Validating Gene Function: A Comprehensive Guide to Plant Virus-Induced Gene Silencing (VIGS) Techniques

Abstract

This article provides a contemporary and practical guide to Virus-Induced Gene Silencing (VIGS) validation techniques for researchers and scientists. It covers the foundational principles of VIGS as a rapid, transient loss-of-function tool, details optimized methodological protocols for diverse plant species including recalcitrant crops, and addresses critical troubleshooting and optimization parameters. A dedicated section on validation and comparative analysis equips researchers with robust methods to confirm silencing efficacy and phenotypic outcomes, ensuring reliable data interpretation for functional genomics and drug discovery research.

The Core Principles of VIGS: From Plant Defense to Functional Genomics Powerhouse

RNA interference (RNAi), also known as Post-Transcriptional Gene Silencing (PTGS), represents a fundamental antiviral defense mechanism in plants. This sophisticated system provides immunity against viral pathogens by detecting and specifically degrading viral RNA, thereby preventing systemic infection. The PTGS pathway operates through a sequence-specific process that is triggered by double-stranded RNA (dsRNA) molecules, which are common replication intermediates for many plant viruses. During viral infection, the plant's innate immune system recognizes these dsRNA structures as foreign and initiates a silencing cascade that ultimately leads to the cleavage of complementary viral RNA sequences [1] [2].

This RNA-based immune response exhibits remarkable specificity and adaptability, allowing plants to target diverse viral pathogens without prior exposure. The discovery of PTGS has not only advanced our understanding of plant-virus interactions but has also paved the way for revolutionary biotechnological applications, most notably Virus-Induced Gene Silencing (VIGS), which leverages this natural antiviral mechanism for functional genomics research. The evolutionary conservation of RNAi pathways across kingdoms underscores their fundamental importance in biological defense systems, while plant-specific adaptations highlight the unique challenges posed by viral pathogens in immobile organisms [3] [4].

Core Molecular Mechanism of Antiviral PTGS

The antiviral PTGS pathway comprises a coordinated sequence of molecular events that begins with viral detection and culminates in targeted RNA degradation. The table below summarizes the core components and their functions in this process.

Table 1: Core Components of the Antiviral PTGS Pathway

Component	Function in Antiviral PTGS
DICER-like (DCL) Proteins	RNase III enzymes that recognize and cleave viral dsRNA into 21-24 nucleotide small interfering RNAs (vsiRNAs) [1]
Argonaute (AGO) Proteins	Central effectors that bind vsiRNAs and form the RNA-Induced Silencing Complex (RISC) [3]
RNA-Dependent RNA Polymerases (RDRs)	Amplify the silencing signal by synthesizing secondary dsRNA using aberrant viral RNA as templates [1]
Small Interfering RNAs (siRNAs)	21-24 nt guide molecules that provide sequence specificity for target recognition [2]
Double-Stranded RNA (dsRNA)	Essential trigger molecule derived from viral replication intermediates or secondary structures [1]

The mechanism begins when viral RNAs form double-stranded structures through inter- or intramolecular base pairing. These viral dsRNAs are recognized as pathogen-associated molecular patterns and cleaved by DICER-like proteins (DCLs), predominantly DCL2, DCL3, and DCL4 in Arabidopsis thaliana, to generate 21-24 nucleotide virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are then loaded onto Argonaute proteins (AGOs), with AGO1 and AGO2 playing primary roles in post-transcriptional silencing. The AGO-vsiRNA complex assembles into the RNA-Induced Silencing Complex (RISC), which performs the effector function of the pathway [1] [4].

The activated RISC complex identifies complementary viral RNA sequences through base-pairing with the guide vsiRNA and cleaves the target, effectively preventing viral translation and replication. To amplify this defense response, plant RNA-dependent RNA polymerases (RDRs), particularly RDR6, synthesize secondary dsRNAs using cleaved viral RNA fragments as templates. These secondary dsRNAs are subsequently processed by DCLs to generate additional vsiRNAs, creating a robust, self-amplifying silencing signal that can spread systemically throughout the plant via plasmodesmata and the vascular system [1] [3].

Diagram 1: Core Mechanism of Antiviral PTGS in Plants. This pathway illustrates the sequence of events from viral infection to RNA degradation, highlighting key steps including vsiRNA biogenesis and systemic signal spread.

Virus-Induced Gene Silencing (VIGS): Harnessing PTGS for Functional Genomics

Virus-Induced Gene Silencing (VIGS) represents a powerful reverse genetics approach that co-opts the natural antiviral PTGS pathway to study gene function in plants. This technology utilizes recombinant viral vectors carrying fragments of host genes to trigger sequence-specific silencing of corresponding endogenous transcripts. The fundamental principle underpinning VIGS is that the plant's RNAi machinery cannot distinguish between viral RNAs and host-derived sequences expressed from viral vectors, leading to degradation of both viral and complementary endogenous mRNAs [2] [3].

The VIGS technique was first established in 1995 when Kumagai and colleagues used a Tobacco Mosaic Virus (TMV) vector carrying a fragment of the phytoene desaturase (PDS) gene from Nicotiana benthamiana to induce silencing, resulting in a characteristic photo-bleaching phenotype. Since this pioneering demonstration, VIGS has been adapted for numerous plant species using various viral vectors, emerging as an indispensable tool for functional genomics, particularly in species recalcitrant to stable transformation [2] [5].

The molecular basis of VIGS involves the same core PTGS machinery employed in antiviral defense. When a recombinant viral vector infects a plant cell, the viral RNA replicase generates dsRNA intermediates during replication. The plant's DCL enzymes recognize these dsRNAs and process them into small interfering RNAs (siRNAs) of 21-24 nucleotides. These siRNAs are then incorporated into RISC complexes that target both viral RNAs and complementary endogenous mRNAs for degradation, resulting in knock-down of the target gene expression [3] [6].

Table 2: Commonly Used VIGS Vectors and Their Applications

Vector Type	Example Viruses	Host Range	Key Features
RNA Virus-Based	Tobacco Rattle Virus (TRV), Tobacco Mosaic Virus (TMV), Potato Virus X (PVX)	Solanaceae species, Arabidopsis	Cytoplasmic replication, efficient systemic movement, rapid silencing induction [2]
DNA Virus-Based	Geminiviruses (Cotton Leaf Crumple Virus, African Cassava Mosaic Virus)	Cotton, Cassava, Monocots	Nuclear replication, longer-lasting silencing, suitable for species resistant to RNA viruses [2] [5]
Satellite Virus-Based	Satellite Tobacco Mosaic Virus, Satellite Tobacco Necrosis Virus	Complementary range to helper viruses	Can be used with helper viruses to extend host range [2]

Experimental Protocol: Implementing VIGS for Gene Function Analysis

The following section provides a detailed methodology for implementing VIGS in plants, specifically adapted from successful applications in Nicotiana benthamiana and Capsicum annuum L. (pepper) using the Tobacco Rattle Virus (TRV) system, one of the most widely employed and versatile VIGS vectors [2] [6].

Vector Design and Clone Preparation

The TRV system utilizes a bipartite genome requiring two separate plasmids: TRV1 and TRV2. The TRV1 plasmid encodes replicase proteins, movement protein, and a weak RNA silencing suppressor, while TRV2 contains the capsid protein gene and a multiple cloning site for inserting target gene fragments [2].

• Target Gene Fragment Selection: Identify a unique 200-500 bp fragment from the target gene's coding sequence. Avoid regions with high sequence similarity to other genes to ensure silencing specificity.
• PCR Amplification: Design gene-specific primers with appropriate restriction enzyme sites for directional cloning into the TRV2 vector.
• Cloning into TRV2 Vector: Ligate the purified PCR product into the corresponding restriction sites of the TRV2 plasmid. Verify successful cloning by colony PCR and sequencing.
• Transformation into Agrobacterium: Introduce the verified TRV2 recombinant plasmid and the TRV1 plasmid separately into Agrobacterium tumefaciens strain GV3101 through electroporation or freeze-thaw transformation [6].

Plant Material Selection and Growth Conditions

• Plant Genotype Selection: Choose plant varieties known to be susceptible to the viral vector. For Solanaceous species like pepper and tomato, specific cultivars with known VIGS efficiency should be selected.
• Growth Conditions: Maintain plants at 22-25°C with a 16/8 hour light/dark photoperiod and 60-70% relative humidity. These conditions optimize both plant health and Agrobacterium infectivity.
• Inoculation Timing: For optimal results, inoculate plants at the 2-4 true leaf stage (approximately 3-4 weeks post-germination) when plants are most susceptible to infection [2].

Agroinoculation Procedure

Two primary methods are commonly employed for VIGS inoculation: agroinfiltration and agrodrench.

Table 3: Comparison of VIGS Delivery Methods

Method	Procedure	Efficiency	Time to Phenotype	Best For
Agroinfiltration	Direct injection of Agrobacterium suspension into leaves using a needleless syringe [6]	60.2 ± 2.9% [6]	7 days [6]	Model plants (N. benthamiana)
Agrodrench	Soil drenching with Agrobacterium suspension around the root zone [6]	10.3 ± 1.5% [6]	14 days [6]	Species with delicate leaves

Standard Agroinfiltration Protocol:

• Prepare Agrobacterium cultures by inoculating single colonies of TRV1 and recombinant TRV2 strains in 5 ml of YEP medium with appropriate antibiotics (kanamycin 50 μg/ml, rifampicin 50 μg/ml). Incubate at 28°C with shaking at 200 rpm for 24 hours.
• Subculture 1 ml of each starter culture into 50 ml of fresh YEP medium with antibiotics and incubate for an additional 16-20 hours until OD600 reaches 1.0-1.5.
• Harvest bacterial cells by centrifugation at 3000 × g for 15 minutes and resuspend in infiltration buffer (10 mM MES, 10 mM MgCl2, 200 μM acetosyringone, pH 5.6) to a final OD600 of 1.0 for each culture.
• Mix the TRV1 and recombinant TRV2 suspensions in a 1:1 ratio and incubate at room temperature for 3-4 hours before infiltration.
• Using a needleless syringe, gently infiltrate the bacterial suspension into the abaxial side of fully expanded leaves. Apply gentle pressure against the leaf with a finger while infiltrating.
• Label infiltrated plants and maintain under normal growth conditions while monitoring for silencing phenotypes [2] [6].

Silencing Validation and Phenotypic Analysis

• Phenotypic Monitoring: Document visible phenotypes regularly. For control experiments, include plants infiltrated with TRV2-PDS (phytoene desaturase) which should exhibit characteristic photo-bleaching.
• Molecular Validation of Silencing:
  • Extract total RNA from silenced tissue using standard protocols.
  • Perform reverse transcription PCR (RT-PCR) using primers designed to amplify a region outside the VIGS target sequence to avoid detection of the viral construct.
  • Compare transcript levels between VIGS-treated and control plants using semi-quantitative RT-PCR or quantitative real-time PCR (qRT-PCR).
• VIGS Efficiency Assessment: Calculate silencing efficiency as the percentage of infiltrated plants showing both molecular evidence of silencing and expected phenotypic changes [6].

Diagram 2: VIGS Experimental Workflow. This diagram outlines the key steps in implementing VIGS, from vector construction to functional analysis of silencing results.

The Scientist's Toolkit: Essential Research Reagents for PTGS Studies

The following table compiles key research reagents and materials essential for investigating PTGS mechanisms and implementing VIGS technology, based on established protocols and recent advancements.

Table 4: Essential Research Reagents for PTGS and VIGS Studies

Reagent/Material	Function/Application	Examples/Specifications
VIGS Vectors	Delivery of target gene sequences to plant cells	TRV (Tobacco Rattle Virus), TMV (Tobacco Mosaic Virus), PVX (Potato Virus X), BBWV2 (Broad Bean Wilt Virus 2) [2]
Agrobacterium Strains	Biological delivery of viral vectors	GV3101, LBA4404, AGL1 [6]
Infiltration Buffer	Medium for Agrobacterium delivery	10 mM MES, 10 mM MgCl₂, 200 μM acetosyringone, pH 5.6 [6]
Plant Growth Regulators	Optimize plant susceptibility	Adjust concentration based on plant species and developmental stage [2]
Antibiotics	Selection of bacterial strains and plasmids	Kanamycin (50 μg/ml), rifampicin (50 μg/ml) for Agrobacterium selection [6]
siRNA Detection Reagents	Validate silencing efficiency	Northern blot reagents, small RNA sequencing kits [1]
Molecular Biology Kits	Clone target genes and validate silencing	RNA extraction kits, RT-PCR kits, cloning kits with restriction enzymes [6]

Advanced Concepts: Non-Canonical RNAi Pathways and Emerging Applications

Recent research has revealed increasing complexity in plant antiviral RNAi pathways, identifying several non-canonical mechanisms that diverge from the established DCL-AGO-RDR paradigm. These alternative pathways expand the defensive arsenal available to plants and offer new opportunities for biotechnological exploitation [1].

One significant non-canonical pathway involves non-canonical RNA-directed DNA methylation (RdDM), where variations in the steps leading to siRNA biogenesis result in transcriptional silencing through DNA methylation. Examples include:

• RDR6 RdDM pathway: 21-22 nt siRNAs produced during PTGS from RNA polymerase II transcripts activate RISCs that engage in RdDM.
• RDR6-DCL3 RdDM pathway: RDR6-mediated dsRNA synthesis followed by DCL3 processing into 24-nt siRNAs that associate with the RdDM pathway.
• miRNA-directed DNA methylation: RNA polymerase II transcripts directly cleaved by DCL3 into 24-nt sRNAs that participate in RdDM [1].

Another emerging concept is the generation of non-canonical small RNAs from externally applied dsRNA. Studies have shown that exogenously supplied dsRNA can generate a ladder of small RNAs ∼18-30 nt in length, rather than the discrete 21- and 22-nt species typically produced during viral infection. These non-canonical sRNAs raise questions about potential alternative processing pathways and their functional significance in antiviral defense [1].

The practical application of PTGS mechanisms has expanded beyond traditional VIGS to include exogenous dsRNA applications for crop protection. Spray-induced gene silencing (SIGS) involves applying dsRNA molecules directly to plants to trigger silencing of essential pathogen genes, offering an environmentally friendly approach to disease management. However, recent studies indicate that externally applied dsRNAs may not always follow canonical PTGS pathways, with some reports showing enrichment of 24-nt siRNAs rather than the expected 21-22 nt species [1].

The emerging field of VIGS-induced heritable epigenetics represents another frontier in PTGS applications. Research has demonstrated that VIGS can induce stable epigenetic modifications that are transgenerationally inherited. For instance, infection with TRV vectors carrying promoter sequences of the FWA gene leads to DNA methylation and transcriptional silencing that persists over multiple generations, opening possibilities for epigenetic breeding strategies [3].

Virus-induced gene silencing (VIGS) is a powerful reverse genetics technique that exploits the plant's natural RNA-mediated antiviral defense mechanism for functional gene analysis [7] [8]. As a form of post-transcriptional gene silencing (PTGS), VIGS operates by degrading mRNA transcripts that share sequence homology with the invading virus, leading to targeted gene knockdown without the need for stable transformation [7]. When a viral vector is modified to carry a fragment of a host gene, the plant's defense system silences both the viral RNA and the corresponding endogenous mRNA, resulting in a loss-of-function phenotype that can be rapidly observed and characterized [8]. The technology has emerged as an indispensable tool for high-throughput functional genomics, enabling researchers to quickly link gene sequences to biological functions in a wide range of plant species [7] [8].

The application of VIGS spans numerous research areas, including disease resistance studies, metabolic engineering, developmental biology, and stress response pathways [9] [10]. Its advantages over traditional transgenic approaches include significantly shorter experimental timelines (typically 3-4 weeks), applicability to plant species recalcitrant to stable transformation, and the ability to screen multiple gene candidates without generating stable lines [7] [11] [12]. Furthermore, VIGS requires only partial sequence information (typically 200-500 bp) to effectively silence target genes, making it particularly valuable for functional characterization in crops with complex genomes [8].

Molecular Mechanism of Virus-Induced Gene Silencing

The molecular machinery of VIGS harnesses the plant's innate RNA silencing pathways, which originally evolved as a defense mechanism against viruses and transposons. The process begins when a recombinant viral vector containing a fragment of a plant gene is introduced into the host plant, typically via Agrobacterium-mediated delivery or in vitro transcripts [7] [8]. Once inside the plant cell, the viral RNA is replicated, forming double-stranded RNA (dsRNA) intermediates through the activity of viral or host RNA-dependent RNA polymerases (RdRps) [7].

These dsRNA molecules are recognized as aberrant by the plant's surveillance system and are cleaved by Dicer-like (DCL) enzymes into short interfering RNAs (siRNAs) of 21-24 nucleotides in length [8]. These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where the guide strand directs the complex to complementary mRNA sequences. The catalytic component of RISC, typically an Argonaute (AGO) protein, cleaves the target mRNA, preventing its translation and effectively "silencing" the corresponding gene [7]. What makes VIGS particularly powerful is the systemic nature of this silencing effect; the initial siRNA population can amplify through the action of host RdRps and move throughout the plant via plasmodesmata and the vascular system, generating secondary siRNAs that propagate the silencing signal to distal tissues [8].

The following diagram illustrates this molecular process:

Figure 1: Molecular Mechanism of Virus-Induced Gene Silencing

Comparative Analysis of Major VIGS Vector Systems

Key Vector Systems and Their Characteristics

Various viral vectors have been engineered for VIGS applications, each with distinct advantages and limitations based on viral genome structure, host range, silencing efficiency, and symptom severity. The most widely used vectors include Tobacco Rattle Virus (TRV), Bean Pod Mottle Virus (BPMV), and Soybean Yellow Common Mosaic Virus (SYCMV), along with other systems such as Apple Latent Spherical Virus (ALSV), Cucumber Mosaic Virus (CMV), and Barley Stripe Mosaic Virus (BSMV) [9] [7] [13]. The selection of an appropriate vector depends on multiple factors, including the target plant species, tissue specificity requirements, and the nature of the biological question being addressed.

TRV-based systems have gained prominence due to their broad host range, efficacy in meristematic tissues, and mild symptomatic effects that minimize interference with phenotypic observations [9] [8]. BPMV vectors have been particularly valuable in legume research, especially soybean, despite technical challenges associated with particle bombardment delivery methods [9]. More recently, SYCMV has emerged as an efficient single-stranded RNA virus vector with simplified cloning procedures and high silencing efficiency across diverse soybean germplasms [13]. Other vectors like ALSV and CMV offer additional options for specific host plants or experimental requirements, though each presents unique advantages and constraints.

Table 1: Comparative Characteristics of Major VIGS Vector Systems

Vector	Virus Type	Primary Host Species	Silencing Efficiency	Key Advantages	Major Limitations
TRV	Positive-sense RNA virus (Tobravirus)	Solanaceous species, Arabidopsis, cotton, soybean [9] [8]	65-95% in soybean [9]	Broad host range, meristem penetration, mild symptoms [9] [8]	Limited efficacy in some monocots
BPMV	Positive-sense RNA virus (Comovirus)	Soybean, common bean [9] [10]	High in susceptible soybean cultivars [9]	Well-established for legumes, reliable silencing [9] [10]	Delivery often requires particle bombardment [9]
SYCMV	Positive-sense single-stranded RNA virus	Soybean (cultivated and wild) [13]	High across various germplasms [13]	Single-component genome, easy cloning, systemic silencing [13]	Relatively new system, less extensively validated
ALSV	RNA virus	Apple, cucurbits, soybean [13]	Effective in multiple species [13]	Broad host range including cucurbits	Not available in all geographic regions [13]
CMV	Positive-sense RNA virus (Cucumovirus)	Cucurbits, soybean [13]	Demonstrated in soybean [13]	Established vector system	Limited to susceptible hosts

Performance Metrics and Experimental Considerations

When evaluating VIGS vectors for specific applications, researchers must consider multiple performance metrics beyond basic host compatibility. Silencing efficiency varies considerably among systems, with TRV demonstrating 65-95% efficiency in soybean under optimized conditions [9], while SYCMV-based systems achieve high efficiency across diverse soybean germplasms including cultivated and wild varieties [13]. The duration of silencing is another critical factor, with most systems maintaining effective gene knockdown for several weeks, sufficient for observing developmental phenotypes or stress response analyses.

The insert size capacity represents an important technical consideration, with most VIGS vectors accommodating fragments of 200-500 base pairs, though optimal sizes vary by system [8]. TRV vectors typically function well with inserts of 300-500 bp [8], while SYCMV has been successfully employed with a 207-bp fragment of GmPDS [13]. Vector stability also differs among systems, with some vectors exhibiting a tendency to lose inserts during viral replication, particularly when containing repetitive sequences or sequences toxic to the virus.

Environmental conditions significantly influence VIGS efficiency across all systems. Research has demonstrated that factors including photoperiod, growth temperature, plant age at inoculation, and inoculation method profoundly impact silencing outcomes [13]. For SYCMV-based systems, optimal conditions include a photoperiod of 16/8 h (light/dark) at approximately 27°C following syringe infiltration to unrolled unifoliolate leaves at the cotyledon stage [13]. Similar optimization is necessary for other vector systems to achieve reproducible and robust silencing.

Table 2: Technical Specifications and Performance Metrics of VIGS Vectors

Parameter	TRV	BPMV	SYCMV	Other Systems
Optimal Insert Size	300-500 bp [8]	Varies, typically 200-300 bp	207 bp demonstrated [13]	200-300 bp for most systems
Delivery Method	Agrobacterium-mediated [9]	Often particle bombardment [9]	Agrobacterium-mediated [13]	Varies by system
Time to Silencing	2-3 weeks [8]	2-3 weeks	~21 days [13]	Typically 2-4 weeks
Tissue Coverage	Whole plant including meristems [8]	Systemic but limited meristem penetration	Entire plant (root, stem, leaves, flowers) [13]	Varies by viral movement capabilities
Key Applications	Functional genomics in broad species [9] [8]	Soybean disease resistance studies [9] [10]	High-throughput screening in soybean [13]	Species-specific applications

Detailed Experimental Protocols for Major VIGS Systems

TRV-Mediated Gene Silencing in Soybean

The TRV-VIGS protocol has been optimized for soybean through Agrobacterium-mediated infection of cotyledon nodes, achieving high silencing efficiency with systemic spread throughout the plant [9]. The procedure begins with vector construction by cloning a 300-500 bp fragment of the target gene into the pTRV2 vector using appropriate restriction sites (e.g., EcoRI and XhoI) or recombination cloning systems [9] [8]. The recombinant plasmid is then transformed into Agrobacterium tumefaciens strain GV3101 through heat shock or freeze-thaw methods [9].

For plant inoculation, surface-sterilized soybean seeds are soaked in sterile water until swollen, then longitudinally bisected to obtain half-seed explants [9]. The fresh explants are immersed in Agrobacterium suspensions containing pTRV1 or pTRV2 derivatives for 20-30 minutes, which represents the optimal duration for infection efficiency [9]. Following inoculation, plants are maintained under high humidity conditions for 24-48 hours before transfer to standard growth conditions. Silencing phenotypes typically become visible within 2-3 weeks post-inoculation, with effective silencing confirmed through phenotypic observation and molecular analysis such as qRT-PCR [9].

Critical optimization parameters for TRV-VIGS include the use of young tissue (cotyledon stage), Agrobacterium optical density (OD600 = 0.6-1.0), and growth temperatures of approximately 25°C [9] [8]. The TRV system has been successfully employed to silence genes involved in disease resistance and metabolic pathways, including the phytoene desaturase (GmPDS) rust resistance gene (GmRpp6907), and defense-related genes (GmRPT4) [9].

SYCMV-Based VIGS Protocol

The SYCMV-VIGS system provides an efficient alternative for soybean functional genomics, with optimization of several environmental and developmental factors to enhance silencing efficiency [13]. The protocol begins with cloning a target gene fragment (approximately 200 bp) into the BsrGI restriction site of the SYCMV vector in the sense orientation [13]. The recombinant plasmid is transformed into Agrobacterium tumefaciens strain GV3101, and positive colonies are selected using appropriate antibiotics.

Agrobacterium cultures are grown overnight at 28°C in Luria-Bertani (LB) medium containing antibiotics (100 μg/ml spectinomycin and 50 μg/ml rifampicin), then centrifuged and resuspended in infiltration buffer (10 mM MES pH 5.6, 10 mM MgCl2, and 200 μM acetosyringone) [13]. The cell suspensions are incubated at room temperature for at least 2 hours before infiltration. For soybean inoculation, unrolled unifoliolate leaves at the cotyledon stage are infiltrated using a needleless syringe with the Agrobacterium suspension adjusted to OD600 = 2.0, which has been identified as optimal for SYCMV [13].

Following infiltration, plants are maintained under a photoperiod of 16/8 h (light/dark) at approximately 27°C with 60% relative humidity [13]. Under these optimized conditions, SYCMV-mediated silencing achieves high efficiency across various soybean germplasms, including cultivated and wild soybeans [13]. The system facilitates silencing in the entire plant, including roots, stems, leaves, and flowers, and can be transmitted to other soybean germplasms via mechanical inoculation [13].

BPMV VIGS Implementation

The BPMV-VIGS system has been extensively used for functional studies of soybean genes, particularly those involved in disease resistance pathways [9] [10]. The protocol involves inserting a target gene fragment into the BPMV RNA2 vector under the control of the cauliflower mosaic virus (CaMV) 35S promoter [9]. For delivery, the recombinant vector is typically introduced via particle bombardment or Agrobacterium infiltration, though the former remains more common despite its technical challenges [9].

In practice, soybean plants at the fully expanded unifoliate stage are inoculated with the BPMV vector, and silencing phenotypes typically emerge within 2-3 weeks post-inoculation [10]. The BPMV system has been successfully employed to study genes involved in soybean mosaic virus (SMV) resistance, such as the Ferredoxin-NADP reductase (GmFNR) gene, where silencing resulted in reduced photosynthetic capacity and altered antioxidant responses [10]. A key advantage of BPMV is its established reliability in soybean systems, though its implementation faces technical hurdles related to delivery methods that can limit its accessibility for some laboratories [9].

The following workflow diagram illustrates the general process for implementing a VIGS experiment:

Figure 2: General Workflow for VIGS Experiment Implementation

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of VIGS technology requires specific reagents and materials optimized for each vector system and plant species. The following table details key components essential for establishing VIGS in laboratory settings:

Table 3: Essential Research Reagents and Materials for VIGS Experiments

Reagent/Material	Specification/Recommended Type	Function/Purpose	Examples/Notes
Viral Vectors	Binary vectors with plant promoters	Carry target gene fragments for silencing	pTRV1/pTRV2 for TRV system [9] [8]; SYCMV vector for soybean [13]
Agrobacterium Strain	GV3101, LBA4404, others	Delivery of viral vectors to plant cells	GV3101 commonly used for TRV and SYCMV [9] [13]
Infiltration Buffer	10 mM MES, 10 mM MgCl2, 200 μM acetosyringone	Agrobacterium resuspension for inoculation	pH adjustment to 5.6-5.8 critical for efficiency [13]
Antibiotics	Spectinomycin, rifampicin, kanamycin	Selection of transformed Agrobacterium	Concentration varies by strain and vector (e.g., 100 μg/ml spectinomycin) [13]
Plant Growth Media	Soil mixtures, sterile tissue culture media	Plant growth and maintenance	Sterile conditions improve infection efficiency [9]
Gene-Specific Primers	Targeting 200-500 bp fragment	Amplification of target gene sequence	Designed to avoid conserved domains; verified for specificity
qRT-PCR Reagents	SYBR Green systems, reverse transcriptase	Validation of silencing efficiency	Reference genes: tubulin, ubiquitin, actin [10]

Applications and Case Studies in Plant Research

Functional Analysis of Disease Resistance Genes

VIGS technology has proven particularly valuable for characterizing genes involved in plant-pathogen interactions, enabling rapid functional assessment without the need for stable transformation. In soybean, TRV-based VIGS has been successfully employed to silence the rust resistance gene GmRpp6907 and the defense-related gene GmRPT4, confirming their roles in disease resistance pathways through observation of significantly altered phenotypic responses [9]. Similarly, BPMV-mediated silencing of the GmFNR gene (Ferredoxin-NADP reductase) in soybean revealed its importance in soybean mosaic virus (SMV) infection responses, with silenced plants exhibiting reduced photosynthetic capacity, decreased catalase (CAT) activity, and increased hydrogen peroxide (H2O2) accumulation in susceptible lines [10]. These findings provided novel insights into the molecular mechanisms of SMV resistance and demonstrated the utility of VIGS for connecting specific genes to physiological responses during pathogen challenge.

The application of VIGS extends beyond soybean to numerous other crop species. In cucurbits, a cucumber fruit mottle mosaic virus (CFMMV)-based VIGS system was used to screen 38 candidate genes related to male sterility in watermelon, resulting in the identification of 8 genes that produced male-sterile flowers with abnormal stamens and no pollen when silenced [11]. This high-throughput approach enabled rapid functional characterization in a species recalcitrant to genetic transformation, highlighting the efficiency of VIGS for candidate gene validation in species with limited genetic tools. Similarly, in Luffa acutangula, a CGMMV-based VIGS system successfully silenced the tendril synthesis-related gene (TEN), resulting in plants with shorter tendrils and altered nodal positions where tendrils developed [12]. These case studies demonstrate the versatility of VIGS for studying diverse biological processes across a broad range of plant species.

Metabolic and Developmental Studies

VIGS has significantly advanced our understanding of metabolic pathways and developmental processes in plants. The classic marker gene phytoene desaturase (PDS) has been silenced in numerous species to validate VIGS systems, resulting in characteristic photobleaching due to disrupted carotenoid biosynthesis [9] [13] [12]. Beyond this marker gene, VIGS has enabled functional analysis of genes involved in diverse metabolic pathways, including hormone biosynthesis, secondary metabolism, and nutrient assimilation. The technology is particularly valuable for studying essential genes that would be lethal in stable knockout lines, as the transient nature of silencing often allows recovery of plants after phenotypic observation.

Recent methodological advances have expanded VIGS applications to include more sophisticated approaches such as virus-induced overexpression (VOX), virus-induced genome editing (VIGE), and delivery of CRISPR/Cas components for targeted gene editing [14] [15]. TRV-based vectors have been engineered to simultaneously express multiple heterologous proteins, enabling more complex functional analyses such as protein-protein interaction studies or complementation assays [14]. These developments position VIGS as a versatile platform not only for gene silencing but also for a broader range of functional genomics applications, further enhancing its utility in plant research.

Technical Considerations and Optimization Strategies

Critical Factors Influencing Silencing Efficiency

The efficiency of VIGS is influenced by multiple factors that researchers must carefully optimize for each experimental system. Plant age at inoculation represents a critical parameter, with most systems achieving highest efficiency when plants are inoculated at early developmental stages. For SYCMV-based systems in soybean, inoculation at the cotyledon stage with unrolled unifoliolate leaves yields optimal results [13], while TRV-based systems also perform best with young, actively growing tissues [9] [8]. Environmental conditions including photoperiod, temperature, and light intensity significantly impact silencing efficiency across all VIGS systems. Research with SYCMV demonstrated that a photoperiod of 16/8 h (light/dark) at approximately 27°C produced the highest silencing efficiency [13], while similar optimization is recommended for other vector systems.

Agrobacterium culture density and infection methods represent additional variables requiring optimization. For TRV-mediated silencing in soybean, immersion of cotyledon node explants for 20-30 minutes in Agrobacterium suspensions achieved infection efficiencies exceeding 80% [9]. In contrast, SYCMV protocols recommend syringe infiltration of unifoliolate leaves with Agrobacterium suspensions adjusted to OD600 = 2.0 [13]. The inclusion of acetosyringone (200 μM) in the infiltration buffer enhances transformation efficiency by activating Vir gene expression in Agrobacterium [13]. Post-inoculation conditions, particularly high humidity maintenance for 24-48 hours, significantly improve infection rates and subsequent silencing efficiency.

Troubleshooting Common Challenges

Several technical challenges commonly arise in VIGS experiments, necessitating systematic troubleshooting approaches. Incomplete or patchy silencing may result from suboptimal inoculation techniques, inadequate viral movement, or insufficient silencing signal amplification. This can be addressed by ensuring thorough infiltration of target tissues, verifying vector integrity, and confirming optimal growth conditions for viral spread. Non-specific phenotypes or symptoms unrelated to target gene silencing may occur due to viral pathogenicity or off-target effects. Including multiple negative controls (empty vector, non-silencing fragments) and replicating experiments with independent plant batches helps distinguish specific silencing effects from non-specific responses.

Molecular validation of silencing efficiency represents an essential step in VIGS experiments. Quantitative RT-PCR analysis typically reveals 40-95% reduction in target gene expression in effective silencing conditions [9] [10]. When silencing efficiency is low, researchers should verify insert stability in the viral vector through PCR amplification from infected tissue, assess potential redundancy in gene family members that might compensate for the silenced gene, and consider alternative target regions within the gene of interest. For genes with high basal expression or rapid turnover, increasing the number of inoculated plants and sampling at multiple time points may be necessary to capture the silencing window and obtain reproducible phenotypes.

Future Perspectives and Emerging Applications

The continuing evolution of VIGS technology promises to expand its applications in plant functional genomics. The integration of VIGS with CRISPR/Cas systems represents a particularly promising direction, enabling both transient gene silencing and precise genome editing within the same experimental framework [14] [15]. TRV-based vectors have already been successfully engineered to deliver Cas nucleases and guide RNAs for targeted mutagenesis, creating opportunities for rapid assessment of gene function through both loss-of-function and precise editing approaches [14]. These integrated platforms offer unprecedented flexibility for functional genomics, particularly in species where traditional transformation methods remain challenging.

Advancements in viral vector design continue to address current limitations and expand technical capabilities. Recent developments include the creation of all-in-one plant virus-based vector toolkits that support streamlined gene silencing, overexpression, and genome editing within unified systems [15]. Vectors with enhanced stability for larger inserts, reduced symptom severity, and improved tissue specificity are expanding the experimental possibilities for plant researchers [14] [8]. Additionally, the discovery and engineering of viral vectors for previously non-host species continues to broaden the applicability of VIGS across the plant kingdom, including monocots, trees, and other recalcitrant species.

As these technological advances mature, VIGS is poised to remain at the forefront of plant functional genomics, enabling increasingly sophisticated studies of gene function, metabolic engineering, and trait development in both model and crop species. The continued refinement of delivery methods, expansion of host ranges, and integration with complementary technologies will further solidify the role of VIGS as an essential component of the plant biologist's toolkit.

Virus-induced gene silencing (VIGS) has emerged as a pivotal reverse genetics tool in plant functional genomics, offering distinct advantages over traditional transformation-based methods. This technique utilizes recombinant viral vectors to trigger the plant's innate RNA interference machinery, enabling rapid, cost-effective, transient knockdown of target genes without requiring stable transformation. The utility of VIGS spans multiple plant species, including recalcitrant crops and perennial woody plants, facilitating high-throughput gene functional analysis. This protocol details the molecular mechanisms, experimental workflows, and key applications of VIGS, with specific emphasis on its implementation in challenging plant systems. We provide comprehensive methodological guidelines, reagent specifications, and quantitative efficiency data to support researchers in implementing this powerful technology for accelerated gene characterization and crop improvement programs.

Virus-induced gene silencing (VIGS) represents an RNA-mediated reverse genetics technology that has evolved into an indispensable approach for analyzing gene function in plants [3]. This technique exploits the plant's post-transcriptional gene silencing (PTGS) machinery, an innate antiviral defense mechanism, to achieve sequence-specific degradation of endogenous mRNAs [3] [2]. First established by Kumagai et al. in 1995 using a Tobacco mosaic virus vector to silence the phytoene desaturase gene in Nicotiana benthamiana, VIGS has since been adapted for numerous plant species with the development of diverse viral vectors [3] [2].

The fundamental advantage of VIGS lies in its ability to transiently knock down gene expression without the need for stable genetic transformation, which remains challenging, time-consuming, and genotype-dependent for many crop species [16] [2] [17]. As a rapid and powerful method for gene functional analysis in plants that pose challenges in stable transformation, VIGS has become particularly valuable for characterizing genes involved in diverse biological processes including development, metabolism, and stress responses [16] [18]. The technology has recently been expanded beyond simple gene silencing to include applications in heritable epigenetic modifications and virus-induced genome editing (VIGE), further broadening its utility in plant biotechnology [3] [19].

Molecular Mechanisms of VIGS

The molecular basis of VIGS centers on the plant's RNA interference pathway, which is naturally activated during viral infection. The process initiates when a recombinant viral vector containing a fragment of the target plant gene is introduced into the host plant [3] [2]. Following inoculation, the viral vector systemically spreads throughout the plant, and the viral RNA replicase generates double-stranded RNA replication intermediates [3] [18].

These dsRNA molecules are recognized by the plant's Dicer-like enzymes (DCL), which process them into small interfering RNAs (siRNAs) of 21-24 nucleotides in length [3] [2]. These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where they serve as guides for sequence-specific identification and cleavage of complementary endogenous mRNA transcripts [3] [18]. The cleavage leads to degradation of the target mRNA, resulting in reduced expression of the corresponding protein and the emergence of observable phenotypes that facilitate functional characterization [3] [2].

Simultaneously, the AGO complex can interact with target DNA molecules in the nucleus, causing transcriptional repression via DNA methylation at the 5' untranslated region, resulting in transcriptional gene silencing [3]. This mechanism forms the basis for VIGS-induced heritable epigenetic modifications, where DNA methylation causes genetically inherited alterations in chromatin structure and gene expression without changing the underlying nucleotide sequence [3].

Figure 1: Molecular Mechanism of Virus-Induced Gene Silencing (VIGS). The process begins with a recombinant viral vector entering plant cells, leading to double-stranded RNA formation during viral replication. DICER-like enzymes process dsRNA into siRNAs, which are loaded into the RISC complex containing AGO proteins. The activated RISC complex cleaves complementary target mRNA, resulting in gene silencing. In the nucleus, the complex can also induce epigenetic modifications through DNA methyltransferases.

Key Advantages of VIGS Technology

Rapid Implementation and High-Speed Functional Analysis

VIGS dramatically accelerates the gene function characterization pipeline compared to conventional stable transformation approaches. While traditional genetic transformation and mutant generation require tedious and laborious efforts spanning multiple generations, VIGS constructs can be assembled in a few days, with silencing phenotypes typically developing within 2-4 weeks post-inoculation [18] [20]. This rapid turnaround enables researchers to quickly progress from gene identification to functional assessment, making it particularly valuable for high-throughput functional genomics screens [2] [18].

The speed advantage of VIGS is especially pronounced in species with long life cycles or challenging transformation systems. For example, in perennial woody plants like Camellia drupifera, where stable transformation systems are largely unavailable, VIGS enables functional gene analysis within a single growing season [16]. Similarly, in species with established transformation protocols but lengthy regeneration times, VIGS provides a rapid preliminary screening tool before committing to more resource-intensive stable transformation experiments [17].

Cost-Effectiveness and Resource Efficiency

VIGS offers significant economic advantages over stable transformation methods, requiring minimal specialized equipment and substantially reduced labor inputs. The technology eliminates the need for expensive tissue culture facilities, selectable markers, and the extensive personnel time required for regenerating and maintaining stable transgenic lines [16] [17]. This cost efficiency makes VIGS particularly accessible for research groups with limited budgets or those working with non-model plant species where established transformation protocols may not exist.

The resource efficiency of VIGS extends beyond direct financial considerations. A single VIGS construct can be applied to multiple plants, and the same viral vector system can often be adapted for related species, further enhancing its cost-effectiveness [18]. Additionally, the small insert size required for VIGS (typically 200-500 bp) simplifies cloning procedures and reduces reagent costs compared to the full-length gene constructs often required for overexpression studies [16] [2].

Bypassing Stable Transformation Challenges

Perhaps the most significant advantage of VIGS is its ability to circumvent the technical challenges associated with stable plant transformation. Many agriculturally important crops, including most monocots and perennial woody species, remain recalcitrant to efficient genetic transformation [16] [17]. Even in transformable species, the process is often genotype-dependent, labor-intensive, and time-consuming, requiring specialized expertise [2] [17].

VIGS overcomes these limitations by utilizing the plant's natural viral infection pathways to deliver gene silencing triggers, eliminating the need for tissue culture and plant regeneration [16] [18]. This approach has enabled functional gene studies in numerous transformation-recalcitrant species, including tea oil camellia [16], soybean [17] [9], taro [21], and various woody perennials [3] [2]. Furthermore, VIGS enables the study of essential genes that would be lethal if constitutively silenced in stable transformants, as the timing and extent of silencing can be controlled through the inoculation protocol [20].

Table 1: Comparative Analysis of VIGS Versus Stable Transformation for Gene Function Studies

Parameter	VIGS Approach	Stable Transformation
Time Required	3-6 weeks [16] [18]	6-12 months [17]
Cost per Gene	Low (minimal reagents) [18]	High (tissue culture, selection) [17]
Technical Expertise	Moderate (basic molecular biology) [16]	Advanced (tissue culture specialization) [17]
Equipment Needs	Standard molecular biology lab [22]	Specialized tissue culture facilities [17]
Species Applicability	Broad range, including recalcitrant species [16] [2]	Limited to transformable genotypes [17]
Genotype Dependence	Low to moderate [2]	High [17]
Silencing Stability	Transient (weeks to months) [18]	Stable (through generations) [17]
Essential Gene Studies	Possible (conditional silencing) [20]	Challenging (lethal mutations) [20]

VIGS Applications and Efficiency Across Plant Species

The versatility of VIGS is demonstrated by its successful implementation across diverse plant families, including both dicots and monocots. Recent methodological advances have extended its application to increasingly challenging species, including those with woody tissues, strong lignification, and robust cuticles that traditionally resist viral infection [16].

Table 2: VIGS Efficiency Across Diverse Plant Species

Plant Species	Viral Vector	Target Gene	Silencing Efficiency	Application Reference
Camellia drupifera (tea oil camellia)	TRV	CdCRY1, CdLAC15	69.8-90.9% [16]	Pericarp pigmentation [16]
Glycine max (soybean)	TRV	GmPDS, GmRpp6907	65-95% [17] [9]	Disease resistance [17]
Styrax japonicus	TRV	PDS	74.19-83.33% [22]	Method optimization [22]
Colocasia esculenta (taro)	TRV	CePDS, CeTCP14	20-27.77% [21]	Starch metabolism [21]
Capsicum annuum (pepper)	TRV, BBWV2, CMV	Multiple	Variable by system [2]	Fruit quality, disease resistance [2]
Nicotiana benthamiana	TRV, TMV, PVX	High-throughput	High efficiency [2] [20]	Model system [2] [20]

VIGS has been successfully employed to characterize genes involved in diverse biological processes:

• Abiotic stress tolerance: Identification of genes involved in drought, salt, oxidative, and nutrient-deficiency stress responses [18]
• Biotic stress resistance: Functional analysis of disease resistance genes against bacterial, oomycete, fungal, and viral pathogens [2] [17]
• Metabolic pathways: Elucidation of genes controlling specialized metabolism, including anthocyanin biosynthesis [16], starch accumulation [21], and capsaicinoid production [2]
• Plant development: Characterization of genes regulating architecture, flowering, and fruit development [2] [20]
• Epigenetic regulation: Induction of heritable epigenetic modifications through RNA-directed DNA methylation [3]

Experimental Protocol: TRV-Mediated VIGS

Vector Construction and Clone Verification

The Tobacco Rattle Virus (TRV)-based system is one of the most widely used VIGS platforms due to its broad host range, efficient systemic movement, and mild viral symptoms [16] [2]. The protocol begins with the identification of a unique 200-500 bp fragment from the target gene coding sequence. This fragment should be screened for specificity using tools such as the SGN VIGS Tool to minimize off-target silencing [16].

Step-by-Step Procedure:

• Target Fragment Selection: Identify a unique 200-300 bp region with no significant homology to non-target genes using sequence analysis tools [16] [17]
• Primer Design: Incorporate appropriate restriction sites (e.g., EcoRI, XhoI) at the 5' ends of gene-specific primers to facilitate directional cloning [17] [9]
• PCR Amplification: Amplify the target fragment from cDNA using high-fidelity DNA polymerase under the following conditions:
  • Initial denaturation: 98°C for 4 minutes
  • 30 cycles of: 98°C for 10s, 59°C for 15s, 72°C for 20s
  • Final extension: 72°C for 5 minutes [16]
• Cloning into TRV2 Vector: Digest both the PCR product and pTRV2 vector with appropriate restriction enzymes, followed by ligation and transformation into E. coli DH5α competent cells [17] [9]
• Sequence Verification: Confirm insert sequence fidelity through Sanger sequencing before proceeding to Agrobacterium transformation [16] [17]

Agrobacterium Preparation and Inoculation

The success of VIGS critically depends on efficient delivery of the viral vector into plant cells. Agrobacterium tumefaciens-mediated transformation represents the most widely used delivery method [16] [17].

Agrobacterium Culture Protocol:

• Transform Agrobacterium: Introduce verified recombinant plasmids (TRV1, TRV2-target gene) into Agrobacterium strain GV3101 using freeze-thaw or electroporation methods [17] [21]
• Starter Culture: Inoculate single colonies into 4 mL YEB medium containing appropriate antibiotics (25 μg/mL kanamycin, 50 μg/mL rifampicin) and incubate at 28°C with shaking at 200-240 rpm for 2 days [16]
• Scale-up Culture: Transfer homogeneous agrobacteria solution to 50 mL YEB medium supplemented with 5 mL MES (pH 5.6, 0.2 M) and 5 μL acetosyringone (0.1 M). Dilute at 1:20 ratio and incubate at 28°C with shaking until OD600 reaches 0.9-1.0 [16]
• Harvest Cells: Centrifuge culture at 5000 rpm for 15 minutes and resuspend pellet in infiltration medium (10 mM MgCl2, 10 mM MES, 200 μM acetosyringone) to final OD600 of 0.6-1.5 (optimize for specific plant species) [16] [22] [21]
• Acclimation: Incubate the resuspended Agrobacterium solution at room temperature for 3-6 hours without shaking before plant inoculation [17]

Plant Inoculation Methods: The optimal inoculation method varies significantly depending on plant species and tissue type:

• Leaf Infiltration: Using a needleless syringe to infiltrate Agrobacterium suspension into the abaxial side of leaves [2] [21]
• Pericarp Cutting Immersion: For firmly lignified capsules as in Camellia drupifera, achieved ~93.94% infiltration efficiency [16]
• Cotyledon Node Immersion: For soybean with thick cuticles and dense trichomes, bisect swollen sterilized seeds and immerse fresh explants for 20-30 minutes, achieving up to 95% infection efficiency [17] [9]
• Vacuum Infiltration: Applied to whole seedlings or specific tissues like taro bulbs under optimized pressure and duration conditions [22] [21]
• Fruit-Bearing Shoot Infusion: For silencing genes during specific developmental stages in perennial species [16]

Figure 2: VIGS Experimental Workflow. The process begins with target fragment selection and cloning into the TRV2 vector, followed by Agrobacterium transformation and culture. Plants are inoculated using various methods optimized for specific species and tissues. After incubation for several weeks, silencing efficiency is analyzed through phenotypic and molecular assessments to validate gene function.

Post-Inoculation Procedures and Silencing Validation

Following inoculation, proper plant maintenance is crucial for achieving efficient systemic silencing:

• Environmental Conditions: Maintain inoculated plants under moderate temperature (20-25°C) and high humidity conditions for 24-48 hours to facilitate infection [2]
• Long-Term Growth: Transfer plants to standard growth conditions appropriate for the species and maintain for 2-6 weeks to allow systemic silencing development [16] [17]
• Phenotypic Monitoring: Document emerging phenotypes compared to control plants inoculated with empty vector [17]

Silencing Validation Methods:

• Quantitative PCR: Measure target gene expression reduction in silenced tissues compared to controls [16] [17]
• Phenotypic Assessment: Document visible phenotypes, such as photobleaching for PDS-silenced plants [17] [21]
• Biochemical Analysis: Assess changes in metabolite levels (e.g., chlorophyll content, starch accumulation) corresponding to target gene function [16] [21]
• Fluorescence Imaging: For vectors incorporating GFP markers, visualize infection efficiency and spatial distribution [17] [9]

Essential Research Reagent Solutions

Successful implementation of VIGS requires carefully selected reagents and vectors optimized for specific applications. The following table details key research reagent solutions for establishing VIGS in various plant systems.

Table 3: Essential Research Reagents for VIGS Implementation

Reagent/Vector	Specifications	Function in VIGS	Application Notes
TRV-Based Vectors	Bipartite system (TRV1, TRV2); TRV1 encodes replicase and movement proteins; TRV2 contains CP and MCS [2]	Primary vector for gene silencing; broad host range	Mild symptoms, efficient systemic movement [2] [17]
Agrobacterium tumefaciens	Strain GV3101; containing pTRV1 and pTRV2-derived plasmids [17] [9]	Delivery vehicle for viral vectors	Optimal OD600 = 0.6-1.5; varies by species [16] [22]
Acetosyringone	0.1 M stock solution in DMSO or ethanol [16]	Vir gene inducer; enhances T-DNA transfer	Critical for efficient transformation; use 200 μM final concentration [16] [22]
Infiltration Medium	10 mM MgCl2, 10 mM MES, 200 μM acetosyringone [16]	Suspension medium for Agrobacterium	Maintains bacterial viability during inoculation [16]
Antibiotics	Kanamycin (25-50 μg/mL), rifampicin (50 μg/mL) [16] [17]	Selection for binary vectors and Agrobacterium strain	Concentrations vary by vector system and strain [16]
PDS Reference Gene	Phytoene desaturase gene fragment (200-500 bp) [17] [21]	Positive control for silencing; causes photobleaching	Universal marker across plant species [17] [21]

Critical Factors for Optimization

The efficiency of VIGS is influenced by multiple experimental parameters that require optimization for each plant system:

• Agrobacterium Concentration: Optimal OD600 ranges from 0.6-1.5, with higher concentrations (OD600 = 1.0) increasing silencing efficiency in taro from 12.23% to 27.77% [21]
• Plant Developmental Stage: Silencing efficiency varies significantly with developmental stage, achieving 69.80% efficiency at early stage versus 90.91% at mid-stage for different genes in Camellia drupifera [16]
• Inoculation Method: Selection of appropriate delivery method is species-dependent, with cotyledon node immersion outperforming leaf infiltration in soybean [17]
• Environmental Conditions: Temperature, humidity, and light intensity significantly impact viral replication and movement, thereby affecting silencing efficiency [2]
• Insert Design: Fragment size (optimal 200-500 bp), specificity, and position within the coding sequence influence silencing efficiency and duration [16] [2]

Virus-induced gene silencing represents a powerful functional genomics tool that combines rapid implementation, cost-effectiveness, and the unique ability to bypass stable transformation. The continued refinement of VIGS protocols, coupled with the development of novel viral vectors and delivery methods, has expanded its application to an increasingly diverse range of plant species. The integration of VIGS with emerging technologies such as virus-induced genome editing and epigenetic modification further enhances its potential for accelerating crop improvement programs. By providing a transient yet highly specific means of gene knockdown, VIGS enables researchers to rapidly characterize gene function and identify valuable traits for molecular breeding, establishing it as an indispensable component of the modern plant biotechnology toolkit.

In the field of functional genomics, forward and reverse genetics represent two complementary philosophies for connecting genotypes to phenotypes. Forward genetics begins with an observable trait and works to identify the underlying genetic cause, while reverse genetics starts with a known gene sequence and investigates its functional role by observing phenotypic consequences of its disruption [23] [24]. These approaches are fundamental to advancing our understanding of biological systems, with Virus-Induced Gene Silencing (VIGS) emerging as a particularly powerful tool for reverse genetics in a wide range of plant species [2].

The integration of these methodologies within plant VIGS validation frameworks has transformed functional genomics, enabling researchers to rapidly characterize gene function without the need for stable transformation. This article provides a comprehensive overview of the ideal applications for forward and reverse genetics, with detailed protocols and resources to facilitate their implementation in both model and non-model species.

Conceptual Foundations and Comparative Analysis

Defining the Approaches

Forward genetics represents a phenotype-driven discovery process. Researchers begin with a mutant phenotype of interest and employ techniques such as genome-wide association studies (GWAS) and quantitative trait locus (QTL) mapping to pinpoint the causal genetic variants [23] [24]. This approach is particularly valuable for identifying novel genes involved in biological processes without prior knowledge of the genetic architecture.

In contrast, reverse genetics is a gene-driven strategy that investigates gene function through targeted disruption or modification. VIGS exemplifies this approach by using recombinant viral vectors to trigger sequence-specific silencing of endogenous genes [2]. This method allows for direct functional assessment of candidate genes identified through genomic studies.

Comparative Advantages and Applications

Table 1: Comparative Analysis of Forward and Reverse Genetic Approaches

Aspect	Forward Genetics	Reverse Genetics
Starting Point	Observable phenotype [23] [24]	Known gene or sequence [23] [2]
Methodology	GWAS, QTL mapping, positional cloning [23] [24]	VIGS, CRISPR/Cas, targeted mutagenesis [23] [25] [2]
Primary Strength	Unbiased discovery of novel genes [23]	Direct functional validation of candidate genes [23] [2]
Typical Timeframe	Longer duration due to mapping requirements [23]	Relatively shorter for initial validation [23] [9]
Key Challenge	Fine-mapping and gene identification [24]	Efficient gene disruption and phenotype interpretation [23] [25]
Ideal Application	Natural variation studies, trait dissection [23] [24]	Functional validation, pathway analysis [23] [9] [16]

Forward Genetics: Principles and Protocols

Genomic Screening in Non-Model Species

Forward genomic screens have proven highly effective in non-model organisms. The Macaque Biobank project exemplifies this approach, where researchers sequenced 919 Chinese rhesus macaques and assessed 52 phenotypic traits. Through genome-wide association analysis, they identified 30 independent loci significantly associated with phenotypic variations, demonstrating the power of forward genetics in species without extensive genetic tools [23].

Key Experimental Protocol: Forward Genetic Screen

Table 2: Key Research Reagents for Forward Genetic Screening

Reagent/Category	Specific Examples	Function/Application
Population Resources	Captive CRM cohort (n=919) [23]	Provides genetic diversity for association studies
Genotyping Technology	High-depth sequencing (~30.47X) [23]	Identifies sequence variations across genome
Phenotypic Assessment	52 quantitatively measured traits [23]	Provides measurable traits for association mapping
Analysis Tools	GWAS pipeline, FRAPPE [23]	Identifies trait-associated loci, infers ancestry
Validation Methods	Loss-of-function variant analysis [23]	Confirms functional impact of associated variants

Procedure:

• Population Selection: Establish a genetically diverse population with sufficient sample size (e.g., 919 individuals) to ensure statistical power [23]
• Deep Sequencing: Perform high-coverage whole-genome sequencing (mean depth ~30.47X) to comprehensively identify genetic variants [23]
• Variant Calling: Implement stringent quality controls to generate high-quality variant sets (84,480,388 variants in macaque study) [23]
• Phenotypic Characterization: Systematically measure quantitative traits relevant to research objectives (52 traits in macaque study) [23]
• Association Analysis: Conduct GWAS using appropriate statistical models to identify significant genotype-phenotype associations [23]
• Functional Validation: Confirm biological significance of associated variants through complementary approaches [23]

Figure 1: Forward Genetics Workflow: This diagram illustrates the phenotype-driven approach of forward genetics, from initial observation to gene identification.

Reverse Genetics: Principles and Protocols

VIGS-Based Approaches in Plants

VIGS has emerged as a powerful reverse genetics tool, particularly valuable for non-model species and those recalcitrant to stable transformation. The technique exploits the plant's post-transcriptional gene silencing (PTGS) machinery, using recombinant viral vectors to trigger systemic suppression of target genes [2]. TRV-based VIGS has been successfully established in multiple plant systems, including soybean [9] and Camellia drupifera [16], with silencing efficiencies reaching 65-95% [9] and even 93.94% in optimized systems [16].

Key Experimental Protocol: TRV-Based VIGS

Table 3: Key Research Reagents for VIGS-Based Reverse Genetics

Reagent/Category	Specific Examples	Function/Application
Viral Vectors	pTRV1, pTRV2 [9] [16]	Bipartite TRV system for infection and silencing
Agrobacterium Strain	GV3101 [9]	Delivery vehicle for viral vectors
Target Gene Constructs	pTRV2-GmPDS, pTRV2-CdCRY1 [9] [16]	Contains target gene fragment for silencing
Infiltration Medium	YEB with antibiotics, MES, acetosyringone [16]	Supports Agrobacterium growth and virulence
Plant Material	Soybean cotyledons, Camellia capsules [9] [16]	Tissue targets for VIGS implementation

Procedure:

• Vector Construction: Clone 200-300 bp fragment of target gene into pTRV2 vector using appropriate restriction sites (EcoRI and XhoI) [9]
• Agrobacterium Preparation: Transform recombinant plasmids into Agrobacterium GV3101; culture in YEB medium with kanamycin (25 μg/mL) and rifampicin (50 μg/mL); induce with acetosyringone (200 μM) [9] [16]
• Plant Material Preparation: For soybean, use cotyledon node explants; for Camellia capsules, select appropriate developmental stages [9] [16]
• Inoculation: Employ optimized delivery methods - cotyledon node immersion for soybean (20-30 minutes), pericarp cutting immersion for Camellia capsules [9] [16]
• Incubation: Maintain inoculated plants under appropriate conditions (temperature, humidity, photoperiod) for systemic silencing [2]
• Phenotypic Assessment: Monitor for visible phenotypes (e.g., photobleaching for GmPDS silencing) at 21 days post-inoculation [9]
• Efficiency Validation: Quantify silencing through qPCR and document phenotypic changes statistically [9] [16]

Table 4: Quantitative Silencing Efficiencies in Various Plant Systems

Plant Species	Target Gene	Silencing Efficiency	Key Optimization Factor
Soybean [9]	GmPDS	65-95%	Cotyledon node immersion method
Camellia drupifera [16]	CdCRY1	~69.80%	Early capsule development stage
Camellia drupifera [16]	CdLAC15	~90.91%	Mid capsule development stage
Soybean [9]	GmRpp6907	High (systemic)	Agrobacterium infection efficiency >80%

Figure 2: Reverse Genetics Workflow: This diagram illustrates the gene-driven approach of reverse genetics using VIGS, from sequence knowledge to phenotypic analysis.

Integrated Applications in Model and Non-Model Species

Synergistic Approaches

The most powerful functional genomics strategies often integrate both forward and reverse genetics. The Macaque Biobank project exemplifies this synergy by combining forward genomic screens to identify phenotype-associated variants with reverse genomic approaches to validate specific candidates like DISC1 (p.Arg517Trp) as a genetic risk factor for neuropsychiatric disorders [23]. This integrated methodology accelerates both gene discovery and functional validation.

Technology Integration for Enhanced Screening

Emerging technologies are further enhancing both approaches. For non-model species with limited genomic resources, pipelines like NEEDLE enable gene discovery by leveraging transcriptomic dynamics to identify key regulatory networks [26]. Similarly, CRISPR/Cas systems are being adapted for non-model organisms through improved genome annotation and guide design strategies [25]. VIGS has been successfully integrated with multi-omics technologies to advance functional genomics studies in various crops, including pepper [2].

Forward and reverse genetics provide powerful, complementary frameworks for functional genomics in both model and non-model species. While forward genetics enables unbiased discovery of novel genes underlying important traits, reverse genetics offers direct functional validation of candidate genes. VIGS has emerged as a particularly valuable tool for reverse genetics in species resistant to stable transformation. The continued refinement of these approaches, along with their integration with emerging technologies, promises to accelerate gene function discovery and facilitate the development of improved crop varieties and biomedical models.

Advanced VIGS Protocols: From Vector Design to Agroinfiltration in Diverse Species

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapid functional analysis of plant genes. This technology exploits the plant's innate RNA-based antiviral defense mechanism, whereby recombinant viral vectors carrying host gene fragments trigger sequence-specific degradation of complementary endogenous mRNAs. The efficiency of VIGS is profoundly influenced by the strategic design of the insert fragment incorporated into the viral vector. This application note synthesizes evidence-based guidelines for optimizing insert design, focusing on three critical parameters: fragment length, positional selection within the target cDNA, and avoidance of homopolymeric regions. Proper consideration of these factors enables researchers to maximize silencing efficiency, minimize viral symptoms, and obtain consistent, interpretable phenotypes in a broad range of plant species, from model organisms to recalcitrant crops.

Core Principles of Optimal Insert Design

Fragment Length

Table 1: Optimal Insert Length Ranges for Different VIGS Vector Systems

Vector System	Optimal Insert Length	Experimental Basis	Host Plant
TRV (Tobacco Rattle Virus)	200–1300 bp [27] [28]	Systematic testing of NbPDS fragments; efficient silencing observed with 192-1304 bp inserts	Nicotiana benthamiana
TRV	~300–500 bp [8]	Common practice for agroinfiltration-based VIGS; balances efficiency and stability	Various Solanaceae
BMV (Brome Mosaic Virus)	~100 nt [29]	Smaller inserts were more stable and provided higher silencing efficiency in wheat	Hexaploid Wheat
TCV (Turnip Crinkle Virus)	~100 nt [30]	Optimal insertion size for the CPB1B vector	Arabidopsis thaliana

The optimal insert length varies significantly depending on the viral vector system employed. For the widely used Tobacco Rattle Virus (TRV) system, a systematic study silencing the phytoene desaturase (PDS) gene in Nicotiana benthamiana demonstrated that inserts ranging from 192 bp to 1304 bp led to efficient silencing, as quantified by leaf chlorophyll a levels [27]. Fragments shorter than 192 bp, particularly those of 103 bp and 54 bp, showed substantially reduced efficiency [27]. In practice, many protocols recommend constructs of 300–500 bp for TRV-based silencing [8], providing a practical balance between silencing efficiency and ease of cloning.

In contrast, for the Brome Mosaic Virus (BMV) system in hexaploid wheat, time-course experiments revealed that smaller inserts of approximately 100 nucleotides were more stable in the BMVCP5 vector and conferred higher silencing efficiency and longer silencing duration compared to larger inserts [29]. Similarly, the optimal insertion size for a novel Turnip Crinkle Virus (TCV) derivative (CPB1B) in Arabidopsis thaliana was found to be around 100 nt [30]. These findings highlight the necessity of vector-specific optimization for fragment length.

Insert Position within the cDNA

The region of the cDNA from which the silencing fragment is derived significantly impacts silencing efficiency. Evidence from TRV-mediated silencing of the NbPDS gene indicates that fragments originating from the middle of the cDNA sequence perform superiorly compared to those from the 5' or 3' ends [27] [28].

This positional effect may be attributed to several factors, including the accessibility of the target mRNA region within the ribonucleoprotein complex, the distribution of effective small interfering RNA (siRNA) target sites, or the secondary structure of the mRNA. Consequently, for the highest probability of successful silencing, researchers should prioritize the amplification of insert fragments from the central coding region of the target gene.

Avoidance of Homopolymeric Regions

The inclusion of homopolymeric sequences, such as poly(A) or poly(G) tracts, is detrimental to VIGS efficiency. Experimental data shows that a 24 bp poly(A) or poly(G) homopolymeric region within an insert can reduce silencing efficiency [27] [28].

Homopolymeric regions can potentially interfere with viral replication or movement, compromise the stability of the recombinant viral genome, or disrupt the generation of a diverse siRNA pool. Therefore, special care must be taken during insert design to exclude homopolymeric stretches and poly(A/T) tails commonly found in conventional oligo(dT)-primed cDNA libraries [27].

Experimental Protocols for Validation

Protocol: Assessing Silencing Efficiency via Visual Marker Genes

Purpose: To empirically determine the optimal insert design parameters using a visual marker gene. Recommended Visual Markers: Phytoene Desaturase (PDS), which causes photobleaching [31], or Chalcone Synthase (CHS), which leads to white pigmentation in floral tissues [31].

• Fragment Amplification: Amplify multiple fragments of your target gene (e.g., PDS) that vary in length (e.g., 100 bp, 200 bp, 500 bp, 1000 bp) and positional origin (5', middle, 3').
• Vector Construction: Clone these fragments into your chosen VIGS vector (e.g., pTRV2) using appropriate cloning techniques (e.g., Gateway recombination [8] or ligation-independent cloning).
• Plant Inoculation:
  • Introduce the recombinant vectors into Agrobacterium tumefaciens.
  • For N. benthamiana, grow plants to the 4-leaf stage [8]. Resuspect Agrobacterium cultures carrying pTRV1 and pTRV2-derived constructs to an OD600 of ~0.5-1.0 in infiltration buffer (10 mM MES, 10 mM MgCl2, 200 μM acetosyringone) [22] [31].
  • Mix the cultures 1:1 and infiltrate into the abaxial side of leaves using a needleless syringe [8].
• Phenotypic Evaluation: Maintain plants under optimal conditions (e.g., 20°C day/18°C night for some species [31]) and monitor for the development of visual silencing phenotypes (e.g., bleaching for PDS) in systemic leaves over 2-4 weeks.
• Efficiency Quantification:
  • Visual Scoring: Rate the extent and intensity of silencing symptoms.
  • Biochemical Assay: Measure chlorophyll a content for PDS silencing as a quantitative metric [27].
  • Molecular Validation: Use quantitative RT-PCR to assess the reduction in endogenous target gene transcript levels.

Protocol: Designing and Testing Inserts for a Target Gene of Interest

Purpose: A generalized workflow for designing effective VIGS constructs for any candidate gene.

• Target Sequence Analysis:
  • Obtain the full-length cDNA sequence of your target gene.
  • Use online tools like the SGN VIGS Tool (https://vigs.solgenomics.net/) to screen for suitable 200-300 bp fragments [16].
• Fragment Selection:
  • Prioritize a fragment from the middle coding region of the gene.
  • Ensure the final length is within the optimal range for your vector (e.g., 200-500 bp for TRV).
  • Avoid fragments containing homopolymeric regions (e.g., polyA tails) or sequences with high similarity to non-target genes (BLAST against the host genome).
• Specificity Check: Perform a homology search (e.g., BLASTN) against the host plant's genome to ensure the selected fragment has minimal off-target potential. Select sequences with high similarity to the target gene and < 40% similarity to other genes [16].
• Construct Assembly: Clone the validated fragment into the VIGS vector and transform into Agrobacterium.
• Functional Validation: Inoculate plants and assess silencing through molecular (qRT-PCR) and phenotypic analysis.

The following diagram illustrates the logical workflow and decision points for designing an optimal VIGS insert.

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Reagent Solutions for VIGS Insert Design and Validation

Reagent / Resource	Function / Purpose	Example / Notes
VIGS Vectors	Delivery of host gene fragment to trigger silencing.	TRV-based vectors (pTRV1, pTRV2): Broad host range, meristem invasion [2] [8]. BMVCP5: For monocots like wheat; superior insert stability [29].
Visual Marker Genes	Empirical optimization and visual assessment of silencing efficiency.	PDS (Phytoene Desaturase): Silencing causes photobleaching [27] [31]. CHS (Chalcone Synthase): Silencing blocks anthocyanin, causing white patches [31].
Bioinformatics Tools	In silico design and specificity check of insert fragments.	SGN VIGS Tool: Designs specific inserts and checks for off-targets [16]. siRNA Target Finder: Predicts potent siRNA sequences within a fragment [30]. BLAST: Confirms fragment specificity against host genome.
Cloning Systems	Efficient and high-throughput insertion of fragments into VIGS vectors.	Gateway Cloning: Uses att site-specific recombination; available for pTRV2 [8]. Ligation-Independent Cloning (LIC): Avoids restriction enzymes; used in TRV-LIC vectors [8].
Agrobacterium Strains	Delivery of T-DNA containing the VIGS vector into plant cells.	GV3101, LBA4404: Common strains for agroinfiltration [16] [31]. Cultured in acetosyringone-containing medium for virulence induction.

The efficacy of Virus-Induced Gene Silencing is profoundly dependent on rational insert design. Empirical evidence dictates that researchers should select fragments from the central region of the target cDNA, tailor the fragment length to the specific viral vector system in use, and rigorously exclude homopolymeric sequences. Adherence to these guidelines, coupled with the use of optimized protocols and reagents detailed in this application note, provides a robust framework for maximizing silencing efficiency. This enables high-confidence functional gene characterization in reverse genetics screens and facilitates the advancement of functional genomics across a diverse spectrum of plant species.

Agrobacterium-mediated transformation is a cornerstone technique in plant biotechnology and is particularly valuable for the initial validation of gene function in virus-induced gene silencing (VIGS) studies. For plant species where stable transformation is challenging or time-consuming, transient transformation systems offer a rapid and effective alternative. This protocol details three efficient methods—cotyledon node immersion, vacuum infiltration, and direct injection—to deliver genetic material into plant tissues. These techniques are indispensable for researchers aiming to quickly validate the function of genes involved in signaling pathways or to silence target genes via VIGS before embarking on more labor-intensive stable transformation.

Method Comparison and Selection

The choice of transformation method depends on several factors, including plant species, developmental stage, and experimental objectives. The table below summarizes the key parameters and applications for each method.

Table 1: Comparative overview of Agrobacterium-mediated transient transformation methods

Method	Optimal Plant Stage	Key Parameters	Transformation Efficiency	Best For
Infiltration (Cotyledon Node Immersion) [32]	3-day-old hydroponically grown seedlings [32]	OD~600~: 0.8; Silwet L-77: 0.02%; Infiltration Time: 2 hours [32]	>90% [32]	High-throughput transformation of young, tender seedlings.
Direct Injection [32]	4 to 6-day-old soil-grown seedlings [32]	OD~600~: 0.8; Silwet L-77: 0.02%; Post-injection dark culture: 3 days [32]	>90% [32]	Targeted delivery into specific tissues like cotyledons.
Ultrasonic-Vacuum [32]	3-day-old Petri dish-cultured seedlings [32]	OD~600~: 0.8; Ultrasonication: 40 kHz, 1 min; Vacuum: 0.05 kPa, 5-10 min [32]	>90% [32]	Difficult-to-transform tissues, enhancing Agrobacterium entry.

Detailed Experimental Protocols

Protocol 1: Cotyledon Node Immersion (Infiltration)

This method is ideal for achieving high transformation efficiency in young seedlings.

• Agrobacterium Culture Preparation: Grow Agrobacterium tumefaciens strain GV3101 carrying your binary vector (e.g., pBI121 with a GUS reporter) in an appropriate antibiotic selection medium. Resuspend the bacterial pellet in an infiltration buffer (e.g., 10 mM MES, 10 mM MgCl~2~, 150 µM acetosyringone) to a final OD~600~ of 0.8 [32].
• Add Surfactant: Supplement the bacterial suspension with 0.02% Silwet L-77 to reduce surface tension and promote infiltration [32].
• Infiltrate Plant Material: Immerse 3-day-old hydroponically grown seedlings entirely in the bacterial suspension for 2 hours with gentle agitation [32].
• Post-Infiltration Culture: Gently remove the seedlings from the suspension, blot off excess liquid, and transfer them to fresh medium or soil. Culture the plants under standard growth conditions. Gene expression can typically be detected for at least 6 days [32].

Protocol 2: Direct Injection

This technique allows for localized transformation of specific plant organs.

• Prepare Bacterial Suspension: Prepare Agrobacterium strain GV3101 at an OD~600~ of 0.8 in an induction buffer with 0.02% Silwet L-77 [32].
• Inject Plant Tissue: Using a needleless syringe, gently press the syringe tip against the surface of the cotyledon of a 4 to 6-day-old soil-grown seedling and slowly inject the bacterial suspension. A small, water-soaked area will appear at the injection site [32].
• Post-Injection Incubation: Following injection, maintain the seedlings in the dark at room temperature for three days to facilitate T-DNA transfer and transgene expression [32].

Protocol 3: Ultrasonic-Vacuum Infiltration

This combined method uses physical forces to enhance Agrobacterium entry, especially useful for more recalcitrant tissues.

• Prepare Seedlings and Bacteria: Place 3-day-old seedlings cultured on Petri dishes in a container with the Agrobacterium suspension (OD~600~ = 0.8 with 0.02% Silwet L-77) [32].
• Apply Ultrasonication: Subject the container to ultrasonication at a frequency of 40 kHz for 1 minute. This creates micro-pores in the plant cell walls [32].
• Apply Vacuum Infiltration: Immediately after sonication, transfer the container to a vacuum desiccator. Apply a vacuum of 0.05 kPa for 5 to 10 minutes. The vacuum forces the bacterial solution into the intercellular spaces, and its rapid release helps drive the bacteria into the tissues [32].
• Recovery: Remove the seedlings from the suspension and culture them under standard conditions.

Experimental Workflow

The following diagram illustrates the logical workflow for selecting and executing the appropriate transformation method, from preparation to analysis.

The Scientist's Toolkit: Research Reagent Solutions

Successful implementation of these protocols relies on key reagents and materials. The following table lists essential items and their functions.

Table 2: Essential reagents and materials for Agrobacterium-mediated transient transformation

Reagent/Material	Function/Application	Example Usage in Protocol
Agrobacterium tumefaciens GV3101	A disarmed strain used as a vector to deliver T-DNA containing the gene of interest (e.g., VIGS construct) into the plant cell [32].	Used in all three methods as the delivery vehicle.
Binary Vector (e.g., pBI121)	Plasmid carrying the T-DNA with the gene to be expressed or the VIGS fragment, along with plant and bacterial selection markers [32].	Engineered to carry the GUS reporter gene or a VIGS cassette in the referenced studies [32].
Silwet L-77	A surfactant that reduces the surface tension of the bacterial suspension, enabling it to spread and infiltrate plant tissues more effectively [32].	Added at 0.02% to the infiltration buffer for all methods [32].
Acetosyringone	A phenolic compound that induces the expression of Agrobacterium's vir genes, enhancing the efficiency of T-DNA transfer [33].	Used at 200 µM in the infiltration buffer for the transformation of Paeonia ostii [33].
GUS Reporter Gene (β-glucuronidase)	A common reporter gene used to visually assess transformation efficiency through a histochemical staining assay [32] [33].	Used to optimize and confirm transformation efficiency in the outlined protocols [32].

The Agrobacterium-mediated delivery methods detailed here—cotyledon node immersion, direct injection, and ultrasonic-vacuum infiltration—provide researchers with a versatile toolkit for rapid transient transformation. By following the optimized parameters and selecting the method appropriate for their plant material, scientists can achieve high transformation efficiencies exceeding 90%. These protocols are directly applicable for the initial, high-throughput validation of gene function in VIGS experiments, significantly accelerating the pace of research in plant molecular biology and functional genomics.

Virus-induced gene silencing (VIGS) has emerged as an indispensable reverse genetics tool for plant functional genomics, particularly for species recalcitrant to stable genetic transformation [2]. This technique leverages the plant's innate RNA interference machinery, using recombinant viral vectors to trigger sequence-specific degradation of target endogenous mRNAs [16]. While VIGS is well-established in model plants, its application to challenging species—including various crops and perennial woody plants—demands extensive protocol optimization encompassing vector delivery, plant developmental stage, and environmental conditions [34]. This application note synthesizes recent methodological advances in adapting VIGS for four challenging systems: soybean, sunflower, tea oil camellia, and woody tissues, providing researchers with standardized protocols for functional gene validation.

Comparative Analysis of VIGS Efficiency Across Species

Table 1: Summary of Optimized VIGS Parameters Across Challenging Species

Plant Species	Optimal Vector	Delivery Method	Developmental Stage	Key Parameters	Silencing Efficiency	Primary Applications
Soybean (Glycine max)	TRV	Cotyledon node immersion	3-4 days post-germination	OD~600~ = 0.9-1.0; 20-30 min immersion	65-95% [9]	Disease resistance (GmRpp6907, GmRPT4) [9]
Sunflower (Helianthus annuus)	TRV	Seed vacuum infiltration	Germinated seeds (coat removed)	6 h co-cultivation; 45% humidity	62-91% (genotype-dependent) [34]	Functional genomics (HaPDS) [34]
Tea Oil Camellia (Camellia drupifera)	TRV	Pericarp cutting immersion	279 days post-pollination	Early stage (CdCRY1); Mid stage (CdLAC15)	~93.94% (infection); ~69.80-90.91% (silencing) [16]	Pigmentation studies (CdCRY1, CdLAC15) [16]
Woody Tissues (General)	TRV	Direct injection/Pericarp cutting	Varies by species	Enhanced VSRs (e.g., C2bN43); Low temperature (20°C)	Varies significantly	Secondary metabolism, stress response [35]

Table 2: Troubleshooting VIGS in Challenging Species

Challenge	Potential Causes	Solutions	Supporting Evidence
Low infection efficiency	Thick cuticle, dense trichomes, lignified tissues	Cotyledon node immersion (soybean); Seed vacuum infiltration (sunflower); Pericarp cutting (camellia)	Soybean: >80% infection efficiency [9]; Sunflower: Up to 91% infection [34]
Inconsistent silencing	Improper developmental stage, suboptimal environmental conditions	Stage-specific inoculation; Temperature control (20°C day/18°C night)	Camellia: Stage-dependent efficiency (69.8-90.91%) [16]; Petunia: Improved silencing at lower temperatures [31]
Viral symptoms in controls	Empty vector toxicity	Use non-plant insert (e.g., GFP) in control vector	Petunia: pTRV2-sGFP eliminates severe necrosis [31]
Limited systemic movement	Restricted vascular transport in woody tissues	Engineer VSRs (e.g., C2bN43) to enhance mobility	Pepper: TRV-C2bN43 enhances systemic silencing [35]
Genotype dependency	Natural variation in susceptibility	Screen multiple genotypes; Optimize per cultivar	Sunflower: 62-91% range across genotypes [34]

Species-Specific Protocol Adaptations

Soybean (Glycine max) VIGS Protocol

Background: Soybean presents unique challenges for VIGS due to its thick leaf cuticle and dense trichomes, which impede conventional infiltration methods [9]. The established TRV-based VIGS protocol achieves efficient systemic silencing through cotyledon node transformation.

Detailed Methodology:

• Vector Construction: Clone 200-300bp target gene fragment (e.g., GmPDS) into pTRV2-GFP vector using EcoRI and XhoI restriction sites [9].
• Agrobacterium Preparation:
  • Transform recombinant plasmids into Agrobacterium tumefaciens GV3101.
  • Culture in YEB medium with antibiotics (25 μg/mL kanamycin, 50 μg/mL rifampicin) at 28°C until OD~600~ reaches 0.9-1.0.
  • Centrifuge at 5,000 rpm for 15 minutes and resuspend in infiltration medium (10 mM MgCl~2~, 10 mM MES, 200 μM acetosyringone) [16] [9].
• Plant Material Preparation:
  • Surface-sterilize soybean seeds and germinate for 3-4 days.
  • Bisect seeds longitudinally to create half-seed explants with intact cotyledonary nodes.
• Inoculation:
  • Immerse explants in Agrobacterium suspension (pTRV1 + pTRV2-target) for 20-30 minutes with gentle agitation.
  • Co-cultivate on medium for 2-3 days in dark conditions.
• Plant Growth and Analysis:
  • Transfer plants to nutrient soil and maintain at 20-22°C.
  • Monitor silencing phenotypes from 14-21 days post-inoculation (dpi).
  • Validate silencing efficiency via qRT-PCR and phenotypic assessment [9].

Validation: This protocol achieves 65-95% silencing efficiency, successfully validating genes including GmPDS (photobleaching), GmRpp6907 (rust resistance), and GmRPT4 (defense response) [9].

Sunflower (Helianthus annuus) VIGS Protocol

Background: Sunflower is traditionally considered a challenging species for genetic transformation [34]. The seed vacuum infiltration method establishes a robust VIGS system without requiring in vitro culture steps, significantly expanding functional genomics capabilities in this important oil crop.

Detailed Methodology:

• Vector Design:
  • Identify optimal silencing fragment (193bp for HaPDS) using pssRNAit software.
  • Clone into TRV2 vector (pYL156) via XbaI and BamHI restriction sites [34].
• Agrobacterium Preparation:
  • Transform TRV constructs (pTRV1, pTRV2-empty, pTRV2-HaPDS) into A. tumefaciens GV3101.
  • Culture as described for soybean until OD~600~ = 0.9-1.0.
• Seed Preparation and Infiltration:
  • Remove seed coats carefully to enhance infiltration.
  • Apply vacuum infiltration to seeds in Agrobacterium suspension for 10-15 minutes.
  • Co-cultivate seeds for 6 hours in dark conditions [34].
• Plant Growth and Analysis:
  • Sow treated seeds directly in soil (peat:perlite, 3:1 ratio).
  • Maintain at 22°C with 18h light/6h dark photoperiod and 45% relative humidity.
  • Document silencing progression through time-lapse photography.
  • Confirm TRV presence and distribution via RT-PCR across different plant regions [34].

Validation: This protocol achieves genotype-dependent infection rates of 62-91%, with efficient systemic TRV movement up to node 9 in sunflower plants [34].

Tea Oil Camellia (Camellia drupifera) VIGS Protocol

Background: Functional genomics studies in tea oil camellia and related woody species are hampered by recalcitrance to stable transformation and firmly lignified tissues [16]. The optimized VIGS system enables gene function analysis in pericarp tissues through orthogonal testing of multiple parameters.

Detailed Methodology:

• Target Selection and Vector Construction:
  • Select target genes with visible phenotypic markers (e.g., CdCRY1 for exocarp pigmentation, CdLAC15 for mesocarp coloration).
  • Screen for specific 200-300bp fragments using SGN VIGS Tool with <40% similarity to non-target genes.
  • Clone fragments into pNC-TRV2 vector via Nimble Cloning [16].
• Agrobacterium Preparation:
  • Prepare Agrobacterium cultures as previously described.
  • Mix TRV1 and TRV2-target cultures in 1:1 ratio before inoculation.
• Inoculation of Capsules:
  • Harvest capsules at specific developmental stages (279 days post-pollination optimal).
  • Use pericarp cutting immersion method: make incisions in pericarp and immerse in Agrobacterium suspension.
  • Alternative methods: peduncle injection, direct pericarp injection, or fruit-bearing shoot infusion [16].
• Phenotypic Analysis:
  • Monitor pericarp pigmentation changes from 14-28 dpi.
  • Quantify silencing efficiency via color assessment and qRT-PCR.

Validation: This approach achieves ~93.94% infiltration efficiency with stage-dependent silencing effects: early stage capsules show ~69.80% efficiency for CdCRY1, while mid-stage capsules reach ~90.91% for CdLAC15 [16].

General Adaptations for Woody Tissues

Background: Woody tissues present exceptional challenges for VIGS due to lignified cell walls, limited vascular connectivity, and complex tissue architecture. Recent advances focus on enhancing viral movement and silencing efficacy through molecular and methodological innovations.

Key Adaptations:

• Viral Suppressor Engineering:
  • Utilize truncated viral suppressors of RNA silencing (VSRs) like C2bN43 from Cucumber Mosaic Virus.
  • These mutants retain systemic suppression activity while abolishing local suppression, enhancing long-distance VIGS efficiency [35].
• Temperature Optimization:
  • Maintain plants at 20°C day/18°C night temperatures post-inoculation.
  • Lower temperatures promote viral spread and enhance silencing efficacy in recalcitrant species [31].
• Developmental Timing:
  • Target younger tissues when possible, as silencing spreads more actively in developing versus mature tissues [34].
• Genotype Screening:
  • Evaluate multiple genotypes for VIGS susceptibility, as significant natural variation exists even within species [34].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for VIGS in Challenging Species

Reagent/Vector	Specifications	Function	Example Applications
TRV Vectors	pTRV1 (pYL192); pTRV2 (pYL156/pNC-TRV2)	Bipartite viral system for replication (TRV1) and silencing induction (TRV2)	Soybean, sunflower, camellia, woody plants [16] [9] [34]
Agrobacterium Strain	GV3101 with pMP90 rifampicin resistance	Efficient plant transformation; T-DNA delivery	All species mentioned [9] [34]
Visual Marker Genes	PDS (phytoene desaturase); CHS (chalcone synthase)	Visual silencing indicators (photobleaching, reduced pigmentation)	Efficiency optimization across species [31]
Enhanced VSRs	C2bN43 (truncated CMV 2b protein)	Augments systemic silencing spread; enhances efficacy in reproductive tissues	Pepper anther silencing [35]
Control Vectors	pTRV2-sGFP (non-plant insert)	Minimizes viral symptomology in control plants	Petunia, soybean [9] [31]
Infiltration Medium	10 mM MgCl~2~, 10 mM MES, 200 μM acetosyringone	Agrobacterium resuspension; enhances T-DNA transfer	Standard across protocols [16] [9]

The protocol adaptations detailed in this application note demonstrate that robust VIGS systems can be established for even the most challenging plant species through methodical optimization of delivery methods, developmental timing, and environmental conditions. The comparative analysis reveals that tissue-specific barriers—whether thick cuticles, lignified tissues, or genotype-dependent susceptibility—can be overcome through strategic approaches such as cotyledon node immersion, seed vacuum infiltration, and pericarp cutting. Furthermore, molecular enhancements including engineered viral suppressors and temperature control significantly expand VIGS efficacy. These standardized protocols provide researchers with essential tools for accelerating functional genomics studies in economically important crops and recalcitrant species, ultimately facilitating gene discovery and trait validation for crop improvement programs.

Using Marker Genes (PDS) for Rapid System Optimization and Efficiency Evaluation

Virus-Induced Gene Silencing (VIGS) is a powerful reverse genetics tool for rapid functional analysis of plant genes. The core principle involves using recombinant viral vectors to carry host gene fragments, triggering sequence-specific mRNA degradation and enabling researchers to link gene function to observable phenotypes. A critical factor in the success of VIGS is the initial optimization of the silencing system, a process greatly accelerated by the use of visual marker genes. The phytoene desaturase (PDS) gene, which is involved in carotenoid biosynthesis, serves as an exceptional visual marker for this purpose. Silencing PDS disrupts chlorophyll synthesis, leading to a characteristic photo-bleached or albino phenotype. This provides a rapid, non-destructive, and easily scorable readout of silencing efficiency, allowing researchers to fine-tune parameters like vector design, inoculation methods, and cultivation conditions before applying the system to genes of unknown function. This Application Note details protocols and data for using PDS as a benchmark for optimizing VIGS across diverse plant species.

Quantitative Data on PDS Silencing Efficiency

The PDS gene has been successfully deployed as a visual marker for VIGS optimization in a wide range of crops. The table below summarizes key quantitative data on its silencing efficiency across different plant species, viral vectors, and inoculation methods.

Table 1: Efficiency of PDS as a Visual Marker in VIGS System Optimization

Plant Species	Viral Vector	Inoculation Method	Silencing Efficiency/Phenotype	Key Optimization Insight	Source
Soybean (Glycine max)	Tobacco Rattle Virus (TRV)	Agrobacterium-mediated, cotyledon node immersion	65% to 95% efficiency; systemic photobleaching	Superior to conventional misting/injection due to thick cuticle and dense trichomes. [9]
Tomato (Solanum lycopersicum)	TRV	Agroinjection into detached fruit	100% silencing frequency in fruit	Effective for studies on fruit-specific processes like ripening and carotenoid biosynthesis. [36]
Ridge Gourd (Luffa acutangula)	Cucumber Green Mottle Mosaic Virus (CGMMV)	Agroinfiltration into leaves	Effective photobleaching in leaves and stems	Establishes CGMMV as a viable VIGS vector for cucurbit species. [37]
Highbush Blueberry (Vaccinium corymbosum)	CRISPR/Cas9 (as proof-of-concept)	Agrobacterium-mediated transformation	Albino phenotypes in edited shoots	Confirms successful genetic transformation and editing protocol; PDS useful beyond VIGS. [38]
Pepper (Capsicum annuum)	TRV, BBWV2, CMV, Geminiviruses	Agroinfiltration	High efficiency; used to study fruit quality and disease resistance	Highlights importance of vector choice and agroinfiltration parameters for different species. [2]

Detailed Experimental Protocols

Protocol: TRV-Based VIGS in Soybean Using Cotyledon Node Immersion

This optimized protocol demonstrates a highly efficient method for VIGS in a challenging plant species [9].

I. Research Reagent Solutions

• Vector System: pTRV1 and pTRV2 (e.g., pTRV2-GFP, pTRV2-GmPDS).
• Agrobacterium Strain: GV3101.
• Plant Material: Sterilized soybean seeds.
• Infiltration Buffer: 10 mM MgCl₂, 10 mM MES (pH 5.6), and 200 µM acetosyringone.
• Culture Media: Luria-Bertani (LB) broth with appropriate antibiotics (e.g., Kanamycin 50 mg/L, Rifampicin 25 mg/L).

II. Step-by-Step Methodology

• Vector Construction: Clone a ~300-500 bp fragment of the GmPDS gene into the multiple cloning site of the pTRV2 vector using restriction enzymes (e.g., EcoRI and XhoI) or homologous recombination.
• Agrobacterium Preparation:
  • Transform recombinant pTRV2-PDS and the helper pTRV1 plasmids separately into Agrobacterium tumefaciens GV3101.
  • Inoculate single colonies in LB broth with antibiotics and incubate at 28°C with shaking (250-300 rpm) until the OD₆₀₀ reaches 0.6-1.0.
  • Pellet the bacterial cells by centrifugation and resuspend in infiltration buffer to a final OD₆₀₀ of 0.8-1.0.
  • Incubate the suspension at room temperature for 2-4 hours with gentle agitation.
• Plant Preparation & Inoculation:
  • Soak sterilized soybean seeds in sterile water until swollen.
  • Bisect the seeds longitudinally to create half-seed explants.
  • Mix the pTRV1 and pTRV2-PDS Agrobacterium suspensions in a 1:1 ratio.
  • Immerse the fresh half-seed explants in the mixed bacterial suspension for 20-30 minutes, ensuring full contact.
• Plant Growth and Phenotyping:
  • Co-cultivate the inoculated explants on tissue culture medium under dark conditions for 2-3 days.
  • Transfer plants to a growth chamber with a 16/8 hour light/dark photoperiod at 24-28°C.
  • Monitor plants systemically for the development of photobleaching, typically appearing in cluster buds and new leaves within 14-21 days post-inoculation (dpi).

III. Troubleshooting and Optimization

• Low Infection Efficiency: Ensure explants are fresh and the immersion time is optimized (20-30 min was found to be ideal for soybean) [9].
• No Silencing Phenotype: Verify the integrity of the PDS insert by sequencing and the concentration of the Agrobacterium inoculum.
• Localized Silencing Only: Maintain high humidity post-inoculation to facilitate systemic viral movement.

Workflow: VIGS Optimization Using a PDS Marker Gene

The following diagram illustrates the logical workflow for optimizing a VIGS system using PDS as a visual marker.

Protocol: Agroinfiltration of Detached Tomato Fruit

This protocol is optimized for studying genes involved in fruit ripening and metabolism [36].

I. Research Reagent Solutions

• Vector System: pTRV1 and pTRV2-PDS (e.g., containing a pepper PDS insert with high homology to tomato PDS).
• Agrobacterium Strain: GV3101.
• Plant Material: Tomato fruits at the mature green stage.
• Inoculation Buffer: 10 mM MgCl₂, 10 mM MES (pH 5.6), and 200 µM acetosyringone.

II. Step-by-Step Methodology

• Prepare the Agrobacterium suspension as described in Section 3.1, resuspending to an OD₆₀₀ of 1.0 in inoculation buffer.
• Harvest healthy, uniform tomatoes at the mature green stage.
• Using a 1 mL syringe, inject the Agrobacterium mixture (pTRV1 + pTRV2-PDS) through the carpopodium (fruit stem).
• Allow the suspension to infiltrate the fruit until it becomes visible in the sepals.
• Place the injected fruits in a growth chamber at 23°C with a 16-hour photoperiod and 70% relative humidity.
• Phenotype Evaluation: Within 14 days post-injection, the successful silencing of PDS will result in a characteristic pale-yellow fruit instead of the normal red coloration, due to inhibited carotenoid biosynthesis.

The Scientist's Toolkit: Key Research Reagent Solutions

The successful implementation of PDS-mediated VIGS optimization relies on a core set of reagents and materials, as detailed below.

Table 2: Essential Research Reagent Solutions for VIGS with PDS Marker

Reagent/Material	Function in VIGS Workflow	Specific Examples & Notes
PDS Gene Construct	Serves as the visual reporter for silencing; its disruption causes photobleaching.	A 300-500 bp fragment from the target plant species, cloned into a VIGS vector (e.g., pTRV2-PDS). Cross-species constructs (e.g., pepper PDS in tomato) can be highly effective [36].
Viral Vectors	Engineered to carry the PDS insert, replicate, and spread systemically, inducing silencing.	TRV: Broad host range, mild symptoms [9] [2]. CGMMV: Effective in cucurbits [37]. BPMV: Well-established for soybean [9].
Agrobacterium tumefaciens	Acts as a delivery vehicle to introduce the viral vector into plant cells.	Strain GV3101 is commonly used for its high transformation efficiency [9] [36] [37].
Infiltration Buffer	A solution that facilitates the transfer of the viral vector from Agrobacterium to plant cells.	Typically contains 10 mM MgCl₂, 10 mM MES (pH 5.6), and 200 µM acetosyringone to induce virulence genes [36] [37].
Antibiotics	Selective agents to maintain the viral vector plasmids in bacterial culture.	Kanamycin (for vector selection), Rifampicin (for Agrobacterium strain selection) [36] [37].

Biochemical Pathway and Phenotypic Outcome

The visual phenotype resulting from PDS silencing is a direct consequence of its role in the carotenoid biosynthesis pathway. The diagram below illustrates this pathway and the effect of PDS silencing.

The silencing of PDS halts the conversion of phytoene to ζ-carotene, preventing the synthesis of colored carotenoids like lycopene and β-carotene. This not only causes the characteristic photobleaching but can also have downstream effects, such as altering the expression of ripening-related genes and ethylene biosynthesis in fruits, which must be considered when interpreting phenotypes [36].

Maximizing Silencing Efficiency: A Troubleshooting Guide for Common VIGS Challenges

Within the framework of plant virus-induced gene silencing (VIGS) validation techniques, controlling the plant's growth environment is not merely a matter of maintaining plant health; it is a fundamental determinant of experimental success and reproducibility. VIGS efficiency hinges on the complex interplay between viral vector accumulation and the plant's innate RNA interference machinery, both of which are profoundly sensitive to ambient conditions [2] [31]. This application note synthesizes recent, evidence-based findings on optimizing photoperiod, temperature, and humidity to achieve robust and consistent gene silencing. The protocols and data summarized herein provide a critical resource for researchers and scientists aiming to standardize and enhance VIGS efficacy across a range of plant species, thereby accelerating functional genomics and drug discovery pipelines in plant biology.

Data compiled from recent optimization studies across diverse species reveal clear trends for temperature, photoperiod, and humidity. The following table provides a consolidated overview of optimized conditions.

Table 1: Optimized Environmental Conditions for VIGS across Plant Species

Plant Species	Optimal Temperature (°C)	Optimal Photoperiod (Light/Dark)	Key Supporting Evidence
Petunia	20 °C Day / 18 °C Night	16h / 8h	A 1.8-fold increase in silencing efficiency compared to higher temperatures [31].
Soybean	20 - 22 °C	Not Specified	Identified as a key optimized factor for effective silencing [9] [17].
Tea Plant	25 °C	Not Specified	Temperature control used in established VIGS protocols [39].
Pepper	20 °C (Post-Inoculation)	16h / 8h	Lower temperature promoted stronger VIGS efficacy [40].
Sunflower	22 °C (Average)	18h / 6h	Successfully applied in a novel seed-vacuum protocol [34].

Humidity is frequently cited as a crucial factor, with studies in petunia and sunflower implementing relative humidity levels of approximately 69% and 45%, respectively [31] [34]. Maintaining high humidity is particularly critical in the first 24-48 hours after inoculation to prevent desiccation of the agroinfiltrated tissues and to facilitate Agrobacterium-mediated delivery of the viral vector [2].

Experimental Protocols for Environmental Optimization

Protocol: Validating the Temperature Gradient for Enhanced Silencing

This protocol is adapted from studies in petunia and pepper, designed to empirically determine the optimal temperature for VIGS in a new species or cultivar [40] [31].

• Objective: To identify the growth temperature that maximizes target gene silencing efficiency while minimizing viral symptom severity.
• Materials:
  • Plant materials (e.g., seeds or uniform seedlings).
  • Constructed TRV vectors (pTRV1 and pTRV2 containing a marker gene like PDS).
  • Agrobacterium tumefaciens strain GV3101.
  • Controlled environment growth chambers with precise temperature control.
  • Equipment for chosen inoculation method (e.g., vacuum infiltration apparatus, syringes).
• Method:
  • Plant Preparation & Inoculation: Inoculate a large batch of uniform plants (e.g., at the 3-4 leaf stage) using a standardized method (e.g., vacuum infiltration or apical meristem inoculation).
  • Temperature Treatment: Immediately post-inoculation, divide the plants into groups and transfer them to separate growth chambers set at different temperature regimes. Key test points often include:
    • 18-20°C Day / 16-18°C Night
    • 23-25°C Day / 18-20°C Night
    • 26-28°C Day / 22-24°C Night
  • Environmental Consistency: Maintain all other environmental factors constant, including photoperiod (e.g., 16h/8h), light intensity (~150-200 μmol m⁻² s⁻¹), and relative humidity (>60%).
  • Monitoring & Data Collection:
    • Phenotypic Assessment: Record the onset and progression of silencing phenotypes (e.g., photobleaching) daily from 10 to 21 days post-inoculation (dpi).
    • Efficiency Quantification: At 21 dpi, calculate silencing efficiency as the percentage of plants showing clear silencing phenotypes.
    • Molecular Validation: Use quantitative RT-PCR on leaf tissue samples to measure the transcript levels of the silenced target gene (e.g., PDS), normalizing to a housekeeping gene.
    • Symptom Scoring: Document any viral symptoms (e.g., stunting, necrosis, leaf mottling) in control (empty vector) plants at each temperature.

The logical workflow and key assessment points for this protocol are outlined in the diagram below.

Figure 1: Experimental workflow for temperature optimization in VIGS.

Protocol: Standardization of Post-Inoculation Photoperiod and Humidity

This protocol establishes consistent light and humidity cycles to ensure reliable VIGS spread and manifestation [31] [34].

• Objective: To define and maintain a photoperiod and humidity regime that supports robust viral systemic movement and gene silencing.
• Materials:
  • Inoculated plants from Protocol 3.1.
  • Growth chambers with programmable photoperiod and humidity control.
  • Data loggers for monitoring environmental parameters.
• Method:
  • Initial Incubation: Post-inoculation, maintain plants in a stable environment with an extended photoperiod (e.g., 16-18 hours of light) for the first 7-10 days to fuel plant growth and viral replication.
  • Humidity Management: Enclose newly inoculated plants in transparent domes or humidity tents for 1-2 days to maintain relative humidity above 80-90%, preventing tissue dehydration.
  • Gradual Acclimation: Gradually reduce humidity to ambient growth levels (e.g., 45-70%) over several days to avoid stress.
  • Long-Term Maintenance: Continue with the established long-day photoperiod (16h/8h) throughout the experimental period to support the plant's metabolic activity, which is essential for the systemic silencing signal propagation.

The Scientist's Toolkit: Essential Research Reagent Solutions

Successful implementation of VIGS relies on a core set of biological and chemical reagents. The following table details these essential components and their functions.

Table 2: Key Research Reagents for VIGS Experiments

Reagent / Solution	Function in VIGS Protocol	Specific Examples & Notes
TRV Viral Vectors	Bipartite vector system for delivering the silencing trigger.	pTRV1 (e.g., pYL192): Encodes viral replication and movement proteins.pTRV2 (e.g., pYL156): Carries the target gene fragment for silencing [8] [34].
Agrobacterium Strain	Mediates the delivery of T-DNA containing the viral vectors into plant cells.	GV3101: A widely used, highly efficient strain for agroinfiltration [9] [34].
Infiltration Buffer	Suspension medium for Agrobacterium to facilitate infection.	Typically contains MgCl₂ and MES buffer to maintain pH, and Acetosyringone to induce Vir gene expression for T-DNA transfer [41] [40].
Selection Antibiotics	Maintains selective pressure for plasmids in bacterial and plant cultures.	Kanamycin, Rifampicin, Gentamicin: Used in LB/YEB media to select for transformed Agrobacterium [41] [34].
Visual Marker Genes	Provides a visual indicator of successful silencing for protocol optimization.	Phytoene Desaturase (PDS): Silencing causes photobleaching [9] [39].Chalcone Synthase (CHS): Silencing causes white patterning on pigmented flowers [31].

The precise control of photoperiod, temperature, and humidity is not an ancillary concern but a critical pillar of reliable VIGS experimentation. As evidenced by recent studies, optimizing these factors can dramatically increase silencing efficiency, improve phenotypic consistency, and reduce confounding viral symptoms. The protocols and data presented here provide a validated roadmap for researchers to systematically incorporate these environmental parameters into their VIGS workflows. Adherence to these optimized conditions will enhance the rigor and reproducibility of functional gene validation, thereby strengthening the foundation of plant science research and its applications in drug development and crop improvement.

Virus-Induced Gene Silencing (VIGS) has emerged as an indispensable reverse genetics tool for rapid functional characterization of plant genes, particularly in species recalcitrant to stable genetic transformation. However, the efficacy of VIGS is significantly constrained by genotype-dependent variation in viral susceptibility and silencing efficiency across plant varieties. This application note synthesizes current methodological advances and provides detailed protocols to overcome genotype dependency, enabling broader application of VIGS in recalcitrant plant varieties for both basic research and drug development applications involving medicinal plants.

The Genotype Dependency Challenge: Evidence and Magnitude

Genotype-dependent variation in VIGS efficiency presents a substantial bottleneck for functional genomics studies across numerous plant families. Quantitative assessments across diverse species reveal significant variability in both infection rates and phenotypic penetration of silencing.

Table 1: Documented Genotype-Dependent VIGS Efficiency Across Plant Species

Plant Species	Genotypes Tested	Efficiency Range	Key Findings	Reference
Sunflower (Helianthus annuus)	6 genotypes including 'Smart SM-64B', 'Buzuluk'	Infection: 62-91%	'Smart SM-64B' showed highest infection (91%) but lowest phenotypic spreading	[34]
Soybean (Glycine max)	Tianlong 1 and others	Silencing: 65-95%	Effective infectivity exceeded 80%, reaching 95% for Tianlong 1	[9]
Primulina	15 species and cultivars	Variable across methods	P. 'Alfonso' and P. carinata most amenable to TRV and CaLCuV systems	[42]
Pepper (Capsicum annuum)	Multiple cultivars	Enhanced by optimized vectors	TRV-C2bN43 system significantly improved efficacy across genotypes	[40]

The molecular basis of genotype dependency involves multiple factors, including variations in RNA silencing machinery components, viral movement constraints, and innate immune responses. Differences in Argonaute protein sequences and functionality between species significantly influence silencing efficiency [2]. Additionally, intercellular and long-distance movement of silencing signals exhibits species-specific variation, affecting systemic propagation of VIGS [2].

Strategic Framework and Molecular Mechanisms for Overcoming Genotype Dependency

The following diagram illustrates the multi-pronged strategic approach to addressing genotype dependency in VIGS systems, connecting specific challenges with their corresponding solutions and molecular mechanisms.

The molecular mechanism of VIGS and strategic enhancement through viral suppressor engineering involves precise manipulation of the plant's RNA silencing machinery, as detailed in the following pathway diagram.

Optimized Experimental Protocols

Seed Vacuum Infiltration Protocol for Recalcitrant Species

This protocol, optimized for sunflower and applicable to other challenging species, achieves up to 91% infection efficiency in susceptible genotypes [34].

Materials

• pTRV1 and pTRV2 vectors (Addgene #148968, #148969)
• Agrobacterium tumefaciens strain GV3101
• Sunflower seeds (genotypes with documented susceptibility)
• Vacuum infiltration apparatus
• Co-cultivation medium (peat:perlite, 3:1 ratio)

Procedure

• Vector Construction: Clone target gene fragment (100-300 bp) into pTRV2 using appropriate restriction sites (XbaI/BamHI)
• Agrobacterium Preparation:
  • Transform recombinant plasmids into GV3101 via electroporation
  • Plate on LB agar with antibiotics (kanamycin 50 µg/mL, gentamicin 10 µg/mL, rifampicin 100 µg/mL)
  • Incubate at 28°C for 36-48 hours
  • Inoculate single colonies in liquid LB with antibiotics, grow to OD600 = 0.8-1.0
  • Resuspend in infiltration medium (10 mM MgCl2, 10 mM MES, 150 µM acetosyringone) to OD600 = 0.4
• Seed Preparation: Partially remove seed coats to enhance infiltration without damaging embryos
• Vacuum Infiltration:
  • Submerge seeds in Agrobacterium suspension
  • Apply vacuum (0.8-1.0 bar) for 5-10 minutes
  • Rapidly release vacuum to ensure bacterial entry
• Co-cultivation:
  • Transfer infiltrated seeds to co-cultivation medium
  • Maintain at 22°C for 6 hours in dark conditions
• Plant Growth:
  • Transfer to greenhouse conditions (22°C, 18h light/6h dark, 45% RH)
  • Monitor for silencing symptoms from 14-21 days post-infiltration

Critical Parameters

• Co-cultivation duration: 6 hours optimal for sunflower
• Bacterial density: OD600 = 0.4 for seed vacuum infiltration
• Seed age: Use freshly harvested or properly stored seeds

Cotyledon Node Method for Soybean and Legumes

This tissue culture-free protocol achieves 65-95% silencing efficiency in soybean [9].

Materials

• Surface-sterilized soybean seeds
• pTRV1 and pTRV2-GFP derivatives
• Agrobacterium suspension medium

Procedure

• Seed Preparation:
  • Surface sterilize seeds with 70% ethanol and sodium hypochlorite
  • Soak in sterile water until swollen (approximately 8-12 hours)
• Explant Preparation: Longitudinally bisect swollen seeds to obtain half-seed explants
• Agroinfiltration:
  • Immerse fresh explants in Agrobacterium suspension (OD600 = 0.5)
  • Infiltrate for 20-30 minutes with gentle agitation
• Recovery and Growth:
  • Transfer to sterile tissue culture medium for 4 days
  • Monitor GFP fluorescence to verify infection efficiency
  • Transplant to soil after 7 days

VIGS Optimization via Viral Suppressor Engineering

The TRV-C2bN43 system enhances silencing in pepper by decoupling local and systemic suppression activities [40].

Materials

• pH7lic4.1 expression vector
• pTRV2-lic vector
• Primers for C2bN43 amplification

Procedure

• Vector Construction:
  • Amplify C2bN43 truncated variant (first 43 amino acids of C2b)
  • Clone into pH7lic4.1 with C-terminal 3×Flag tag
  • Fuse with PEBV subgenomic RNA promoter
  • Insert into pTRV2-lic to generate pTRV2-C2bN43
• Plant Infection:
  • Combine pTRV1 with pTRV2-C2bN43-target construct
  • Infiltrate pepper plants at 4-6 leaf stage
  • Maintain at 20°C post-inoculation to enhance silencing

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Research Reagent Solutions for VIGS in Recalcitrant Varieties

Reagent/Resource	Function	Application Notes	Reference
TRV Vectors (pTRV1/pTRV2)	Bipartite viral vector system	Most versatile; efficient systemic movement; mild symptoms	[34] [2]
CFMMV Vector (pCF93)	Tobamovirus-based vector	Optimal for cucurbits; 93 bp upstream of CP for high efficiency	[43]
CaLCuV Vector	DNA virus-based system	Alternative for TRV-recalcitrant Primulina species	[42]
C2bN43 Truncated Suppressor	Enhanced VIGS efficiency	Retains systemic but not local silencing suppression	[40]
Agrobacterium GV3101	Vector delivery	High transformation efficiency; compatible with pTRV system	[34] [9]
Acetosyringone	Vir gene inducer	150 µM in infiltration medium enhances T-DNA transfer	[34]

Addressing genotype dependency in VIGS requires integrated optimization of delivery methods, vector systems, and viral counter-defense strategies. The protocols outlined herein provide a roadmap for adapting VIGS to challenging plant varieties, with particular relevance for functional studies in medicinal plants where rapid gene validation can accelerate drug development pipelines. Future directions include developing genotype-specific viral vectors through high-throughput susceptibility screening and engineering synthetic viral suppressors tailored to particular plant families. As VIGS increasingly interfaces with CRISPR-based functional genomics [44], overcoming genotype dependency will remain crucial for unlocking the full potential of plant reverse genetics in both basic and applied research.

Virus-induced gene silencing (VIGS) has emerged as a powerful tool for rapid functional genomics in plants, but its application is often constrained by physical barriers such as thick cuticles, dense trichomes, and lignified tissues. These structures naturally protect plants from environmental threats and pathogens, but they also impede the delivery of VIGS vectors, limiting the technique's efficacy across diverse species, particularly perennial and woody plants. For researchers and drug development professionals working with recalcitrant species, overcoming these barriers is crucial for accelerating gene function validation and harnessing plant biodiversity for pharmaceutical discovery. This protocol details optimized, evidence-based methods to achieve efficient gene silencing in challenging plant systems, enabling high-throughput functional genomics in previously inaccessible species.

Physical Barriers and VIGS Efficiency: Quantitative Challenges

The efficacy of VIGS is intrinsically linked to the successful delivery and systemic movement of viral vectors, processes directly hampered by specific plant morphological traits. The table below summarizes the primary physical barriers and their documented impact on VIGS efficiency.

Table 1: Physical Barriers to VIGS and Their Documented Impact

Physical Barrier	Affected VIGS Process	Example Species	Reported Impact on Efficiency
Dense Trichomes [45] [9]	Initial Infiltration	Soybean (Glycine max) [9]	Acts as a physical block; conventional spray/infiltration "impeded liquid penetration" [9].
Thick Cuticles [16]	Viral Penetration & Initial Infection	Tea Oil Camellia (Camellia drupifera) [16]	Creates a waxy, impermeable layer that prevents Agrobacterium or viral entry into epidermal cells.
Lignified Tissues [16]	Viral Systemic Movement & Delivery	Woody capsules of C. drupifera [16]	Lignified cell walls hinder viral movement, requiring specialized inoculation methods for fruit tissues.

Optimized Inoculation Protocols for Recalcitrant Tissues

Standard agroinfiltration protocols often fail against robust plant structures. The following optimized methods have been empirically demonstrated to overcome these challenges.

Cotyledon Node Immersion for Species with Dense Trichomes

Background: Soybean leaves are protected by dense trichomes, making standard leaf infiltration methods like spraying or injection ineffective [9]. This protocol uses the cotyledon node as a direct conduit for Agrobacterium delivery.

Materials:

• Agrobacterium tumefaciens strain GV3101 containing pTRV1 and pTRV2-derived vectors [9].
• Induction Medium: YEB with MES buffer (0.2 M, pH 5.6) and acetosyringone (0.1 M) [16].
• Infiltration Medium: Fresh induction medium resuspension to OD₆₀₀ = 0.9–1.0 [9] [16].

Step-by-Step Protocol:

• Seed Preparation: Surface-sterilize soybean seeds and soak in sterile water until swollen.
• Explant Preparation: longitudinally bisect the swollen seeds to create half-seed explants, exposing the cotyledonary node.
• Agrobacterium Preparation: Mix the pTRV1 and pTRV2 (e.g., pTRV2-GmPDS) cultures in a 1:1 ratio.
• Immersion Inoculation: Submerge the fresh half-seed explants completely in the Agrobacterium suspension for 20-30 minutes with gentle agitation.
• Co-cultivation & Growth: Blot the explants dry and transfer to sterile tissue culture conditions for 3-4 days before moving to soil.
• Efficiency Validation: Monitor systemic silencing (e.g., photobleaching for GmPDS) at ~21 days post-inoculation (dpi). This method achieved an infection efficiency of >80%, up to 95%, and successful silencing of target genes [9].

Pericarp Cutting Immersion for Lignified Fruits

Background: The lignified capsules of Camellia drupifera are impervious to standard injection methods. This protocol uses precise wounding combined with immersion to facilitate Agrobacterium entry [16].

Materials:

• As in Protocol 2.1.
• Sharp, sterile scalpel or razor blade.

Step-by-Step Protocol:

• Plant Material Selection: Harvest C. drupifera capsules at the optimal developmental stage (e.g., early stage for CdCRY1, mid stage for CdLAC15).
• Wounding: Create precise, shallow cuts on the pericarp surface without detaching the fruit.
• Immersion Inoculation: Immerse the wounded capsules in the Agrobacterium suspension (OD₆₀₀ ~1.0) for 20-30 minutes.
• Post-inoculation Care: Maintain plants under optimal growth conditions (e.g., ~20°C).
• Phenotypic Analysis: Observe pericarp pigmentation phenotypes (e.g., fading). This method achieved an infiltration efficiency of ~94% and strong observable silencing phenotypes [16].

Critical Co-Factors for Enhancing VIGS Efficacy

Beyond the inoculation method, several co-factors are critical for robust silencing.

Table 2: Key Co-Factors for Optimizing VIGS in Recalcitrant Systems

Co-Factor	Optimal Condition / Strategy	Experimental Rationale & Impact
Plant Developmental Stage [31] [16]	Inoculate at 3-4 weeks after sowing (soybean) [9]; use stage-specific fruit (Camellia) [16].	Younger tissues are more susceptible to infection and support better viral movement.
Temperature [31]	20°C day/18°C night cycles.	Cooler temperatures reduce plant defense responses and slow viral replication, minimizing symptom severity and allowing for more effective systemic silencing.
Vector & Insert Design [31] [9]	Use pTRV2 with a non-plant insert (e.g., sGFP) as control; ensure target insert is 200-500 bp with low homology to non-target genes.	An empty pTRV2 vector can cause severe necrosis and plant death. A non-plant insert eliminates these symptoms, providing a healthy control. Specificity prevents off-target silencing.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for VIGS

Research Reagent	Function in VIGS Protocol
pTRV1 & pTRV2 Vectors [2] [9]	Bipartite RNA viral vector system; TRV1 encodes replication and movement proteins, TRV2 carries the target gene insert for silencing.
Agrobacterium tumefaciens GV3101 [9] [16]	Standard disarmed strain for delivering TRV vectors into plant cells via T-DNA transfer.
Acetosyringone [16]	A phenolic compound that induces the Agrobacterium vir genes, essential for efficient T-DNA transfer.
Visual Marker Genes (PDS, CHS) [31] [9]	PDS silencing causes photobleaching; CHS silencing leads to loss of pigmentation (white patches). Used as positive controls to visualize silencing.

Experimental Workflow for Barrier Overcoming

The following diagram illustrates the decision-making workflow for selecting and applying the appropriate protocol based on the primary barrier presented by the plant species.

Within plant functional genomics, Virus-Induced Gene Silencing (VIGS) has emerged as an indispensable reverse genetics tool for rapid gene function characterization. This technology leverages the plant's innate post-transcriptional gene silencing machinery, using recombinant viral vectors to trigger systemic suppression of target gene expression [2]. The Agrobacterium-mediated delivery of VIGS constructs is widely adopted, yet its efficiency is profoundly influenced by specific inoculation parameters [34]. This Application Note delineates optimized protocols for fine-tuning three critical parameters—Agrobacterium optical density (OD600), co-cultivation duration, and plant developmental stage—to achieve robust, reproducible silencing across diverse plant species, providing a standardized framework for research and drug development professionals.

Optimizing VIGS requires a balanced interplay of biological material, chemical environment, and physical conditions. The summarized data below provide a foundation for developing species-specific protocols.

Table 1: Optimized Inoculation Parameters for Various Plant Species

Plant Species	Optimal Developmental Stage	Optimal OD600	Optimal Co-cultivation Time	Key Infiltration Method	Reported Efficiency	Primary Reference
Tomato (Solanum lycopersicum)	No-apical-bud stem section with 1-3 cm axillary bud [46]	1.0 [46]	8 days (post-infiltration observation) [46]	Injection of No-Apical-Bud Stem (INABS) [46]	56.7% VIGS, 68.3% virus inoculation [46]	[46]
Sunflower (Helianthus annuus)	Germinated seeds (seed vacuum infiltration) [34]	Information missing	6 hours [34]	Seed Vacuum Infiltration [34]	Up to 77% infection, 91% in some genotypes [34]	[34]
Madagascar Periwinkle (Catharanthus roseus)	5-day-old etiolated seedlings [47]	1.0 [47]	Integrated into vacuum infiltration [47]	Cotyledon-based Vacuum Infiltration [47]	Significant gene downregulation & phenotype onset in 6 days [47]	[47]
Taro (Colocasia esculenta)	Mature plants (leaf injection); Bulbs (vacuum) [21]	1.0 (superior to 0.6) [21]	Information missing	Leaf Injection; Bulb Vacuum Treatment [21]	27.77% silencing plant rate at OD600=1.0 [21]	[21]
Wheat & Maize	(Germinated) seeds [48]	Information missing	Information missing	Vacuum Infiltration of (Germinated) Seeds [48]	Whole-plant level silencing [48]	[48]
Tea Oil Camellia (Camellia drupifera)	Early to mid-stage capsules (fruits) [16]	0.9-1.0 [16]	Information missing	Pericarp Cutting Immersion [16]	~93.94% infiltration efficiency [16]	[16]

Table 2: General Effect of Parameter Variation on VIGS Efficiency

Parameter	Too Low/Too Early	Optimal Range	Too High/Too Late	Impact on Efficiency
Agrobacterium OD600	< 0.5 - 0.6 [46] [21]	0.6 - 1.5 (Species-dependent, often 1.0 is optimal) [46] [21]	> 1.5 [46]	Low bacterial density fails to establish robust infection; excessive density causes plant stress and cell death [46].
Co-cultivation Time	< 6 hours [34]	6 hours - Several days (Method dependent) [34]	Excessively long durations	Insufficient time limits T-DNA transfer; prolonged exposure can overstress tissue [34].
Developmental Stage	Overly juvenile tissues	Species-specific ideal stage (e.g., young seedlings, specific stem sections) [47] [46]	Overly mature, lignified tissues	Young tissues are more susceptible, but very early stages may not support systemic spread; mature tissues are recalcitrant [47] [16].

Detailed Experimental Protocols

Protocol A: Cotyledon-Based VIGS in Medicinal Plants

This protocol, optimized for Catharanthus roseus and applicable to Glycyrrhiza inflata and Artemisia annua, is ideal for species where metabolites of interest are produced in early seedlings [47].

• Plant Material Preparation:

  • Germinate surface-sterilized seeds on moist filter paper or agar in the dark at 22-25°C [47].
  • Utilize 5-day-old etiolated seedlings where cotyledons have fully emerged for optimal susceptibility [47].

• Agrobacterium Culture Preparation:

  • Transform the pTRV1 and pTRV2 (carrying gene-of-interest fragment) vectors into Agrobacterium tumefaciens strain GV3101 [47].
  • Initiate cultures from single colonies in LB medium with appropriate antibiotics (e.g., kanamycin, rifampicin, gentamicin) and grow at 28°C with shaking for ~48 hours [47] [34].
  • Sub-culture the bacteria into fresh induction medium (e.g., containing acetosyringone and MES) and grow until the OD600 reaches 0.9-1.0 [47] [16].
  • Pellet the bacteria by centrifugation (e.g., 5000 rpm for 15 minutes) and resuspend in an infiltration solution (e.g., containing MgCl₂, MES, and acetosyringone) to a final OD600 of 1.0. Incubate the suspension at room temperature for 3-4 hours before use [47] [48].

• Vacuum Infiltration:

  • Immerse the batches of etiolated seedlings in the prepared Agrobacterium suspension within a vacuum desiccator.
  • Apply a vacuum (e.g., 250-500 mmHg) for a short duration (e.g., 30 minutes). The rapid release of vacuum facilitates the infiltration of bacteria into the intercellular spaces of the cotyledons [47].

• Co-cultivation and Plant Care:

  • Following infiltration, blot the seedlings and transfer them to soil or cultivation medium.
  • Maintain the plants in the dark for ~3 days before transferring to a standard light regime (e.g., 16h light/8h dark) at 22-25°C [47].
  • Silencing phenotypes, such as yellowing of cotyledons when targeting ChlH, can be observed as early as 6 days post-infiltration [47].

Protocol B: Injection of No-Apical-Bud Stem Sections (INABS) in Tomato

This protocol is highly efficient for Solanaceous plants and generates silenced transformants rapidly [46].

• Plant Material Preparation:

  • Grow tomato plants until they develop "Y-type" stem sections (1-3 cm in length) that lack an apical bud but contain an axillary bud [46].

• Agrobacterium Preparation:

  • Prepare Agrobacterium cultures harboring TRV vectors as described in Protocol A, resuspending to an OD600 of 1.0 [46].

• Stem Injection:

  • Using a plastic syringe and needle, slowly inject 100-200 µL of the Agrobacterium suspension directly into the bare stem of the no-apical-bud section. Infiltration is successful when a film of the liquid is visible at the top of the injected stem [46].

• Co-cultivation and Observation:

  • No explicit co-cultivation period in a different medium is required. Maintain plants under standard greenhouse conditions.
  • Bleaching in newly grown leaves from the axillary bud can be evident within 6-10 days, with maximum silencing efficiency observed at 8 days post-infiltration (dpi) [46].

Protocol C: Seed-Vacuum Infiltration in Sunflower

This simple protocol is valuable for recalcitrant species like sunflower, bypassing the need for in vitro culture steps [34].

• Seed Preparation:

  • Partially remove the seed coats ("peeling") to enhance infiltration. No surface sterilization is required [34].

• Agrobacterium and Vacuum Infiltration:

  • Prepare Agrobacterium suspension as previously described.
  • Submerge the peeled seeds in the suspension and apply vacuum. Specific parameters (pressure, time) should be optimized for the target species [34].

• Co-cultivation:

  • After vacuum infiltration, subject the seeds to a 6-hour co-cultivation period in the Agrobacterium suspension [34].
  • Following co-cultivation, sow the seeds directly in soil and cultivate under standard greenhouse conditions. Silencing symptoms will appear in the first true leaves [34].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for VIGS Experiments

Reagent / Material	Function / Role in VIGS	Example Usage & Notes
TRV Vectors (pTRV1, pTRV2)	Bipartite viral vector system. pTRV1 encodes replication/movement proteins. pTRV2 carries the capsid protein and the insert for target gene silencing [2].	The most widely used VIGS vector due to broad host range and mild symptoms [47] [46] [2].
Agrobacterium tumefaciens GV3101	A disarmed Ti-plasmid strain used to deliver the engineered TRV vectors into plant cells via its Type IV Secretion System [49].	Preferred for its high transformation efficiency in many dicot species [47] [34].
Acetosyringone	A phenolic compound that activates the Agrobacterium vir genes, which are essential for T-DNA transfer into the plant cell [49].	Added to the bacterial induction and infiltration media to maximize transformation efficiency [16] [48].
Antibiotics (Kanamycin, Rifampicin, Gentamicin)	Selective agents to maintain binary vectors in Agrobacterium and control bacterial contamination [34].	Concentrations must be optimized for the specific Agrobacterium strain and vector system [34].
Marker Genes (PDS, ChlH)	Visual reporter genes. Silencing PDS causes photobleaching; silencing ChlH (magnesium chelatase) leads to yellowing due to blocked chlorophyll synthesis [47] [2].	Used as positive controls to visually confirm successful VIGS and optimize system parameters [47] [46] [21].

Workflow and Parameter Interplay Visualization

VIGS Parameter Optimization Workflow

Key Parameter Effects on VIGS Outcome

The precise calibration of Agrobacterium OD600, co-cultivation time, and plant developmental stage is fundamental to successful VIGS outcomes. As evidenced by the protocols and data herein, optimal parameters are highly species-specific, requiring empirical validation. The continued refinement of these parameters, coupled with an understanding of the underlying biological interactions [49], will further solidify VIGS as a cornerstone technology in functional genomics and accelerator for the discovery of valuable therapeutic compounds in medicinal plants [47].

Confirming Silencing Efficacy: Phenotypic, Molecular, and Comparative Validation Techniques

Phenotypic validation is a critical component of functional genomics, enabling researchers to link gene sequences to biological functions. Within plant research, Virus-Induced Gene Silencing (VIGS) has emerged as a powerful reverse genetics tool that facilitates this process through rapid gene knockdown and subsequent phenotypic observation [2]. This application note details standardized protocols for documenting visible markers, with a particular emphasis on photobleaching and altered disease susceptibility, within the context of VIGS experiments. The precise documentation of these phenotypes provides crucial evidence for validating gene function in plant defense mechanisms, stress responses, and metabolic pathways.

The fundamental principle of VIGS relies on the plant's natural post-transcriptional gene silencing (PTGS) machinery. When recombinant viral vectors carrying fragments of plant genes are introduced, they trigger a sequence-specific RNA degradation mechanism that suppresses expression of the corresponding endogenous genes [2]. This system enables researchers to observe the phenotypic consequences of gene knockdown without the need for stable transformation, which is particularly valuable for species with challenging transformation systems or long life cycles [50]. The effectiveness of VIGS depends on multiple factors, including viral vector selection, inoculation methodology, plant genotype, and environmental conditions [2] [31].

Table 1: Key Visible Markers for VIGS Phenotypic Validation

Phenotypic Marker	Target Gene	Observed Phenotype	Biological Process Affected
Photobleaching	Phytoene Desaturase (PDS)	White or bleached leaf areas [9] [50] [34]	Carotenoid biosynthesis [31]
Altered Pigmentation	Chalcone Synthase (CHS)	White floral tissues or reduced pigmentation [31]	Anthocyanin biosynthesis [31]
Enhanced Disease Susceptibility	Rpp6907 (Soybean)	Compromised rust resistance [9]	Disease immunity [9]
Altered Starch Accumulation	CeTCP14 (Taro)	Significant reduction in starch content [21]	Corm development and metabolism [21]
Anther Color Change	CaAN2 (Pepper)	Loss of anthocyanin accumulation in anthers [40]	Flavonoid biosynthesis [40]

Figure 1: The molecular mechanism of Virus-Induced Gene Silencing (VIGS) illustrates how introduced viral vectors trigger a silencing cascade that ultimately leads to observable phenotypic changes, enabling gene function validation.

The Scientist's Toolkit: Essential Research Reagents

Implementing a successful VIGS experiment requires careful selection of molecular tools and biological materials. The following table outlines the essential research reagent solutions necessary for establishing an effective VIGS system for phenotypic validation.

Table 2: Key Research Reagent Solutions for VIGS Experiments

Reagent/Material	Function/Purpose	Examples/Specifications
TRV Vectors	Bipartite viral vector system for inducing silencing	pTRV1 (Replicase/Movement), pTRV2 (Target gene insertion) [9] [2]
Agrobacterium tumefaciens Strain	Delivery vehicle for TRV vectors	GV3101 [9] [50] [34]
Visual Marker Genes	Positive controls for silencing efficiency	PDS (photobleaching), CHS (pigmentation loss) [9] [31]
Infiltration Buffer	Facilitates Agrobacterium delivery into plant tissues	10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂, 0.03% Silwet-77 [50]
Viral Suppressors of RNA Silencing (VSRs)	Enhance silencing efficiency by countering plant defenses	C2bN43 (retains systemic but not local suppression) [40]
Plant Genotypes	Host materials with varying VIGS susceptibility	Soybean 'Tianlong 1', Pepper 'L265', Petunia 'Picobella Blue' [9] [40] [31]

Documenting Key Visible Markers in VIGS Experiments

Photobleaching as a Visual Silencing Marker

Photobleaching represents one of the most reliable and easily detectable visible markers for assessing VIGS efficiency. This phenomenon occurs when the phytoene desaturase (PDS) gene, which encodes a critical enzyme in the carotenoid biosynthesis pathway, is successfully silenced [31]. Carotenoids play an essential role in protecting chlorophyll from photooxidation, and their depletion results in characteristic white or bleached leaf tissues due to chlorophyll degradation [50].

In a recent soybean study utilizing an optimized TRV-based VIGS system, researchers observed distinct photobleaching in leaves inoculated with pTRV:GmPDS at 21 days post-inoculation (dpi), while control plants showed no such phenotype [9]. The photobleaching initially manifested in the cluster buds before spreading to developing leaves, demonstrating the systemic nature of VIGS. The silencing efficiency ranged from 65% to 95%, as confirmed by both phenotypic observation and expression analysis [9]. Similar results were documented in Atriplex canescens, where systemic photobleaching appeared in newly emerged leaves at approximately 15 dpi, accompanied by a 40-80% reduction in AcPDS transcript levels according to qRT-PCR validation [50].

The documentation protocol for photobleaching should include regular visual inspection beginning at 10-14 dpi, with particular attention to newly emerging leaves and apical meristems where the phenotype typically first appears. Photographic documentation should be performed at regular intervals (e.g., weekly) under consistent lighting conditions to track phenotype progression. For quantitative assessment, chlorophyll content measurements using a SPAD meter or spectrophotometric analysis of leaf extracts provide objective data to complement visual observations [21].

Altered Disease Susceptibility as a Functional Marker

Beyond easily visible markers like photobleaching, VIGS enables researchers to investigate gene function in plant-pathogen interactions through altered disease susceptibility phenotypes. This approach is particularly valuable for identifying and characterizing genes involved in plant defense pathways. When defense-related genes are silenced, plants typically exhibit enhanced susceptibility to specific pathogens, manifesting as more severe disease symptoms compared to control plants [9].

In soybean functional genomics, the TRV-VIGS system has been successfully employed to validate the role of the GmRpp6907 gene in conferring resistance to rust. Silencing this gene resulted in compromised immunity, confirming its critical function in disease resistance [9]. Similarly, the bean pod mottle virus (BPMV)-mediated VIGS system has been used to demonstrate that silencing of Rpp1 significantly reduced soybean rust resistance [9]. These findings highlight how VIGS can directly link specific genetic sequences to defense mechanisms through observable changes in disease susceptibility.

Documenting altered disease susceptibility requires standardized pathogen inoculation protocols and systematic scoring of disease symptoms. Researchers should establish appropriate negative controls (empty vector) and positive controls (previously validated susceptibility genes) to ensure accurate interpretation. Disease assessment typically includes measurement of lesion size, quantification of pathogen biomass through qPCR, and scoring of symptom severity using established rating scales specific to the pathogen under investigation.

Additional Visible Markers for Specialized Applications

While photobleaching and disease susceptibility represent the most common visible markers, VIGS enables the investigation of diverse biological processes through various other phenotypic indicators:

Altered floral pigmentation resulting from silencing of anthocyanin biosynthesis genes (e.g., CHS) provides a visually striking marker in ornamental species and fruits [31]. In pepper, suppression of CaAN2, an anther-specific MYB transcription factor, led to coordinated downregulation of structural genes in the anthocyanin biosynthesis pathway and complete abolition of anthocyanin accumulation in anthers [40]. This established CaAN2 as an essential regulator of pigmentation and demonstrated the utility of anther color as a reliable phenotypic marker for VIGS efficiency in reproductive tissues.

Developmental phenotypes including altered plant architecture, modified leaf morphology, and changes in starch accumulation offer additional visible markers. In taro, silencing of CeTCP14, a transcription factor gene, resulted in significantly reduced starch content in corms (70.88%-80.61% of control levels), linking this gene to starch metabolism and storage organ development [21].

Standardized Experimental Protocols

VIGS Implementation Workflow

A standardized, optimized workflow is essential for achieving consistent and reliable VIGS results. The following diagram illustrates the key stages in implementing an effective VIGS system for phenotypic validation.

Figure 2: Standardized workflow for implementing VIGS experiments, outlining key stages from vector construction to phenotypic documentation and data analysis.

TRV Vector Construction and Agrobacterium Preparation

The initial phase of VIGS experimentation involves meticulous preparation of genetic constructs and transformation of the delivery vehicle:

Vector Construction: Select a 300-400 bp fragment of the target gene with high sequence specificity to minimize off-target silencing [50]. Utilize the SGN-VIGS online tool (https://vigs.solgenomics.net/) for predicting optimal nucleotide target regions [50]. Amplify the fragment using gene-specific primers containing appropriate restriction enzyme sites (e.g., EcoRI and XhoI) [9]. Ligate the purified PCR product into the pTRV2 vector, which had been digested with the corresponding restriction enzymes. Transform the ligation product into competent E. coli cells (e.g., DH5α), select positive clones, and verify insert sequence through sequencing [9].

Agrobacterium Preparation: Introduce the recombinant pTRV2 and pTRV1 plasmids into Agrobacterium tumefaciens strain GV3101 using freeze-thaw transformation or electroporation [50] [34]. Plate transformed bacteria on YEP agar containing appropriate antibiotics (50 mg/L kanamycin, 50 mg/L rifampicin) and incubate at 28°C for 48 hours [50]. Inoculate individual colonies into YEP liquid medium with the same antibiotics and culture at 28°C with shaking at 200 rpm until reaching the mid-logarithmic growth phase (OD₆₀₀ = 0.6-1.0) [50] [21]. 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) to the desired OD₆₀₀ [50]. Combine equal volumes of TRV1 and TRV2-derived Agrobacterium suspensions and incubate at room temperature in darkness for 3 hours prior to inoculation to induce virulence gene expression [50].

Plant Inoculation and Cultivation

The inoculation method significantly influences VIGS efficiency and must be optimized for specific plant species:

Vacuum Infiltration: For species with challenging transformation, such as sunflower and Atriplex canescens, vacuum infiltration of germinated seeds represents an effective approach [50] [34]. Submerge prepared materials in Agrobacterium suspension and apply vacuum (0.5 kPa) for 5-10 minutes [50]. This method achieved approximately 16.4% silencing efficiency in A. canescens and up to 91% infection rate in certain sunflower genotypes [50] [34].

Cotyledon Node Method: In soybean, high silencing efficiency (65-95%) was achieved through Agrobacterium-mediated infection of cotyledon nodes [9]. Bisect sterilized, swollen soybeans to obtain half-seed explants, then infect fresh explants by immersion in Agrobacterium suspension for 20-30 minutes [9]. Fluorescence microscopy at 4 days post-infection confirmed successful infection in over 80% of cells in the cotyledonary node [9].

Alternative Methods: Additional inoculation techniques include direct injection of Agrobacterium into tissues using needleless syringes and apical meristem inoculation, which proved effective in petunia [31]. Following inoculation, maintain plants under controlled environmental conditions, with research indicating that 20°C day/18°C night temperatures induce stronger gene silencing than higher temperatures in some species [31].

Table 3: Quantitative Assessment of VIGS Efficiency Across Plant Species

Plant Species	Target Gene	Silencing Efficiency	Key Optimized Parameters
Soybean (Glycine max)	GmPDS	65-95% [9]	Cotyledon node inoculation [9]
Pepper (Capsicum annuum)	CaPDS	Enhanced with TRV-C2bN43 [40]	C2bN43 suppressor, 20°C growth [40]
Atriplex (A. canescens)	AcPDS	16.4% (up to 80% transcript reduction) [50]	Vacuum infiltration (0.5 kPa, 10 min) [50]
Sunflower (Helianthus annuus)	HaPDS	62-91% (genotype-dependent) [34]	Seed vacuum, 6h co-cultivation [34]
Taro (Colocasia esculenta)	CePDS	27.77% (OD₆₀₀=1.0) [21]	Leaf injection, high bacterial density [21]
Petunia (Petunia × hybrida)	CHS	69% area increase after optimization [31]	Meristem inoculation, 20°C/18°C [31]

Phenotypic Documentation and Validation

Robust phenotypic documentation requires both qualitative and quantitative approaches to ensure reliable data collection and interpretation:

Visual Documentation: Begin regular visual inspection at 10-14 days post-inoculation, with more frequent monitoring as phenotypes develop. For photobleaching, document the first appearance, spatial pattern (patchy vs. systemic), and progression of white or bleached areas [9] [50]. Capture high-quality photographs under standardized lighting conditions at regular intervals (e.g., weekly). For disease susceptibility phenotypes, record the timing of symptom appearance, lesion number and size, and disease progression using established rating scales [9].

Molecular Validation: Confirm silencing at the molecular level through quantitative real-time PCR (qRT-PCR) to measure transcript abundance of target genes [9] [40]. In A. canescens, qRT-PCR revealed 40-80% reduction in AcPDS transcript levels in photobleached tissues [50]. For disease-related genes, additional validation may include measurement of pathogen biomass through pathogen-specific qPCR assays and expression analysis of defense marker genes [9].

Additional Quantitative Measures: Supplement visual observations with objective measurements specific to the target pathway. For photobleaching phenotypes, quantify chlorophyll content using a SPAD meter or through spectrophotometric analysis of leaf extracts [21]. In taro, silencing of CeTCP14 led to significantly reduced starch content, which was quantified through chemical analysis to validate the phenotypic observation [21].

The strategic documentation of visible markers, particularly photobleaching and altered disease susceptibility, provides critical evidence in VIGS-based gene function validation. The protocols outlined in this application note emphasize standardized approaches for consistent and reproducible phenotypic assessment across diverse plant species. Key factors for success include selecting appropriate visible marker genes, implementing species-optimized inoculation methods, maintaining controlled environmental conditions, and employing both qualitative and quantitative documentation methods. As VIGS technology continues to evolve with enhancements such as engineered viral suppressors and novel vector systems, the precise documentation of visible phenotypes remains fundamental to advancing our understanding of gene function in plants.

Virus-induced gene silencing (VIGS) has become an indispensable reverse genetics tool for rapid functional analysis of plant genes. The technology leverages the plant's native RNA interference machinery to target endogenous mRNAs for degradation, enabling researchers to study gene function without stable transformation [2]. However, the effectiveness of VIGS experiments hinges on proper molecular validation to confirm successful viral spread and target gene knockdown. This application note provides detailed protocols for using reverse-transcription quantitative PCR (RT-qPCR) to quantify transcript knockdown and conventional RT-PCR to monitor viral spread, ensuring reliable interpretation of VIGS phenotypes in plant systems.

The Scientist's Toolkit: Essential Research Reagents

Table 1: Essential reagents and materials for VIGS molecular confirmation

Reagent/Material	Function/Application	Examples & Specifications
TRV VIGS Vectors	Bipartite viral vector system for inducing silencing	pTRV1 (RNA1: Replicase/movement proteins), pTRV2 (RNA2: Capsid protein + target insert) [50] [9]
Agrobacterium Strain	Delivery vehicle for TRV vectors into plant tissues	GV3101 with appropriate antibiotic resistance (e.g., kanamycin, rifampicin) [50] [51]
Infiltration Buffer	Resuspension medium for Agrobacterium induction	10 mM MES, 200 µM acetosyringone, 10 mM MgCl₂ [50] [51]
Stable Reference Genes	Endogenous controls for RT-qPCR normalization	GhACT7, GhPP2A1 (validated for stability in VIGS experiments) [51]
RNA Extraction Kit	Isolation of high-quality total RNA from silenced tissues	Spectrum Total RNA Extraction Kit or equivalent [51]
Reverse Transcriptase	cDNA synthesis from RNA templates	High-efficiency enzymes for first-strand cDNA synthesis [9]
qPCR Master Mix	Fluorescence-based quantitative PCR	SYBR Green or probe-based mixes compatible with your detection system

The following diagram illustrates the comprehensive workflow for VIGS experimentation and subsequent molecular validation, integrating both viral spread detection and transcript knockdown analysis.

Protocol: qRT-PCR for Transcript Knockdown Validation

Experimental Design and Tissue Sampling

For accurate assessment of gene silencing, collect leaf tissue from both VIGS-treated plants (experimental and empty vector controls) and non-infiltrated wild-type plants. Sample newly emerged leaves where systemic silencing is most evident, typically 14-21 days post-inoculation [50]. For temporal studies, include multiple time points to track silencing progression. Immediately freeze tissue in liquid nitrogen and store at -80°C until RNA extraction.

RNA Extraction and cDNA Synthesis

Extract total RNA using a commercial kit with DNase I treatment to eliminate genomic DNA contamination. Verify RNA integrity and purity by spectrophotometry (A260/280 ratio ~2.0) and gel electrophoresis. Synthesize cDNA using 1μg of total RNA and a high-efficiency reverse transcriptase according to manufacturer protocols. Include a no-reverse transcriptase control for each sample to confirm absence of genomic DNA amplification.

Reference Gene Selection and Validation

Reference gene stability is paramount for accurate normalization in VIGS experiments. Cotton-herbivore studies demonstrate that commonly used reference genes like GhUBQ7 and GhUBQ14 show poor stability under VIGS conditions, while GhACT7 and GhPP2A1 maintain consistent expression [51]. Validate at least two reference genes for your system using stability algorithms (geNorm, NormFinder) before target gene analysis.

Table 2: qRT-PCR validation of successful VIGS in various plant systems

Plant Species	Target Gene	Silencing Efficiency	Key Methodological Details	Citation
Atriplex canescens	AcPDS	40-80% reduction	Phenotypic observation (photobleaching) at 15 dpi; vacuum infiltration of germinated seeds	[50]
Soybean (Glycine max)	GmPDS	65-95% range	Silencing evident at 21 dpi; Agrobacterium-mediated cotyledon node infection	[9]
Cotton (Gossypium hirsutum)	GhHYDRA1	Significant upregulation detected	Proper normalization with GhACT7/GhPP2A1 crucial for detecting expression changes	[51]

qPCR Reaction Setup and Data Analysis

Prepare reactions containing 1× SYBR Green Master Mix, gene-specific primers (200-400nM each), and cDNA template (10-100ng equivalent) in a total volume of 10-20μL. Perform triplicate technical replicates for each biological sample. Use the following cycling conditions: initial denaturation at 95°C for 3-5min, followed by 40 cycles of 95°C for 10-15s and 60°C for 30-60s, concluding with a melt curve analysis to verify amplification specificity.

Calculate gene expression using the comparative ΔΔCt method, normalizing target gene Ct values to the geometric mean of validated reference genes. Report knockdown efficiency as percentage reduction compared to empty vector control samples.

Protocol: RT-PCR for Viral Spread Confirmation

Primer Design and Optimization

Design primers specific to viral vector components (TRV1 RNA-dependent RNA polymerase or TRV2 coat protein genes) to distinguish from endogenous plant sequences. Ensure amplicon size of 150-300bp for clear resolution on agarose gels. Validate primer specificity using positive control plasmids and non-inoculated plant tissue as negative control.

PCR Amplification and Detection

Amplify viral sequences using 1-2μL of cDNA template in a standard PCR reaction with 25-30 cycles to maintain semi-quantitative conditions. Include controls for: (1) non-inoculated plants, (2) empty vector VIGS plants, and (3) experimental VIGS plants. Separate amplification products on 1.5-2% agarose gels with appropriate molecular weight markers. Successful viral spread is confirmed by clear amplification bands in both empty vector and experimental VIGS samples, but not in non-inoculated controls.

Troubleshooting Common Experimental Challenges

• Low Silencing Efficiency: Optimize inoculation methods (vacuum infiltration often superior to soaking), Agrobacterium density (OD600 = 0.8-1.0), and plant growth conditions (temperature 20-23°C) [50] [31].
• High Variability in qPCR Results: Implement rigorous reference gene validation specifically for VIGS conditions [51] and ensure consistent tissue sampling from comparable leaf positions.
• Unacceptable Viral Symptoms: Use control vectors containing non-plant inserts (e.g., GFP) instead of empty vectors to minimize viral pathogenicity [31].
• Patchy or Inconsistent Silencing: Inoculate at optimal developmental stages (young seedlings often more susceptible) and ensure proper viral systemic movement conditions.

Robust molecular confirmation is fundamental to reliable VIGS experimentation. The integrated protocols presented here for qRT-PCR-based transcript quantification and RT-PCR-based viral tracking provide a comprehensive framework for validating gene silencing experiments. Proper implementation of these techniques, with particular attention to reference gene validation and appropriate controls, ensures accurate interpretation of gene function from VIGS phenotypes and advances functional genomics research in diverse plant species.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetics tool for rapidly characterizing gene function in plants, bypassing the need for stable transformation. This technology leverages the plant's innate RNA-based antiviral defense mechanism to silence endogenous genes by introducing recombinant viral vectors carrying host gene fragments [3]. The application of VIGS has revolutionized functional genomics, particularly for species with complex genomes or those recalcitrant to genetic transformation [2]. This article presents contemporary case studies and detailed protocols for applying VIGS in plant disease resistance and abiotic stress tolerance research, providing a practical framework for researchers investigating plant stress response mechanisms.

VIGS Mechanism and Workflow

Molecular Mechanisms of VIGS

VIGS operates through the plant's post-transcriptional gene silencing (PTGS) pathway, an evolutionarily conserved RNA silencing mechanism. When a recombinant viral vector containing a fragment of a plant gene is introduced into the host, the virus replicates and produces double-stranded RNA (dsRNA) intermediates during its life cycle [3]. These dsRNA molecules are recognized by the plant's Dicer-like (DCL) enzymes, which process them into 21-24 nucleotide small interfering RNAs (siRNAs). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), which uses the siRNA as a guide to identify and cleave complementary endogenous mRNA transcripts, resulting in targeted gene silencing [3]. This mechanism enables researchers to effectively knock down expression of target genes and observe resulting phenotypic consequences.

Generalized VIGS Experimental Workflow

The diagram below illustrates the standard experimental workflow for implementing VIGS in functional gene analysis:

Diagram 1: Generalized VIGS experimental workflow for functional gene analysis.

Application Notes: Disease Resistance

Case Study 1: Soybean Rust Resistance Validation

Recent research has demonstrated the successful application of TRV-based VIGS for validating genes involved in soybean rust resistance. The study established a highly efficient TRV-VIGS system utilizing Agrobacterium tumefaciens-mediated infection through cotyledon nodes, achieving systemic silencing throughout the plant with efficiency ranging from 65% to 95% [9].

Key Findings:

• Silencing of the rust resistance gene GmRpp6907 resulted in compromised resistance to soybean rust, confirming its functional role in disease resistance.
• The defense-related gene GmRPT4 was successfully silenced, leading to enhanced susceptibility, validating its importance in defense signaling.
• The cotyledon node immersion method proved highly effective, with fluorescence imaging showing successful infection in over 80% of cells, reaching up to 95% for the Tianlong 1 cultivar [9].

Case Study 2: Cotton Verticillium Wilt Resistance

A short-chain dehydrogenase/reductase (SDR) gene was identified as conferring resistance to Verticillium wilt in cotton through VIGS-mediated functional validation [52]. Silencing of this GhSDR500 gene increased susceptibility to Verticillium dahliae infection, confirming its crucial role in cotton defense mechanisms against fungal pathogens.

Case Study 3: Cotton Leaf Curl Disease Resistance

Functional analysis of nucleotide-binding site (NBS) domain genes in cotton revealed their involvement in resistance to cotton leaf curl disease (CLCuD). Virus-induced gene silencing of GaNBS (OG2) in resistant cotton demonstrated its putative role in virus tittering, establishing its importance in antiviral defense mechanisms [53].

Quantitative Data on Disease Resistance Genes Validated via VIGS

Table 1: Disease resistance genes functionally validated using VIGS technology

Gene Name	Plant Species	Pathogen	Silencing Effect	Silencing Efficiency	Citation
GmRpp6907	Soybean (Glycine max)	Soybean rust	Compromised resistance	65-95%	[9]
GmRPT4	Soybean (Glycine max)	General defense	Enhanced susceptibility	65-95%	[9]
GhSDR500	Cotton (Gossypium hirsutum)	Verticillium dahliae	Increased susceptibility	Not specified	[52]
GaNBS (OG2)	Cotton (Gossypium arboreum)	Cotton leaf curl virus	Increased virus tittering	Not specified	[53]
SITL5	Tomato (Solanum lycopersicum)	Not specified	Decreased disease resistance	95-100%	[54]
SITL6	Tomato (Solanum lycopersicum)	Not specified	Decreased disease resistance	95-100%	[54]

Application Notes: Abiotic Stress Tolerance

Comparative Analysis of Drought Response in Cotton Diploids

A phylogeny-based comparative analysis of gene expression modulation upon drought stress across three cotton diploids (G. arboreum, G. stocksii, and G. bickii) revealed significant variation in drought response mechanisms. The study employed transcriptomic approaches to identify conserved and species-specific drought response pathways [55].

Key Findings:

• G. bickii showed the widest range of expression modulation with 5,584 differentially expressed genes (DEGs), compared to 4,484 in G. arboreum and 2,147 in G. stocksii [55].
• Up-regulated genes in stressed G. arboreum and G. bickii were enriched in protein phosphorylation and dephosphorylation processes.
• G. stocksii showed enrichment in hormone-related signaling pathways, including ethylene-activated and salicylic acid-mediated signaling [55].
• A core set of 287 genes exhibited conserved regulatory responses to drought across species, highlighting pathways including starch and sucrose metabolism, chlorophyll catabolite degradation and synthesis, and hormone-mediated signal transduction [55].

VIGS Applications in Abiotic Stress Research

While VIGS has been extensively applied to study disease resistance, its implementation in abiotic stress tolerance research is expanding. The technology enables rapid functional validation of candidate genes identified through transcriptomic analyses under various stress conditions [56]. The integration of VIGS with multi-omics approaches provides a powerful platform for dissecting complex abiotic stress response networks in plants.

Experimental Protocols

Cotyledon Node Agroinoculation Method for Soybean

Application: This protocol is particularly effective for plant species with thick cuticles and dense trichomes that impede liquid penetration [9].

Materials:

• pTRV1 and pTRV2 vectors
• Agrobacterium tumefaciens GV3101
• Soybean seeds
• Sterilization solution (e.g., 70% ethanol)
• Acetosyringone stock solution
• MES buffer
• Antibiotics: kanamycin, rifampicin

Procedure:

• Vector Construction: Clone target gene fragment (300-500 bp) into pTRV2 vector using appropriate restriction enzymes (e.g., EcoRI and XhoI) [9].
• Agrobacterium Preparation:
  • Transform recombinant plasmids into Agrobacterium tumefaciens GV3101.
  • Culture positive clones in LB medium with appropriate antibiotics (50 μg/mL kanamycin, 25 μg/mL rifampicin) at 28°C for 2 days.
  • Prepare infiltration solution (10 mM MgCl₂, 10 mM MES pH 5.6, 150 μM acetosyringone) and resuspend cultures to OD₆₀₀ = 0.8.
  • Incubate in dark at 28°C for 3 hours [54].
• Plant Material Preparation:
  • Surface sterilize soybean seeds.
  • Soak in sterile water until swollen.
  • longitudinally bisect to obtain half-seed explants.
• Agroinfiltration:
  • Immerse fresh explants in Agrobacterium suspension containing pTRV1 and pTRV2 derivatives for 20-30 minutes [9].
  • Co-cultivate on medium for 2-3 days.
• Plant Growth:
  • Transfer treated plants to growth chambers with controlled conditions (16h light/8h dark, 25°C).
  • Monitor silencing phenotypes from 14-21 days post-inoculation.

Root Wounding-Immersion Method

Application: This method is suitable for multiple plant species including Nicotiana benthamiana, tomato, pepper, eggplant, and Arabidopsis thaliana, with reported silencing rates of 95-100% for PDS in N. benthamiana and tomato [54].

Materials:

• pTRV1 and pTRV2-GFP vectors
• Agrobacterium GV1301
• Plant seedlings (3-4 weeks old with 3-4 real leaves)
• LB medium with appropriate antibiotics

Procedure:

• Agrobacterium Preparation:
  • Culture Agrobacterium containing pTRV2-GFP-PDS and pTRV1 overnight.
  • Measure OD₆₀₀ and ensure >1.0.
  • Prepare infiltration solution as described in section 5.1.
• Root Treatment:
  • Carefully remove seedlings from soil, preserving roots.
  • Wash roots with pure water to remove soil.
  • Using sterilized blade, remove approximately 1/3 of root length.
• Inoculation:
  • Concurrent Inoculation: Immerse wounded roots in TRV1:TRV2 mixed solution for 30 minutes.
  • Successive Inoculation: Immerse in TRV1 for 15 minutes, then transfer to TRV2 for 15 minutes [54].
• Transplanting:
  • Transplant treated seedlings to fresh soil.
  • Maintain under controlled environmental conditions.

Validation and Analysis

Silencing Efficiency Assessment:

• Phenotypic Monitoring: For genes like PDS, photobleaching serves as visible marker, typically appearing 14-21 days post-inoculation [9] [57].
• Molecular Validation:
  • Quantitative PCR (qPCR) to measure transcript levels of target genes.
  • GFP fluorescence imaging to visualize infection efficiency [9] [54].
• Statistical Analysis: Include appropriate replicates and statistical tests to ensure reliability.

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key research reagents for VIGS experiments

Reagent/Vector	Function/Purpose	Examples/Specifications
TRV Vectors	Bipartite viral vector system for VIGS	pTRV1 (replicase proteins), pTRV2 (capsid protein + MCS) [2]
Alternative Vectors	For species where TRV is less effective	BPMV (soybean), CymMV (orchids), ALSV, CMV [9] [58]
Agrobacterium Strains	Delivery of viral vectors into plant cells	GV3101, GV1301 [9] [54]
Selection Antibiotics	Maintain vector integrity in bacterial cultures	Kanamycin (50 μg/mL), Rifampicin (25 μg/mL) [54]
Induction Compounds	Enhance T-DNA transfer from Agrobacterium	Acetosyringone (150-200 μM) [9] [54]
Marker Genes	Visual assessment of silencing efficiency	PDS (photobleaching), GFP (fluorescence) [9] [57]
Infiltration Buffer	Maintain Agrobacterium viability during inoculation	10 mM MgCl₂, 10 mM MES (pH 5.6) [54]

Signaling Pathways in Plant Stress Responses

The diagram below illustrates key signaling pathways involved in plant responses to biotic and abiotic stresses, highlighting potential targets for VIGS-based functional analysis:

Diagram 2: Key signaling pathways in plant stress responses, showing interconnected networks that can be investigated using VIGS.

VIGS has established itself as an indispensable tool for functional genomics, enabling rapid characterization of genes involved in plant disease resistance and abiotic stress tolerance. The case studies and protocols presented here demonstrate the versatility and efficiency of VIGS across multiple plant species. Recent advances in vector development, inoculation methods, and integration with multi-omics approaches have further expanded its applications. The cotyledon node immersion and root wounding-immersion methods represent significant improvements in efficiency, particularly for species traditionally recalcitrant to VIGS. As plant functional genomics continues to evolve, VIGS will play an increasingly important role in validating candidate genes and elucidating complex stress response networks, ultimately contributing to the development of improved crop varieties with enhanced stress resilience.

Within plant functional genomics, Virus-Induced Gene Silencing (VIGS) serves as a powerful reverse genetics tool for rapid gene function analysis. This application note details integrated methodologies employing GFP reporter tracking to quantitatively assess the systemic spread and durability of VIGS. The protocols are framed within a broader thesis research context aimed at standardizing VIGS validation techniques across plant species, addressing critical challenges in silencing efficiency and long-distance movement of silencing signals. We present a comparative analysis of established and emerging technologies, including traditional VIGS vectors and novel nanoparticle-based delivery systems, providing researchers with a standardized framework for evaluating spatial and temporal silencing dynamics.

Key Research Reagent Solutions

Table 1: Essential research reagents for VIGS tracking and durability studies

Reagent/Solution	Function in Experiment	Key Characteristics & Applications
TRV-based VIGS Vectors (e.g., pTRV1, pTRV2)	RNA virus vector for inducing gene silencing; pTRV2 carries the target gene fragment.	Versatile vector for dicots and some monocots; enables systemic spread and transient silencing [59] [44].
GFP Reporter Gene	Visual marker for tracking virus movement and assessing silencing efficacy via fluorescence.	Allows real-time, non-destructive monitoring of systemic spread and silencing durability using fluorescence imaging [59] [60].
Phytoene Desaturase (PDS) Gene	Endogenous reporter gene; silencing causes visible photobleaching, validating system efficacy.	Provides a rapid, scorable visual phenotype without requiring specialized equipment [59] [44].
Gu+-siRNA Nanoparticles	Novel nanocarrier for species-independent delivery of siRNA, enabling long-distance silencing.	Synthetic, non-viral delivery system; protects siRNA from degradation, facilitates vascular transport [61].
Agrobacterium tumefaciens (Strain GV3101)	Bacterial delivery vehicle for introducing VIGS constructs into plant cells via agroinfiltration.	Standard method for in-planta delivery of VIGS vectors; provides high transformation efficiency [59] [62].

Quantitative Data on Silencing Spread and Durability

Table 2: Comparative quantitative data from different gene silencing delivery systems

Delivery System / Vector	Target Plant Species	Silencing Onset	Peak Silencing / Max Efficiency	Systemic Spread Evidence	Key Durability Findings
TRV-based VIGS [59]	Iris japonica	-	36.67% (in 1-year-old seedlings)	Observed via photobleaching in tissues distant from inoculation site.	Efficiency highly dependent on seedling age; optimized for transient silencing.
TRV-based VIGS [44]	Nicotiana benthamiana	~10 days	Most pronounced at 30-45 dpi	Photobleaching in leaves, stems, axillary buds, and sepals.	Silencing persisted for ~4 months in some experiments, but efficacy declined over time.
GALV-based Vector [62]	N. benthamiana	-	Clear silencing phenotype on systemic leaves	Successful knockdown of ChlH gene in systemic leaves.	Vectors stable through three serial passages, indicating moderate persistence.
Gu+-siRNA Nanoparticles [61]	Arabidopsis thaliana, Rice	1 hour (FITC signal detection)	~98% GFP restoration in ex vivo bone marrow cells [60]	FITC signal detected in root tips, root growth zones, and leaves after root immersion.	Enabled sustained gene silencing; proposed for long-term applications due to high stability.

Experimental Protocols

Protocol 1: TRV-Based VIGS with GFP Reporter for Systemic Spread Analysis

This protocol utilizes a Tobacco Rattle Virus (TRV) vector to silence an endogenous gene while using a co-delivered GFP reporter to visually track the systemic spread of the virus and the silencing signal throughout the plant.

Materials:

• Agrobacterium tumefaciens strain GV3101
• Recombinant pTRV1 and pTRV2 vectors (pTRV2 contains target gene fragment, e.g., IjPDS [59])
• pTRV2-GFP vector for reporter tracking [59]
• Injection buffer (10 mM MES, 10 mM MgCl₂, 200 µM acetosyringone, pH 5.6)
• Sterile syringes (1 mL) without needle

Methodology:

• Vector Preparation: Transform the pTRV1, pTRV2 (with target insert), and pTRV2-GFP vectors separately into Agrobacterium.
• Agrobacterium Culture: Inoculate single colonies of each construct in 5 mL of LB medium with appropriate antibiotics and incubate at 28°C for 24 hours with shaking.
• Induction Culture: Sub-culture the bacteria into fresh LB medium with antibiotics, 10 mM MES, and 200 µM acetosyringone. Grow to an OD₆₀₀ of 0.5-1.0.
• Cell Pellet Harvesting: Centrifuge the cultures and resuspend the pellets in injection buffer to a final OD₆₀₀ of 1.0-2.0.
• Agroinfiltration Mixture: Combine the pTRV1 suspension with either pTRV2 (target) or pTRV2-GFP suspensions in a 1:1 ratio. Allow the mixture to incubate in the dark for 3-4 hours at room temperature.
• Plant Inoculation: Using a sterile syringe, press the tip against the abaxial side of leaves from 1-year-old Iris japonica seedlings (or other optimized age) and gently infiltrate the bacterial mixture.
• GFP Tracking & Phenotyping: Monitor GFP fluorescence daily using a long-wave UV light or a laser confocal microscope in a dark room. In parallel, observe and record the photobleaching phenotype of PDS silencing in systemic tissues over 2-4 weeks.
• Molecular Confirmation: Validate silencing efficiency by quantifying the reduction in target gene mRNA levels in systemic leaves using RT-qPCR.

Protocol 2: Assessing Long-Term Silencing Durability

This protocol outlines the methods for quantifying the duration and stability of the VIGS response over time, which is critical for functional studies of genes involved in prolonged developmental processes.

Materials:

• Plant material with established VIGS
• RNA extraction kit
• cDNA synthesis kit
• RT-qPCR reagents
• Equipment for fluorescence imaging (e.g., UV lamp, confocal microscope)

Methodology:

• Temporal Sampling: After VIGS establishment, systematically collect leaf discs or tissue samples from silenced and control plants every 7-10 days over a period of 2-4 months.
• Phenotypic Monitoring: At each time point, visually document the extent and intensity of the silencing phenotype (e.g., photobleaching area). For GFP-based systems, capture fluorescence images under standardized exposure settings.
• Molecular Quantification:
  • Extract total RNA from each sample.
  • Synthesize cDNA and perform RT-qPCR using primers specific for the target gene (e.g., IjPDS) and reference housekeeping genes (e.g., Actin).
• Data Analysis: Calculate the relative expression level of the target gene at each time point compared to the control. Plot the gene expression level and/or phenotypic strength against time to visualize the decay profile of the silencing effect.
• Efficiency Determination: The silencing durability can be expressed as the time taken for the target gene expression to return to 50% of its pre-silencing level, or the duration for which a visible phenotype is maintained.

Protocol 3: Nanoparticle-Mediated siRNA Delivery for Long-Distance Silencing

This protocol describes the use of novel guanidinium-containing disulfide nanoparticles (Gu+-siRNA NPs) for species-independent, long-distance gene silencing, an emerging alternative to viral vectors.

Materials:

• Guanidinium (Gu+)-containing disulfide molecule (GDM)
• Synthetic siRNA targeting gene of interest
• Nuclease-free water
• Arabidopsis or rice seedlings

Methodology:

• Nanoparticle Synthesis: Mix the GDM adjuvant with the siRNA at an N/P ratio ([Gu+]/[PO₄⁻]) of 15:1 in nuclease-free water. Allow the mixture to self-assemble into Gu+-siRNA nanoparticles (NPs) for 30 minutes at room temperature.
• Stability Verification: Characterize the formed NPs using Dynamic Light Scattering (DLS) to confirm a monodisperse size distribution averaging 200 nm.
• Plant Treatment: For root uptake, immerse the root tips of intact seedlings (e.g., Arabidopsis thaliana) in the Gu+-siRNA NP solution. Ensure the aerial parts are not in direct contact with the solution.
• Systemic Movement Tracking: As early as 1 hour post-treatment, detect the systemic movement of fluorescently labeled (FITC) siRNA using confocal microscopy in the root growth zone and distal leaves.
• Silencing Assessment: Harvest shoot and root tissues separately at 48-72 hours post-treatment. Extract RNA and protein to evaluate target gene knockdown at the transcript and protein level via RT-qPCR and Western blot, respectively.

Signaling Pathways and Workflows

Figure 1: VIGS Mechanism and GFP Reporter Tracking Workflow. The core virus-induced gene silencing (VIGS) pathway leads to target mRNA degradation (Post-Transcriptional Gene Silencing, PTGS). Concurrently, the movement of the virus, and by proxy the silencing signal, is tracked in real-time using a co-delivered GFP reporter (red path), allowing for non-destructive correlation of viral spread with functional silencing outcomes [59] [44].

Figure 2: Decision Workflow for Selecting a Gene Silencing Delivery System. This flowchart compares the primary considerations when choosing between traditional viral vectors and novel nanoparticle-based systems for gene silencing experiments. The choice depends on the target species, required durability, and experimental constraints [62] [61] [44].

Conclusion

VIGS has firmly established itself as an indispensable tool for rapid gene function validation, dramatically accelerating functional genomics in a wide range of plant species. The key to its successful application lies in a thorough understanding of its foundational principles, coupled with meticulous protocol optimization and robust, multi-faceted validation. As research advances, the integration of VIGS with multi-omics technologies and its adaptation to an even broader host range, including plants producing valuable pharmaceutical compounds, will further solidify its role in driving discoveries in plant biology and the development of novel therapeutic agents. Future efforts should focus on standardizing protocols, enhancing silencing durability, and developing novel vectors to overcome existing genotype limitations.

References

• 1. Antiviral RNA interference in plants: Increasing complexity ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12546453/]
• 2. Virus-Induced Gene Silencing (VIGS) in Functional Genomics [https://www.mdpi.com/2311-7524/11/11/1297]
• 3. Virus-Induced Gene Silencing (VIGS): A Powerful Tool for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10057534/]
• 4. Comprehensive Mechanism of Gene Silencing and Its Role ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.705249/full]
• 5. Virus-induced gene silencing: A versatile tool for discovery ... [https://www.sciencedirect.com/science/article/abs/pii/S0981942809001934]
• 6. A virus-induced gene silencing (VIGS) system for functional ... [https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-10-16]
• 7. Virus-Induced Gene Silencing, a Post Transcriptional ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC2699436/]
• 8. A Methodological Advance of Tobacco Rattle Virus ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.671091/full]
• 9. Efficient Virus-Induced Gene Silencing (VIGS) Method for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12115190/]
• 10. Functional Analysis of A Soybean Ferredoxin-NADP ... [https://www.mdpi.com/2073-4395/11/8/1592]
• 11. Virus-induced gene silencing for in planta validation of ... [https://pubmed.ncbi.nlm.nih.gov/35944218/]
• 12. Establishment of virus-induced gene silencing (VIGS) ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-023-01064-4]
• 13. Optimization of a Virus-Induced Gene Silencing System ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4853101/]
• 14. Development of tobacco rattle virus-based platform for dual ... [https://www.sciencedirect.com/science/article/abs/pii/S0168945222003168]
• 15. An all-in-one plant virus-based vector toolkit for ... [https://www.sciencedirect.com/science/article/pii/S2590346225001415]
• 16. Development of a robust and efficient virus-induced gene ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-024-01320-1]
• 17. Efficient Virus-Induced Gene Silencing (VIGS) Method for ... [https://www.mdpi.com/2223-7747/14/10/1547]
• 18. Virus-induced gene silencing is a versatile tool for ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2014.00323/full]
• 19. Virus-Induced Genome Editing (VIGE): One Step Away ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12111327/]
• 20. Virus-induced gene silencing in plants [https://www.sciencedirect.com/science/article/abs/pii/S1046202303000379]
• 21. Construction and application of a virus-induced gene ... [https://www.maxapress.com/article/doi/10.48130/tp-0024-0025]
• 22. an efficient virus induced gene silencing (VIGS) system [https://www.sciencedirect.com/science/article/pii/S2468014123000833]
• 23. Forward and reverse genomic screens enhance the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12485125/]
• 24. Beyond the genome: the role of functional markers in ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2025.1637299/full]
• 25. Roadmap and Considerations for Genome Editing in a Non ... [https://www.mdpi.com/1422-0067/25/23/12530]
• 26. Article A network-enabled pipeline for gene discovery and ... [https://www.sciencedirect.com/science/article/pii/S2667237524003539]
• 27. Optimized cDNA libraries for virus-induced gene silencing ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC2254408/]
• 28. Optimized cDNA libraries for virus-induced gene silencing ... [https://pubmed.ncbi.nlm.nih.gov/18211705/]
• 29. An Efficient Brome mosaic virus-Based Gene Silencing ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.685187/full]
• 30. Simultaneous silencing of two different Arabidopsis genes ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-020-00701-6]
• 31. An Optimized Protocol to Increase Virus-Induced Gene ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3893464/]
• 32. Establishment of Agrobacterium-Mediated Transient ... [https://www.mdpi.com/2223-7747/14/15/2412]
• 33. An Efficient Agrobacterium-Mediated Transient ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12388925/]
• 34. Advancing virus-induced gene silencing in sunflower [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-024-01241-z]
• 35. Truncated CMV2bN43 enhances virus-induced gene ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12542452/]
• 36. Silencing of the phytoene desaturase (PDS) gene affects ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6777038/]
• 37. Establishment of virus-induced gene silencing (VIGS) ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10470258/]
• 38. Knockout of phytoene desaturase gene using CRISPR ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.1074541/full]
• 39. Enhancing virus-induced gene silencing efficiency in tea ... [https://www.sciencedirect.com/science/article/abs/pii/S0304423824007398]
• 40. Truncated CMV2b N43 enhances virus-induced gene ... [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-025-01446-w]
• 41. Development of a robust and efficient virus-induced gene ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11697828/]
• 42. Efficient virus-induced gene silencing in Primulina using ... [https://www.sciencedirect.com/science/article/abs/pii/S0304423824009816]
• 43. Virus-induced gene silencing for in planta validation of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9706489/]
• 44. Application and Expansion of Virus-Induced Gene ... [https://www.mdpi.com/2311-7524/9/8/934]
• 45. Unraveling the Complexity of Plant Trichomes - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC12294984/]
• 46. A rapid, simple, and highly efficient method for VIGS and in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8605592/]
• 47. A Cotyledon-based Virus-Induced Gene Silencing (Cotyledon ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10860238/]
• 48. Vacuum and Co-cultivation Agroinfiltration of (Germinated) ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.00393/full]
• 49. Agrobacterium-mediated plant transformation: biology and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6501860/]
• 50. based virus-induced gene Silencing in Atriplex canescens [https://plantmethods.biomedcentral.com/articles/10.1186/s13007-025-01427-z]
• 51. Selection of stable reference genes for accurate reverse- ... [https://www.nature.com/articles/s41598-025-08940-0]
• 52. A short-chain dehydrogenase/reductase gene confers ... [https://www.sciencedirect.com/science/article/pii/S2667064X25002398]
• 53. Comparative analysis, diversification, and functional ... [https://www.nature.com/articles/s41598-024-62876-5]
• 54. Establishment and application of a root wounding ... [https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1336726/full]
• 55. Phylogeny-based comparative analysis of gene expression ... [https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-025-06049-0]
• 56. Advances in Functional Genomics for Exploring Abiotic ... [https://www.mdpi.com/2223-7747/14/16/2459]
• 57. Development and application of virus-induced gene ... [https://www.sciencedirect.com/science/article/abs/pii/S030442382300763X]
• 58. Virus-induced gene silencing (VIGS) for functional analysis ... [https://www.sciencedirect.com/science/article/abs/pii/S0304423822006057]
• 59. Developing a homology-driven gene silencing system in < ... [https://www.maxapress.com/article/id/68c8b5d4fa6c58419c48f3c2]
• 60. GFP-on mouse model for interrogation of in vivo gene editing [https://www.nature.com/articles/s41467-025-61449-y]
• 61. Enabling long‐distance gene silencing in plants via ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11933838/]
• 62. Development of Virus-Induced Gene Expression and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4968648/]