Introduction

Functional genomic research on several model and crop plants has benefitted from the wealth of sequence information now available. Bioinformatics tools, genome sequences, expressed sequence tags, shotgun sequencing, next-generation sequencing, RNA-seq and microarrays have all made major contributions to progress in this area of research1,2. However, these technologies do not provide a complete understanding of the functional relevance of gene sequences. Generation and characterization of stable plant mutant lines and downregulation of gene expression are useful for understanding gene function. However, only Arabidopsis3 and a few other plant species have cloning-friendly insertional mutant resources (Supplementary Table 1). In addition, targeting induced local lesions in genomes (TILLING)4 can only be used for mutant identification in plant species in which the genome sequences are available and well annotated. RNA interference (RNAi)5,6 and gene overexpression studies are currently limited to plant species that are amenable to genetic transformation, and these studies are time-consuming. Artificial microRNA-mediated gene silencing7 and microRNA-induced gene silencing8 can be used for targeted gene silencing in plants, but they require stable plant transformation, which is time consuming and unsuitable for high-throughput studies. Similarly, zinc-finger nucleases (ZFNs)9, transcription activator–like effector nucleases (TALENs)10 and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system11,12 can be used to generate specific gene knockouts in plants, but these methods are mainly suitable as 'reverse genetics' tools, and they too require time-consuming stable plant transformation.

Targeted gene silencing using VIGS

VIGS is a PTGS method13 used by plants as a defense mechanism against invading viruses14. During viral replication, double-stranded RNA (dsRNA) is produced by an RNA-dependent RNA polymerase, and this triggers PTGS13,14. One of the major aspects of this process involves Dicer-like enzyme-mediated cleavage of dsRNA to produce siRNA14. This siRNA binds to and activates the RNA-induced silencing complex (RISC), which cleaves the viral RNA in a homology-dependent manner14. This plant defense strategy has been exploited to develop a method for silencing endogenous target genes in plants15. To silence a specific plant gene, a fragment of the target gene is cloned into a modified virus vector and delivered into the plant15,16. The target gene fragment inserted in the virus vector is multiplied by the virus replication machinery in the plant cell, and the transcripts are systemically spread throughout the plant15. When PTGS is induced, siRNA homologous to the target gene will be generated, which will direct silencing of the endogenous plant gene.

VIGS has been widely used to study the function of plant genes for various biological processes17 after it was first reported in the late 1990s (refs. 15,18). VIGS not only overcomes many of the above-mentioned hurdles in gene functional analyses, but also has added advantages; namely, it can be used to study the function of genes where mutations are embryo-lethal or result in a severely deformed plant19. In addition, VIGS is suitable for high-throughput gene function analysis16,20, is relatively easy to perform in a shorter time compared with other PTGS methods available to date and does not alter the plant genome21. It can be used for both forward- and reverse-genetics–based gene identification and functional characterization22,23.

Using TRV as a VIGS vector

Many VIGS vectors are available for silencing in various plant species21,24. However, there are 22 commonly used VIGS vectors that have gene silencing ability in more than one plant species (Supplementary Table 2). TRV-based VIGS vectors (Supplementary Fig. 1) are widely used in several plant species, especially in Solanaceae family plants such as N. benthamiana and tomato21. Other VIGS vectors have been reported to cause gene silencing in N. benthamiana21, including barley stripe mosaic virus (BSMV), apple latent spherical virus (ALSV), potato virus-X (PVX), tobacco mosaic virus (TMV) and tomato golden mosaic virus (TGMV). Among these, TRV- and PVX-based vectors are commonly used to silence target gene expression in N. benthamiana. TRV can infect meristem tissues25,26, whereas many other VIGS vectors cannot. The PVX-based VIGS vector15,27,28 causes virus-associated symptoms such as chlorosis, leaf distortion and localized cell death in infected plants, sometimes making the silencing phenotype difficult to interpret26. In contrast, TRV-VIGS vector–infected plants show milder symptoms.

TRV has two genomes: TRV1 (i.e., RNA1) and TRV2 (i.e., RNA2; ref. 29). TRV1 is essential for viral movement30. TRV1 has genes encoding 134- and 194-kDa replicase proteins, a 29-kDa movement protein and a 16-kDa cysteine-rich protein whose function is not fully known29. The TRV2 genome varies among different isolates of this virus and has genes encoding the coat protein and nonstructural proteins29. These nonstructural proteins are implicated in nematode transmission of this virus31, but they are not essential for plant infection in the laboratory. Therefore, for use as a VIGS vector, the two nonstructural protein–encoding genes in TRV2 are replaced with multiple cloning sites for inserting fragments of the target gene to be silenced26 (Supplementary Fig. 1). Both TRV1- and TRV2-derived constructs are then cloned into a binary vector for Agrobacterium-mediated plant inoculation25,26.

Modifications to the TRV-VIGS system

TRV-VIGS was first reported for endogenous gene silencing in N. benthamiana26, and this vector system was later modified25. The TRV2 vector was enabled for gateway-based32 or ligation-independent33 cloning (LIC) of plant gene inserts. We developed the Agrodrench method of inoculation of TRV vectors in N. benthamiana34. Further, we demonstrated the possibility of using gene fragments from different plant species for silencing homologs in N. benthamiana35,36. We also demonstrated that VIGS can be used for silencing genes for a long duration (∼2 years) and for transmission of silencing to progeny seedlings37. In addition, cDNA libraries have been developed in the TRV vector38,39 for forward genetics and large-scale silencing to identify plant genes that have a role during plant development and stress responses23,39,40,41,42. Recently, TRV1 has been modified to insert plant gene fragments, and this vector invoked gene silencing without TRV2 (ref. 43). Taken together, the studies cited here have contributed to improving the versatility of TRV-VIGS in N. benthamiana and are expected to provide more efficient TRV-based VIGS systems in the future.

Advantages of TRV-VIGS

By using TRV-VIGS, a specific gene can be silenced in 3–4 weeks, and the silenced plants can be maintained for more than 2 years for performing various analyses37 (Fig. 1). TRV vectors are used in a broad host range with high silencing efficiency. Some gene-silenced plants can be vegetatively propagated via cuttings to maintain silencing in the clones. Leaf punches from gene-silenced plants can be used to maintain gene silencing in the calli derived through tissue culture39,44. TRV-VIGS can silence genes in most plant parts19,34,45, even in detached fruits46. These advantages are specific to TRV-VIGS, and other VIGS vectors have not been reported to possess these features. In addition to these advantages, gene silencing is also transmitted to a small percentage of progeny seedlings, facilitating target gene silencing during seed germination and early stages of seedling development37. VIGS has also been used for silencing genes during early stages of seedling growth by using the sprout-vacuum method47. Only a few other VIGS vectors (e.g., BSMV and ALSV) are suitable for gene silencing in progeny seedlings. Taken together, these abilities make TRV-VIGS one of the best methods for both forward23,39,40,48 and reverse22,33 screens for high-throughput gene function analyses. TRV-mediated VIGS has been successfully used in various plant species21,34,35. In this manuscript, we describe the protocol for TRV-mediated VIGS in N. benthamiana.

Figure 1: Overview of the VIGS protocol in N. benthamiana.
figure 1

A GOI is cloned into the TRV2 vector by using conventional restriction digestion–based or gateway-based or ligation-independent cloning, depending on the vector. Selection of the appropriate gene fragment is important for invoking efficient gene silencing and to avoid off-target gene silencing. Transform the construct into Agrobacterium, and co-inoculate the culture along with Agrobacterium carrying the TRV1 vector onto 3-week-old plants. Silencing usually starts from 2 to 3 weeks. Plants are confirmed for downregulation of the endogenous target gene, and if needed the plants are also tested for virus titer by leaf lesion assay. Silenced plants can be used for analysis from 3 weeks after TRV inoculation. Silencing can be maintained for longer periods by booster inoculations with TRV1 and TRV2::GOI. Seeds of the silenced plants that harbor VIGS vector can be used to study the inheritance of VIGS in progeny seedlings. Dark-green leaves represent nonsilenced leaves, and light-green leaves represent gene-silenced leaves.

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Applications of TRV-VIGS

The foremost application of this method is to silence a specific plant gene by a reverse-genetics approach to study its function during a particular biological process in planta. Various aspects of plant development, biosynthesis of metabolites, biotic and abiotic stress tolerance, and plant evolution have been studied using TRV-VIGS17,19, and this information is compiled in Supplementary Figure 2. Another application of TRV-VIGS is to identify plant genes required for a particular biological process by forward-genetics screening48. To cite a few examples, we applied this method for the identification of plant genes involved in Agrobacterium-mediated plant transformation39, nonhost disease resistance23,40 and a phytotoxin (coronatine)-induced chlorosis41. In addition to the basic applications of any VIGS vector–based method17,19,21, the TRV-VIGS–based method described in this manuscript can be used to specifically study the target gene function during seed germination, early seedling establishment and meristem growth. In addition, TRV-mediated VIGS can be maintained for a long period, which enables various assays to be performed on the same plant25,34,37. Protocols described for TRV-VIGS in this manuscript can be applied to several other plant species with minor modifications (Supplementary Table 3). We also describe various methods of TRV inoculation (Supplementary Table 4) in this manuscript; hence, researchers have an option to choose the approach appropriate for their experiment.

Limitations

First, in most cases VIGS does not result in a complete loss of gene expression. If the study demands a complete loss of target gene expression, it is advisable to choose an alternative method (e.g., mutants) for functional analysis. Second, off-target gene silencing can occur. Thus, it is important to choose a fragment of a gene that produces specific siRNAs targeting only the gene of interest (GOI). Third, TRV may not only alter plant metabolism or plant gene expression, but may also cause mild virus-associated phenotypic changes in the gene-silenced plants. In some cases, it may be difficult to nullify the 'virus effect'. Fourth, gene silencing is not always uniform. Hence, a large number of replicate plants need to be analyzed to obtain consistent results. Note that many of these limitations are inherent to all VIGS vectors and are not specific to TRV. Ways to overcome some of these limitations are described below in the PROCEDURE section of this manuscript and also in a recent review21. Nevertheless, appropriate vector controls and inclusion of many replicates are important for meaningful VIGS experiments. In addition, efficient experimental design, including the use of uniform plant age and prestandardized environmental growth conditions37,48, is important for obtaining consistent results.

Overview of the procedure

To silence a target gene, TRV1 vector and gene-specifically modified TRV2 vector are individually mobilized into disarmed Agrobacterium tumefaciens for agroinoculation49. Agrobacterium cultures are grown overnight to an appropriate optical density (OD) and virulence is induced with acetosyringone49. Induction of Agrobacterium virulence (Vir) genes by using acetosyringone49 is important for efficient transfer of T-DNA carrying the virus into the plant cell. Agrobacterium strains carrying TRV1 and TRV2 derivatives are then co-inoculated into the lower leaves of the target plant. The method chosen to deliver the Agrobacterium solution carrying VIGS constructs into the plant is dependent on the planned downstream studies (Experimental design). Agrobacterium-mediated delivery of the virus vectors is the mode of choice because this method is efficient, easy and cost-effective compared with direct inoculation of the virus particles13,49. Gene silencing usually occurs from 2 weeks after inoculation, and plants are then analyzed. To maintain gene silencing for longer periods, booster inoculations can be done37. Long-duration silencing will be useful for studying the relevance of target genes until the terminal growth stage of the plant. In addition, owing to transmission of the virus to seeds, TRV-VIGS is also transmitted to a small number of progeny seedlings37.

Target gene silencing is assessed by reverse-transcription quantitative PCR (RT-qPCR), using primers that amplify an endogenous target gene, as described previously37. An average of ∼80% downregulation in endogenous transcript levels of the target gene compared with vector control plants can be achieved by TRV-VIGS. However, the silencing efficiency can vary depending on several factors, including virus infection, systemic spread, threshold of virus titer, homology of target gene fragment in the vector with the endogenous target gene mRNA and environmental factors during plant growth21,35,37. The optimum level of transcript reduction to observe a desired phenotype or change in expected biological process can vary depending on the gene. If severely deformed plants are observed, a leaf lesion assay37,50 is performed, by using Chenopodium amaranticolor plants, to determine whether the presence of excess TRV contributes to the phenotype37. An overview of the TRV-VIGS protocol is depicted in Figure 1.

Experimental design

Construct design. The first step involved in designing an efficient TRV2 VIGS construct is selection of the target gene fragment; the aim is to choose a gene fragment that produces efficient siRNAs with minimal predicted off-target gene silencing. Candidate fragments of 200–400 nt should be selected from a coding or untranslated region (UTR) of the gene. This is the optimum length for efficient VIGS with minimal off-target effects. However, fragments with fewer than 100 nt and as many as 1.5 kb can also invoke TRV-VIGS. Gene fragments with fewer than 100 nt may have reduced silencing efficiency, whereas fragments that are more than 1,300 nt can become unstable in TRV, and inserts larger than 400 bp also increase the chance of off-target silencing38. Poly-A tail sequences should be avoided. Bioinformatics tools (e.g., siRNA scan, http://bioinfo2.noble.org/RNAiScan.htm or http://plantgrn.noble.org/pssRNAit/) can be used for predicting siRNA sequences51. These tools can be used to design vectors for silencing all or many genes in a gene family. Alternatively, a more specific region (e.g., UTR) that does not share homology with other genes can also be chosen for silencing a particular gene in a gene family by using these bioinformatics tools. It is essential to make sure that the selected fragment does not produce similar 21-nt siRNAs that can match other genes; this can be tested by using the Basic Local Alignment Search Tool (BLAST) or siRNA prediction software. Alternative fragments should be selected if the software reveals possible off-target effects.

The second step is selection of appropriate plant tissue (e.g., leaf) at the appropriate age or particular time in the day or night for RNA extraction, depending on the expression pattern of the target gene. Some target genes may be temporally or spatially regulated, or controlled by circadian rhythm or environmental stress.

The third step is to clone the gene fragment into a TRV2-based vector. Currently, TRV2 vectors are available for conventional, gateway-ready and ligation-independent cloning. Both sense and antisense orientations of the insert in the TRV2 vector have been shown to invoke similar VIGS38. For forward-genetic screening, a cDNA library can be constructed, as described previously38.

Controls. Plants co-inoculated with TRV2::GFP and TRV1 should be used as a vector control, and results from the target gene–silenced plants should be compared with this vector control to rule out effects of TRV infection. Previous studies showed that a TRV2 vector carrying an insert (e.g., a GFP fragment34) is a better vector control than an empty vector (TRV2::00) (refs. 52,53). As a positive control, the phytoene desaturase (PDS) gene or Mg-protoporphyrin chelatase (ChlH) gene can be targeted for VIGS. PDS-silenced plants show photobleaching (albino phenotype) of aerial plant parts owing to disruption of phytoene desaturation, which is an important step in the β-carotene biosynthesis pathway35,37,54. ChlH gene–silenced plants show a yellowing phenotype owing to disruption of chlorophyll biosynthesis55.

Inoculation methods. We include protocols for Agrobacterium-mediated delivery of TRV1 and TRV2 constructs into target plants by syringe inoculation36, agrodrench33 and prick inoculation23 with toothpicks (Fig. 2 and Supplementary Table 4). Similar protocols are described in other publications56,57,58. Syringe inoculation and agrodrench are useful for inoculating a few plants, and toothpick-mediated inoculation is used for large-scale experiments such as forward-genetics screening. One of the advantages of the syringe inoculation method is that it can deliver a precise amount of inoculum and it invokes efficient and consistent gene silencing across various replicate plants. The agrodrench method not only is easy to perform but also improves the efficiency of gene silencing at the whole plant level, including roots, compared with the syringe-mediated leaf infiltration method. However, the syringe inoculation and agrodrench methods are not feasible for inoculating a large number of plants. Agrodrench can increase cross-contamination with neighboring plants when placed in the same container during watering. Prick inoculation by toothpicks is easy and quick to perform, and it is cost-effective compared with the syringe inoculation method. However, the gene-silencing efficiency by the prick inoculation method is lower compared with the syringe inoculation or agrodrench methods.

Figure 2: Different methods of TRV inoculation in N. benthamiana plants.
figure 2

(a) Agrobacterium cultures carrying TRV1 and TRV2 containing the target gene fragment (TRV2::GOI) can be inoculated into leaves with a needleless syringe. (b) Another method of inoculation called agrodrench involves drenching the culture at the crown region of the plant. These two methods are used for silencing one or a few genes in a reverse-genetics approach. (c) Another method used during forward-genetics screening is pricking the leaves with a toothpick dipped in an Agrobacterium colony carrying TRV2::GOI at the site of infiltration of the Agrobacterium culture carrying TRV1.

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Materials

REAGENTS

  • Luria-Bertani (LB) medium agar capsules (MP Biomedicals, cat. no. 113002031)

  • LB medium liquid capsules (MP Biomedicals, cat. no. 113002031)

  • Antibiotics (gentamicin, rifampicin and kanamycin)

  • 2-(N-Morpholino) ethanesulfonic acid (MES) buffer (VWR, cat. no. 97062)

  • Acetosyringone (Sigma-Aldrich, cat. no. D134406), see Reagent Setup

  • K2HPO4 (JT Baker, cat. no. 4012-01)

  • KH2PO4 (JT Baker, cat. no. 3246-01)

  • DMSO (Sigma-Aldrich, cat. no. 472301)

  • Carborundum (Sigma-Aldrich, cat. no. 05-1700)

  • Glycerol (JT Baker, cat. no. 2136-01)

  • Bleach (Clorox, available from local stores)

  • PCR master mix GoTaq (Promega, cat. no. M7122)

  • RNeasy mini plant RNA extraction kit (Qiagen, cat. no. 74104)

  • cDNA synthesis superscript-III RT kit (Life Technologies, cat. no. 11752)

  • Plasmid Plus mini kit (Qiagen, cat. no. 12123)

  • KiCqStart SYBR Green qPCR ready mix (Sigma, cat. no. KCQS00)

  • Custom-synthesized gene-specific primer pairs for semiquantitative RT-PCR and RT-qPCR analysis (IDT DNA Technologies), see Reagent Setup for design considerations and Supplementary Table 5 for details of commonly used primer sequences

  • Metro-Mix 350 (Sun Gro horticulture distribution) plant growth medium. Other soil or soil-less plant growth medium can also be used

  • Peters Professional 20-10-20 Peat-Lite Special (Everris NA) at a rate of 0.5 grams per liter in tap water. Other nutrient or fertilizer sources can also be used

  • TRV1 vector plasmid DNA (Arabidopsis Biological Resource Center (ABRC) stock no. CD3-1039, vector name YL192), TRV1 National Center for Biotechnology Information (NCBI) accession no. AF406990

  • Gateway-compatible TRV2 vector plasmid DNA (ABRC stock no. CD3-1041, vector name YL279) harboring target gene sequence (TRV2::GOI), TRV2 NCBI accession no. AF406991

  • TRV2 control vector. Example, TRV2::GFP (Reagent Setup). GFP gene NCBI accession no. U87625

  • N. benthamiana plants, grown as described in Reagent Setup. Individually potted 3-week-old plants are used in the PROCEDURE

  • C. amaranticolor plants, grown as described in Reagent Setup. Individually potted 2-week-old plants are used for the leaf lesion assay described in Box 1

  • A. tumefaciens strain GV2260

    Critical

    Other disarmed Agrobacterium strains, such as GV3101, EHA105 and LBA4404, can also be used. However, GV2260 is the most suitable strain for N. benthamiana and other Solanaceae family plants.

  • Escherichia coli–competent cells, strain JM109 (Promega, cat. no. P9751)

EQUIPMENT

  • Growth chamber for seed germination and initial seedling growth (Percival Scientific, cat. no. CU32L)

  • Geranium pots, 11.4 cm (Dillon Products, cat. no. GPR450GT4)

  • 3-inch pots (Dillon Products)

  • Nursery supplies classic one-gallon pots (TO plastics, cat. no. 300)

  • Nursery supplies classic three-gallon pots (TO plastics, cat. no. 1000)

  • Flats for seed sowing (Dillon Products, cat. no. GPPF288CE4)

  • Shaker (Thermolyne Bigger Bill platform shaker M49235)

  • Centrifuge (Eppendorf, 5810R)

  • Benchtop pH meter (VWR sympHony, cat. no. B20PI 89231-692) with probe (cat. no. 89231-580)

  • Spectrophotometer (Jenway, cat. no. 6320D)

  • MicroPulser electroporator (Bio-Rad, cat. no. 165-2100)

    Critical

    Set the instrument to 2,500 V before electroporation. Electroporation should be performed according to the manufacturer's instructions.

  • Mini Vortexer VM-3000 (VWR, cat. no. 58816-121)

  • Fume cupboard

  • Real-time PCR detection system (Bio-Rad, cat. no. CFX96)

  • 96 Solid Pin Multi-blot replicator (V&P Scientific, cat. no. VP409)

  • Mortar and pestle (VWR, cat. no. 89037)

  • Face shield with head gear (Cole Parmer, cat. no. EW-81690-00)

  • Cuvette for Gene Pulser/MicroPulser (Bio-Rad, cat. no. 165-2089)

  • Needleless syringe, 1 ml (Becton Dickinson, cat. no. 301025)

  • Wooden toothpicks (Diamond toothpicks, available from local stores)

  • U-bottom plates, 96 well (Becton Dickinson Labware, cat. no. 35-3077)

  • Lids Seal alum foil (VWR, cat. no. BK538619). Appropriate seals suitable for cold storage from any other supplier can also be used

REAGENT SETUP

Primer design

  • Forward and reverse primers for the quantification of the target gene fragment by RT-qPCR can be designed with PrimerQuest software by IDT technologies (http://www.idtdna.com/Primerquest). Any other similar primer design software can also be used. Some preferred primer characteristics are as follows: Tm ∼60 °C, 18–25 nt length and 40–60% GC content. Product length can be in the range of 60–150 nt. Primers for checking the downregulation of a predicted off-target gene(s) can be designed by following similar parameters. Some of the commonly used primers are given in Supplementary Table 5.

    Critical

    At least one primer should be designed outside the region of the gene that is used in the VIGS vector to elicit gene silencing. Primers should not form dimers and should be specific for the target gene without cross-amplification of nontarget genes. This is especially important for amplification of a specific gene from a gene family.

TRV2::GFP vector

  • The GFP-encoding gene fragment was amplified from transgenic N. benthamiana plants expressing the jellyfish (Aequorea victoria) GFP gene59. This GFP fragment was cloned into the TRV2 vector by gateway-based cloning, as described previously34, and this construct is referred to as TRV2::GFP. The targets of siRNAs expected to be generated from the GFP (from the 101–552-bp NCBI accession no. U87625) fragment that was selected for cloning into TRV2 were analyzed by siRNA scan software by using an N. benthamiana draft genome60 and expressed sequence tag sequences. No off-targets were predicted in this analysis. Confirm successful cloning by sequencing the insert. TRV2::GFP is used to develop vector control plants.

TRV2::GOI vector

  • About 200–400 nt fragments of the target GOI are cloned into TRV2 vector by conventional or gateway-based or LIC-independent cloning methods25,32,33. Confirm successful cloning by sequencing the insert. This vector is referred to as TRV2::GOI.

cDNA library

  • cDNA library in the TRV2 vector can be prepared by following previously described methods38. The library mentioned as an example in this manuscript was constructed from the RNA extracted from leaf tissues by following the subtractive hybridization method48. Verification of the insert in the TRV2 vector is as previously described48.

Preparation of Agrobacterium-competent cells for transformation

  • Competent cells are needed for the transformation of plasmids into bacteria. Competent cells for A. tumefaciens strain GV2260 can be prepared according to previously described procedures61. Other similar protocols can also be used for competent cell preparation.

LB liquid medium

  • LB medium liquid capsules are added to sterile water by following the supplier (MP Biomedicals) recommendations and autoclaved. This can be stored at room temperature (∼21 °C) for 3–4 weeks.

LB agar plates

  • LB medium agar capsules are added to sterile water according to the supplier's recommendations and then autoclaved. If needed, antibiotics are added, warm medium is poured into appropriately sized Petri plates and the agar is allowed to solidify. The plates can be stored at 4 °C for 3–4 d.

96-well plate containing glycerol-liquid LB medium

  • LB medium liquid capsules are added to sterile water according to the supplier's (MP Biomedicals) recommendations. Add glycerol (10% (vol/vol)) and autoclave. Required antibiotics can be added after the medium reaches room temperature, and just before use. About 220 μl (for wells of 230-μl capacity) can be dispensed into all 96 U-bottom wells by using a multichannel pipette.

Glycerol stock preparation for long-term storage of bacteria

  • LB medium liquid capsules are added to sterile water by following the supplier's (MP Biomedicals) recommendations. Add glycerol (50% (vol/vol)) and autoclave. Add the bacterial culture growing at exponential growth phase (750 μl) to the 50% (vol/vol) glycerol solution (750 μl) at a 1:1 (vol/vol) ratio in a 2-ml screw-top tube or cryovial. Freeze the tubes in liquid nitrogen after shaking them 4–5 times, and store them at –80 °C. The final concentration of the glycerol in the tubes should be 25% (vol/vol).

Sowing N. benthamiana

  • Sow seeds on soil-less potting medium Metro-Mix 350 in a flat tray, and then incubate them in a growth chamber maintained at a temperature of 28 °C and a relative humidity of 60–70% for 1 week. Thin the seedlings, and place them at 30 °C day/24 °C night at a 16-h day length for 2 more weeks. Water the seedlings in the growth chamber with Peters 20-10-20 Peat-Lite Special at the rate of 0.5 grams per liter in tap water as and when needed. This protocol is also described in previous literature48 and demonstrated in Supplementary Video 1.

Sowing C. amaranticolor

  • Sow seeds in a potting medium (Metro-Mix 350) in geranium pots tray, and incubate them in a growth chamber maintained at a temperature of 28 °C and a relative humidity of 60–70% for 1 week. Then move the seedlings to a greenhouse maintained at 23 °C. Apply Peters 20-10-20 Peat-Lite Special as a fertilizer at the rate of 0.5 grams per liter in tap water as and when needed.

    Critical

    Potting medium should have optimum moisture content for seed imbibition. Optimum temperature (28 °C) and relative humidity (∼70%) are important for efficient seed germination. Potting medium should be free of pathogen inoculum to prevent diseases (e.g., damping off).

Acetosyringone stock

  • Dissolve 39.24 mg of acetosyringone powder in 1 ml of DMSO, and vortex the mixture thoroughly to prepare a 200 mM solution. Store the stock at −20 °C.

    Caution

    DMSO should be handled in a fume cupboard, and protective clothing should be worn when handling it. Use pipette tips that are safe to handle solvents or use glass pipettes.

    Critical

    Acetosyringone stock should be used within 3 months of the date of preparation.

Agrobacterium induction buffer

  • Dissolve 0.195 g of MES in 100 ml of water (10 mM), and then add 200 μl (200 mM stock) of acetosyringone stock. Adjust the pH to 5.5. 100 ml of buffer is sufficient for Agrobacterium cultures for ∼10 constructs.

    Critical

    Freshly prepare the buffer for every experiment.

Infiltration buffer

  • Dissolve 0.0976 g of MES in 100 ml of water (5 mM). Adjust the pH to 5.5. 10 ml of buffer is sufficient for syringe inoculation of ∼5 plants. 60 ml of buffer is sufficient for agrodrench inoculation of ∼10 plants.

    Critical

    Freshly prepare the buffer for every experiment.

Phosphate buffer

  • Add 13.2 ml (1 M stock) of K2HPO4 into 86.8 ml (1 M stock) of KH2PO4 to get 0.1 mM phosphate buffer. Adjust the pH to 6.0.

    Critical

    After autoclaving, the buffer stock can be stored at room temperature for up to 2 months. Do not use the buffer if a precipitate has formed.

Procedure

Caution

All cultures, plants, plant growth medium and apparatus used for handling cultures must be disposed of by duly complying with institutional biosafety regulations. Some routine lab practices include autoclaving all material that comes in contact with Agrobacterium-containing TRV before disposal. Accidental spillage of Agrobacterium cultures and TRV-infected plant parts should be handled as per standard operating procedures. Institutions are expected to develop standard operating procedures based on national guidelines (e.g., US National Institutes of Health and/or United States Department of Agriculture) for handling virus-infected plant materials in the greenhouse and growth chambers. Biosafety level (e.g., Biosafety level 1 in the United States)-certified laboratories and greenhouses are required for performing VIGS experiments. TRV can infect more than 400 plant species; hence, environmental release of this virus must be prevented. Shipment of virus vectors, virus-infected plant materials and progeny seeds obtained from VIGS plants should be carried out only after obtaining appropriate permits (e.g., APHIS 2000 for interstate shipments within the United States).

Preparation of plants for inoculation

Timing 2–3 d

  1. 1

    Transplant 3-week-old individual N. benthamiana seedlings into 3-inch pots containing Metro-Mix 350, and grow them in the greenhouse until TRV inoculation (Step 10). Water them with Peters Professional 20-10-20 Peat-Lite Special fertilizer at a rate of 0.5 grams per liter in tap water as and when needed.

    Critical Step

    TRV inoculation should only be performed 2–3 d after transplantation. This time period helps plants overcome the transplantation shock and establish better roots before virus infection. We have observed that VIGS is not efficient in plants older than 4 weeks. Recommended environmental conditions in the greenhouse are as follows: temperature 21 ± 2 °C, relative humidity 45–60% and light intensity 350–450 μmol/m2/s with 14 h day/10 h night.

    Troubleshooting

Transforming Agrobacterium with TRV constructs

Timing ∼3–10 d

  1. 2

    Transform TRV-VIGS constructs into competent Agrobacterium (Reagent Setup). Transformation of individual TRV2::GOI constructs is described under option A, and the transformation of a cDNA library is described under option B.

    1. A

      Transformation of individual TRV constructs

      1. i

        Prepare plasmid DNA for TRV2::GOI (silencing construct, Reagent Setup), TRV2::GFP (vector control, Reagent Setup) and TRV1 constructs by using the Plasmid Plus mini kit according to the manufacturer's (Qiagen) instructions.

      2. ii

        Separately transform 1 μl of plasmid DNA (concentration 15 ng/μl) of each construct into A. tumefaciens strain GV2260 (50 μl of competent cells per plasmid) by the electroporation method by using a MicroPulser Electroporator according to the manufacturer's (Bio-Rad) instructions.

      3. iii

        Separately set up 1 ml of culture in LB liquid medium (without antibiotics, Reagent Setup) for each transformation event. Grow the cultures at 28 °C in a shaker at 250 r.p.m. for 2 h.

      4. iv

        Separately spread 100 μl of each culture on LB agar plates (Reagent Setup) containing rifampicin (10 μg/ml) and kanamycin (50 μg/ml). Incubate the plates at 28 °C for 2 d.

        Pause point

        Individual bacterial colonies carrying each construct can be streaked on an LB agar plate containing antibiotics and grown as described in this step; after bacterial growth, these plates can be stored for 1 week at 4 °C. Liquid cultures of bacteria can be stored as glycerol stocks (Reagent Setup) for several years.

      5. v

        Proceed to Step 3 to set up liquid cultures from individual colonies.

        Troubleshooting

      Timing ∼3 d

    2. B

      Transformation of a cDNA library

      1. i

        Transform 200 ng of the cDNA library pool (Reagent Setup) into A. tumefaciens strain GV2260 (50 μl of competent cells per 15 ng of cDNA) by electroporation by using a MicroPulser Electroporator according to the manufacturer's (Bio-Rad) instructions.

        Critical Step

        cDNA quantity is approximate, and the values mentioned here are based on the initial RNA concentration used for making cDNA.

      2. ii

        Separately set up 1 ml of culture in LB liquid medium (without antibiotics) for each transformation event. Grow the cultures at 28 °C in a shaker at 250 r.p.m. for 2 h.

      3. iii

        Spread 50 μl of the liquid culture on LB agar plates containing rifampicin (10 μg/ml) and kanamycin (50 μg/ml). Incubate the plates at 28 °C for 2 d. Repeat this step for additional plates depending on the number of clones required for the experiment.

        Critical Step

        Periodically monitor the appearance of bacterial colonies on the plate. Remove the plate from the 28 °C incubator before individual colonies merge.

        Troubleshooting

      4. iv

        Pick individual colonies with toothpicks and inoculate them into individual wells of a 96-well plate. The wells contain glycerol–liquid LB medium (Reagent Setup) with rifampicin (10 μg/ml) and kanamycin (50 μg/ml).

        Critical Step

        Label the plates to enable their identification.

      5. v

        Incubate the cultures at 28 °C in an incubator for 2–4 d.

        Critical Step

        Make sure that the culture is fully grown, as determined by the turbidity of the medium (OD600 = ∼0.2). Because of the presence of glycerol, growth may sometimes take more than 3 d.

        Troubleshooting

      6. vi

        Seal the plate with aluminum foil sealing film, and then close the lid. Store the 96-well plates as glycerol stocks at −80 °C.

        Pause point

        Plates can be stored at −80 °C for several years.

      Timing ∼10 d

Growing Agrobacterium cultures and preparation for agroinoculation

Timing ∼2 d

  1. 3

    If you intend to perform inoculation by needleless syringe (Step 10A) or by agrodrench (Step 10B), set up separate 2-ml cultures for TRV1, TRV2::GOI and TRV2::GFP. If you intend to perform inoculation by pricking (Step 10C), only a TRV1 culture is required. Set up the cultures by inoculating 2 ml of LB liquid medium containing rifampicin (10 μg/ml) and kanamycin (50 μg/ml) with a single Agrobacterium colony carrying the appropriate construct (e.g., from Step 2A(iv)). Grow the cultures at 28 °C in a shaker at 250 r.p.m. for 10–12 h.

  2. 4

    Subculture 0.5 ml of each 2-ml culture from Step 3 into 10 ml of LB liquid medium containing rifampicin (10 μg/ml) and kanamycin (50 μg/ml), and grow the culture at 28 °C with shaking at 250 r.p.m. for 5–6 h.

    Critical Step

    The duration of the culture growth should be decided on the basis of the OD required at the next step. Agrobacterium carrying TRV1 sometimes grow slower than TRV2 cultures. In such cases, use freshly transformed Agrobacterium for inoculation.

  3. 5

    Measure the absorbance of each culture with a spectrophotometer (OD600). An OD600 of 0.5–0.6 is preferred.

    Critical Step

    Use LB liquid medium along with antibiotics as a blank for measuring the OD of Agrobacterium cultures.

  4. 6

    When the appropriate OD is reached, collect the cells from each culture by centrifugation (3,000g) for 5 min at room temperature.

    Caution

    The supernatant should be treated with an equal volume of bleach (20% (vol/vol)) for 30 min before discarding it in the drain.

  5. 7

    For each culture, resuspend the cells in 10 ml of Agrobacterium induction buffer (Reagent Setup), and incubate them at room temperature in a shaker (50 r.p.m.) for 3 h.

  6. 8

    Collect the cells by centrifugation (3,000g) for 5 min at room temperature, and then resuspend the cultures for each construct independently in ∼5 ml of infiltration buffer (Reagent Setup).

    Critical Step

    Decide the appropriate volume of infiltration buffer for resuspending the cells on the basis of the OD value required. The recommended range for syringe inoculation (Step 10A) is OD600 = 0.2–0.6; for agrodrench (Step 10B) it is ∼1.0; and for the pricking method (TRV1 only, Step 10C) it is ∼0.3. OD has to be optimized for each experiment, depending on the target tissue and plant analysis. Avoid using dead cells by harvesting the cells at the logarithmic growth phase of the bacteria.

  7. 9

    Measure the absorbance with a spectrophotometer. Adjust the absorbance of each culture to the same OD600, depending on the chosen mode of infection.

    Critical Step

    Use infiltration buffer as a blank for taking the OD of individual cultures.

    Troubleshooting

TRV inoculation into target plants

Timing ∼5 min–5 d

  1. 10

    Inoculate the plants with the appropriate Agrobacterium cultures via one the following options: by syringe inoculation into the leaves (option A), by the agrodrench method (option B) or by pricking the leaves with a toothpick (option C). Options A and B are most suited for reverse-genetics studies using a few TRV2::GOI constructs, whereas option C is suited for forward-genetics studies using a library of TRV2::GOI constructs.

    1. A

      Inoculation by needleless syringe

      1. i

        Mix TRV1+TRV2::GOI cultures (from Step 9) at a 1:1 (vol/vol) ratio. Repeat this for the TRV1+TRV2::GFP control construct.

        Critical Step

        The final absorbance of the mixed culture should not exceed OD600 = 0.7.

      2. ii

        Select 3-week-old N. benthamiana plants grown in individual 3-inch pots (from Step 1).

      3. iii

        Use a needleless syringe to inoculate the abaxial side of the lower leaves with the TRV1+TRV2::GOI mixture of Agrobacterium cultures (from Step 10A(i)). About 0.5 ml of culture is delivered into each leaf, and three or four leaves per plant are inoculated (Supplementary Video 1). Repeat for the TRV1+TRV2::GFP construct in another plant. Use two plants for each construct.

        Caution

        Cultures can accidentally produce spray during infiltration. Use a face shield or other appropriate face protection, and wear a lab coat.

        Critical Step

        Do not allow the cultures to contact neighboring plants (i.e., those that are assigned for a different construct or experiment), as this will lead to cross-contamination.

        Troubleshooting

      4. iv

        Maintain the inoculated plants in the greenhouse at 21 ± 2 °C until the observation period is reached. Relative humidity of 45–60% and light intensity of 350–450 μmol/m2/s with 14 h day/10 h night are optimum for plant growth and VIGS. Check the plants for changes in phenotype starting from 10 d after inoculation.

        Troubleshooting

      Timing ∼5 min

    2. B

      Agrodrench method of inoculation

      1. i

        Mix TRV1+TRV2::GOI cultures from Step 9 at a 1:1 (vol/vol) ratio. The final OD can be ∼1.0. Similarly, mix TRV1+TRV2::GFP.

      2. ii

        Select 3-week-old N. benthamiana plants grown in individual 3-inch pots (from Step 1).

        Critical Step

        Do not water the plants for a few hours before and after the planned inoculation.

      3. iii

        Dispense ∼6 ml of the TRV1+TRV2::GOI Agrobacterium culture mix (from Step 10B(i)) onto the crown region of the plant (Supplementary Video 1). Repeat with TRV1+TRV::GFP culture in another plant. Inoculate two or more plants for each construct.

        Critical Step

        Plants that are drenched with Agrobacterium should be separated in trays according to treatment, and they should be bottom-watered to avoid the spread of Agrobacterium to neighboring plants.

      4. iv

        Maintain inoculated plants in the greenhouse with the conditions described in Step 10A(iv) until the observation period is reached.

        Caution

        Water drained from the pot should be sterilized before discarding to prevent the release of bacterial cultures harboring TRV.

        Troubleshooting

      Timing ∼5 min

    3. C

      Prick inoculation

      1. i

        Thaw the cDNA library (96-well plates from the freezer, Step 2B(vi) to room temperature. Inoculate individual Agrobacterium colonies from the plate onto LB agar medium by using a 96-pin replicator. Also pick Agrobacterium colony carrying TRV2::GFP (from Step 2A(iv)) and inoculate it on LB agar medium. Incubate the plates at 28 °C for 2 d.

        Caution

        Wear cryogenic gloves to protect your hands from injury while handling the frozen plates.

        Critical Step

        Do not refreeze the plate once it has thawed, as freezing will reduce the viability of the bacteria. Four replicate colonies for each clone are made so as to provide adequate inoculum during inoculation. After completing inoculation, cDNA library clones in the plate can be replicated (Step 2B(iv–vi)) on a new plate and stored at −80 °C.

      2. ii

        Select 3-week-old N. benthamiana plants grown in individual 3-inch pots (from Step 1).

      3. iii

        Use the TRV1 culture only (OD600 = ∼0.3) from Step 9 to syringe-infiltrate the abaxial side of three or four leaves as described above in Step 10A(iii).

        Critical Step

        Prick inoculation of the Agrobacterium colony that carries the TRV1 construct along with prick inoculation of the TRV2 construct is not recommended, as it does not provoke efficient VIGS.

      4. iv

        Pick each of the four replicate Agrobacterium colonies carrying the same TRV2::GOI construct from the library (from Step 10C(i)) with a toothpick, and prick the toothpick a few times into the TRV1-infiltrated spots of two or three leaves (Supplementary Video 1). Inoculate two or more plants for each construct. Repeat this for each construct in the library and for the TRV2::GFP vector control.

        Caution

        Do not exert high pressure during pricking, as it may not only damage the leaf but it may also hurt the finger underneath the leaf. Discard the toothpick in an appropriate container and autoclave it.

        Critical Step

        Agrobacterium with TRV2::GOI should be inoculated soon after TRV1 inoculation. Change gloves for every Agrobacterium construct to prevent cross-contamination during inoculation.

      5. v

        Maintain the inoculated plants in the greenhouse with the conditions described in Step 10A(iv) until the observation period is reached.

        Critical Step

        As the plant growth increases, maintain adequate spacing between plants inoculated with different TRV2 constructs to avoid rubbing of leaves. TRV can be transmitted through mechanical injury, and this may lead to contamination.

      Timing ∼5 d

    Critical Step

    Temperature and other environmental growth conditions, as well as plant nutrition, are important, as they influence VIGS efficiency and efficacy. The efficiency of VIGS is reduced at higher (24–26 °C) environmental temperatures. More than 27 °C is not suitable for VIGS experiments in N. benthamiana37.

Phenotypic observation of inoculated plants

Timing 2–3 weeks

  1. 11

    Check plants for changes in phenotype starting ∼10 d after inoculation. Typically, changes in phenotype due to target gene silencing are expected to occur 2–3 weeks after inoculation. If TRV2::GOI–inoculated plants show a more severe growth retardation (or similar severe growth alteration) phenotype than vector control (TRV::GFP) plants, then proceed to Box 1 to carry out a leaf lesion assay. This assay will help determine whether the phenotype is caused by target gene silencing or whether it is caused by loss of plant immunity against TRV owing to target gene silencing (and, hence, higher multiplication of this virus in TRV2::GOI plants). An alternative to the leaf lesion assay is to perform quantification of TRV2 by RT-PCR with TRV2 coat protein–specific primers (Supplementary Table 5), by using standard methods.

    Critical Step

    Change in visible phenotype due to target gene silencing is not expected for every gene-silenced plant. Hence, confirmation of gene silencing should also be based on the downregulation of the target gene and its expected molecular or biochemical changes.

Validation of gene silencing

Timing 2–3 d

  1. 12

    Extract RNA from the plant tissue with the RNeasy mini kit according to the manufacturer's (Qiagen) instructions. Any other plant RNA extraction protocol can also be used.

    Caution

    Reagents used for RNA extraction (e.g., 2-mercaptoethanol) should be handled in a fume cupboard.

    Critical Step

    Use appropriate plant tissue produced after TRV inoculation for RNA extraction. For example, a newly emerged leaf after the inoculation should be used. Do not use the TRV-inoculated leaf. Confirm RNA quality by measuring the OD at 260 and 280 nm with a spectrophotometer before proceeding to cDNA synthesis.

  2. 13

    Perform cDNA synthesis with the SuperScript III RT cDNA synthesis kit as per the manufacturer's (Life Technologies) instructions. Any other suitable cDNA synthesis method can also be used.

  3. 14

    By using gene-specific primers, perform RT-qPCR as described by the real-time PCR machine manufacturer (e.g., real-time PCR detection system from Bio-Rad) and according to commonly used guidelines62. A typical reaction setup with the composition of cDNA primers and reaction kit supplied by a manufacturer (buffer, dNTPs, Mg2+ and DNA polymerase) is given below. However, this information should only be used as a starting point, and this step should be customized according to the method used, intended target gene, manufacturer of the real-time PCR machine and reagents.

    Table 2

    Component

    Amount (μl)

    Final concentration

    cDNA (∼100 ng/μl)

    1

    10 ng

    Reaction mix (SYBR Green master mix 2×, Sigma)

    5

    1×

    Primer mix (5 μM each, forward and reverse)

    0.4

    0.2 μM each primer

    Sterile water

    3.6

    —

    Total volume (per one reaction)

    10

    —

    Critical Step

    Mix the reagents from the kit (e.g., SYBR Green from Sigma-Aldrich) with the cDNA and the primers directly in the plate used for performing the PCR. cDNA quantity mentioned in the table is based on the RNA concentration used.

    Critical Step

    If the construct used for VIGS has been predicted to cause any off-target gene silencing on the basis of a bioinformatics analysis (Experimental design), the transcript levels of predicted off-target genes should be assessed by RT-qPCR to determine whether (and to what extent) unintended downregulation of those genes has occurred. This will facilitate the interpretation of results of subsequent plant analysis.

  4. 15

    Perform real-time PCR by using the following conditions as a starting point; conditions can be further optimized depending on the primers and target gene intended for amplification:

    Table 3

    Cycle number

    Denature

    Anneal

    Extend

    1

    95 °C, 10 min

    		  

    2–40

    95 °C, 15 s

    60 °C, 1 min

    —

  5. 16

    Calculate the fold difference in the target gene transcript levels compared with the vector control. The relative expression of the target gene can be calculated on the basis of the cycle threshold (Ct) values of the target gene and reference gene transcripts63. Relative expression values are used to calculate the fold difference. TRV-VIGS in N. benthamiana plants is expected to silence 80% or more of the target gene transcripts.

    Troubleshooting

Analysis of VIGS plants

Timing 3 months–2 years

  1. 17

    Analyze plants that show downregulation of the target gene for the transient effects of VIGS on a specific trait or on a set of parameters by using appropriate methods. If the aim of the study is long-term silencing of the target gene, maintain the plants and perform booster inoculations as described in option A before performing further analyses. If the aim is to study target gene silencing in progeny seedlings, follow option B.

    1. A

      Long-duration VIGS

      1. i

        Move the plants to 1-gallon pots and maintain them under greenhouse growth conditions described in Step 10A(iv).

        Critical Step

        Remove the dried leaves and stems. When possible, cut back the plant to avoid overgrowth. Avoid accidental mechanical inoculation of TRV between plants. Larger plants can be moved to bigger pots (3-gallon size) with potting mixture by following standard greenhouse plant maintenance procedures. Water the plants only when necessary. New growth is essential for the maintenance of VIGS.

      2. ii

        About 1 month after the first inoculation, perform a booster TRV inoculation by using the same constructs; both the syringe inoculation method (Step 10A) and the agrodrench method (Step 10B) should be used simultaneously. A second booster inoculation can be performed ∼1 month after the first booster inoculation.

        Critical Step

        VIGS can be influenced by environmental conditions and plant vigor. Gene-silenced plants should be healthy in order for booster inoculation to facilitate long-duration silencing. Booster inoculation is not required (or can be postponed) if silencing by initial TRV inoculation persists for the required duration.

      3. iii

        Analyze target parameters (e.g., assessing stress tolerance) ∼2 weeks after booster inoculation by using methods appropriate for the study.

        Troubleshooting

      Timing 4–24 months

    2. B

      VIGS in progeny seedlings

      1. i

        Allow plants to flower. VIGS in progeny seedlings is mainly due to virus vector carried over to these seedlings through seed transmission, thereby silencing the target gene transcripts. However, transcriptional gene silencing of the target gene is also possible owing to methylation of the genomic DNA. VIGS-mediated PTGS is referred to here.

        Critical Step

        Longer day length (16 h light) will enhance flowering and subsequent seed formation.

      2. ii

        Confirm the persistence of gene silencing in the top leaves of flowering plants by repeating RT-qPCR as described in Steps 12–16.

      3. iii

        Maintain the plants that show persistent gene silencing until seed maturity. Collect mature and dried seeds.

        Pause point

        Seeds can be stored in a desiccator at 4 °C for ∼1 year or at room temperature for ∼3 months.

      4. iv

        Sow the seeds as described in Reagent Setup.

      5. v

        Determine which seedlings contain the virus by performing the leaf lesion assay (Box 1). Alternatively, perform semiquantitative RT-PCR with TRV2 coat protein–specific primers to specifically confirm the presence of TRV sequences.

      6. vi

        Select plants for further downstream analyses. The presence of TRV constructs is essential for VIGS; use seedlings that are positive for the virus to study the effect of VIGS. Use seedlings without the virus to study any long-term effect of target gene silencing in the previous generation that is inherited by the progeny seedlings.

        Troubleshooting

      Timing 3–4 months

Troubleshooting

Troubleshooting advice can be found in Table 1.

Table 1 Troubleshooting table.
Full size table

Timing

Step 1, preparation of plants for inoculation: 2–3 d

Step 2, transforming Agrobacterium with TRV constructs: ∼3–10 d

Steps 3–9, growing Agrobacterium cultures and preparation for agroinoculation: ∼2 d

Step 10, TRV inoculation into target plants: ∼5 min–5 d

Step 11, phenotypic observation of inoculated plants: 2–3 weeks

Steps 12–16, validation of gene silencing: 2–3 d

Step 17, analysis of VIGS plants: 3 months–2 years

Box 1, leaf lesion assay in C. amaranticolor: 3–4 d

Anticipated results

Silencing of a particular target gene may result in changes in the plant phenotype. For example, NbPDS gene silencing invokes a photobleaching phenotype in plants (Fig. 3a). The NbPDS gene is involved in carotenoid biosynthesis18. Silencing this gene in plants leads to a reduction in carotenoids that protect photosystems and hence invokes photo-oxidation18which leads to photobleaching of aerial plant parts when they are grown under normal light intensity18,26.

Figure 3: Silencing the NbPDS gene in N. benthamiana plants by TRV-VIGS.
figure 3

(a) Agrobacterium carrying TRV1 and TRV2::NbPDS were co-inoculated by using a needleless syringe onto the abaxial side of the lower leaves of 3-week-old plants. TRV2::GFP was used as a vector control. The GFP sequence does not have any homology to plant DNA, and therefore it will not cause silencing. The plant was photographed at ∼2 weeks after inoculation. (b) The reduction in endogenous NbPDS transcript levels in the silenced plant was quantified using RT-qPCR. Error bars show standard error. (c) Plants were maintained by cut-back, and the new growth from the trimmed plant continued to show photobleaching. Booster inoculations of Agrobacterium cultures were done both by the agrodrench method and syringe inoculation at 4 months after the first inoculation. The photograph was taken at ∼5 months post inoculation. (d) Seeds collected from these plants were sown in potting medium, and a couple of progeny seedlings showed photobleaching, indicating the transmittance of gene silencing. (e) C. amaranticolor leaf lesion assay was done by using the leaf samples from NbPDS-silenced N. benthamiana progeny seedlings (photobleached) and the seedlings from wild-type control plants. Red arrows show the lesions caused by TRV.

Full size image

To confirm gene silencing, endogenous transcript levels of target genes should be evaluated by RT-qPCR by using gene-specific primers. The extent of gene silencing varies, depending on several factors. Some of these factors include the extent of virus infection, native expression levels of the target gene and time since the VIGS vector inoculation. TRV::NbPDS-inoculated plants showed ∼90% reduction in transcript levels (Fig. 3b), and the photobleaching phenotype was observed ∼10 d after inoculation. The visual phenotype depends on the extent of target gene silencing and the target gene function. Many gene-silenced plants may not show changes in the visible phenotype compared with the vector controls. Generally, when there is ∼80% or greater downregulation of the endogenous target gene transcripts, we consider VIGS effective in plants. However, this depends on the nature of the gene and its function. VIGS plants have been shown to maintain silencing for a couple of years (Fig. 3c). In addition, a small percentage of progeny seedlings from seeds of these plants have been shown to inherit TRV vectors and, hence, gene silencing (Fig. 3d); the leaf lesion assay can be used to assess the presence of the virus (Fig. 3e).