species and may include leaf infiltration, vacuum infiltration, agrodrench, or the novel root wounding-immersion technique [18]. Following inoculation, plants are maintained under specific environmental conditions (often lower temperatures of 18-22°C) to optimize viral spread and silencing efficiency [2]. Silencing phenotypes typically manifest within 2-4 weeks post-inoculation, and the efficiency is validated through molecular techniques such as qRT-PCR and phenotypic observation [10].

TRV Host Range and Application Across Plant Species

Documented Host Range of TRV-VIGS

The versatility of TRV as a VIGS vector is demonstrated by its successful application across a wide spectrum of plant families, from model organisms to agriculturally important crops and recalcitrant species. The following table summarizes the documented host range of TRV-VIGS based on recent research:

Table 1: Documented Host Range of TRV-VIGS Across Plant Species

Plant Species	Family	Key Applications	Silencing Efficiency	Optimal Inoculation Method	Special Considerations
Nicotiana benthamiana	Solanaceae	Model plant for protocol optimization [12]	90-100% [18]	Leaf infiltration, root wounding-immersion [18]	Reference species for method development
Arabidopsis thaliana	Brassicaceae	Defense genes, metabolic pathways [2]	~100% (optimal conditions) [2]	Agroinfiltration (2-3 leaf stage) [2]	Requires young seedlings, long-day conditions (16h light) [2]
Tomato (Solanum lycopersicum)	Solanaceae	Fruit development, disease resistance [18]	95-100% [18]	Root wounding-immersion, agrodrench	Well-established model for Solanaceae
Soybean (Glycine max)	Fabaceae	Disease resistance genes (GmRpp6907, GmRPT4) [10]	65-95% [10]	Cotyledon node immersion	Thick cuticle and dense trichomes challenge leaf infiltration [10]
Walnut (Juglans regia)	Juglandaceae	Abiotic stress research [3]	Up to 48% [3]	Stem injection, vacuum infiltration	Recalcitrant transformation system; 255bp fragment optimal [3]
Sunflower (Helianthus annuus)	Asteraceae	Flower development, stress responses [19]	62-91% (genotype-dependent) [19]	Seed vacuum infiltration	Genotype-dependent efficiency; 'Smart SM-64B' most susceptible (91%) [19]
Petunia (Petunia hybrida)	Solanaceae	Flower pigmentation, scent genes [15]	Improved by 28-69% after optimization [15]	Apical meristem inoculation	Optimal at 20°C day/18°C night; 3-4 weeks after sowing [15]
Pepper (Capsicum annuum)	Solanaceae	Disease resistance, WRKY transcription factors [18]	High (protocol established) [18]	Root wounding-immersion	Species-specific PDS fragment required [18]

Factors Influencing TRV Host Range and Silencing Efficiency

Several critical factors determine the success of TRV-VIGS across diverse plant species. Plant genotype significantly impacts susceptibility to TRV infection and silencing efficiency, as demonstrated in sunflower where infection rates varied from 62% to 91% across different cultivars [19]. The developmental stage at inoculation is crucial, with younger plants generally showing higher silencing efficiency—in Arabidopsis, seedlings at the two-to-three-leaf stage showed nearly 100% silencing efficiency compared to a 50% reduction in older plants [2]. Environmental conditions, particularly temperature, profoundly affect VIGS efficiency, with lower temperatures (18-22°C) often enhancing silencing spread and duration [15]. The length and specificity of the inserted gene fragment must be optimized for each species, with fragments of 200-300 bp typically generating the strongest silencing [3] [19]. Additionally, inoculation method selection must consider the morphological characteristics of each species—soybean's thick cuticle and dense trichomes make leaf infiltration challenging, necessitating alternative approaches like cotyledon node immersion [10].

Protocols for Recalcitrant Species

Root Wounding-Immersion Method for Solanaceous Plants

The root wounding-immersion technique represents a significant advancement for TRV-VIGS application in multiple Solanaceous species, achieving 95-100% silencing efficiency in tomato and Nicotiana benthamiana [18]. This protocol leverages the efficient vascular connectivity between roots and shoots to enhance systemic viral spread.

Materials: pTRV1 and pTRV2 vectors with target gene insert; Agrobacterium strain GV1301; appropriate antibiotics; acetosyringone; 10 mM MgCl₂; 10 mM MES buffer; 3-4 week-old plants with well-developed roots.

Procedure:

• Prepare Agrobacterium cultures containing pTRV1 and pTRV2 constructs separately in LB medium with appropriate antibiotics. Grow overnight at 28°C with shaking at 200 rpm.
• Centrifuge bacterial cultures and resuspend in infiltration buffer (10 mM MgCl₂, 10 mM MES pH 5.6, 150 μM acetosyringone) to OD600 = 0.8.
• Mix the pTRV1 and pTRV2 suspensions in 1:1 ratio and incubate in dark at 28°C for 3 hours.
• Carefully remove plants from soil and wash roots to remove impurities.
• Using a sterilized blade, remove approximately one-third of the root system lengthwise to create wound sites.
• Immerse the wounded roots in the Agrobacterium suspension for 30 minutes with gentle agitation.
• Replant treated seedlings in fresh soil and maintain at 22°C with high humidity for 2 days.
• Transfer plants to normal growth conditions (22-25°C, 16h light/8h dark) and monitor for silencing phenotypes over 3-4 weeks.

Applications: This method has been successfully applied to silence PDS genes in tomato, pepper, eggplant, and Arabidopsis, and disease resistance genes (SITL5 and SITL6) in tomato cultivar CLN2037E [18].

Seed Vacuum Infiltration for Sunflower

Sunflower has traditionally been considered a recalcitrant species for genetic transformation, but a recently developed seed vacuum infiltration protocol has achieved up to 91% infection efficiency in certain genotypes [19].

Materials: Sunflower seeds; pTRV1 and pTRV2-HaPDS vectors; Agrobacterium strain GV3101; vacuum infiltration system; Murashige and Skoog (MS) medium; appropriate antibiotics.

Procedure:

• Partially remove seed coats to enhance infiltration efficiency.
• Prepare Agrobacterium cultures as described in section 4.1 and resuspend to OD600 = 1.5 in infiltration medium.
• Place seeds in Agrobacterium suspension and apply vacuum (0.8-0.9 bar) for 10 minutes.
• Release vacuum gradually and continue immersion for 30 minutes.
• Transfer seeds to co-cultivation medium (MS medium with acetosyringone) for 6 hours.
• Plant treated seeds directly in soil (no in vitro recovery needed).
• Maintain plants at 22°C with 18h light/6h dark photoperiod and approximately 45% relative humidity.
• Monitor for photobleaching symptoms beginning at 14-21 days post-inoculation.

Key Considerations: Silencing efficiency is genotype-dependent, with 'Smart SM-64B' showing the highest infection rate (91%) [19]. The protocol does not require surface sterilization or in vitro recovery steps, significantly simplifying the process compared to previous methods.

Cotyledon Node Immersion for Soybean

Soybean's thick cuticle and dense trichomes present challenges for traditional leaf infiltration methods. The cotyledon node immersion protocol overcomes these barriers, achieving 65-95% silencing efficiency [10].

Materials: Soybean seeds; pTRV1 and pTRV2-GFP derivatives; Agrobacterium strain GV3101; sterilization reagents; tissue culture supplies.

Procedure:

• Surface-sterilize soybean seeds and soak in sterile water until swollen.
• Longitudinal bisect seeds to obtain half-seed explants with intact embryonic axes.
• Prepare Agrobacterium cultures as previously described and adjust to OD600 = 1.0.
• Immerse fresh explants in Agrobacterium suspension for 20-30 minutes (optimal duration).
• Co-cultivate explants on regeneration medium for 3 days in dark.
• Transfer to selection medium and monitor GFP fluorescence to verify infection.
• Photobleaching typically appears in cluster buds at 21 days post-inoculation.

Validation: Infection efficiency can be assessed by GFP fluorescence at the hypocotyl region 4 days post-infection, with successful transformation showing fluorescence in over 80% of cells [10].

The Scientist's Toolkit: Essential Research Reagents

Table 2: Essential Research Reagents for TRV-VIGS Experiments

Reagent/Vector	Specifications	Function	Examples/Notes
TRV Vectors	pTRV1 (RNA1 functions); pTRV2 (insert location) [12]	Viral genome components for replication and carrying target gene	pYL192 (TRV1), pYL156 (TRV2) [19]; pTRV2-GFP for tracking [18]
Agrobacterium Strains	GV3101, GV1301, GV2260 [18]	Delivery of TRV vectors into plant cells	GV3101 most common; specific strains may work better in certain species
Selection Antibiotics	Kanamycin (50μg/mL), Rifampicin (25-100μg/mL) [18]	Selection of transformed Agrobacterium	Concentrations vary by strain and vector system
Induction Compounds	Acetosyringone (150-200μM) [18]	Enhances T-DNA transfer from Agrobacterium	Critical for efficient infection; requires dark incubation
Infiltration Buffers	10 mM MgCl₂, 10 mM MES (pH 5.6-5.7) [18]	Maintains bacterial viability during inoculation	Optimal pH mimics plant apoplastic environment
Visual Marker Genes	PDS (photobleaching), CHS (white flowers) [15]	Visual indicators of silencing efficiency	Phytoene desaturase (PDS) most common across species
Positive Control Vectors	TRV2-PDS (species-specific) [3]	Protocol validation and optimization	Must be species-specific; e.g., JrPDS255 for walnut [3]
Empty Vector Controls	pTRV2-empty or pTRV2-GFP [15]	Distinguish viral symptoms from silencing phenotypes	pTRV2-empty can cause severe symptoms; pTRV2-sGFP recommended [15]

Technical Considerations and Troubleshooting

Optimizing Silencing Efficiency Across Species

Maximizing TRV-VIGS efficiency requires addressing species-specific challenges through systematic optimization. For woody plants like walnut, key parameters include using younger seedlings with 5-10 true leaves, employing stem injection or vacuum infiltration rather than spraying, and optimizing insert length to 255 bp for maximum efficiency [3]. In cereals and grasses, adjusting bacterial density (OD600 = 1.5-2.0) and maintaining lower temperatures (18-20°C) post-inoculation significantly enhances silencing spread [12]. For species with natural transformation barriers like soybean's thick cuticle, alternative inoculation methods such as cotyledon node immersion bypass these limitations [10]. Even within species, genotype selection is critical—in sunflower, cultivar 'Smart SM-64B' showed 91% infection rate compared to 62% in other genotypes [19]. When designing experiments, include both positive controls (TRV-PDS) and appropriate negative controls (TRV-GFP or TRV-empty) to distinguish true silencing phenotypes from viral symptoms or experimental artifacts [15].

Advanced Applications and Future Directions

The versatility of TRV continues to expand with technological advancements. Recent developments include TRV-based CRISPR/Cas9 delivery systems that utilize Csy4 ribonucleases to process gRNAs from the viral genome, enabling targeted genome editing without stable transformation [20]. The integration of fluorescent markers like GFP into TRV vectors allows real-time tracking of viral spread and silencing progression [18]. For functional genomics, tissue-specific promoters drive Cas9 expression in particular cell types when combined with TRV-gRNA delivery [20]. The demonstrated mobility of TRV into meristematic tissues enables studies of gene function in reproductive development and early developmental processes [12]. As these technologies mature, TRV-based systems are increasingly being adapted for high-throughput functional screening in non-model crops, significantly accelerating the identification of genes controlling agronomic traits in species lacking established transformation systems [3] [19].

Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool for rapidly elucidating gene function in plants. Within Tobacco Rattle Virus (TRV)-based VIGS protocols, the validation of silencing efficiency presents a significant experimental challenge. The phytoene desaturase (PDS) gene has become a cornerstone visual reporter for addressing this challenge, providing researchers with an easily scorable phenotypic marker for successful silencing events. As a key enzyme in the carotenoid biosynthesis pathway, PDS catalyzes the conversion of phytoene to ζ-carotene [3]. Its silencing disrupts carotenoid production, which protects chlorophyll from photo-oxidative degradation, resulting in a characteristic photobleaching phenotype [21]. This visible white or yellow bleaching serves as an intrinsic visual indicator, confirming successful infection, systemic spread of the TRV vector, and effective activation of the plant's silencing machinery without requiring expensive reagents or specialized equipment [4] [21].

The integration of PDS as a visual reporter is particularly valuable within TRV-VIGS systems, where silencing efficiency can be heterogeneous across tissues [22]. This article provides comprehensive application notes and protocols for using PDS to validate and optimize TRV-mediated VIGS, presenting standardized methodologies, quantitative efficiency data across species, and practical tools for implementing this critical validation system in plant functional genomics research.

Biological Rationale: PDS as an Ideal Visual Reporter

The Carotenoid Biosynthesis Pathway and PDS Disruption

Phytoene desaturase occupies a critical position in the carotenoid biosynthesis pathway, catalyzing the first desaturation step that converts colorless phytoene into colored carotenoids [3]. This conversion is essential for photoprotection and photosynthetic efficiency. When PDS expression is silenced via TRV-VIGS, the carotenoid biosynthesis pathway is blocked at this early stage, leading to the accumulation of colorless phytoene and the failure to produce carotenoid pigments that typically absorb light energy and quench chlorophyll excitons [21]. The consequent absence of photoprotective carotenoids renders chlorophyll susceptible to photo-oxidative damage upon light exposure, ultimately manifesting as the characteristic photobleaching phenotype [21]. This biochemical cascade makes PDS silencing both non-lethal and visually detectable, unlike the silencing of many essential genes.

Advantages Over Alternative Reporter Systems

The PDS gene offers several distinct advantages as a visual reporter for VIGS validation compared to alternative systems. Unlike exogenous reporters like β-glucuronidase (GUS) or Green Fluorescent Protein (GFP), which require specialized substrates, specific wavelengths of light, or expensive imaging equipment [22], PDS photobleaching is easily visible to the naked eye under normal growth conditions. Furthermore, as an endogenous gene, PDS does not require the development of transgenic reporter lines, significantly accelerating experimental timelines compared to systems relying on transformed visual markers such as anthocyanin regulators [22] [23]. The PDS reporter system also benefits from its broad phylogenetic conservation and functional stability across diverse plant species, enabling protocol standardization and cross-species comparisons [4] [19] [21].

Figure 1: Mechanism of PDS Silencing Phenotype in TRV-VIGS. The diagram illustrates the molecular pathway from TRV vector inoculation to visible photobleaching, highlighting the key stages of viral spread, siRNA processing, and carotenoid biosynthesis disruption.

Comparative Performance Metrics of PDS Across Plant Systems

The utility of PDS as a visual reporter has been extensively validated across diverse plant species, with documented variations in silencing efficiency and phenotypic presentation. These differences reflect species-specific responses to TRV infection and silencing mechanisms.

Table 1: Quantitative PDS Silencing Efficiency Across Plant Species in TRV-VIGS Systems

Plant Species	Infiltration Method	Time to Phenotype (days)	Silencing Efficiency	Key Optimization Factors	Citation
Atriplex canescens	Vacuum infiltration (germinated seeds)	15	16.4% (plant level)	Fragment position (5' end), OD~600~=0.8	[4]
Abelmoschus manihot	Back-of-blade injection (cotyledons)	16	54.4% (plant level)	Double injection, OD~600~=1.0	[21]
Juglans regia (Walnut)	Stem scratching	28	48% (leaf level)	Fragment length (255 bp), OD~600~=1.5	[3]
Helianthus annuus (Sunflower)	Seed vacuum infiltration	14	77% (infection rate)	6h co-cultivation, genotype selection	[19]
Castilleja tenuiflora	Cocultivation (Tc-AII)	32	80% (photobleaching area)	A. tumefaciens strain C58C1	[24]

The data presented in Table 1 demonstrates that while PDS serves as a reliable visual reporter across species, optimization of protocol parameters is essential for achieving high silencing efficiency. The observed variation in time to phenotype emergence reflects differences in viral mobility and replication rates across plant species, with herbaceous species generally showing more rapid phenotype development than woody plants [3] [4]. Silencing efficiency is influenced by multiple factors, including the specific PDS fragment selected, inoculation method, plant developmental stage, and bacterial density during infiltration [3] [4] [19].

Experimental Protocols for PDS-Based VIGS Validation

Protocol 1: TRV Vector Construction for PDS Silencing

Principle: The TRV2 vector is modified to include a fragment of the PDS gene, which will trigger sequence-specific silencing when co-delivered with TRV1 into plant tissues.

Materials:

• pTRV1 and pTRV2 binary vectors
• Specific primers for PDS fragment amplification
• Restriction enzymes (e.g., EcoRI, BamHI) or Gateway BP Clonase II enzyme mix
• Agrobacterium tumefaciens strain GV3101

Procedure:

• PDS Fragment Selection: Identify a 200-400 bp fragment from the 5' end, central region, or 3' end of the PDS coding sequence using tools like SGN-VIGS (https://vigs.solgenomics.net/) [4]. For walnut, a 255 bp fragment provided optimal efficiency [3].
• Fragment Amplification: Amplify the selected fragment from cDNA using gene-specific primers with incorporated restriction sites (e.g., EcoRI and BamHI) [4].
• Vector Ligation: Digest both the pTRV2 vector and PCR product with appropriate restriction enzymes. Purify fragments and ligate using T4 DNA ligase [19]. Alternatively, use Gateway BP Clonase II enzyme mix for recombination cloning.
• Transformation: Introduce the constructed pTRV2-PDS and pTRV1 vectors separately into A. tumefaciens strain GV3101 using freeze-thaw or electroporation methods [19].
• Confirmation: Verify positive clones by colony PCR and sequencing to ensure correct insert orientation and sequence.

Protocol 2: Plant Inoculation for PDS Silencing

Principle: Agrobacterium harboring TRV vectors is introduced into plant tissues to initiate viral infection and subsequent PDS silencing.

Materials:

• Agrobacterium cultures containing pTRV1 and pTRV2-PDS
• Infiltration buffer (10 mM MES, 200 μM acetosyringone, 10 mM MgCl₂)
• Silwet L-77 surfactant (0.03%)
• Plant materials (seeds, seedlings, or specific plant parts)

Procedure:

• Culture Preparation: Inoculate single colonies of Agrobacterium containing pTRV1 and pTRV2-PDS separately in YEP medium with appropriate antibiotics. Grow at 28°C with shaking (200 rpm) until OD₆₀₀ reaches 0.6-1.5 [3] [4] [19].
• Cell Harvesting: Pellet bacterial cultures by centrifugation at 6000 rpm for 8 minutes. Resuspend in infiltration buffer supplemented with acetosyringone (200 μM) to the desired OD₆₀₀ [4].
• Mixture Preparation: Combine equal volumes of pTRV1 and pTRV2-PDS suspensions. Incubate the mixture at room temperature in darkness for 3 hours to induce virulence gene expression [4].
• Plant Inoculation: Apply the bacterial suspension using one of these optimized methods:
  • Vacuum Infiltration: Submerge germinated seeds or seedlings in bacterial suspension and apply vacuum (0.5 kPa) for 5-10 minutes [4] [19].
  • Stem Scratching: Gently scratch stems near the apical meristem and apply bacterial suspension with a sterile brush [3].
  • Leaf Infiltration: Inject bacterial suspension into the abaxial side of leaves using a needleless syringe [21].
• Post-Inoculation Care: Maintain inoculated plants at 22-24°C with high humidity for 24-48 hours, then transfer to normal growth conditions [4] [19].

Protocol 3: Phenotypic and Molecular Validation of PDS Silencing

Principle: Successful PDS silencing is confirmed through visual assessment of photobleaching and molecular quantification of PDS transcript reduction.

Materials:

• TRIzol reagent for RNA extraction
• cDNA synthesis kit
• Quantitative PCR system with SYBR Green chemistry
• PDS-specific qRT-PCR primers
• Reference genes (e.g., Actin, GAPDH, UBQ)

Procedure:

• Phenotypic Monitoring:
  • Observe plants daily for development of photobleaching symptoms, typically appearing 14-32 days post-inoculation depending on species (Table 1).
  • Document the spatial distribution and progression of photobleaching using digital photography.
  • Quantify the percentage of plants showing photobleaching and estimate the affected leaf area [24] [19].

• Molecular Validation:

  • Extract total RNA from silenced (photobleached) and control tissues using TRIzol reagent.
  • Synthesize cDNA using reverse transcriptase according to manufacturer protocols.
  • Perform quantitative RT-PCR using PDS-specific primers and reference gene primers.
  • Calculate relative PDS expression using the 2^(-ΔΔCt) method [4].

• Efficiency Assessment:

  • Successful silencing typically shows 40-80% reduction in PDS transcript levels compared to empty vector controls [4].
  • Correlate the degree of transcript reduction with the extent of visual photobleaching.

Figure 2: Experimental Workflow for PDS-Based VIGS Validation. The diagram outlines the key steps from vector construction to data interpretation, highlighting both phenotypic and molecular validation pathways.

Advanced Applications and Technical Considerations

PDS in Composite VIGS Reporter Systems

Recent advancements have integrated PDS with additional visual markers to create more sophisticated reporter systems. For example, a dual visual reporter system combines PDS-mediated photobleaching with anthocyanin accumulation markers, enabling more precise discrimination of silenced sectors [22]. In tomato, the Del/Ros1 system expressing anthocyanin transcription factors produces purple fruit, while co-silencing with PDS restores red pigmentation in non-silenced areas, providing both positive and negative visual controls [22]. These composite systems are particularly valuable for quantifying non-visual phenotypes in metabolic studies, as they allow precise dissection of silenced versus non-silenced tissues for subsequent chemometric analysis [22].

Troubleshooting Common Challenges

Low Silencing Efficiency: If photobleaching is weak or patchy, optimize the PDS fragment length and position. Fragments of 250-400 bp from the 5' end often yield higher efficiency [3] [4]. Additionally, ensure bacterial OD₆₀₀ is optimized for the specific plant species (typically 0.8-1.5) [3] [4] [19].

Delayed or No Phenotype: Extend the observation period, as some species require 4+ weeks for phenotype manifestation [3]. Verify TRV systemic movement by testing for viral coat protein via PCR in newly emerged leaves [19].

Plant Development Effects: The photobleaching caused by PDS silencing can stress plants. Include appropriate controls and minimize light intensity during phenotype development to reduce photo-oxidative damage [21].

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Research Reagents for PDS-Based VIGS Experiments

Reagent/Vector	Specification	Function in PDS-VIGS	Example Source
pTRV1 Vector	Contains RNA1 genes for replication and movement	Essential viral component for TRV system	Addgene #148968 [19]
pTRV2 Vector	Contains cloning site for insert fragments	Carries PDS fragment to trigger silencing	Addgene #148969 [19]
A. tumefaciens GV3101	Disarmed helper strain with Ti plasmid	Delivers TRV vectors into plant cells	Multiple distributors
Acetosyringone	200 μM in infiltration buffer	Induces Agrobacterium virulence genes	Sigma-Aldrich [4]
Silwet L-77	0.03% in infiltration buffer	Surfactant that enhances tissue penetration	Multiple distributors [4]
PDS-specific primers	Designed for target species	Amplifies PDS fragment for cloning	Custom synthesis [4]

The phytoene desaturase gene serves as an indispensable visual reporter system for validating TRV-based VIGS protocols across diverse plant species. Its easily scorable photobleaching phenotype, coupled with molecular validation through qRT-PCR, provides researchers with a robust system for optimizing silencing efficiency and confirming successful gene knockdown. The standardized protocols and comparative metrics presented here enable researchers to implement and troubleshoot PDS-based validation in both model and non-model plant systems. As VIGS technology continues to evolve, particularly with emerging innovations like engineered viral suppressors to enhance silencing efficiency [25] and encapsidated TRV1 replicons for simplified application [26], the role of PDS as a reliable visual reporter remains fundamental to advancing plant functional genomics and accelerating the characterization of gene function in species resistant to stable transformation.

A Step-by-Step TRV-VIGS Protocol: From Vector Construction to Agroinfiltration

Within the broader research on Tobacco Rattle Virus (TRV)-based Virus-Induced Gene Silencing (VIGS) protocols, the strategic cloning of target gene fragments into the pTRV2 vector is a critical foundational step. This process enables researchers to systematically investigate gene function by directing the viral machinery to silence specific endogenous plant genes [15]. The efficiency of the entire VIGS experiment is profoundly influenced by the careful selection of the target gene fragment and its precise insertion into the viral delivery vector [4]. This application note provides a detailed protocol for selecting optimal target sequences and executing their successful cloning into the pTRV2 vector, thereby ensuring robust and interpretable silencing outcomes.

Target Gene Fragment Selection

The initial and most crucial design phase involves selecting an effective fragment from the gene of interest for cloning into the pTRV2 vector. Not all fragments of a gene are equally capable of inducing efficient silencing.

Selection Criteria and Bioinformatics Analysis

The selection process should prioritize unique, conserved regions of the gene to maximize silencing efficiency and minimize off-target effects. Table 1 outlines the key parameters for optimal fragment selection.

Table 1: Criteria for Selecting Target Gene Fragments for pTRV2 Cloning

Parameter	Optimal Characteristic	Rationale & Practical Consideration
Fragment Length	300–400 base pairs (bp) [4]	Balances efficient cloning and potent triggering of the silencing machinery.
Sequence Specificity	Unique to the target gene, with no significant homology to other genes [4]	Minimizes off-target silencing; verified using BLASTN against the host genome.
Region of ORF	5' end, central, or 3' end of the coding sequence (CDS) [4]	Testing multiple non-overlapping regions is recommended to identify the most effective fragment.
Sequence Features	Avoids regions of high GC content (>70%) or repetitive sequences	Ensures efficient cloning and prevents secondary structures that hinder silencing.

In Silico Tools for Fragment Design

Leverage bioinformatics tools to streamline the selection process:

• SGN-VIGS Tool: A dedicated online resource (https://vigs.solgenomics.net/) for predicting optimal nucleotide target regions for VIGS [4].
• Nucleotide BLAST: Perform a final specificity check of the selected fragment against the plant's genome or transcriptome to rule out unintended targets [4].

Molecular Cloning into pTRV2

The standard method for inserting the selected fragment into the pTRV2 vector is traditional restriction enzyme-based cloning. This involves preparing both the vector and the insert with compatible ends for ligation.

The following diagram illustrates the comprehensive workflow for cloning a target fragment into the pTRV2 vector.

Protocol: Restriction Enzyme Cloning into pTRV2

Primer Design and Amplification of Insert

• Primer Design: Design forward and reverse primers to amplify the 300-400 bp target fragment. Add appropriate restriction enzyme sites (e.g., EcoRI, BamHI, XbaI) to the 5' ends of the primers. These sites should be compatible with the Multiple Cloning Site (MCS) of your pTRV2 vector [4] [10].
• PCR Amplification: Amplify the fragment from a cDNA template using a high-fidelity DNA polymerase. Purify the PCR product using standard methods [10].

Restriction Digestion

• Double Digest: Simultaneously digest both the purified PCR product (insert) and the pTRV2 plasmid vector with the two selected restriction enzymes.
  • Reaction Setup:
    • Nuclease-Free Water: to 20 µl
    • 10X Restriction Buffer: 2 µl
    • Acetylated BSA (1mg/ml): 2 µl
    • DNA (Insert ~100 ng or Vector ~1 µg): 1 µl
    • Restriction Enzyme 1 (10 U): 1 µl
    • Restriction Enzyme 2 (10 U): 1 µl
    • Final Volume: 20 µl [27]
  • Incubation: Mix by pipetting and incubate at the enzymes' optimal temperature (typically 37°C) for 1–2 hours.
• Critical Note on pTRV2 Vector: Some widely distributed pTRV2-MCS vectors contain an unannotated ~1 kb E. coli genomic insertion that introduces extra restriction sites. Always verify your vector sequence through diagnostic digest or sequencing before cloning. For example, an unexpected ~300 bp band after an NcoI/BamHI digest indicates this issue; switching to enzymes like XbaI can resolve it [28].

Purification of Vector and Insert

• Gel Purification: Resolve the digested products on an agarose gel. Excise the linearized pTRV2 vector band and the target insert band.
• DNA Recovery: Purify the DNA from the gel slices using a commercial gel extraction kit, following the manufacturer's instructions [27]. Elute in nuclease-free water or a low-EDTA buffer. Quantify the purified DNA.

Ligation

• Reaction Setup: Combine the digested and purified vector and insert in a ligation reaction.
  • Molar Ratio: Use a 1:1 to 5:1 molar ratio of insert to vector. A 3:1 ratio is a common starting point.
  • Reagent:
    • Purified, digested pTRV2 vector
    • Purified, digested insert fragment
    • T4 DNA Ligase Buffer (with ATP)
    • T4 DNA Ligase
    • Nuclease-Free Water to final volume (e.g., 10–20 µl) [29]
• Incubation: Incubate the reaction at room temperature for 1 hour or at 16°C overnight.

Transformation and Screening

• Transformation: Transform the ligation reaction into competent E. coli cells (e.g., DH5α) via heat shock or electroporation [30] [10]. Plate the cells on LB agar containing the appropriate antibiotic (e.g., Kanamycin, 50 mg/L).
• Colony Screening: Select individual colonies and screen for positive clones.
  • PCR Screening: Use colony PCR with gene-specific or vector-specific primers to confirm the presence of the insert.
  • Diagnostic Restriction Digest: Isolate plasmid DNA from liquid cultures and perform a restriction digest to release the insert, verifying its correct size via gel electrophoresis [31].
  • Sequencing: Finally, sequence the cloned insert using MCS-flanking primers to confirm the correct sequence and orientation.

Troubleshooting Common Cloning Issues

The following flowchart provides a systematic approach to diagnosing and resolving common problems encountered during the cloning process.

The Scientist's Toolkit: Essential Reagents for Cloning

Table 2: Key Research Reagent Solutions for pTRV2 Cloning

Reagent / Tool	Function in Protocol	Specific Examples & Notes
pTRV2 Vector	Viral vector for delivering the target gene fragment into the plant.	Obtain from reputable sources (e.g., Addgene). Always verify the sequence of the MCS [28].
Restriction Enzymes	Create specific cuts in the vector and insert for assembly.	Use high-quality enzymes (e.g., from Thermo Fisher Scientific, NEB). Confirm compatibility for double digests [27].
T4 DNA Ligase	Joins the compatible ends of the insert and the linearized vector.	Requires ATP and Mg²⁺ in its buffer. Crucial for forming the recombinant plasmid [29].
High-Fidelity DNA Polymerase	Amplifies the target gene fragment from cDNA with low error rates.	Phusion or similar polymerases ensure accurate amplification of the insert [30].
Gel Extraction Kit	Purifies the digested vector and insert fragments from agarose gels.	Kits based on silica columns (e.g., Zymoclean) provide high purity and yield for ligation [30] [27].
Competent E. coli Cells	Host for propagating the ligated plasmid after cloning.	Select strains with high transformation efficiency (e.g., DH5α). For methylation-sensitive enzymes, use dam-/dcm- strains [27] [29].

A meticulously planned and executed cloning strategy is the cornerstone of successful TRV-based VIGS research. By applying the principles of rational fragment design, adhering to a rigorous molecular cloning protocol, and utilizing the appropriate toolkit of reagents, researchers can consistently generate high-quality pTRV2 silencing constructs. This reliability directly translates into more efficient and interpretable functional genomics experiments, accelerating the discovery of gene function in a wide range of plant species.

Agrobacterium Strain Preparation and Infiltration Buffer Composition

Within the framework of Tobacco Rattle Virus (TRV)-based Virus-Induced Gene Silencing (VIGS) research, the preparation of competent Agrobacterium tumefaciens cells and the formulation of optimal infiltration buffers are critical foundational steps. This protocol details standardized methodologies for strain selection, culture preparation, and the composition of infiltration solutions, which are essential for ensuring high-efficiency T-DNA delivery and robust gene silencing phenotypes in a broad range of plant hosts [12]. The reproducibility and success of the entire VIGS workflow are contingent upon the precise execution of these initial procedures.

Key Research Reagent Solutions

The following table catalogues the essential reagents and materials required for the preparation of Agrobacterium strains and infiltration buffers in a TRV-VIGS workflow.

Table 1: Essential Research Reagents for Agrobacterium-Mediated VIGS

Reagent/Material	Function/Application	Key Details
Agrobacterium Strains	T-DNA delivery vector	Common strains: GV3101, AGL1, C58C1, LBA4404 [32] [4] [33].
Antibiotics	Selection of transformed Agrobacterium	e.g., Kanamycin, Rifampicin, Gentamycin; concentration varies by strain and vector [34] [35].
Acetosyringone	Virulence gene inducer	Critical for activating Agrobacterium's T-DNA transfer machinery; typically used at 100-200 µM [32] [36] [34].
Silwet L-77	Surfactant	Enhants wetting and penetration of infiltration solution into plant tissues [32] [4].
MgCl₂	Washing and infiltration buffer base	Provides essential ions for bacterial viability and osmotic balance [36] [35].
MES Buffer	pH stabilization	Maintains optimal pH (5.5-6.0) for virulence induction [36] [33].
Sucrose	Osmoticum in infiltration buffer	Maintains osmotic potential, improving bacterial viability during infiltration [32] [33].

Infiltration Buffer Composition

The infiltration buffer serves as the delivery medium for the Agrobacterium suspension, and its components are crucial for facilitating bacterial survival and T-DNA transfer. Multiple formulations have been successfully employed in the literature, with slight variations tailored to specific plant species or inoculation methods.

Table 2: Comparative Formulations of Agrobacterium Infiltration Buffers

Component	Standard Formulation [32]	Alternative Formulation 1 [36]	Alternative Formulation 2 [33]
Base Salts	1.1 g/L Murashige and Skoog (MS) medium	10 mM MgCl₂	5 g/L MS Basal Salts
Buffer	-	5 mM MES, pH 5.6	10 mM MES
Inducer	100 µM Acetosyringone	150 µM Acetosyringone	200 µM Acetosyringone
Surfactant	0.01% (v/v) Silwet L-77	-	-
Osmoticum	1% (w/v) Sucrose	-	20 g/L Sucrose
Notes	pH adjusted to 6.0; must be freshly prepared.	Also referred to as Agrobacterium Infiltration Media (AIM).	Used for vacuum and syringe infiltration in multiple species.

Buffer Preparation Protocol

• Standard Formulation (1 L) [32] [35]:
  1. Dissolve 1.1 g of Murashige and Skoog (MS) medium and 10 g of sucrose in approximately 800 mL of distilled water.
  2. Add 1 mL of a 0.1 M stock solution of acetosyringone (prepared in DMSO).
  3. Adjust the pH to 6.0 using 1 M KOH.
  4. Add 100 µL of Silwet L-77 and gently stir to avoid foaming.
  5. Make up the final volume to 1 L with distilled water. It is recommended to prepare this solution fresh on the day of infiltration.
• Washing Solution (1 L) [35]:
  1. Combine 10 mL of 1 M MgCl₂ and 1 mL of 0.1 M acetosyringone in a graduated cylinder.
  2. Add distilled water to a final volume of 1 L. This solution can be used to wash and resuspend bacterial pellets prior to final dilution in the infiltration buffer.

Agrobacterium Strain Preparation

Strain Selection and Transformation

Commonly used Agrobacterium strains for TRV-VIGS include GV3101, AGL1, and C58C1 [32] [4] [10]. The TRV binary vectors (pTRV1 and pTRV2 containing the gene of interest) are first introduced into Agrobacterium cells via electroporation or the freeze-thaw method [34].

Culture and Induction Protocol

• Primary Culture: Streak the transformed Agrobacterium from a glycerol stock onto a solid YEP or LB agar plate containing the appropriate antibiotics (e.g., 50 µg/mL Kanamycin, 25 µg/mL Gentamycin, 50 µg/mL Rifampicin) [4] [34]. Incubate the plate at 28°C for 48 hours.
• Secondary Liquid Culture: Pick a single colony and inoculate a 5-50 mL liquid culture of YEP or LB medium with the same antibiotics. Supplement the medium with 10 mM MES and 20 µM acetosyringone to begin virulence induction. Grow the culture overnight at 28°C with constant shaking (200-250 rpm) [34] [10].
• Harvesting and Washing: When the bacterial culture reaches the mid-logarithmic phase (OD600 ≈ 0.6 - 1.0), pellet the cells by centrifugation at 3,000 - 4,000 × g for 5-15 minutes [34] [10]. Gently resuspend the pellet in the pre-prepared washing solution or infiltration buffer to remove residual antibiotics and metabolites.
• Final Resuspension and Induction: Resuspend the washed bacterial pellet in the chosen infiltration buffer (see Section 3) to the desired final OD600. The optimal density is species-dependent but typically ranges from 0.5 to 1.5 [2] [34] [10].
• Pre-infiltration Incubation: Allow the final Agrobacterium suspension to incubate at room temperature in the dark for 3-6 hours to fully activate the virulence genes [34] [33].
• Inoculum Mixing: For VIGS, mix the induced suspensions of Agrobacterium carrying pTRV1 and pTRV2 (or its derivative with the target gene insert) in a 1:1 ratio immediately before infiltration [34] [33].

Integrated Workflow

The diagram below illustrates the complete, integrated workflow for preparing Agrobacterium and infiltration buffers for a TRV-VIGS experiment.

Within the framework of Tobacco Rattle Virus (TRV)-mediated Virus-Induced Gene Silencing (VIGS) research, selecting an optimal inoculation method is paramount for achieving high silencing efficiency. This technique serves as a powerful reverse-genetics tool for analyzing gene function, particularly in non-model plant species that are recalcitrant to stable transformation [37] [19]. The inoculation process directly influences the initial infection rate and subsequent systemic spread of the TRV vector, thereby determining the success and reliability of functional genomics studies. This application note provides a detailed comparative analysis of the two predominant Agrobacterium-mediated inoculation techniques: syringe infiltration and vacuum infiltration. We evaluate these methods based on quantitative efficiency data, outline standardized protocols, and discuss their suitability for different plant species and research objectives, thereby offering a practical guide for researchers in plant science and drug development who utilize VIGS for gene function validation.

Mechanism of TRV-VIGS and Inoculation Fundamentals

The fundamental principle of TRV-VIGS relies on the plant's innate post-transcriptional gene silencing (PTGS) mechanism, which is activated upon viral infection [37] [12]. The process begins when a recombinant TRV vector, carrying a fragment of the target plant gene, is introduced into plant cells. The viral RNA genome is replicated, leading to the formation of double-stranded RNA (dsRNA) by the viral RNA-dependent RNA polymerase (RdRp). This dsRNA is recognized and cleaved by the plant's Dicer-like enzymes into small interfering RNAs (siRNAs). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), which guides the sequence-specific degradation of complementary endogenous mRNA transcripts, resulting in gene silencing [37] [12]. A key visual marker for successful silencing is the photobleaching phenotype observed when silencing the phytoene desaturase (PDS) gene, which is involved in carotenoid biosynthesis [38] [15].

The inoculation method is a critical first step that determines the efficiency of this entire process. Both syringe and vacuum infiltration aim to deliver Agrobacterium tumefaciens, harboring the TRV vectors (pTRV1 and pTRV2), into the intercellular spaces (apoplast) of plant tissues. The choice of method affects the number of initially infected cells, the uniformity of infection, and the potential for systemic viral movement, thereby directly influencing the observed silencing efficiency and phenotype stability [37] [19].

Diagram: A workflow comparing the fundamental mechanisms and primary applications of syringe and vacuum infiltration methods in TRV-VIGS.

Comparative Efficiency Analysis

Extensive research across diverse plant species has demonstrated that vacuum infiltration generally achieves higher and more consistent whole-plant silencing efficiency compared to syringe infiltration, particularly in recalcitrant species. The quantitative data below summarizes key performance metrics.

Table 1: Quantitative Comparison of Silencing Efficiency Across Plant Species

Plant Species	Inoculation Method	Silencing Efficiency/ Phenotype Description	Key Experimental Parameters	Source
Cannabis sativa	Syringe Infiltration	Localized photobleaching, primarily in leaf veins	Infiltration of abaxial leaf surface with needleless syringe	[39]
	Vacuum Infiltration	Dramatically increased photobleaching; intense, widespread phenotype	Whole-plant vacuum infiltration of 3-week-old rooted cuttings	[39]
Atriplex canescens	Vacuum Infiltration	~16.4% silencing efficiency; systemic photobleaching in new leaves at 15 dpi	Germinated seeds, OD~600~=0.8, 0.5 kPa for 10 min	[38]
Sunflower	Seed Vacuum Infiltration	Up to 77% infection rate; TRV detected in leaves up to node 9	Vacuum of decorticated seeds + 6h co-cultivation	[19]
Petunia	Apical Meristem Inoculation	69% increased area of CHS silencing; 28% increased PDS silencing	Mechanically wounded shoot apical meristems	[15]
Taro	Syringe Infiltration (OD~600~=0.6)	12.23% silencing plant rate	Leaf injection method	[40]
	Syringe Infiltration (OD~600~=1.0)	27.77% silencing plant rate	Leaf injection method	[40]
Ilex dabieshanensis	Syringe Infiltration	Yellow-leaf phenotype at 21 dpi; significant reduction in ChlH transcripts	Leaf syringe-infiltration, OD~600~=1.8	[41]

The data reveals a clear trend where vacuum-based methods, particularly those applied to young plantlets or seeds, facilitate more extensive systemic movement of the virus, leading to silencing phenotypes that manifest throughout the entire plant [38] [19] [39]. In contrast, syringe infiltration often results in more localized silencing, as seen in cannabis and initial taro experiments, unless optimized with higher Agrobacterium densities [39] [40]. The developmental stage of the plant is also a critical factor, with younger tissues often showing higher susceptibility to viral movement and gene silencing [15] [42].

Table 2: Method Suitability and Technical Comparison

Parameter	Syringe Infiltration	Vacuum Infiltration
Core Principle	Manual pressure application forces Agrobacterium suspension into leaf mesophyll.	Vacuum removes air from intercellular spaces; atmospheric pressure drives infiltration.
Best Suited For	Mature leaves, established plants, localized silencing studies.	Seedlings, germinated seeds, whole-plant systemic silencing, recalcitrant species.
Typical Silencing Pattern	Often localized near infiltration sites.	Frequently whole-plant and systemic.
Technical Complexity	Low; requires minimal equipment.	Moderate; requires access to a vacuum chamber and pump.
Throughput	Lower; leaves are infiltrated individually.	Higher; multiple plants/seeds can be processed simultaneously.
Species Success	Robust in model plants like N. benthamiana and tomato.	Effective in monocots (wheat, maize) and dicots (sunflower, Atriplex). [43]

Detailed Experimental Protocols

Protocol for Syringe Infiltration

This protocol is adapted from methods successfully used in Ilex dabieshanensis [41] and Cannabis sativa [39].

1. Agrobacterium Preparation: - Transform Agrobacterium tumefaciens strain GV3101 with pTRV1 and pTRV2 (or pTRV2 containing the target gene fragment) separately [38] [41]. - Inoculate single colonies into LB medium containing appropriate antibiotics (e.g., 50 µg/mL kanamycin, 50 µg/mL rifampicin) and grow overnight at 28°C with shaking. - Sub-culture the starters into fresh, antibiotic-containing LB medium supplemented with 10 mM MES and 20 µM acetosyringone. Grow until the cultures reach an OD~600~ of 0.6-1.8 [39] [41] [40]. - Pellet the bacterial cells by centrifugation (e.g., 6000 rpm for 10 min) and resuspend in an infiltration buffer (10 mM MgCl~2~, 10 mM MES, 200 µM acetosyringone, pH 5.6) to the desired OD~600~. The optimal density is species-dependent and should be optimized (e.g., OD~600~=1.8 for Ilex [41], OD~600~=1.0 for taro [40]). - Incubate the resuspended mixtures at room temperature for 3-4 hours in the dark [41]. - Combine the pTRV1 and pTRV2 suspensions in a 1:1 ratio immediately before infiltration [38] [41].

2. Plant Preparation: - Use well-watered plants at an appropriate developmental stage, typically with 4-6 true leaves [15]. Avoid water-stressed plants. - Gently pierce the abaxial (lower) surface of the target leaves with a fine needle, if necessary, to facilitate infiltration.

3. Infiltration: - Using a 1 mL needleless syringe, gently press the tip against the abaxial leaf surface at the pierced or stomata-rich site. - Slowly depress the plunger to infiltrate the leaf mesophyll. A successful infiltration is indicated by the appearance of a dark, water-soaked area. - Infiltrate multiple spots per leaf to increase the likelihood of systemic spread.

4. Post-Inoculation Care: - Maintain inoculated plants in moderate light and temperature conditions (e.g., 22-25°C) with high humidity for the first 1-2 days. - Monitor plants for the development of viral symptoms and the desired silencing phenotype, which typically appears 2-4 weeks post-inoculation [37] [41].

Protocol for Vacuum Infiltration

This protocol is adapted from highly efficient methods established for Atriplex canescens [38], sunflower [19], and cereals [43].

1. Agrobacterium Preparation: - Prepare the Agrobacterium suspension as described in Section 4.1, adjusting the final OD~600~ to the species-optimized concentration (e.g., OD~600~=0.8 for Atriplex [38]).

2. Plant Material Preparation: - For germinated seeds: Surface-sterilize and germinate seeds until the radicle reaches 1-3 cm in length [38]. For some species like sunflower, decortication (removing the seed coat) is recommended prior to infiltration [19]. - For young seedlings: 5-day-old etiolated seedlings with fully emerged cotyledons have proven highly effective for VIGS in periwinkle, licorice, and wormwood [42].

3. Vacuum Infiltration: - Submerge the plant material (germinated seeds or seedlings) completely in the prepared Agrobacterium suspension inside a vacuum desiccator or chamber. - Apply a vacuum of 0.5 to 0.7 kPa (or 500-700 mbar) for a duration of 5 to 30 minutes. Specific parameters vary by protocol (e.g., 0.5 kPa for 10 min for Atriplex [38]; 30 min for periwinkle cotyledons [42]). - Co-cultivation: Following vacuum infiltration, a co-cultivation period in the Agrobacterium suspension for several hours (e.g., 6 hours for sunflower [19]) can significantly enhance infection rates. - Gently release the vacuum to allow the atmospheric pressure to force the suspension into the plant tissues.

4. Post-Inoculation Care: - After co-cultivation, rinse the plant materials gently with sterile distilled water to remove excess Agrobacterium [38]. - Transplant germinated seeds or seedlings into pots with a sterile soil mix or vermiculite. - Grow plants under controlled environmental conditions. Silencing phenotypes in new leaves can appear as early as 15 days post-inoculation [38].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Reagents and Materials for TRV-VIGS Experiments

Item	Function/Description	Example Use Case
TRV Vectors (pTRV1 & pTRV2)	Binary T-DNA vectors containing viral genomes. pTRV1 encodes replication/movement proteins; pTRV2 carries the coat protein and the cloned target gene fragment.	The backbone for all TRV-VIGS experiments; available as MCS or Gateway clones [37] [12].
Agrobacterium tumefaciens (GV3101)	A disarmed strain used to deliver T-DNA from binary vectors into plant cells.	The standard workhorse for Agrobacterium-mediated VIGS protocols [38] [41].
Infiltration Buffer	A solution to maintain Agrobacterium viability and facilitate T-DNA transfer. Typically contains MgCl~2~, MES buffer, and the virulence gene inducer acetosyringone.	Used to resuspend bacterial pellets before inoculation [38] [41].
Marker Genes (PDS, ChlH)	Endogenous plant genes whose silencing produces a visual phenotype (photobleaching or yellowing), used to optimize and validate the VIGS system.	Essential positive controls for testing and optimizing any new VIGS protocol [38] [15] [41].
Vacuum Infiltration Apparatus	Consists of a vacuum desiccator/chamber and a pump capable of reaching and holding pressures of 0.5-0.7 kPa.	Critical for performing vacuum-based inoculation of seeds or seedlings [38] [19] [43].

The choice between syringe and vacuum infiltration for TRV-VIGS is not a matter of one being universally superior, but rather depends on the specific research goals and plant material.

• Syringe Infiltration is recommended for studies on mature plants where localized silencing is sufficient, or for species where high-throughput stable transformation is not the limiting factor. Its low technical barrier makes it ideal for initial tests in model plants.
• Vacuum Infiltration is unequivocally recommended for achieving high-efficiency, whole-plant systemic silencing, especially in recalcitrant species, plants with difficult-to-infiltrate leaves, or when working with young seedlings and germinated seeds [38] [19] [39]. The sprout vacuum infiltration method represents a significant advancement for functional genomics in crop plants, enabling rapid systemic gene silencing from the earliest developmental stages.

For researchers aiming to establish a robust VIGS protocol, particularly in a non-model species, beginning with the optimization of a vacuum-based method applied to young plant material is likely to yield the most reliable and consistent results for high-throughput reverse-genetics screens.

Optimizing Plant Developmental Stage for Maximum Silencing Efficiency

Within Tobacco Rattle Virus-mediated Virus-Induced Gene Silencing (TRV-VIGS) protocols, the developmental stage of the plant at the time of inoculation is a critical determinant of success. This parameter significantly influences the efficiency of viral spread and the potency of the resulting silencing phenotype. The optimization of this factor is particularly crucial for functional genomics research in non-model plants and crops recalcitrant to stable transformation. This Application Note synthesizes recent research to provide a standardized protocol for identifying and utilizing the optimal developmental window for TRV-VIGS across diverse plant species, contextualized within a broader thesis on TRV-VIGS protocol refinement.

The following table consolidates empirical findings on the relationship between plant developmental stage and achieved silencing efficiency across various species.

Table 1: Impact of Plant Developmental Stage on TRV-VIGS Efficiency

Plant Species	Optimal Developmental Stage/Inoculation Material	Key Silencing Efficiency Metric	Reference
Nepeta cataria (Catmint)	Cotyledons (Silencing effect spread to first two pairs of true leaves)	Silencing efficiency reached 84.4%	[44]
Camellia drupifera (Tea Oil Camellia)	Early-stage capsules: For silencing CdCRY1 (affecting exocarp pigmentation)Mid-stage capsules: For silencing CdLAC15 (affecting mesocarp pigmentation)	Infiltration efficiency: ~93.94%Optimal VIGS effect: ~69.80% (Early stage), ~90.91% (Mid stage)	[13]
Atriplex canescens	Germinated seeds (radicle length 1–3 cm)	Average silencing efficiency: ~16.4%AcPDS transcript reduction: 40–80%	[4]
Glycine max (Soybean)	Cotyledon node of half-seed explants (longitudinally bisected, swollen seeds)	Effective infectivity efficiency: >80%, up to 95% for specific cultivars	[5]
Juglans regia (Walnut)	Seedlings (for spray infiltration)	Silencing efficiency up to 48% with optimized parameters	[45]

Detailed Experimental Protocols for Stage-Specific VIGS

Protocol A: High-Efficiency Cotyledon-Based VIGS in Dicots

This protocol, adapted for catmint (Nepeta spp.), is designed for rapid, high-throughput functional validation and achieves exceptional efficiency by targeting the cotyledon stage [44].

Key Reagents & Materials:

• Plant Material: Nepeta cataria or N. mussinii seeds.
• VIGS Vectors: pTRV1 and pTRV2 vectors.
• Agrobacterium Strain: A. tumefaciens GV3101.
• Infiltration Buffer: 10 mM MES, 10 mM MgCl₂, 200 µM Acetosyringone.

Step-by-Step Workflow:

• Seed Sowing and Growth: Sow seeds at a depth of 1 cm in compost. Cultivate plants under a 16/8-hour light/dark photoperiod at 25°C/22°C (day/night) until the cotyledons are fully expanded.
• Vector Construction: Clone a 200-400 bp fragment of the target gene (e.g., G8H, 288 bp) into the multiple cloning site of the pTRV2 vector. For visual monitoring, a fragment of the ChlH gene (329 bp) can be co-cloned or used in a control vector to induce photobleaching.
• Agrobacterium Preparation: Transform the recombinant pTRV2 and the helper pTRV1 plasmids into Agrobacterium. Culture single colonies in YEP medium with appropriate antibiotics (e.g., Kanamycin, Rifampicin) until the OD₆₀₀ reaches 0.9-1.0. Centrifuge the culture and resuspend the pellet in infiltration buffer to a final OD₆₀₀ of 0.8-1.5.
• Agroinfiltration: Mix the pTRV1 and pTRV2-derived Agrobacterium suspensions in a 1:1 ratio. Immerse the cotyledons of the seedlings in the bacterial suspension. For enhanced efficiency, apply a brief vacuum infiltration (0.5 kPa for 5-10 minutes) [4] [44].
• Post-Inoculation Care: Post-inoculation, rinse the seedlings with sterile water and transplant them into pots with vermiculite or a suitable growth medium. Maintain the plants under the same controlled environmental conditions (22-25°C, 16/8h light/dark).
• Phenotype Observation and Validation: The silencing phenotype, such as photobleaching in ChlH-silenced plants, typically appears in the first two pairs of true leaves within 3 weeks post-inoculation. Validate silencing efficiency via qRT-PCR analysis of target gene transcript levels [44].

Protocol B: Optimized VIGS for Recalcitrant Woody Tissues

This protocol is tailored for challenging systems like Camellia drupifera capsules, where the developmental stage of the specific target organ is paramount [13].

Key Reagents & Materials:

• Plant Material: Camellia drupifera capsules at defined developmental stages (e.g., 279 days post-pollination).
• VIGS Vectors: pNC-TRV2 (a modified pTRV2) and pTRV1.
• Agrobacterium Strain: A. tumefaciens (e.g., GV3101).

Step-by-Step Workflow:

• Plant Material Selection: Precisely stage the capsules based on Days After Pollination (DAP). For genes involved in early pigmentation (CdCRY1), use early-stage capsules. For genes active later in development (CdLAC15), use mid-stage capsules [13].
• Target Fragment Selection: Use online tools like the SGN VIGS Tool to identify a unique 200-300 bp fragment from the target gene's coding sequence. Verify specificity via BLAST against the host genome to minimize off-target effects.
• Agrobacterium Preparation and Inoculation: Prepare the Agrobacterium culture as described in Protocol A. The recommended inoculation method for firm, lignified capsules is Pericarp Cutting Immersion.
  • Make careful incisions on the pericarp without damaging the seeds.
  • Immerse the cut capsules in the Agrobacterium suspension (OD₆₀₀ ~1.0).
  • This method achieved an infiltration efficiency of approximately 93.94% in C. drupifera [13].
• Incubation and Analysis: Following inoculation, co-cultivate the capsules and monitor for phenotypic changes (e.g., fading exocarp or mesocarp color). Quantify silencing efficiency through qRT-PCR.

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Research Reagent Solutions for TRV-VIGS Optimization

Reagent/Material	Function/Application in VIGS Protocol	Specific Examples / Notes
pTRV1 & pTRV2 Vectors	Bipartite TRV genome; TRV2 contains MCS for target gene insertion.	Modified versions exist (e.g., pTRV2-lic, pTRV2e, pNC-TRV2) for enhanced expression or reporting [25] [46] [13].
Agrobacterium tumefaciens	Mediates delivery of TRV vectors into plant cells.	GV3101 is a commonly used, disarmed strain [5] [4] [44].
Infiltration Buffer	Suspension medium for Agrobacterium; induces virulence.	Standard components: 10 mM MES, 10 mM MgCl₂, 200 µM Acetosyringone. Silwet L-77 (a surfactant) can be added (e.g., 0.03%) to enhance penetration [4].
Visual Marker Genes	Provides a rapid, visible indicator of silencing success for system validation.	Phytoene Desaturase (PDS):
		Induces photobleaching [5] [4] [45].Mg-chelatase subunit H (ChlH):
		Induces chlorosis [44].
Viral Suppressor of RNA Silencing (VSR)	Enhances VIGS efficacy by countering plant antiviral RNAi.	Truncated CMV 2b (C2bN43):
		Engineered to retain systemic suppression while abolishing local suppression, significantly enhancing VIGS in pepper [25].

Workflow and Pathway Diagrams

The following diagram illustrates the logical and experimental workflow for optimizing plant developmental stage in a TRV-VIGS experiment, integrating the key protocols and reagents described.

Figure 1: TRV-VIGS Developmental Stage Optimization Workflow.

The core mechanism of VIGS is an RNA silencing pathway. The following diagram details the signaling pathway from viral vector delivery to target gene silencing, highlighting key molecular components.

Figure 2: TRV-VIGS RNA Silencing Molecular Pathway.

Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse-genetics tool for rapid functional analysis of plant genes. Within this domain, the Tobacco Rattle Virus (TRV)-based VIGS system has gained prominence due to its wide host range, efficacy in meristematic tissues, and ability to induce robust silencing with mild viral symptoms [12]. This protocol research is framed within a broader thesis investigating the optimization and application of TRV-VIGS across diverse plant species, particularly those recalcitrant to stable genetic transformation. The ability to rapidly validate gene function is crucial for advancing molecular breeding and drug development, especially in species with complex genomes or long life cycles. This article details the systemic application and validation of TRV-VIGS protocols in three distinct species: soybean (Glycine max), cannabis (Cannabis sativa), and Atriplex (Atriplex canescens), providing standardized application notes and methodologies for the research community.

Comparative Performance of TRV-VIGS Across Species

The TRV-VIGS system was systematically validated in three plant species, demonstrating variable but effective silencing efficiencies. Key quantitative outcomes are summarized in the table below.

Table 1: Comparative Summary of TRV-VIGS Application in Soybean, Cannabis, and Atriplex

Plant Species	Target Gene(s)	Optimal Inoculation Method	Silencing Efficiency	Key Phenotypic Observations	Time to Phenotype (Post-Inoculation)
Soybean (Glycine max)	GmPDS, GmRpp6907, GmRPT4	Agrobacterium-mediated cotyledon node immersion [5]	65% - 95% [5]	Systemic photobleaching; compromised rust resistance [5]	21 days [5]
Cannabis (Cannabis sativa)	PDS, ChlI	Vacuum infiltration of leaves [47]	~70% transcript reduction (CLCrV system) [48]	Localized photobleaching, spotted leaf bleaching [48] [47]	7-14 days [47]
Atriplex (Atriplex canescens)	AcPDS, AcTIP2;1, AcPIP2;5	Vacuum-assisted agroinfiltration of germinated seeds [4]	~16.4% (phenotypic); 40-80% transcript reduction [4]	Systemic photobleaching in new leaves [4]	15 days [4]

Detailed Experimental Protocols

TRV-VIGS in Soybean (Glycine max)

Principle: This protocol utilizes Agrobacterium tumefaciens to deliver TRV vectors directly into the cotyledonary nodes of soybean, enabling efficient systemic infection and silencing [5].

Materials:

• Plant Material: Soybean seeds, cultivar 'Tianlong 1' [5].
• Agrobacterium Strain: GV3101 harboring pTRV1 and pTRV2-derived vectors (e.g., pTRV2-GFP, pTRV2-GmPDS) [5].
• Key Solutions: Infiltration buffer (10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂), YEP medium with appropriate antibiotics [5].

Procedure:

• Seed Preparation: Surface-sterilize soybean seeds and soak in sterile water until swollen. Bisect seeds longitudinally to obtain half-seed explants [5].
• Agrobacterium Preparation:
  • Inoculate single colonies of Agrobacterium containing pTRV1 and pTRV2 derivatives into YEP liquid medium with antibiotics (50 mg/L kanamycin, 50 mg/L rifampicin).
  • Culture at 28°C with shaking (200 rpm) until OD₆₀₀ reaches 0.6-0.8.
  • Pellet cells by centrifugation (6000 rpm, 8 min) and resuspend in infiltration buffer to a final OD₆₀₀ of 0.8-1.0 [5] [4].
  • Mix equal volumes of pTRV1 and pTRV2 Agrobacterium suspensions and incubate at room temperature in darkness for 3 hours [4].
• Inoculation: Immerse the fresh half-seed explants in the Agrobacterium suspension for 20-30 minutes, ensuring full contact [5].
• Co-cultivation and Plant Growth:
  • Post-inoculation, transfer explants to tissue culture media or soil under sterile conditions.
  • Maintain plants in a greenhouse at 22-25°C with a 16/8 hour light/dark cycle [5] [4].
• Efficiency Assessment: Monitor GFP fluorescence at the infection site 4 days post-inoculation (dpi) using a fluorescence microscope. Silencing phenotypes (e.g., photobleaching) typically appear systemically by 21 dpi [5].

TRV-VIGS in Cannabis (Cannabis sativa)

Principle: For cannabis, which has leaves with thick cuticles and dense trichomes, vacuum infiltration enhances the penetration of TRV vectors, leading to more effective infection and silencing [47].

Materials:

• Plant Material: Rooted cuttings from selected cannabis lines (e.g., 'MF-219') [47].
• Agrobacterium Strain: GV3101 containing pTRV1 and pTRV2-PDS (e.g., pTRV2-PDS310) [47].
• Key Equipment: Vacuum chamber [47].

Procedure:

• Agrobacterium Preparation: Prepare cultures as described in section 3.1, resuspending to OD₆₀₀ = 0.8 in infiltration buffer [47].
• Plant Preparation: Use 3-week-old rooted cannabis cuttings. Gently abrade the leaf surface if necessary to improve infiltration.
• Vacuum Infiltration:
  • Submerge whole leaves or entire above-ground parts of the plant in the Agrobacterium suspension.
  • Place the container in a vacuum chamber and apply a vacuum of 0.5 kPa for 5-10 minutes.
  • Rapidly release the vacuum to force the bacterial suspension into the intercellular spaces.
  • Repeat for two cycles [4] [47].
• Post-Inoculation Care: Rinse plants gently with sterile water and grow under controlled conditions (22°C day/18°C night, long-day photoperiod) [48].
• Phenotype Monitoring: Localized photobleaching in veins and leaf mesophyll typically appears within 7-14 days post-infiltration [47].

TRV-VIGS in Atriplex (Atriplex canescens)

Principle: This protocol is optimized for germinated seeds of the halophyte model Atriplex canescens, using vacuum infiltration to achieve systemic gene silencing, overcoming the lack of a stable transformation system [4].

Materials:

• Plant Material: Atriplex canescens seeds, acid-scarified (50% H₂SO₄, 8 h) and germinated on vermiculite until radicles reach 1-3 cm [4].
• Agrobacterium Strain: GV3101 with pTRV1 and pTRV2 vectors (e.g., TRV2:AcPDS) [4].

Procedure:

• Seed Preparation: Decorticate germinated seeds to expose the folded cotyledons [4].
• Agrobacterium Preparation: Follow the standard preparation outlined in section 3.1.
• Vacuum Infiltration:
  • Submerge the decorticated, germinated seeds in the Agrobacterium suspension.
  • Apply a vacuum of 0.5 kPa for 10 minutes (e.g., two cycles of 5 minutes each) [4].
• Plant Growth: After infiltration, transplant seeds to individual pots with vermiculite. Grow in a greenhouse under controlled conditions (25°C, 16/8 hour light/dark cycle, light intensity 150 µM m⁻² s⁻¹). Irrigate with ½-strength Hoagland solution weekly [4].
• Evaluation: Systemic photobleaching in newly emerged leaves is observable from 15 dpi. Silencing efficiency can be quantified by qRT-PCR, showing 40-80% reduction in target transcript levels [4].

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Research Reagents for TRV-VIGS Experiments

Reagent / Material	Function / Application	Examples & Notes
TRV Vectors	Core silencing vehicle; bipartite system (RNA1 & RNA2)	pTRV1 (replication/movement), pTRV2 (contains target gene fragment) [12]. pTRV2-GFP allows visual tracking [5] [4].
Agrobacterium tumefaciens Strain	Delivery vector for T-DNA containing TRV constructs	GV3101 is widely used for its high transformation efficiency and disarmed pathogenicity [5] [4] [47].
Infiltration Buffer	Medium for Agrobacterium suspension during inoculation	Standard composition: 10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂. Acetosyringone induces virulence genes [4].
Marker Genes	Visual assessment of silencing efficiency	PDS (photobleaching), ChlI (yellowing), POR (photobleaching). Phenotypes confirm system functionality [5] [4] [49].
Vacuum Infiltration System	Enhances Agrobacterium delivery into plant tissues	Critical for species with tough leaves (cannabis) or specific tissues (germinated seeds of Atriplex) [4] [47].

Workflow and Mechanism of TRV-VIGS

The following diagram illustrates the core workflow and mechanism of the TRV-VIGS system, from vector construction to phenotypic analysis.

TRV-VIGS Workflow: From Vector Construction to Phenotypic Analysis

Discussion and Concluding Remarks

The validation of the TRV-VIGS protocol in soybean, cannabis, and Atriplex underscores its adaptability and power as a reverse-genetics tool. Key factors influencing efficiency include the selection of target gene fragment (typically 300-500 bp, avoiding untranslated regions) [4] [47], plant growth conditions, and the method of inoculation, which must be tailored to the specific plant morphology and tissue accessibility.

For soybean, the cotyledon node immersion method proved highly effective, achieving up to 95% silencing efficiency [5]. In contrast, cannabis required vacuum infiltration to overcome barriers posed by its thick leaf cuticle and dense trichomes [47]. Atriplex, a non-model halophyte, was successfully silenced using germinated seeds as starting material, highlighting the protocol's utility in species lacking established transformation systems [4].

While TRV is a versatile vector, alternative viruses can be considered. For instance, the Cotton Leaf Crumple Virus (CLCrV) has also been successfully deployed in cannabis [48], and the Bean Pod Mottle Virus (BPMV) is well-established in soybean research [5]. The choice of vector depends on the host plant and specific research requirements.

This collection of detailed, species-specific protocols provides a solid foundation for researchers and drug development professionals to implement the TRV-VIGS system, accelerating the functional characterization of genes involved in agronomically and pharmacologically important traits.

Optimizing TRV-VIGS Efficiency: Critical Factors and Troubleshooting Guide

The functional characterization of genes in non-model plant species is often hampered by the absence of efficient and stable genetic transformation systems. This challenge is particularly acute for recalcitrant species—those resistant to standard in vitro transformation and regeneration protocols. Virus-Induced Gene Silencing (VIGS), mediated by the Tobacco Rattle Virus (TRV), has emerged as a powerful reverse genetics tool to circumvent this limitation, enabling rapid functional analysis without the need for stable transformation [4] [3]. However, the efficacy of TRV-VIGS is inherently dependent on the efficient delivery of the viral vector into plant cells.

Traditional inoculation methods, such as syringe infiltration or simple soaking, often yield inconsistent results and low efficiency, especially in species with physical barriers like thick cuticles or dense trichomes [50] [5]. Vacuum infiltration has been identified as a critical solution to this problem. This technique involves submerging plant tissues in an Agrobacterium suspension containing the TRV vectors and applying a controlled vacuum. The sudden release of pressure forces the bacterial solution into intercellular spaces, achieving more widespread and uniform infection compared to manual methods [51] [4]. This Application Note details the transformative impact of vacuum infiltration on enhancing TRV-VIGS efficiency in recalcitrant species, providing validated protocols and quantitative data to guide researchers.

The Recalcitrance Problem and the VIGS Solution

Recalcitrance in plants can stem from multiple biological factors:

• Complex Ploidy and Heterozygosity: Many perennial grasses and woody species are polyploid and highly heterozygous, masking recessive alleles and complicating traditional breeding and transformation [50].
• Self-Incompatibility: This trait reduces seed set and limits the availability of explants like immature embryos, which are crucial for conventional transformation [50].
• Physical Barriers: Tough seed coats, waxy cuticles, and dense trichomes can physically impede the entry of Agrobacterium [5].

TRV-VIGS offers a rapid alternative, operating on the principle of post-transcriptional gene silencing (PTGS). The TRV vector is engineered to carry a fragment of the host plant's target gene. Upon infection and viral replication, double-stranded RNA intermediates are recognized by the plant's RNAi machinery, leading to the degradation of homologous endogenous mRNA sequences. This results in a transient but potent knock-down of gene function, allowing for phenotypic assessment [4] [3]. The success of this process hinges entirely on the initial delivery and systemic spread of the virus, a bottleneck that vacuum infiltration effectively alleviates.

Evidence of Efficacy: Quantitative Data from Multiple Species

Recent studies across diverse recalcitrant species have consistently demonstrated the superiority of vacuum infiltration over other inoculation techniques. The table below summarizes key findings from the literature, highlighting the dramatic improvements in silencing efficiency.

Table 1: Impact of Vacuum Infiltration on VIGS Efficiency in Recalcitrant Species

Plant Species	Infiltration Parameters	Silencing Efficiency	Key Observations	Citation
Atriplex canescens (Halophytic shrub)	-0.5 kPa for 10 min (germinated seeds)	~16.4% (phenotypic); 40-80% reduction in AcPDS transcripts	Superior to soaking method; systemic photobleaching in 15 dpi.	[4]
Walnut (Juglans regia L.)	-0.08 MPa for 3 min (seedlings)	Up to 48% (phenotypic)	Most effective of three tested methods; produced clear photobleaching.	[3]
Soybean (Glycine max L.)	Not specified (cotyledon node immersion)	65% to 95% (molecular and phenotypic)	Overcomes barriers of thick cuticle and dense trichomes.	[5]
Melon (Cucumis melo L.)	-1.0 kPa for 90 s (explants)	Strongest GFP signal vs. other methods	Used in combination with sonication and micro-brushing.	[52]

The data unequivocally shows that vacuum infiltration significantly boosts VIGS performance. In walnut, it was the only method among several tested that produced a clear and quantifiable silencing phenotype [3]. In Atriplex, the efficiency achieved via vacuum was sufficient to reliably silence non-visual marker genes like aquaporins, confirming the method's robustness for functional genomics [4].

Detailed Protocol: TRV-VIGS via Vacuum Infiltration for Recalcitrant Species

The following protocol is optimized for recalcitrant species such as Atriplex canescens and walnut, synthesizing the most effective steps from recent publications [4] [3].

Research Reagent Solutions and Essential Materials

Table 2: Key Research Reagent Solutions for TRV-VIGS

Item	Function/Description	Example or Concentration
pTRV1 and pTRV2 Vectors	Binary TRV vectors for VIGS; pTRV2 carries the target gene fragment.	N/A
Agrobacterium tumefaciens GV3101	Standard strain for delivering TRV vectors into plant cells.	N/A
Infiltration Buffer	Solution for suspending Agrobacterium during inoculation.	10 mM MES, 200 µM AS, 10 mM MgCl₂, 0.03% Silwet-77 [4]
Acetosyringone (AS)	Phenolic compound that induces Agrobacterium virulence genes.	200 µM [4]
Silwet-77	Surfactant that reduces surface tension, improving tissue wettability and infiltration.	0.03% (v/v) [4]
Phytoene Desaturase (PDS) Gene	Visual marker gene; silencing causes photobleaching, used to optimize system.	AcPDS, JrPDS, GmPDS [4] [5] [3]

Step-by-Step Workflow

• Vector Construction: Clone a 250-400 bp fragment of the target gene (e.g., PDS for optimization) into the multiple cloning site of the pTRV2 vector. Use tools like the SGN-VIGS predictor to select a highly specific fragment [4] [3].
• Agrobacterium Preparation:
  • Transform the recombinant pTRV2 and the helper pTRV1 plasmids into A. tumefaciens strain GV3101.
  • Culture positive colonies in YEP or LB medium with appropriate antibiotics (e.g., kanamycin, rifampicin) until the culture reaches the mid-logarithmic phase (OD₆₀₀ = 0.6-0.8) [4] [5].
  • Pellet the bacteria by centrifugation and resuspend in infiltration buffer to a final OD₆₀₀ of 0.8-1.0.
  • Incubate the suspension at room temperature in the dark for 3-4 hours to induce virulence [4].
• Plant Material Preparation:
  • Seeds: Surface-sterilize and pre-germinate. For species with hard seed coats (e.g., Atriplex), scarification with sulfuric acid may be necessary. Germinated seeds with radicles of 1-3 cm are ideal [4].
  • Seedlings: Alternatively, use young seedlings with 5-10 true leaves [3].
• Vacuum Infiltration:
  • Submerge the prepared plant materials completely in the Agrobacterium suspension inside a vacuum desiccator.
  • Apply a vacuum of 0.5 kPa for 5-10 minutes [4]. For walnut, a stronger vacuum of -0.08 MPa (~80 kPa) for 3 minutes has been used effectively [3].
  • Release the vacuum abruptly. This rapid release is critical, as it forces the suspension into the intercellular spaces of the plant tissue.
  • Gently blot the materials to remove excess suspension and transfer them to a growth medium (e.g., vermiculite or soil) [4].
• Post-Inoculation Care and Analysis:
  • Maintain plants in a growth chamber or greenhouse at species-appropriate temperatures. Studies in petunia suggest that cooler temperatures (e.g., 20°C day/18°C night) can enhance silencing efficiency [15].
  • Monitor for silencing phenotypes. For PDS, photobleaching typically appears systemically in new leaves within 15-21 days post-inoculation (dpi) [4] [5].
  • Validate silencing efficiency quantitatively using qRT-PCR to measure the reduction in target gene transcript levels [4].

The following workflow diagram visualizes the key steps of this protocol.

Critical Parameters for Optimization

Successful implementation requires careful attention to several key parameters:

• Optical Density (OD₆₀₀): The density of the Agrobacterium culture is critical. Both excessively low and high densities can reduce efficiency. An OD₆₀₀ between 0.8 and 1.0 is frequently optimal, ensuring a high enough titer for infection without causing excessive stress to the plant tissue [4] [3].
• Vacuum Pressure and Duration: The optimal vacuum strength and duration are species-dependent. A mild vacuum (0.5 kPa) for a longer duration (10 min) worked well for Atriplex [4], while a stronger vacuum (-0.08 MPa) for a shorter time (3 min) was effective for walnut [3]. Excessive pressure can damage tissues, so empirical testing is recommended.
• Developmental Stage: The age and type of plant material significantly influence success. Germinated seeds and young seedlings are often more susceptible than mature plants, as their tissues are more amenable to infiltration and the virus can establish itself before extensive plant development [4] [15].
• Use of Additives: The inclusion of a surfactant like Silwet-77 in the infiltration buffer is essential. It reduces surface tension, allowing the bacterial suspension to spread evenly and penetrate stomata and other microscopic openings more effectively [4].

Vacuum infiltration has proven to be a transformative methodology for applying TRV-VIGS in recalcitrant plant species. By enabling uniform and deep delivery of the viral vector, it overcomes the primary physical barriers that render many species resistant to conventional genetic analyses. The quantitative data from species like walnut, Atriplex, and soybean demonstrate that this technique can elevate silencing efficiencies from negligible to levels sufficient for robust functional genomics studies (e.g., up to 48-95%). The provided protocol and optimization guidelines offer researchers a reliable framework to implement this powerful technique, accelerating the discovery of gene functions in a broader range of plants and contributing to crop improvement and drug discovery from plant-based resources.

Optimal Growth Conditions: The Critical Role of Temperature and Light

Application Notes and Protocols for TRV-Based VIGS

---

Within functional genomics research, the Tobacco Rattle Virus (TRV)-based Virus-Induced Gene Silencing (VIGS) protocol serves as a powerful reverse genetics tool for rapid gene function analysis. The efficacy of this system is profoundly influenced by the host plant's growth conditions. This document outlines the critical role of temperature and light in optimizing TRV-VIGS efficiency, providing a synthesized summary of quantitative data and detailed methodologies. This guidance is framed within a broader thesis on standardizing and enhancing TRV-VIGS protocols to ensure reproducible and high-throughput results for researchers and scientists in plant biology and drug development.

Quantitative Impact of Temperature and Light on VIGS Efficiency

Environmental factors, particularly temperature, directly impact the replication and movement of the TRV vector, thereby influencing the efficiency of gene silencing. The data summarized in the table below, compiled from recent studies, provides a benchmark for optimal conditions.

Table 1: Quantitative Data on the Impact of Temperature and Light on TRV-VIGS Efficiency

Plant Species	Optimal Temperature	Effect on Silencing Efficiency	Light Cycle	Citation
Populus euphratica & P. × canescens	28 °C	Increased silencing frequency to 65-73% and efficiency to 83-94%, compared to lower temperatures (e.g., 18 °C).	Not Specified	[53]
Capsicum annuum (Pepper)	20 °C (Post-Inoculation)	Used for maintaining plants after inoculation; lower temperatures can facilitate viral spread and silencing.	16h Light / 8h Dark	[54]
Atriplex canescens	25 °C (Germination)	Standard growth temperature for plant material preparation prior to agroinfiltration.	Not Specified	[4]
Nicotiana benthamiana	25 °C (Pre-Inoculation)	Standard growth temperature for plant material preparation prior to agroinfiltration.	Not Specified	[54]

The data unequivocally demonstrates that temperature is a decisive factor. For instance, in Populus species, a 10 °C increase from 18 °C to 28 °C resulted in a high silencing frequency and efficiency [53]. Furthermore, a consistent photoperiod is routinely employed to maintain plant health and support the systemic development of silencing phenotypes, as seen in protocols for pepper which use a 16-hour light/8-hour dark cycle [54].

Detailed Experimental Protocol for Optimized TRV-VIGS

The following protocol integrates the optimal environmental conditions into a standard TRV-VIGS workflow, suitable for adaptation in various plant species.

Reagent Preparation

• Agrobacterium Strain: A. tumefaciens GV3101 [5] [4] [54].
• Vectors: Binary TRV vectors, pTRV1 (encoding viral replication proteins) and pTRV2 (containing the target gene fragment) [12] [11].
• Infiltration Buffer: 10 mM MES, 200 µM Acetosyringone, 10 mM MgCl₂. The use of additives like 0.03% Silwet-77 is recommended for enhancing infiltration [4].
• Agrobacterium Culture: Resuspend bacterial pellets in infiltration buffer to a final OD₆₀₀ between 0.5 and 1.0. A 1:1 mixture of pTRV1 and pTRV2 cultures is incubated for 3-4 hours at room temperature before inoculation [4].

Inoculation Procedure

Multiple inoculation methods can be employed, with the choice depending on the plant species.

• Syringe Infiltration: The most common method for leaves of model plants like N. benthamiana. Use a needleless syringe to infiltrate the abaxial side of leaves [53].
• Vacuum Infiltration: Ideal for germinated seeds or seedlings. Submerge materials in the Agrobacterium suspension and apply a vacuum (0.5 kPa for 5-10 minutes) [4].
• Seed Soak Inoculation (SSI): Soak decorticated or germinated seeds in Agrobacterium suspension for 20-90 minutes [5] [55].

Post-Inoculation Incubation Conditions

This is the most critical phase for environmental control.

• Immediately after inoculation, transfer plants to a controlled environment growth chamber or greenhouse.
• Set the temperature to the species-specific optimum. For many species, including Populus, 28 °C is highly effective [53]. For others, like pepper, 20 °C is used post-inoculation [54].
• Maintain a consistent photoperiod. A 16-hour light/8-hour dark cycle is widely used to support robust plant growth and systemic silencing [54].
• Maintain plants under these conditions for the duration of the experiment. Visible silencing phenotypes, such as photobleaching from PDS gene silencing, typically appear 14-21 days post-inoculation [5] [4].

The Molecular Pathway of TRV-VIGS

The following diagram illustrates the molecular mechanism of TRV-VIGS, from agroinfiltration to the observable silenced phenotype, emphasizing steps influenced by environmental conditions.

Diagram Title: TRV-VIGS Mechanism with Key Optimization Points

Research Reagent Solutions

The following table lists essential materials and their functions for establishing a TRV-VIGS system.

Table 2: Key Research Reagents for TRV-VIGS Experiments

Reagent/Material	Function & Application in TRV-VIGS	Examples & Specifications
Agrobacterium tumefaciens	Delivery vehicle for transferring T-DNA containing TRV vectors into plant cells.	Strain GV3101 is commonly used for its high transformation efficiency [5] [4] [54].
TRV Binary Vectors	Engine viral genomes that carry and express the target gene fragment to trigger silencing.	pTRV1 (RNA1 genes) and pTRV2 (CP and MCS for gene insert) [12] [11].
Acetosyringone	A phenolic compound that induces the Agrobacterium virulence genes, crucial for efficient T-DNA transfer.	Used in infiltration buffers at concentrations of 200 µM [4].
Silwet-77 (Surfactant)	Reduces surface tension of the infiltration buffer, allowing it to spread and penetrate plant tissues more effectively.	Typically used at 0.01-0.03% (v/v) in infiltration buffer [4].
Marker Gene Constructs	Used as positive controls to visually confirm the success and efficiency of the VIGS system.	Phytoene Desaturase (PDS): Silencing causes photobleaching [53] [4]. Cloroplastos Alterados (CLA1): Causes albino phenotype [55].

The precise control of growth conditions, most notably temperature, is not merely beneficial but essential for the success of TRV-VIGS experiments. The quantitative data presented herein provides a clear rationale for maintaining species-specific temperatures, often near 28 °C, to maximize viral activity and systemic silencing. By integrating these optimized environmental parameters with robust molecular protocols, researchers can achieve high-efficiency gene silencing, thereby accelerating functional genomics studies and the validation of candidate genes for drug development and crop improvement.

Within tobacco rattle virus (TRV)-based Virus-Induced Gene Silencing (VIGS) protocols, a critical challenge is distinguishing true phenotypic consequences of gene silencing from non-specific symptoms caused by viral infection or experimental procedures. The use of GFP-labeled TRV vectors as safer controls addresses this issue directly. These control vectors express a neutral marker, Green Fluorescent Protein (GFP), which is not endogenous to plants, allowing researchers to monitor viral spread and infection efficiency without triggering defensive or confounding phenotypic changes in the host.

This application note details the methodology for employing these controls, framed within the context of a broader thesis on TRV-VIGS protocol research. By providing a robust baseline, GFP-labeled controls enhance the reliability of gene functional analysis in plants, a technique particularly valuable for species recalcitrant to stable genetic transformation [5] [13].

The Role of GFP-Labeled Controls in VIGS Experiments

Problem: Non-Specific Viral Symptoms

In VIGS experiments, plants can exhibit symptoms such as chlorosis, stunting, or leaf curling due to the viral vector's presence or the mechanical stress of inoculation. When the target gene is silenced, these non-specific effects can mask, mimic, or confound the true silencing phenotype, leading to erroneous interpretations [5].

Solution: GFP as a Neutral Reporter

The core principle of using a GFP-labeled control is to isolate the variable of interest. The pTRV2-GFP vector contains a GFP gene insert instead of a target plant gene fragment.

• Infection Control: Plants inoculated with TRV1 + pTRV2-GFP experience the same viral infection and handling stress as plants inoculated with the target gene construct (TRV1 + pTRV2-Target).
• Phenotype Baseline: Any symptoms appearing in the GFP-control plants are attributable to the viral infection process itself, not specific gene silencing.
• Efficiency Monitoring: GFP fluorescence provides a visual and quantifiable measure of successful vector delivery and systemic spread, confirming the experiment is technically sound before phenotyping [5] [10].

Table 1: Key Comparisons in a VIGS Experiment Utilizing GFP-Labeled Controls

Experimental Component	Test Group (pTRV2-Target)	GFP Control Group (pTRV2-GFP)	Purpose of Comparison
Genetic Construct	TRV1 + TRV2 with target gene insert	TRV1 + TRV2 with GFP insert	Isolates the effect of the target gene fragment from the viral backbone
Expected Phenotype	Specific phenotype from target gene silencing + any viral symptoms	No specific silencing phenotype; only viral/handling symptoms	Identifies the true, gene-specific silencing phenotype
Infection Monitoring	May be inferred from phenotype	Directly visualized via GFP fluorescence	Confirms uniform infection efficiency across experimental groups

The following workflow diagram illustrates the experimental design and decision-making process when using these controls.

Detailed Protocol for Using TRV-GFP Controls

This protocol is adapted from established TRV-VIGS methods in soybean and tea oil camellia, which have been successfully optimized for high-efficiency silencing [5] [10] [13].

Vector Construction and Agrobacterium Preparation

Materials:

• Plasmids: pTRV1 (RNA1 component), pTRV2-GFP (RNA2 component with GFP insert) [5] [13].
• Agrobacterium tumefaciens strain GV3101.
• YEB medium with appropriate antibiotics (e.g., kanamycin, rifampicin).

Method:

• Transform Agrobacterium: Introduce the pTRV1 and pTRV2-GFP plasmids into separate A. tumefaciens GV3101 competent cells.
• Culture Agrobacteria:
  • Pick a single colony for each construct and incubate in 4 mL of YEB medium with antibiotics at 28°C for 48 hours with shaking.
  • Subculture this starter into a larger volume (e.g., 50 mL) of fresh YEB medium with antibiotics, 10 mM MES (pH 5.6), and 20 μM acetosyringone.
  • Incubate at 28°C with shaking until the OD₆₀₀ reaches 0.9-1.0 [13].
• Harvest and Resuspend:
  • Centrifuge the bacterial culture at 5,000 rpm for 15 minutes.
  • Discard the supernatant and resuspend the pellet in an infiltration buffer (e.g., 10 mM MgCl₂, 10 mM MES, 200 μM acetosyringone).
  • Adjust the final OD₆₀₀ to 0.5-1.0 for each Agrobacterium culture.
• Mix Cultures: Combine the pTRV1 and pTRV2-GFP suspensions in a 1:1 ratio. Let the mixture stand in the dark at room temperature for 3-4 hours before inoculation [5] [13].

Plant Inoculation and Infection Monitoring

The inoculation method must be tailored to the plant species. The optimized method for soybean via the cotyledon node is described below, which achieves high efficiency.

Materials:

• Sterilized soybean seeds.
• Agrobacterium mixture from Step 3.1.

Method:

• Prepare Explants:
  • Soak sterilized soybean seeds in sterile water until swollen.
  • longitudinally bisect the seeds to obtain half-seed explants, ensuring the cotyledon node is intact [5] [10].
• Agro-infiltration:
  • Immerse the fresh half-seed explants in the Agrobacterium mixture (pTRV1 + pTRV2-GFP) for 20-30 minutes with gentle agitation.
  • Alternatively, for other species or tissues, methods like direct pericarp injection or peduncle injection can be used [13].
• Co-cultivation and Plant Growth:
  • After infiltration, transfer the explants to sterile tissue culture media or directly to soil.
  • Maintain plants under standard growth conditions (e.g., 24°C, 16/8 hour light/dark cycle) [5].
• Monitor GFP Fluorescence:
  • Timing: Begin observation 4 days post-inoculation (dpi) and continue regularly.
  • Procedure: Under a fluorescence microscope, examine the inoculation site (e.g., cotyledon node, hypocotyl). Successful infection is indicated by clear green fluorescence.
  • Efficiency Check: In soybean, transverse sections of the hypocotyl should show >80% of cells exhibiting fluorescence, indicating high infection efficiency [5] [10].

Table 2: Quantitative Silencing Efficiency of TRV-VIGS in Various Crops

Plant Species	Target Gene	Infiltration Method	Silencing Efficiency	Reference
Soybean (Glycine max)	GmPDS	Cotyledon node immersion	65% - 95%	[5] [10]
Tea Oil Camellia (C. drupifera)	CdCRY1	Pericarp cutting immersion	~69.8%	[13]
Tea Oil Camellia (C. drupifera)	CdLAC15	Pericarp cutting immersion	~90.9%	[13]
Walnut (Juglans regia)	JrPDS	Seedling vacuum infiltration	Up to 48%	[3]

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for TRV-VIGS with GFP Controls

Reagent/Item	Function/Description	Example/Specification
pTRV1 Plasmid	Encodes viral RNA1 for replication and movement.	Essential component of the TRV system.
pTRV2-GFP Plasmid	RNA2 vector with GFP insert; serves as the neutral control.	Modified pTRV2 vector (e.g., pNC-TRV2-GFP) [13].
Agrobacterium tumefaciens	Delivery vehicle for TRV plasmids into plant cells.	Strain GV3101 is commonly used [5] [13].
Acetosyringone	Phenolic inducer of Agrobacterium virulence genes.	Critical for enhancing T-DNA transfer efficiency.
Infiltration Buffer	Medium for suspending Agrobacterium during inoculation.	Typically contains MgCl₂ (10 mM) and MES (10 mM).
Fluorescence Microscope	Essential equipment for visualizing GFP fluorescence to monitor infection.	Used to confirm systemic spread and quantify efficiency.

Safety Considerations and Best Practices

Working with viral vectors, even in a research context, requires adherence to biosafety protocols. While TRV is a plant pathogen, standard safety practices are mandatory.

• Containment: VIGS work is typically conducted at Biosafety Level 1 (BSL-1) or higher, depending on the host plant and specific regulations [56] [57].
• Personal Protective Equipment (PPE): Always wear gloves and a lab coat. Eye protection is required whenever there is a risk of splashes [56].
• Decontamination: Decontaminate all liquid waste and surfaces that contact the Agobacterim/virus. Plastics should be treated with 10% household bleach. Note that AAVs are resistant to ethanol, and bleach is recommended [56] [57].
• Disposal: Follow your institution's biohazard disposal policies for all single-use plastics and plant waste [56].

The following diagram outlines the key safety assessment and decision pathway for a VIGS project.

Selecting the Ideal Target Gene Fragment for Effective Silencing

Within the framework of Tobacco Rattle Virus (TRV)-mediated Virus-Induced Gene Silencing (VIGS), the selection of an appropriate target gene fragment is a critical determinant for achieving high-efficiency gene knockdown. This process leverages the plant's innate post-transcriptional gene silencing (PTGS) machinery, where sequence-specific small interfering RNAs (siRNAs) guide the degradation of complementary endogenous mRNA transcripts [11] [12]. The efficacy of this entire system hinges on the strategic design of the fragment inserted into the TRV2 vector. This protocol details a systematic, evidence-based approach for selecting, designing, and validating optimal target gene fragments to ensure robust and reliable silencing in functional genomics studies.

Core Principles of Fragment Selection

The selection of a target fragment is not arbitrary; it must satisfy several key criteria to maximize silencing efficiency and specificity.

• Sequence Specificity: The chosen fragment must be unique to the target gene to prevent unintended silencing of non-target genes, especially critical in plant species with large gene families. Tools like Nucleotide-BLAST should be used to verify the specificity of the candidate fragment against the host genome [4].
• Fragment Length: Empirical studies across diverse species consistently indicate that an insert size between 255 bp and 500 bp is optimal for efficient silencing. Fragments within this range are readily processed into siRNAs, balancing silencing efficiency with stable maintenance in the viral genome [5] [3] [12].
• Avoiding Conserved Domains: While targeting conserved regions can be useful for silencing multiple members of a gene family, for specific gene knockdown, fragments should avoid highly conserved functional domains to ensure specificity.
• Positional Considerations: Research indicates that fragments located at the 3'-end or central regions of the coding sequence (CDS) often yield higher silencing efficiency compared to those at the 5'-end [4].

Table 1: Optimized Target Fragment Parameters Based on Empirical Data

Parameter	Recommended Specification	Experimental Basis
Fragment Length	255 - 500 bp	Effective in soybean, walnut, and other models; balances siRNA production and viral stability [5] [3].
GC Content	~40-60%	Avoids extreme values that hinder PCR amplification or vector construction.
Target Region	3'-end or central CDS	Showed higher silencing efficiency compared to the 5'-end in Atriplex canescens [4].
Sequence Validation	BLAST analysis for specificity	Used to verify fragment specificity and avoid off-target silencing [4].

Computational Design andIn SilicoAnalysis

Before laboratory work, comprehensive in silico analysis is essential for rational fragment design.

Protocol: Target Fragment Identification and Primer Design

Principle: Bioinformatics tools are used to identify a unique, optimal fragment within the target gene's CDS and to design primers for its amplification.

Materials:

• Target gene CDS sequence.
• Access to online bioinformatics tools (SGN-VIGS, NCBI BLAST).

Procedure:

• Retrieve Sequence: Obtain the full-length CDS of your target gene from genomic or transcriptomic databases.
• Fragment Selection:
  • Use the SGN-VIGS online tool (https://vigs.solgenomics.net/) to predict optimal nucleotide target regions for silencing [4].
  • Alternatively, manually identify a 255-500 bp region within the CDS.
• Specificity Check: Perform a BLASTN search against the host plant's genome database to ensure the selected fragment is unique and lacks significant homology with non-target genes [4].
• Primer Design: Design primers to amplify the selected fragment. Incorporate appropriate restriction enzyme sites (e.g., EcoRI, BamHI, XhoI) at the 5' ends of the forward and reverse primers to facilitate subsequent cloning into the pTRV2 vector [4] [5].
• In Silico PCR: Verify that the primers specifically amplify only the intended fragment.

Experimental Workflow for VIGS Vector Construction

The following diagram and protocol outline the key experimental steps from sequence selection to the creation of functional Agrobacterium strains ready for plant inoculation.

Protocol: Cloning Target Fragment into TRV2 Vector

Principle: The amplified and verified target fragment is cloned into the multiple cloning site of the pTRV2 vector, which is then transformed into Agrobacterium tumefaciens for plant delivery.

Materials:

• pTRV1 and pTRV2 binary vectors.
• E. coli competent cells (e.g., DH5α).
• A. tumefaciens strain GV3101.
• Restriction enzymes and corresponding buffers.
• T4 DNA Ligase.
• LB agar and liquid media with appropriate antibiotics (Kanamycin, Rifampicin, Gentamicin).

Procedure:

• Amplify and Purify Fragment: Perform PCR using gene-specific primers and purify the resulting amplicon.
• Digest Fragment and Vector: Digest both the purified PCR product and the pTRV2 vector with the selected restriction enzymes (e.g., EcoRI and BamHI). Purify the digested products.
• Ligation: Ligate the target fragment into the prepared pTRV2 backbone using T4 DNA Ligase.
• E. coli Transformation: Transform the ligation product into E. coli competent cells. Select positive colonies on LB agar plates containing Kanamycin.
• Plasmid Verification: Isolate plasmid DNA from positive colonies and verify the correct insertion of the fragment by colony PCR and/or restriction digestion. Confirm the sequence by Sanger sequencing.
• Agrobacterium Transformation: Introduce the validated recombinant pTRV2 plasmid and the pTRV1 plasmid separately into A. tumefaciens strain GV3101 using the freeze-thaw method [4] [58].
• Select Agrobacterium Clones: Plate transformed Agrobacterium on YEP or LB agar medium supplemented with Kanamycin and Rifampicin. Incubate at 28°C for 48 hours [4].

Validation and Efficiency Assessment

Using a Reporter Gene for System Optimization

Phytoene desaturase (PDS) is a critical enzyme in carotenoid biosynthesis, and its silencing results in a characteristic photobleaching phenotype due to chlorophyll degradation. Using PDS as a target allows for visual assessment of VIGS efficiency and optimization of the entire protocol, including inoculation methods and growth conditions, before silencing genes of interest with less obvious phenotypes [4] [3] [15].

Procedure:

• Construct a TRV2 vector harboring a fragment of the host's PDS gene (e.g., AcPDS, JrPDS).
• Inoculate plants with the TRV2:PDS construct alongside empty vector (TRV2:0) controls.
• Monitor plants for the development of photobleaching in newly emerged leaves, typically appearing 15-21 days post-inoculation (dpi) [4] [5].
• Record the proportion of plants showing systemic photobleaching to calculate silencing efficiency.

Molecular Validation by Reverse-Transcription Quantitative PCR (RT-qPCR)

Principle: Quantifying the reduction in target gene mRNA levels provides direct, quantitative evidence of successful silencing.

Materials:

• RNA extraction kit.
• DNase I.
• Reverse transcriptase.
• SYBR Green qPCR master mix.
• Real-time PCR system.
• Validated, stable reference genes (e.g., GhACT7, GhPP2A1 for cotton) [58].

Procedure:

• Sample Collection: Harvest tissue from systemically silenced leaves (e.g., showing photobleaching for PDS) and corresponding leaves from control plants.
• RNA Extraction: Isolate total RNA and treat with DNase I to remove genomic DNA contamination.
• cDNA Synthesis: Synthesize first-strand cDNA using a reverse transcriptase.
• qPCR Analysis: Perform qPCR using primers specific to the target gene and at least two stable reference genes.
• Data Analysis: Calculate the relative expression level using the comparative 2^(-ΔΔCt) method. Successful silencing is confirmed by a significant reduction (e.g., 40-80% [4] or higher [5]) in transcript abundance in VIGS plants compared to controls.

Table 2: Silencing Efficiency Achieved in Various Plant Species

Plant Species	Target Gene	Fragment Length	Key Optimization Factor	Silencing Efficiency / Outcome
Atriplex canescens	AcPDS	300-400 bp	Vacuum infiltration of germinated seeds	~16.4% silencing efficiency; 40-80% transcript reduction [4]
Soybean (Glycine max)	GmPDS	~300-400 bp	Agrobacterium-mediated cotyledon node infection	65-95% silencing efficiency; photobleaching at 21 dpi [5]
Walnut (Juglans regia)	JrPDS	255 bp	Seedling age & agroinfiltration method	Up to 48% silencing efficiency [3]
Iris japonica	IjPDS	Not specified	Use of one-year-old seedlings	36.67% silencing efficiency [14]

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents for TRV-VIGS Vector Construction and Plant Inoculation

Reagent / Material	Function / Purpose	Example / Specification
pTRV1 & pTRV2 Vectors	Binary T-DNA vectors containing viral genomes for replication (TRV1) and carrying the target insert (TRV2).	pYL192 (TRV1), pYL156 (TRV2) [58] [12]
Agrobacterium tumefaciens	Bacterial strain used to deliver the T-DNA containing TRV vectors into plant cells.	Strain GV3101 [4] [5] [58]
Restriction Enzymes	Molecular scissors for digesting the PCR fragment and pTRV2 vector to create compatible ends for ligation.	EcoRI, BamHI, XhoI [4] [5]
T4 DNA Ligase	Enzyme that catalyzes the joining of the target DNA fragment into the linearized pTRV2 vector.	-
Infiltration Buffer	Solution for resuspending and inducing Agrobacterium before plant inoculation.	10 mM MES, 200 µM Acetosyringone, 10 mM MgCl₂ [4] [58]
Antibiotics	Selection for bacteria containing the plasmid vectors.	Kanamycin (for TRV vectors), Rifampicin (for GV3101), Gentamicin [4] [58]

Troubleshooting and Concluding Remarks

Even with a well-designed fragment, silencing efficiency can be influenced by several factors. If efficiency is low, consider:

• Inoculation Method: Vacuum infiltration can be more efficient than simple soaking [4]. The developmental stage of the plant is also critical [15].
• Agrobacterium Density: The optical density (OD600) of the Agrobacterium culture used for inoculation, typically between 0.8 and 1.5, can impact infection efficiency [4] [3] [58].
• Growth Conditions: Post-inoculation temperatures can significantly affect viral spread and silencing; cooler temperatures (e.g., 20°C) often enhance efficiency [15].

In conclusion, selecting the ideal target gene fragment is a foundational step in TRV-VIGS that combines strategic bioinformatic design with rigorous experimental validation. By adhering to the principles and protocols outlined herein—focusing on fragment size, specificity, and position, and employing PDS as a visual reporter—researchers can establish a robust and efficient VIGS system to accelerate functional genomics studies in a wide range of plant species.

Cultivar-Specific Responses and Genotype Selection

Table 1: Cultivar-Specific Silencing Efficiencies and Optimal Parameters in TRV-VIGS Studies

Plant Species	Cultivar/Variety	Optimal Inoculation Method	Key Experimental Parameters	Silencing Efficiency	Primary Evidence/Readout
Soybean (Glycine max)	Tianlong 1 [5]	Agrobacterium-mediated cotyledon node immersion [5]	Incubation: 20-30 min; Evaluation: 21 dpi [5]	65% - 95% [5]	GFP fluorescence; Photobleaching; qPCR on GmPDS, GmRpp6907, GmRPT4 [5]
Walnut (Juglans regia)	Qingxiang, Xiangling [3]	Vacuum infiltration (germinated seeds) [3]	Agrobacterium OD~600~: 0.8; Fragment size: ~255 bp [3]	Up to 48% [3]	Visible photobleaching; qPCR on JrPDS [3]
Atriplex (Atriplex canescens)	N/A [4]	Vacuum-assisted agroinfiltration (decoritcated seeds) [4]	Pressure: 0.5 kPa; Duration: 10 min; OD~600~: 0.8 [4]	~16.4% (Phenotype), 40-80% (qPCR) [4]	Systemic photobleaching; qPCR on AcPDS, AcTIP2;1, AcPIP2;5 [4]

Detailed Experimental Protocols

Protocol 1: Agrobacterium-Mediated Cotyledon Node Infection for Soybean

This protocol is optimized for legumes and plants with thick cuticles and dense trichomes that impede liquid penetration [5].

• Vector Construction: Clone a target gene fragment (e.g., 300-400 bp) into the multiple cloning site of a pTRV2 vector using restriction enzymes (e.g., EcoRI and XhoI) or a Gateway recombination system [5] [37].
• Agrobacterium Preparation:
  • Transform recombinant pTRV2 and pTRV1 vectors into Agrobacterium tumefaciens strain GV3101 [5] [4].
  • Culture single colonies in YEP liquid medium with appropriate antibiotics (e.g., 50 mg/L kanamycin, 50 mg/L rifampicin) at 28°C with shaking until OD~600~ reaches 0.6-0.8 [4].
  • Pellet cells by centrifugation and resuspend in infiltration buffer (10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂) to a final OD~600~ of 0.8-1.0. Add a surfactant like Silwet-77 (0.03%) to enhance infiltration [5] [4].
  • Mix equal volumes of the pTRV1 and pTRV2 Agrobacterium suspensions and incubate in darkness at room temperature for 3 hours before inoculation [4].
• Plant Material Preparation & Inoculation:
  • Surface-sterilize soybean seeds and soak in sterile water until swollen. Bisect the seeds longitudinally to obtain half-seed explants [5].
  • Immerse the fresh cotyledon node explants in the prepared Agrobacterium suspension for 20-30 minutes, with gentle agitation [5].
  • After inoculation, briefly rinse the explants with sterile water and co-cultivate on medium or in vermiculite under high-humidity conditions [5] [4].
• Efficiency Evaluation:
  • Early Screening: At 4 days post-inoculation (dpi), examine the hypocotyl and cotyledon nodes under a fluorescence microscope for GFP signals to confirm successful infection [5].
  • Phenotypic Assessment: Monitor for systemic silencing phenotypes, such as photobleaching in the case of PDS, from 15-21 dpi onwards [5].
  • Molecular Confirmation: Quantify the transcript levels of the target gene using qRT-PCR (e.g., using the 2^(-ΔΔC_T) method) on tissue samples from newly emerged leaves [3] [4].

Protocol 2: Vacuum Infiltration of Germinated Seeds for Walnut and Woody Species

This protocol is effective for hard-to-transform plants like woody trees and halophytic shrubs [3] [4].

• Vector Construction & Agrobacterium Preparation: As described in Protocol 1.
• Plant Material Preparation:
  • Walnut: Soak seeds in water for 7 days, then germinate in sand [3].
  • Atriplex: Treat seeds with 50% (v/v) H₂SO₄ to weaken the hard seed coat, then rinse and germinate on moist vermiculite in darkness at 25°C [4].
  • Select germinated seeds with radicle lengths of 1-3 cm. For some species like Atriplex, decortication (removing the seed coat to expose cotyledons) significantly improves efficiency [4].
• Inoculation:
  • Submerge the germinated seeds in the Agrobacterium suspension.
  • Place the container in a vacuum chamber and apply a vacuum of 0.5 kPa for 5-10 minutes [3] [4].
  • Release the vacuum slowly to allow the suspension to infiltrate the plant tissues thoroughly.
• Post-Inoculation Care & Evaluation:
  • Transplant the inoculated materials into pots with vermiculite or a suitable growth medium.
  • Grow plants in a controlled environment (e.g., 22-24°C, 16h light/8h dark photoperiod) and irrigate with a nutrient solution like ½-strength Hoagland [4].
  • Evaluate silencing efficiency phenotypically and molecularly as in Protocol 1.

Experimental Workflow and Logical Relationships

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents and Materials for TRV-VIGS Experiments

Item Name	Function/Application in TRV-VIGS	Key Considerations & Examples
TRV Vector System	Engineered viral genome to carry and deliver target gene fragments into host plants, initiating silencing [37].	Most common: pTRV1 (RNA1 functions) and pTRV2 (RNA2 with MCS for insert). Variants: pYL156 (TRV2-MCS), pYL279 (TRV2-GATEWAY for high-throughput cloning) [37].
Agrobacterium Strain	Delivery vehicle; transfers T-DNA containing the TRV vector from the binary plasmid into the plant cell [5].	Commonly used: GV3101. Culture requires appropriate antibiotics (e.g., Kanamycin, Rifampicin) and induction with Acetosyringone for virulence [5] [4].
Infiltration Buffer	Suspension medium for Agrobacterium, maintaining cell viability and facilitating T-DNA transfer during inoculation [4].	Standard composition: 10 mM MES (pH buffer), 10 mM MgCl₂, 200 µM Acetosyringone (vir gene inducer). Often includes surfactant (e.g., 0.03% Silwet-77) to reduce surface tension [4].
Marker Gene (PDS)	Endogenous reporter gene; silencing causes visible photobleaching, providing a rapid, visual assessment of VIGS efficiency and spread [3] [4].	Phytoene desaturase is a key enzyme in carotenoid biosynthesis. Used to optimize parameters (infiltration method, fragment length) before targeting genes of interest [3] [4].
qRT-PCR Reagents	Gold-standard method for molecular quantification of silencing efficiency by measuring the reduction in target gene mRNA levels [3] [4].	Requires RNA extraction kits, reverse transcriptase, sequence-specific primers, and fluorescent DNA intercalating dye (e.g., SYBR Green). Data analyzed via 2^(-ΔΔC_T) method [3] [4].

Validating Silencing and Comparing TRV-VIGS with Other Functional Genomic Tools

Virus-induced gene silencing (VIGS) mediated by Tobacco Rattle Virus (TRV) has emerged as a powerful reverse genetics tool for rapid functional genomics studies in a wide range of plant species. This application note provides a comprehensive framework for validating VIGS efficacy through integrated phenotypic and molecular analyses. We detail standardized protocols for visualizing silencing phenotypes—using photobleaching of phytoene desaturase (PDS) as a primary marker—and for quantifying gene knockdown efficiency via reverse-transcription quantitative PCR (RT-qPCR). Within the broader context of TRV-VIGS protocol research, this guide addresses critical considerations including reference gene selection, experimental timing, and optimized inoculation methods across diverse plant systems to ensure reliable and reproducible results for researchers and drug development professionals.

Phenotypic Validation of VIGS Efficiency

Visible phenotypic changes provide the first evidence of successful gene silencing in VIGS experiments. The most widely used visual marker is the photobleaching phenotype resulting from silencing of the phytoene desaturase (PDS) gene, which plays a critical role in carotenoid biosynthesis [3] [59].

The PDS Photobleaching Assay

Mechanism: PDS is a key rate-limiting enzyme in carotenoid biosynthesis that catalyzes the conversion of colorless phytoene into colored carotenoids [3] [59]. Silencing of PDS disrupts this pathway, leading to chlorophyll photooxidation and the characteristic white or bleached appearance in leaves and other tissues due to the absence of photoprotective carotenoids [3].

Experimental Workflow and Timeline: The diagram below illustrates the typical workflow and timeline for a TRV-VIGS experiment using PDS as a visual marker.

Table 1: Documented Photobleaching Phenotypes Across Plant Species Using TRV-VIGS

Plant Species	Time to Phenotype (dpi)	Silencing Efficiency	Key Observations	Citation
Atriplex canescens	15	16.4% (by plant count)	Systemic photobleaching in new leaves	[4]
Walnut (J. regia)	8	Up to 88% (transcript reduction)	Complete photobleaching in fruits	[59]
Soybean (G. max)	21	65-95%	Photobleaching initially in cluster buds	[5]
Pepper (C. annuum)	-	Significantly enhanced	Improved anther-specific silencing	[25]
Primulina species	14-21	~47% (by plant count)	Variegated patterns in leaves	[60]

Advanced Phenotypic Markers Beyond PDS

While PDS serves as an excellent initial marker, other visible phenotypes can validate VIGS efficacy:

• Anther pigmentation: In pepper, silencing of CaAN2 (an anther-specific MYB transcription factor) resulted in abolished anthocyanin accumulation, establishing its regulatory role in pigmentation [25].
• Fruit browning: In walnut, silencing of polyphenol oxidase (JrPPO1 and JrPPO2) genes significantly reduced fruit browning phenotype, with transcript levels decreasing by 67% and 80% respectively [59].

Molecular Validation of Gene Silencing

Molecular confirmation is essential to correlate observed phenotypes with specific gene knockdown and quantify silencing efficiency.

RNA Extraction and Quality Control

Protocol: Total RNA is extracted from silenced tissues using commercial kits (e.g., Spectrum Total RNA Extraction Kit, Plant RNA Kit R6827) [58] [59]. For walnut and other phenolic-rich tissues, protocols may include additional purification steps. RNA quality should be verified by spectrophotometry (A260/A280 ratio of ~2.0) and integrity confirmed by gel electrophoresis [58].

Critical Consideration: Sample collection should account for tissue age and heterogeneity of VIGS establishment. For aphid herbivory studies in cotton, pooling tissues from the 2nd and 4th true leaves controlled for within-plant variation [58].

Reverse Transcription Quantitative PCR (RT-qPCR)

Experimental Workflow: The diagram below outlines the key steps in the qRT-PCR workflow for validating VIGS efficiency, highlighting critical validation points.

Table 2: Documented Silencing Efficiencies for Non-PDS Genes Using TRV-VIGS

Target Gene	Plant Species	Silencing Efficiency	Biological Effect	Citation
CaAN2	Pepper (C. annuum)	Coordinated downregulation of anthocyanin pathway genes	Abolished anthocyanin accumulation in anthers	[25]
JrPPO1, JrPPO2	Walnut (J. regia)	67-80% transcript reduction	Significant reduction in fruit browning	[59]
GmRpp6907	Soybean (G. max)	Significant phenotypic changes	Compromised rust resistance	[5]
AcTIP2;1, AcPIP2;5	Atriplex canescens	60.3-69.5% knockdown	Validation of abiotic stress genes	[4]
GhHYDRA1	Cotton (G. hirsutum)	Significant upregulation detected	Response to aphid herbivory	[58]

Reference Gene Selection: Proper normalization is critical for accurate RT-qPCR results. A comprehensive study in cotton under VIGS and biotic stress conditions revealed significant variation in reference gene stability [58]:

• Most stable: GhACT7 and GhPP2A1
• Least stable: GhUBQ7 and GhUBQ14 (commonly used but unstable under these conditions)

Normalization with unstable reference genes can completely mask biological significant changes; using GhUBQ7 reduced sensitivity to detect expression changes of GhHYDRA1 in response to aphid herbivory [58].

qRT-PCR Protocol:

• cDNA Synthesis: 1-2μg total RNA using reverse transcriptase with random hexamers or oligo-dT primers [59]
• Reaction Setup: 10μL reactions including SYBR Green Master Mix, gene-specific primers, and cDNA template [25]
• Amplification Conditions: 95°C for 30s, followed by 40 cycles of 95°C for 10s and 55-60°C for 30s [59]
• Data Analysis: Calculate relative expression using the 2^(-ΔΔCt) method [25]

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents for TRV-VIGS Phenotypic and Molecular Validation

Reagent/Resource	Specification/Function	Application Notes	Citation
TRV Vectors	pTRV1 (RNA1) + pTRV2 (RNA2 with target insert)	pTRV2 derivatives with gene-specific fragments (200-400bp)	[5] [4]
Agrobacterium Strain	GV3101 with pSoup helper plasmid	Standard for VIGS; resuspended in induction buffer (OD600=0.5-1.0)	[58] [4]
Infiltration Buffer	10mM MES, 200μM acetosyringone, 10mM MgCl₂	Essential for virulence gene induction; 3h incubation pre-infiltration	[58] [4]
RNA Extraction Kit	Spectrum Total RNA Kit or equivalent	Include DNase treatment; assess quality by spectrophotometry	[58] [59]
RT-qPCR Master Mix	SYBR Green-based (e.g., ChamQ SYBR, Hifair II)	Ensure consistent lot-to-lot performance	[25] [61]
Reference Genes	Species-specific validated genes (e.g., GhACT7, GhPP2A1)	Always validate stability under experimental conditions	[58]
Visual Marker	PDS gene fragment (200-300bp)	Positive control for silencing efficiency	[4] [59]

Protocol Optimization for Enhanced Efficiency

Inoculation Methods

Comparative studies across species reveal optimal inoculation approaches:

• Vacuum Infiltration: Most effective for Atriplex canescens germinated seeds (0.5 kPa, 10 min, 16.4% efficiency) [4]
• Cotyledon Node Method: Optimal for soybean using bisected half-seed explants immersed for 20-30 min [5]
• Co-culture Inoculation: Effective for walnut fruits, inducing complete photobleaching [59]

Engineering Enhanced VIGS Systems

Recent advances include engineered viral suppressors of RNA silencing (VSRs) to enhance VIGS efficacy. Structure-guided truncation of Cucumber mosaic virus 2b (C2b) created a C2bN43 mutant that retained systemic silencing suppression while abrogating local suppression activity, significantly enhancing VIGS efficacy in pepper [25].

Integrated phenotypic and molecular validation is essential for robust VIGS experiments. The photobleaching assay using PDS silencing provides an excellent visual marker for initial efficiency assessment, while RT-qPCR with properly validated reference genes offers quantitative confirmation of target gene knockdown. The protocols and considerations outlined here provide a standardized framework for researchers employing TRV-VIGS in functional genomics studies, with particular relevance for non-model species where stable transformation remains challenging. As VIGS technology continues to evolve with enhanced vectors and optimized protocols, it offers an increasingly powerful tool for rapid gene function characterization across diverse plant species.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional characterization of genes in plants, particularly in species where stable genetic transformation remains challenging [37]. The tobacco rattle virus (TRV) has become one of the most widely used VIGS vectors due to its broad host range, effective systemic movement, and mild symptomatic impact on host plants [37] [3]. However, establishing the broad applicability of any VIGS system requires demonstration of its effectiveness beyond canonical marker genes like phytoene desaturase (PDS).

This case study details the experimental approach and provides protocols for utilizing aquaporin genes as secondary validation targets to confirm the robustness and broad applicability of a TRV-based VIGS system. The methodology is adapted from a successful implementation in Atriplex canescens, where silencing two aquaporin genes (AcTIP2;1 and AcPIP2;5) achieved 60.3–69.5% knockdown efficiency, thereby validating the system's utility for functional genomics [4].

Background and Scientific Rationale

TRV-VIGS as a Functional Genomics Tool

TRV is a positive-sense RNA virus whose genome is divided into two components: RNA1, encoding proteins for replication and movement, and RNA2, which can be modified to carry host-derived sequences [37]. In the VIGS process, recombinant TRV vectors containing fragments of plant target genes are delivered into plant cells, typically via Agrobacterium tumefaciens-mediated transformation. Once inside, the viral RNA replicates, triggering the plant's post-transcriptional gene silencing (PTGS) machinery. This leads to the production of small interfering RNAs (siRNAs) that guide the sequence-specific degradation of homologous endogenous mRNA transcripts, resulting in a loss-of-function phenotype [37] [62].

Why Aquaporins are Ideal Secondary Validation Targets

• Diverse Physiological Roles: Aquaporins are membrane-channel proteins that facilitate the movement of water and other small neutral solutes. They play critical roles in root water uptake, leaf transpiration, cell elongation, and responses to abiotic stresses [63].
• Quantifiable Phenotypes: Silencing specific aquaporins can lead to measurable physiological changes, such as altered water transport efficiency or stress response phenotypes, providing functional readouts beyond molecular confirmation [64] [63].
• Broad Phylogenetic Distribution: Aquaporin genes are present across all plant species, making them applicable for VIGS validation in diverse plant systems [63].

The following diagram illustrates the experimental workflow for using TRV-VIGS to silence aquaporin genes, from vector construction to phenotypic analysis:

Experimental Design and Workflow

Target Selection and Vector Construction

The first step involves selecting appropriate aquaporin gene fragments for silencing. In the referenced case study, AcTIP2;1 (a tonoplast intrinsic protein) and AcPIP2;5 (a plasma membrane intrinsic protein) were targeted [4].

Protocol: Target Fragment Selection and Cloning

• Identify Target Sequences: Using transcriptome data or known sequences, identify the coding sequences of target aquaporin genes (e.g., TIP2;1 and PIP2;5).
• Fragment Design: Utilize online tools like the SGN-VIGS tool (https://vigs.solgenomics.net/) to select unique, highly specific 300-400 bp fragments from the open reading frame. Avoid regions of high homology with other genes to ensure silencing specificity [4].
• Verify Specificity: Perform a Nucleotide-BLAST against the host genome to confirm the selected fragment's specificity for the intended target [4].
• Primer Design: Design gene-specific primers with added terminal restriction sites (e.g., EcoRI and BamHI) to facilitate directional cloning.
  • Example primer format: Forward 5'-[restriction site]+[gene-specific 18-22 bp]-3' [4] [5].
• Amplification and Cloning: Amplify the target fragment from cDNA and clone it into the corresponding sites of the pTRV2 vector (or a derivative like pTRV2-GFP for visual tracking) to generate the recombinant vector (e.g., TRV2:AcTIP2;1, TRV2:AcPIP2;5) [4] [65] [5].

Plant Material and Inoculation

The choice of plant material and inoculation method significantly impacts silencing efficiency.

Protocol: Agrobacterium Preparation and Inoculation

• Vector Transformation: Introduce the recombinant pTRV2 vectors and the pTRV1 helper vector into Agrobacterium tumefaciens strain GV3101 [4] [5] [58].
• Agro-culture Preparation:
  • Grow single colonies of Agrobacterium containing pTRV1 and the recombinant pTRV2 in YEP or LB medium with appropriate antibiotics (e.g., 50 mg/L kanamycin, 50 mg/L rifampicin) at 28°C with shaking until OD600 reaches 0.6-1.0 [4] [5].
  • Pellet the bacterial cells by centrifugation (e.g., 6000 rpm for 8 min) and resuspend in an infiltration buffer (10 mM MgCl2, 10 mM MES, 200 µM acetosyringone) to a final OD600 of 0.8-1.5 [4] [58].
  • Mix the pTRV1 and recombinant pTRV2 suspensions in a 1:1 ratio and incubate in the dark at room temperature for 3-4 hours to induce virulence gene expression [4] [65].
• Plant Inoculation:
  • Plant Material: Use germinated seeds with radicle lengths of 1-3 cm or young seedlings at the cotyledon stage [4] [5].
  • Optimal Method - Vacuum Infiltration: Submerge plant materials in the Agrobacterium suspension and apply a vacuum of 0.5 kPa for 5-10 minutes. This method achieved approximately 16.4% silencing efficiency in A. canescens [4]. For soybean, a 20-30 minute immersion of bisected cotyledonary nodes proved highly effective, with infection efficiency exceeding 80% [5].
  • Alternative Method: For some species, syringe infiltration into leaves or cotyledons can be used, though it may be less efficient for seeds or whole seedlings [58].

Molecular Validation of Silencing

Confirming successful gene silencing at the molecular level is crucial before phenotypic assessment.

Protocol: Efficiency Analysis by qRT-PCR

• Tissue Sampling: Collect systemic leaves (newly emerged after inoculation) at 14-21 days post-inoculation (dpi) [4].
• RNA Extraction: Isolate total RNA using a standard kit (e.g., Spectrum Total RNA Extraction Kit).
• cDNA Synthesis: Synthesize first-strand cDNA using a reverse transcriptase kit.
• Quantitative PCR (qPCR):
  • Design qPCR primers that amplify a region of the aquaporin gene outside the fragment used for VIGS to avoid amplification of the viral vector.
  • Select and validate stable reference genes for normalization. In VIGS studies under stress conditions, GhACT7 and GhPP2A1 have been identified as stable, whereas commonly used genes like GhUBQ7 can be unstable and lead to inaccurate results [58].
  • Perform qPCR reactions in triplicate. Calculate the relative expression level of the target aquaporin gene in TRV2:aquaporin plants compared to control plants (infected with empty TRV2 vector) using the 2^(-ΔΔCt) method [4] [58]. Successful silencing is indicated by a significant reduction (e.g., 40-80%) in transcript abundance [4].

Key Data and Results

The table below summarizes quantitative results from a successful application of this protocol for validating a VIGS system in Atriplex canescens [4].

Table 1: Summary of Key Validation Data from Aquaporin Gene Silencing in Atriplex canescens

Parameter	AcPIP2;5 Gene	AcTIP2;1 Gene	Experimental Context
Knockdown Efficiency	60.3%	69.5%	Confirmed by qRT-PCR analysis of transcript levels [4]
Inoculation Material	\multicolumn{2}{c	}{Germinated seeds}	Seeds treated with H2SO4, germinated on vermiculite [4]
Infiltration Method	\multicolumn{2}{c	}{Vacuum-assisted agroinfiltration}	0.5 kPa for 10 min (2 cycles of 5 min) [4]
Agro-OD600	\multicolumn{2}{c	}{0.8}	Optical density of the final Agrobacterium suspension [4]
Phenotype Onset	\multicolumn{2}{c	}{~15 days post-inoculation}	Observed in newly emerged, systemic leaves [4]

The Scientist's Toolkit

The following table lists essential reagents and their applications for implementing this protocol.

Table 2: Essential Research Reagents and Materials for TRV-VIGS with Aquaporins

Reagent / Material	Specification / Example	Critical Function in Protocol
TRV Vectors	pTRV1 (pYL192); pTRV2 (pYL156) or pTRV2-GFP	Binary vectors for viral replication (pTRV1) and carrying the target gene insert (pTRV2) [37] [5] [58].
Agrobacterium Strain	GV3101	Standard strain for delivering T-DNA containing the TRV vectors into plant cells [4] [5] [58].
Infiltration Buffer	10 mM MgCl₂, 10 mM MES, 200 µM Acetosyringone	Buffer for suspending Agrobacterium; acetosyringone induces virulence [4] [58].
Restriction Enzymes	EcoRI, BamHI	For directional cloning of the aquaporin gene fragment into the pTRV2 vector [4] [5].
Reference Genes	GhACT7, GhPP2A1	Stable internal controls for accurate normalization in qRT-PCR validation of silencing [58].

Critical Factors for Success

• Insert Length and Design: For TRV vectors, inserts of 200-500 bp typically yield high silencing efficiency. Avoid homopolymeric regions (e.g., polyA tails) and select fragments from the middle of the coding sequence for optimal results [66].
• Visual Tracking: Using a modified pTRV2-GFP vector allows for non-destructive monitoring of viral spread and helps predict silencing success, especially in non-model plants [65] [5].
• Experimental Controls: Always include plants inoculated with an empty TRV2 vector (TRV2:0) as a negative control and with TRV2:PDS as a positive control for visual assessment of silencing efficacy [4] [3].

This case study provides a validated blueprint for employing aquaporin gene silencing as a robust method to confirm the broad applicability of a newly established TRV-VIGS system. The detailed protocols for vector construction, plant inoculation, and molecular validation enable researchers to move beyond proof-of-concept with PDS and demonstrate their system's capability for functional analysis of a wider range of genes involved in critical physiological processes.

{# The User's Request}

TRV-VIGS vs. BPMV-VIGS: A Comparative Analysis of Efficiency and Symptoms

{# The Context of a Broader Thesis on TRV-VIGS Protocol Research}

Virus-Induced Gene Silencing (VIGS) has become an indispensable reverse genetics tool for rapid functional gene analysis in plants. For researchers investigating the Tobacco Rattle Virus (TRV)-VIGS protocol, understanding its performance relative to other well-established systems, such as the Bean Pod Mottle Virus (BPMV)-VIGS, is crucial for experimental design, particularly in legume species. This application note provides a comparative analysis of these two prominent VIGS systems, focusing on quantitative efficiency, symptomatic responses, and optimized protocols to guide their application in functional genomics studies.

{## Introduction to VIGS Systems}

VIGS is a powerful technique that leverages a plant's innate antiviral RNA-silencing machinery to target homologous endogenous genes for post-transcriptional silencing [2]. Among the various viral vectors developed, TRV and BPMV have emerged as leading systems for dicotyledonous plants and legumes, respectively. The TRV system is celebrated for its broad host range and ability to infect meristematic tissues [5] [2], while the BPMV-based system is a well-adapted tool for legumes like soybean and common bean, which are often recalcitrant to stable transformation [67] [68] [69]. This analysis directly compares their operational parameters to inform their use in high-throughput genetic screens.

{## Comparative Analysis: TRV-VIGS vs. BPMV-VIGS}

The choice between TRV-VIGS and BPMV-VIGS involves trade-offs between silencing efficiency, symptom severity, host range, and technical accessibility. The table below summarizes a direct comparison of key performance metrics based on recent studies.

Table 1: A comparative overview of TRV-VIGS and BPMV-VIGS systems.

Feature	TRV-VIGS	BPMV-VIGS
Viral Genome	Bipartite positive-sense single-stranded RNA [3] [70]	Bipartite positive-sense RNA [67] [68]
Primary Hosts	Solanaceous species (e.g., tomato, tobacco), Arabidopsis, and an expanding list including soybean and woody species [5] [2] [3]	Primarily legumes (soybean, common bean) [67] [68]
Typical Inoculation Method	Agrobacterium tumefaciens-mediated delivery (e.g., agroinfiltration, vacuum infiltration, seed immersion) [5] [4]	Direct rub-inoculation of infectious plasmid DNA ("one-step" vector) or Agrobacterium-mediated delivery [67] [68]
Silencing Efficiency	65% - 95% in soybean [5]; up to 100% in Arabidopsis [2]; ~48% in walnut [3]; ~16% in Atriplex [4]	Up to 100% in susceptible common bean cultivars [67]
Viral Symptom Profile	Generally mild or asymptomatic in vectors with inserts, minimizing phenotype interference [5] [15]. Severe necrosis can occur with empty vectors in some hosts [15].	Ranges from mild to moderate mosaic and mottling symptoms, depending on the RNA1 construct used [67] [68].
Key Advantages	Broad host range; infection of meristems; mild symptoms with inserts [5] [2]	Highly efficient and stable in legumes; "one-step" plasmid rubbing simplifies inoculation [67] [68]
Limitations	Efficiency can be highly dependent on plant species, cultivar, and optimization of protocol [3] [4]	Host range is largely restricted to legumes; viral symptoms can sometimes interfere with phenotyping [67]

{## Visualizing the Vector Architectures and Workflows}

The functional mechanisms of TRV and BPMV vectors can be understood through their engineered structures and the sequential steps of a standard VIGS protocol. The following diagrams illustrate these core concepts.

Diagram 1: TRV and BPMV VIGS Vector Architecture

Diagram 2: Generalized Workflow for VIGS

{## Detailed Experimental Protocols}

TRV-VIGS Protocol for Soybean

The following protocol, adapted from a 2025 study, establishes a highly efficient TRV-VIGS system in soybean using cotyledon node inoculation [5].

• Vector Construction: Clone a ~255-400 bp fragment of the target gene (e.g., GmPDS) into the pTRV2 vector using appropriate restriction sites (e.g., EcoRI and XhoI) [5] [3]. The construct is then transformed into Agrobacterium tumefaciens strain GV3101.
• Plant Material Preparation: Surface-sterilize soybean seeds and imbibe them in sterile water until swollen. Bisect the seeds longitudinally to create half-seed explants [5].
• Agrobacterium Preparation: Culture the Agrobacterium strains containing pTRV1 and the recombinant pTRV2. Resuspend the bacterial pellets in an infiltration buffer (10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂) to a final OD₆₀₀ of 0.8-1.5 [5] [4]. Mix the pTRV1 and pTRV2 suspensions in a 1:1 ratio and incubate in the dark for 3-4 hours.
• Plant Inoculation: Immerse the fresh half-seed explants in the Agrobacterium suspension for 20-30 minutes, with or without vacuum infiltration [5] [4].
• Post-Inoculation Care: Co-cultivate the inoculated explants on sterile medium or vermiculite. Transplant treated seedlings to soil and maintain them in a growth chamber at 20-22°C to optimize silencing efficiency [15] [70].
• Phenotype and Efficiency Validation: Silencing phenotypes, such as photobleaching, typically appear systemically in new leaves 15-21 days post-inoculation (dpi). Confirm silencing efficiency by quantifying the downregulation of target gene mRNA using qRT-PCR [5] [4].

BPMV-VIGS Protocol for Common Bean

This protocol for common bean utilizes the efficient "one-step" BPMV vector system, which allows for direct plasmid rubbing [67].

• Vector Preparation: The BPMV VIGS vector (pBPMV-IA-V2) carries the target gene fragment (e.g., a 132-391 bp fragment of PvPDS) inserted after the stop codon of the RNA2 polyprotein, enabling the silencing of non-coding sequences [67] [68].
• Plasmid DNA Isolation: Propagate and purify the pBPMV-IA-R1M (RNA1 with moderate symptoms) and the recombinant pBPMV-IA-V2 (RNA2 with insert) plasmids from E. coli [67].
• Inoculum Preparation: Mix the two plasmid DNAs in a 1:1 molar ratio. For rub-inoculation, use 5 µg of each plasmid per plant [67].
• Plant Inoculation: Dust primary leaves of 10-day-old common bean seedlings (e.g., cultivar 'Black Valentine') with carborundum. Gently rub the leaf surface with a gloved finger or a cotton swab dipped in the plasmid DNA mix [67].
• Growth Conditions: Maintain inoculated plants in a greenhouse or growth chamber. Viral symptoms and silencing phenotypes typically become evident in systemic leaves 2-3 weeks post-inoculation [67].
• Efficiency Analysis: Similar to the TRV protocol, silencing efficiency is scored by visual assessment of phenotypes and confirmed by molecular analysis such as qRT-PCR.

{## The Scientist's Toolkit: Essential Research Reagents}

Table 2: Key reagents and materials for implementing TRV-VIGS and BPMV-VIGS.

Reagent/Material	Function in VIGS	Example Use Case
pTRV1 & pTRV2 Vectors	Binary TRV vectors; pTRV1 contains replication genes, pTRV2 is for inserting target fragments [5] [3].	The backbone for creating TRV-VIGS constructs in a wide range of plants, from soybean to walnut [5] [3] [71].
pBPMV-IA-R1M & pBPMV-IA-V2	DNA-based BPMV vectors; R1M induces moderate symptoms for easy tracking, V2 is the insert-carrying vector [67] [68].	The "one-step" system for high-throughput VIGS in soybean and common bean, allowing direct plasmid rubbing [67].
Agrobacterium tumefaciens GV3101	A disarmed strain used to deliver binary TRV vectors into plant cells via T-DNA transfer [5] [4].	Standard strain for agroinfiltration-based inoculation in TRV-VIGS protocols [5] [3] [4].
Infiltration Buffer (with Acetosyringone)	A buffer to prepare Agrobacterium suspensions; acetosyringone induces virulence genes, enhancing T-DNA transfer [5] [4].	Used in the final resuspension step before inoculating plants via immersion, infiltration, or vacuum [5] [4].
Phytoene Desaturase (PDS) Gene	A visual marker gene for VIGS; its silencing disrupts carotenoid biosynthesis, causing photobleaching [5] [67] [4].	Standard positive control to optimize and validate a new VIGS protocol in any plant species [5] [67] [3].

{## Conclusion}

The decision to employ a TRV-VIGS or BPMV-VIGS system is fundamentally guided by the host plant species and the specific experimental requirements. TRV-VIGS offers a versatile and broad-host-range tool with minimal symptomatic interference, making it excellent for exploratory studies in non-model plants, including optimized protocols for soybean [5]. Conversely, BPMV-VIGS remains the specialized and highly efficient vector of choice for legume functional genomics, with its simplified "one-step" inoculation providing a significant advantage for high-throughput screens [67] [68]. Researchers must weigh these factors of efficiency, symptomology, and technical feasibility to successfully harness these powerful silencing technologies.

Tobacco Rattle Virus (TRV) has emerged as a premier viral vector for Virus-Aided Gene Expression (VAGE) and Virus-Induced Gene Silencing (VIGS), providing researchers with a powerful reverse-genetics tool for rapid functional genomics in plants. As a positive-sense RNA virus with a bipartite genome, TRV can be engineered to carry foreign gene sequences and systematically silence target genes through the plant's innate RNA-based defense mechanisms [12]. The TRV-based system is particularly valuable because it offers a transient alternative to stable genetic transformation, enabling high-throughput gene function characterization without the need for lengthy transformation procedures [4] [5]. This technical advance has accelerated gene discovery across numerous plant species, from model organisms to crops with complex genetics.

The fundamental principle underlying TRV-VIGS involves harnessing the plant's post-transcriptional gene silencing (PTGS) pathway. When recombinant TRV vectors containing host-derived sequences infect plants, the viral RNA replicates, generating double-stranded RNA intermediates that the plant's Dicer-like enzymes recognize and cleave into small interfering RNAs (siRNAs). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), which guides sequence-specific degradation of complementary endogenous mRNA transcripts [12]. This process results in targeted gene knockdown, allowing researchers to observe loss-of-function phenotypes within weeks rather than the months or years required for traditional mutagenesis approaches.

TRV Vector Systems and Engineering

TRV Genome Organization and Vector Construction

The TRV genome consists of two RNA components: RNA1 and RNA2. RNA1 encodes essential viral replication and movement proteins, including a 134KDa replicase, a 194KDa replicase, a movement protein (MP), and a 16KDa cysteine-rich protein (16K) that functions as a suppressor of RNA silencing [12]. RNA2 typically contains the coat protein (CP) gene and non-essential 29.4K and 32.8K proteins that can be replaced with foreign sequences for VIGS applications [12]. Modern TRV vector systems maintain RNA1 and RNA2 as separate T-DNA binary vectors under the control of Cauliflower Mosaic Virus (CaMV) 35S promoters for Agrobacterium-mediated delivery.

Significant engineering advancements have optimized TRV vectors for enhanced usability and efficiency. The current generation of TRV vectors incorporates several key features: (1) duplicated CaMV 35S promoters for high-level transcription in plant cells, (2) ribozyme sequences for precise processing of viral RNA transcripts, and (3) versatile multiple cloning sites or recombination sites for simplified insertion of target gene fragments [12]. The development of GATEWAY-compatible TRV vectors (e.g., pTRV2-attR1-attR2) has significantly streamlined the cloning process, allowing efficient directional recombination of PCR products without traditional restriction enzyme digestion and ligation [12]. Additional modifications include the incorporation of fluorescent markers such as GFP, enabling visual tracking of viral spread and infection efficiency [5] [12].

Advanced Vector Modifications for Specialized Applications

Recent vector modifications have expanded TRV-VIGS applications to previously challenging tissues, particularly root systems. Conventional TRV vectors lacking the RNA2-encoded 2b protein show limited invasion of meristematic tissues and roots. However, TRV-2b vectors that retain this helper protein demonstrate significantly enhanced capacity to invade root tips and meristems, achieving up to 55% infection efficiency in Nicotiana benthamiana roots compared to 29% with Δ2b vectors [72]. This improvement has enabled functional studies of root development genes and soil-borne pathogen resistance mechanisms [72]. Additional specialized vectors include TRV-LIC (Ligation-Independent Cloning) vectors that further simplify insert cloning and TRV-GFP vectors that fuse GFP with the coat protein to monitor viral movement systemically [12].

Experimental Protocols and Workflows

Vector Preparation andAgrobacteriumTransformation

The following protocol details the establishment of an efficient TRV-VIGS system, incorporating optimal parameters from recent studies:

Step 1: Target Gene Fragment Selection and Amplification

• Select a 300-500 bp gene-specific fragment with minimal homopolymeric regions to ensure high silencing efficiency [12].
• Use bioinformatics tools (e.g., SGN-VIGS) to identify unique target regions and verify specificity against the host genome [4].
• Design primers with appropriate restriction sites (e.g., EcoRI, BamHI) or recombination sites (attB1/attB2 for GATEWAY cloning) for directional cloning [4] [12].
• Amplify the fragment from cDNA using high-fidelity DNA polymerase to minimize mutations.

Step 2: Vector Construction

• For traditional cloning: Digest pTRV2 vector and insert fragment with appropriate restriction enzymes, then ligate [4].
• For GATEWAY cloning: Perform BP recombination reaction between attB-flanked PCR product and attP-containing donor vector, then LR recombination with pTRV2-attR1-attR2 destination vector [12].
• Transform recombinant plasmids into E. coli, screen positive colonies, and verify insert sequence by colony PCR and sequencing.

Step 3: Agrobacterium Preparation

• Introduce verified pTRV1, pTRV2-empty (control), and pTRV2-target recombinant plasmids into Agrobacterium tumefaciens strain GV3101 via freeze-thaw transformation [4] [5].
• Plate transformed Agrobacterium on YEP agar containing appropriate antibiotics (50 mg/L kanamycin, 50 mg/L rifampicin) and incubate at 28°C for 48 hours [4].
• Inoculate single colonies into YEP liquid medium with antibiotics and culture at 28°C with shaking (200 rpm) until OD600 reaches 0.6-0.8 [4].
• Pellet bacterial cells by centrifugation (6000 rpm, 8 minutes) and resuspend in infiltration buffer (10 mM MES, 200 μM acetosyringone, 10 mM MgCl2, 0.03% Silwet-77) to final OD600 of 0.8-1.0 [4] [73].
• Incubate the suspension at room temperature in darkness for 3 hours to induce virulence gene expression [4].

Plant Inoculation Methods

Multiple inoculation methods have been optimized for different plant species and experimental requirements:

Vacuum Infiltration Method (Highly Efficient for Seeds)

• Subject germinated seeds (radicle length 1-3 cm) to vacuum infiltration (0.5 kPa) for 5-10 minutes while submerged in Agrobacterium suspension [4].
• Repeat vacuum cycle twice for maximum efficiency [4].
• Transplant inoculated materials to vermiculite or soil and maintain under controlled conditions (22°C, 16h light/8h dark, 150 μM m⁻² s⁻¹ light intensity) [4].

Cotyledon Node Method (Optimized for Soybean)

• Surface-sterilize seeds and soak in sterile water until swollen [5] [10].
• Longitudinally bisect seeds to obtain half-seed explants with intact cotyledonary nodes [5] [10].
• Immerse fresh explants in Agrobacterium suspension for 20-30 minutes with gentle agitation [5] [10].
• Co-culture explants on medium for several days before transferring to soil [5].

Leaf Infiltration Method (Standard for N. benthamiana)

• Use a needleless syringe to infiltrate Agrobacterium suspension into the abaxial side of fully expanded leaves [12].
• Apply gentle pressure until the infiltration zone becomes water-soaked [12].
• Maintain plants under standard growth conditions with high humidity for 48 hours post-infiltration [12].

The experimental workflow below illustrates the complete TRV-VIGS process from vector construction to phenotypic analysis:

Efficiency Optimization Parameters

Multiple factors influence TRV-VIGS efficiency and require optimization for different plant systems. The table below summarizes key parameters and their optimal ranges based on recent studies:

Table 1: Optimization Parameters for TRV-VIGS Efficiency

Parameter	Optimal Range	Impact on Efficiency	Species Validated
Acetosyringone Concentration	150-200 μM	Enhances T-DNA transfer	S. japonicus, A. canescens [4] [73]
Agrobacterium OD₆₀₀	0.5-1.0	Balanced infection vs. phytotoxicity	S. japonicus, Soybean [5] [73]
Vacuum Pressure/Duration	0.5 kPa, 5-10 min	Enhances suspension penetration	A. canescens [4]
Co-culture Period	2-3 days	Allows T-DNA transfer	Soybean, N. benthamiana [5]
Plant Growth Temperature	20-22°C	Optimal viral movement & silencing	A. canescens, Tomato [4] [12]
Target Fragment Size	300-500 bp	Minimizes recombination, maintains efficiency	Multiple species [12]

Applications in Functional Genomics

Gene Function Validation in Diverse Species

TRV-VIGS has been successfully established in numerous plant species, enabling rapid functional characterization of genes involved in various biological processes. In the halophyte model Atriplex canescens, TRV-VIGS achieved approximately 16.4% silencing efficiency when targeting AcPDS, with photobleaching phenotypes appearing in new leaves at 15 days post-inoculation and 40-80% reduction in AcPDS transcript levels [4]. The system was further validated by silencing two aquaporin genes (AcTIP2;1 and AcPIP2;5), achieving 60.3-69.5% knockdown efficiency [4]. In soybean, an optimized TRV-VIGS protocol utilizing cotyledon node inoculation achieved remarkable 65-95% silencing efficiency, successfully targeting GmPDS (resulting in photobleaching), the rust resistance gene GmRpp6907, and the defense-related gene GmRPT4 [5] [10].

The technology has proven particularly valuable for studying root biology and soil-borne pathogen resistance. The TRV-2b vector, which retains the nematode-transmission helper protein, demonstrates significantly enhanced root invasion capability, enabling functional studies of genes involved in root development (e.g., IRT1, TTG1, RHL1, β-tubulin) and nematode resistance (e.g., Mi gene in tomato) [72]. This represents a critical advancement as roots have traditionally been challenging targets for virus-based vectors due to inefficient invasion.

Quantitative Assessment of Silencing Efficiency

The table below summarizes silencing efficiencies achieved with TRV-VIGS across various plant species and target genes:

Table 2: Silencing Efficiencies in Various Plant Species

Plant Species	Target Gene	Silencing Efficiency	Phenotypic Observations	Citation
Atriplex canescens	AcPDS	40-80% transcript reduction	Systemic photobleaching at 15 dpi	[4]
Atriplex canescens	AcTIP2;1, AcPIP2;5	60.3-69.5% knockdown	Confirmed system applicability	[4]
Soybean	GmPDS	65-95%	Photobleaching at 21 dpi	[5] [10]
Soybean	GmRpp6907	65-95%	Compromised rust resistance	[5]
Styrax japonicus	Endogenous genes	74.19-83.33%	Method-dependent efficiency	[73]
N. benthamiana (roots)	IRT1, TTG1	55% infection rate	Root development defects	[72]

The Scientist's Toolkit: Essential Research Reagents

Successful implementation of TRV-VIGS requires specific biological materials and reagents. The following table outlines key components and their functions:

Table 3: Essential Research Reagents for TRV-VIGS

Reagent/Component	Specifications	Function	Examples/Alternatives
TRV Binary Vectors	pTRV1 (RNA1), pTRV2 (RNA2 with MCS)	Viral genome components for silencing	pYL156, pYL279 [12]
Agrobacterium Strain	GV3101 with appropriate resistance	T-DNA delivery to plant cells	LBA4404, AGL1 [4] [5]
Infiltration Buffer	10 mM MES, 200 μM AS, 10 mM MgCl₂, 0.03% Silwet-77	Facilitates bacterial entry into plant tissue	MES buffer, acetosyringone [4]
Selection Antibiotics	Kanamycin (50 mg/L), Rifampicin (50 mg/L)	Maintain plasmid selection in bacteria	Spectinomycin for alternative vectors
Plant Growth Media	Vermiculite, ½-strength Hoagland solution	Optimal plant growth post-inoculation	Soil mixtures, MS media [4]
Validation Primers	Gene-specific qRT-PCR primers	Confirm silencing at transcript level	Reference gene primers required [4]

Troubleshooting and Technical Considerations

Despite its robustness, several factors can affect TRV-VIGS efficiency and require attention. In species with hard seed coats or dense trichomes (e.g., soybean), conventional inoculation methods like leaf spraying or injection often yield low efficiency due to limited suspension penetration [5] [10]. In such cases, vacuum infiltration of germinated seeds or cotyledon node inoculation significantly improves results [4] [5]. Viral movement limitations can restrict silencing in certain tissues, particularly meristems and roots. The TRV-2b vector, which retains the helper protein 2b, shows enhanced meristem invasion and root silencing capability compared to conventional Δ2b vectors [72].

The timing of phenotypic analysis is crucial, as silencing is transient and may peak at specific time points post-inoculation. For most species, initial silencing phenotypes appear 10-15 days post-inoculation, with maximal effects at 3-4 weeks [4] [5]. Including appropriate controls is essential for valid interpretation: empty vector (TRV1+TRV2:0) controls account for viral infection effects, while a marker gene like PDS provides visual confirmation of silencing efficiency [4]. Molecular validation through qRT-PCR should accompany phenotypic observations to quantify transcript reduction, as phenotypes alone may not fully reflect silencing efficiency [4] [5].

TRV-based VAGE and VIGS represent a versatile and efficient platform for functional genomics across diverse plant species. The continuous optimization of vector systems, inoculation methods, and experimental parameters has significantly expanded applications from basic gene characterization to agricultural trait improvement. As research progresses, further refinements in tissue specificity, silencing persistence, and expansion to additional species will solidify TRV's position as an indispensable tool in plant biotechnology and drug discovery research.

Virus-Induced Gene Silencing (VIGS) using the Tobacco Rattle Virus (TRV) vector has become an indispensable reverse-genetics tool in plant functional genomics. This powerful technique leverages the plant's own post-transcriptional gene silencing (PTGS) machinery, triggered by recombinant viral vectors, to systematically suppress endogenous gene expression. The resulting phenotypic changes enable researchers to characterize gene function without the need for stable transformation. However, the broad application of TRV-VIGS is constrained by two significant and interconnected limitations: the transient nature of silencing effects and the variable efficiency of viral invasion into meristematic tissues. This application note details these challenges within the context of an optimized sunflower VIGS protocol and presents targeted strategies for mitigation, providing researchers with a framework for improving experimental outcomes in recalcitrant species.

Quantitative Analysis of Key Limitations

The practical efficacy of TRV-VIGS is governed by several quantifiable factors. The data below, synthesized from recent studies, highlights the core challenges related to silencing persistence and tissue invasion.

Table 1: Quantified Limitations in TRV-VIGS Efficacy

Limitation Factor	Experimental Findings	Impact on Silencing	Supporting Reference
Silencing Duration	Systemic photobleaching phenotypes appeared ~15 days post-inoculation in Atriplex canescens [38].	Transient effect; may be insufficient for studying long-term developmental processes.	[38]
Genotype Dependency	Infection rates varied from 62% to 91% across six different sunflower genotypes [19].	High variability in efficiency complicates protocol standardization across species/cultivars.	[19]
Meristem & Root Invasion	A modified TRV vector retaining the 2b protein showed extensive replication in whole plants, including meristems, and triggered systemic VIGS in roots [74].	Standard TRV vectors often show poor silencing in root and shoot apical meristems, limiting studies on development.	[74]
Viral Mobility vs. Phenotype	TRV was detected in leaves up to node 9 in sunflower, but its presence was not limited to tissues with observable silencing symptoms [19].	Indicates that efficient viral spread does not always correlate with strong phenotypic manifestation of silencing.	[19]

Detailed Experimental Protocols for Assessing Limitations

The following protocols are adapted from recent, high-efficiency VIGS studies and are designed to help researchers systematically evaluate and overcome the challenges of silencing duration and meristem invasion.

Protocol: Seed Vacuum Agroinfiltration for Enhanced Meristem Invasion

This protocol, optimized for sunflower, leverages an early developmental stage inoculation to maximize systemic spread, including into meristematic regions [19].

• Step 1: Agrobacterium Preparation

  • Transform recombinant TRV vectors (pTRV1 and pTRV2 with target insert) into Agrobacterium tumefaciens strain GV3101.
  • Streak transformed bacteria from glycerol stocks onto LB-agar plates with appropriate antibiotics (50 µg/mL kanamycin, 10 µg/mL gentamicin, 100 µg/mL rifampicin). Incubate at 28°C for 1.5–2 days.
  • Inoculate a single colony into YEP liquid medium with the same antibiotics. Shake at 200 rpm at 28°C until the culture reaches the mid-logarithmic phase (OD600 = 0.6–0.8).
  • Pellet bacterial cells by centrifugation (6000 rpm for 8 min). Resuspend in infiltration buffer (10 mM MES, 200 µM acetosyringone, 10 mM MgCl2) to a final OD600 of 0.8–1.0 [38].
  • Combine equal volumes of the pTRV1 and pTRV2 Agrobacterium suspensions. Incubate the mixture at room temperature in darkness for 3–4 hours to induce virulence.

• Step 2: Seed Preparation and Vacuum Infiltration

  • For sunflower: Partially remove the seed coat to facilitate infiltration [19].
  • For Atriplex canescens: Treat seeds with 50% (v/v) H2SO4 to weaken the hard seed coat, then germinate on moist vermiculite until radicles reach 1–3 cm in length [38].
  • Submerge the prepared seeds or germinated sprouts in the Agrobacterium suspension in a vacuum desiccator.
  • Apply a vacuum of 0.5 kPa for 10 minutes [38].
  • Gently release the vacuum to allow the suspension to fully penetrate the plant tissues.

• Step 3: Co-cultivation and Plant Growth

  • Following infiltration, subject the plant material to a 6-hour co-cultivation period [19].
  • Rinse the seeds/sprouts with sterile water to remove residual Agrobacterium.
  • Transfer the inoculated materials to pots filled with a peat-perlite mixture (3:1) or vermiculite.
  • Grow plants in a controlled greenhouse with a 16/8 or 18/6 light/dark photoperiod at approximately 22°C. Water as needed and irrigate weekly with a ½-strength Hoagland nutrient solution [19] [38].

Protocol: Evaluating Silencing Duration and Meristematic Spread

This analytical protocol provides a methodology for quantifying the key limitations discussed.

• Step 1: Phenotypic Monitoring

  • Record the first appearance of silencing phenotypes (e.g., photobleaching in PDS-silenced plants) in true leaves.
  • Systemically document the spread and intensity of the phenotype over time using time-lapse photography. Note the appearance of symptoms in newly emerged leaves, indicating systemic spread.
  • Quantify the phenotype by measuring the percentage of photo-bleached leaf area using image analysis software (e.g., ImageJ) at regular intervals until the phenotype diminishes.

• Step 2: Molecular Confirmation

  • Tissue Sampling: At the peak of phenotypic expression, collect tissue samples from different plant parts: young leaves (near the apical meristem), mature leaves with strong silencing symptoms, mature leaves without symptoms, and root tips.
  • RNA Extraction and qRT-PCR: Extract total RNA from all sampled tissues. Synthesize cDNA and perform quantitative RT-PCR (qRT-PCR) to measure the transcript abundance of the target gene (e.g., PDS). Use reference genes for normalization.
  • Viral Detection via RT-PCR: Using the same cDNA, perform standard RT-PCR with primers specific to the TRV vector (e.g., targeting the viral coat protein) to confirm the presence and distribution of the virus.

• Step 3: Data Analysis

  • Correlate the target gene knockdown (from qRT-PCR) with the observed phenotypic strength in different tissues.
  • Compare the presence of the TRV vector (from RT-PCR) in green versus bleached tissues and, critically, in young leaves and root tips to assess meristem invasion.

Visualization of Workflow and Challenges

The following diagram illustrates the optimized protocol and the key points where limitations in silencing duration and meristem invasion can be assessed and addressed.

Diagram 1: TRV-VIGS workflow with key challenges.

The Scientist's Toolkit: Essential Research Reagents & Materials

Successful implementation of TRV-VIGS and investigation into its limitations require a specific set of biological and chemical reagents.

Table 2: Key Research Reagent Solutions for TRV-VIGS

Reagent / Material	Function / Role in Protocol	Example & Notes
TRV Viral Vectors	Bipartite vector system for delivering silencing fragments.	pYL192 (TRV1), pYL156 (TRV2) [19]. TRV2-2b for enhanced meristem invasion [74].
Agrobacterium Strain	Delivery vehicle for introducing TRV vectors into plant cells.	A. tumefaciens strain GV3101 [19] [38].
Infiltration Buffer	Suspension medium that induces virulence and facilitates infection.	10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂. Add Silwet-77 (0.03%) as a surfactant [38].
Marker Gene Construct	Positive control for visualizing silencing efficiency and spread.	TRV2-PDS (Phytoene Desaturase). Silencing causes photobleaching [19] [38].
qRT-PCR Reagents	For molecular quantification of target gene knockdown and viral titer.	Primers for target gene (e.g., HaPDS, AcPDS) and TRV coat protein. Used to correlate phenotype with molecular data [19] [38].

The challenges of transient silencing duration and incomplete meristem invasion remain significant hurdles in TRV-VIGS research. However, as evidenced by the protocols and data herein, these limitations can be systematically quantified and mitigated. Strategic optimization, including the use of seed vacuum infiltration, prolonged co-cultivation, and potentially engineered TRV vectors (e.g., TRV-2b), significantly enhances viral spread and persistence. A rigorous approach that combines phenotypic observation with molecular validation of both target gene knockdown and viral distribution is crucial for accurately interpreting VIGS results. By adopting these refined methodologies, researchers can push the boundaries of the TRV-VIGS system, enabling more reliable functional gene analysis, particularly in non-model and recalcitrant plant species.

Conclusion

The TRV-VIGS protocol has firmly established itself as a rapid, efficient, and versatile reverse genetics platform, overcoming the limitations of stable transformation, especially in recalcitrant plant species. Key advancements in vector design, optimized inoculation methods like vacuum infiltration, and a refined understanding of critical parameters such as temperature and plant developmental stage have significantly boosted its reliability and silencing efficiency. As a high-throughput functional screening tool, TRV-VIGS is poised to accelerate the discovery and validation of genes controlling agronomically important traits, from disease resistance to specialized metabolism. Future directions should focus on extending its application to a wider range of non-model species, improving systemic movement and silencing persistence, and integrating it with emerging technologies like CRISPR for comprehensive gene function analysis, ultimately expediting crop improvement and biotechnological innovation.

References

• 1. Establishing a Virus-Induced Gene Silencing System in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10346259/]
• 2. Efficient Virus-Induced Gene Silencing in Arabidopsis - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC1557620/]
• 3. Developing and applying a virus-induced gene silencing ... [https://www.sciencedirect.com/science/article/abs/pii/S0378111924009685]
• 4. based virus-induced gene Silencing in Atriplex canescens [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-025-01427-z]
• 5. Efficient Virus-Induced Gene Silencing (VIGS) Method for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12115190/]
• 6. Regulation of Glandular Size and Phytoalexin Biosynthesis ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11321651/]
• 7. Tobacco Rattle Virus - an overview [https://www.sciencedirect.com/topics/immunology-and-microbiology/tobacco-rattle-virus]
• 8. Genus: Tobravirus [https://ictv.global/report/chapter/virgaviridae/virgaviridae/tobravirus]
• 9. Characterization of an unusual tobacco rattle virus isolate and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12326667/]
• 10. Efficient Virus-Induced Gene Silencing (VIGS) Method for ... [https://www.mdpi.com/2223-7747/14/10/1547]
• 11. Virus-Induced Gene Silencing (VIGS) in Functional Genomics [https://www.mdpi.com/2311-7524/11/11/1297]
• 12. A Methodological Advance of Tobacco Rattle Virus ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.671091/full]
• 13. Development of a robust and efficient virus-induced gene ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-024-01320-1]
• 14. Developing a homology-driven gene silencing system in < ... [https://www.maxapress.com/article/id/68c8b5d4fa6c58419c48f3c2]
• 15. An Optimized Protocol to Increase Virus-Induced Gene ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3893464/]
• 16. Prediction of VIGS efficiency by the Sfold program and its ... [https://www.sciencedirect.com/science/article/abs/pii/S2095927316301530]
• 17. Enhancement of Virus-induced Gene Silencing in Tomato ... [https://www.sciencedirect.com/science/article/pii/S1016847823129141]
• 18. Establishment and application of a root wounding ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1336726/full]
• 19. Advancing virus-induced gene silencing in sunflower [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-024-01241-z]
• 20. Development of a Csy4-Processing TRV -Based... [https://link.springer.com/article/10.1007/s11105-024-01459-0]
• 21. Establishment of a Virus-Induced Gene Silencing System ... [https://www.mdpi.com/2223-7747/14/2/150]
• 22. A Visual Reporter System for Virus-Induced Gene Silencing in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC2705029/]
• 23. A native visual screening reporter-assisted CRISPR/Cas9 ... [https://molhort.biomedcentral.com/articles/10.1186/s43897-025-00151-5]
• 24. Agrobacterium-mediated transformation for virus-induced ... [https://pubmed.ncbi.nlm.nih.gov/40730887/]
• 25. Truncated CMV2bN43 enhances virus-induced gene ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12542452/]
• 26. Engineering Encapsidated TRV1 as a Complete VIGS ... [https://pubmed.ncbi.nlm.nih.gov/40438032/]
• 27. Molecular Cloning Guide [https://www.promega.com/resources/guides/nucleic-acid-analysis/subcloning/]
• 28. Take care when cloning the pTRV2 vector for Virus-induced ... [https://lcwheeler.github.io/2018-03-01-VIGS/]
• 29. Traditional Cloning Basics | Thermo Fisher Scientific - ES [https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/cloning/traditional-cloning-basics.html]
• 30. Heritable CRISPR-Cas9 editing of plant genomes using ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9943877/]
• 31. Protocols for Molecular Biology, Plasmid Cloning, and Viral ... [https://www.addgene.org/protocols/]
• 32. A Highly Efficient Agrobacterium-Mediated Method for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7747990/]
• 33. Agrobacterium inoculation and optimization of TRV-based ... [https://bio-protocol.org/exchange/minidetail?id=3205823&type=30]
• 34. Agrobacterium-Mediated Virus-Induced Gene Silencing ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3217629/]
• 35. Agrobacterium-Mediated Transient Gene Expression ... [https://en.bio-protocol.org/en/bpdetail?id=4964&type=0]
• 36. Acetosyringone - an overview | ScienceDirect Topics [https://www.sciencedirect.com/topics/chemistry/acetosyringone]
• 37. A Methodological Advance of Tobacco Rattle Virus-Induced ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8212136/]
• 38. based virus-induced gene Silencing in Atriplex canescens [https://pmc.ncbi.nlm.nih.gov/articles/PMC12330136/]
• 39. Tobacco Rattle Virus as a Tool for Rapid Reverse-Genetics ... [https://www.mdpi.com/2223-7747/11/3/327]
• 40. Construction and application of a virus-induced gene ... [https://www.maxapress.com/article/doi/10.48130/tp-0024-0025]
• 41. Efficient Virus-Induced Gene Silencing in Ilex ... [https://www.mdpi.com/1999-4907/14/3/488]
• 42. A Cotyledon-based Virus-Induced Gene Silencing (Cotyledon ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-024-01154-x]
• 43. Vacuum and Co-cultivation Agroinfiltration of (Germinated) ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.00393/full]
• 44. Integrating cotyledon-based virus-induced gene silencing ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1514614/full]
• 45. Developing and applying a virus-induced gene silencing ... [https://pubmed.ncbi.nlm.nih.gov/39542283/]
• 46. Virus-Based MicroRNA Silencing in Plants - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC3875814/]
• 47. Tobacco Rattle Virus as a Tool for Rapid Reverse-Genetics ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8838890/]
• 48. Virus-induced gene silencing (VIGS) in Cannabis sativa L - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC6931244/]
• 49. Establishment of a Virus-Induced Gene-Silencing (VIGS) ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9820744/]
• 50. In planta transformation methods to accelerate the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12310747/]
• 51. Vacuum-Assisted Transient Agrobacterium tumefaciens ... [https://pubmed.ncbi.nlm.nih.gov/40146508/]
• 52. Optimised Agrobacterium-Mediated Transformation and ... [https://www.mdpi.com/2073-4425/14/7/1432]
• 53. High rates of virus-induced gene silencing by tobacco rattle ... [https://pubmed.ncbi.nlm.nih.gov/26209619/]
• 54. Truncated CMV2b N43 enhances virus-induced gene ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-025-01446-w]
• 55. A novel virus induced gene silencing method and its ... [https://www.cottongen.org/node/8287799]
• 56. Viral Vectors 101: Virus Safety [https://blog.addgene.org/viral-vectors-101-virus-safety]
• 57. Viral Vectors - Research Safety [https://researchsafety.uky.edu/biological-safety/biohazardous-materials/viral-vectors]
• 58. Selection of stable reference genes for accurate reverse- ... [https://www.nature.com/articles/s41598-025-08940-0]
• 59. An Efficient Virus-Induced Gene Silencing System for ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.661633/full]
• 60. Efficient virus-induced gene silencing in Primulina using ... [https://www.sciencedirect.com/science/article/abs/pii/S0304423824009816]
• 61. An Efficient Rice Virus-Induced Gene Silencing System ... [https://www.mdpi.com/2076-3417/15/11/5818]
• 62. Protocol for triggering plant gene silencing by viral delivery ... [https://pubmed.ncbi.nlm.nih.gov/40966092/]
• 63. Versatile Roles of Aquaporins in Plant Growth and ... [https://www.mdpi.com/1422-0067/21/24/9485]
• 64. 2 to regulate reversible flower opening in Gentiana scabra [https://pmc.ncbi.nlm.nih.gov/articles/PMC9252468/]
• 65. TRV–GFP: a modified Tobacco rattle virus vector for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3883300/]
• 66. Optimized cDNA libraries for virus-induced gene silencing ... [https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-4-5]
• 67. The "one-step" Bean pod mottle virus (BPMV)-derived vector ... [https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-014-0232-4]
• 68. The Development of an Efficient Multipurpose Bean Pod ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC2862437/]
• 69. Development of Bean pod mottle virus-based vectors for ... [https://www.sciencedirect.com/science/article/pii/S0042682205005611]
• 70. California TRV-based VIGS vectors mediate gene silencing ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8607596/]
• 71. Virus-Induced Gene Silencing (VIGS) in Chinese Jujube [https://www.mdpi.com/2223-7747/12/11/2115]
• 72. Efficient Virus-Induced Gene Silencing in Roots Using a ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC535832/]
• 73. an efficient virus induced gene silencing (VIGS) system [https://www.sciencedirect.com/science/article/pii/S2468014123000833]
• 74. Efficient virus-induced gene silencing in roots using a modified... [https://pubmed.ncbi.nlm.nih.gov/15591447/]