New Dimensions for VIGS in Plant Functional Genomics

Supplementary Material

New dimensions for VIGS in plant functional genomics

Muthappa Senthil-Kumar and Kirankumar S. Mysore

Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA.

Corresponding author:Mysore, K.S.().

Table S1. Virus-induced gene silencing (VIGS) vectors: A critical analysis on extending current VIGS protocols to new crop species
A / B / C / D / E / F / G
Sl. No. / Name of VIGS vector / Currently VIGS-amenable target plant species or familyand [closely related VIGS-amenable model plant to the species listed in column D and other crops] / Currently VIGS-recalcitrant crop plant, but can benefit by performing heterologous VIGS in a species listed in column C / Potential target crop species (or family) suitable for VIGS protocol standardization using VIGS vectors mentioned in column B / Extent of seed transmission in a natural host+ / Refs
RNA viruses
1 / Apple latent spherical virus (ALSV) / Nicotiana benthamiana, cucurbits (Cucumis spp.), apple (Malus spp.), pear (Pyrus spp.), tobacco (Nicotiana spp.), tomato (Solanum spp.) and [soybean(Glycine max)] / Legumes [e.g. peanut (Arachis hypogaea)and alfalfa (Medicago sativa)] / Pea (Pisum sativum) / Low (20–30%) seed-transmitted (through embryo) / [S1-S3]
2 / Alternanthera mosaic virus (AltMV) / N. benthamiana and [Arabidopsis] / Canola (Brassica spp.), cauliflower, cucumber (Cucumis sativus) and flax (Linum spp.) / Soybean, sunflower (Helianthus annuus), Phlox species, Portulaca, Angelonia, Crossandra and Torenia / Less than 5% transmission through seed coat / [S4]
3 / Brome mosaic virus (BMV) / N. benthamiana, [maize (Zea mays), barley (Hordeum vulgare), sorghum(Sorghum bicolor), rice (Oryza sativa) and foxtail millet (Setaria italica)] / Switchgrass (Panicum virgatum), sugarcane (Saccharumspp.) and other Panicoideae members / Switchgrass / Less than 20% of seed coat transmission / [S5-S8] and Dr Rick Nelson, personal communication
4 / Barley stripe mosaic virus (BSMV) / N. benthamiana, barley, oat (Avena sativa), Haynaldia villosa, ginger (Zingiber officinale), [false brome (Brachypodium distachyon)and wheat (Triticum spp.)] / Switchgrass / Panicoideae (millets) / Highly (more than 80%) seed transmitted (through embryo) / [S5,S9]
5 / Bean pod mottle virus (BPMV) / N. benthamiana, common bean (Phaseolus vulgaris)and [soybean] / Alfalfa and other beans / Stizolobium herb and crimson clover / Moderately (about 50%) transmitted through seed coat / [S10]
6 / *Cucumber mosaic virus (CMV) / N. benthamiana and [soybean] / Legumes (e.g. peanut, bean and alfalfa) / Tomato / About 30% transmission through seed coat / [S11]
7 / Cymbidium mosaic virus (CymMV) / [Orchids(Phalaenopsisspp.)] / Other orchid species / Vanilla and other orchids (e.g. Phalaenopsis spp.) / No report of seed transmission / [S5,S12,S13]
8 / *Potato virus X (PVX) / Tomato, [Arabidopsis, potato (Solanum spp.) and N. benthamiana] / Other Solanum clade members and canola (Brassica spp.) / Solanaceae plants and oil seed (e.g. rape) / Very little (<10%), sometimes peripherally attached to seed / [S14-S16]
9 / Potato virus A (PVA) / N. benthamiana / – / – / No report of seed transmission / [S17]
10 / Pea early browning virus (PEBV) / N. benthamiana, [pea (Pisum sativum), lotus(Lathyrus spp.) and barrel medic (Medicagotruncatula)] / Alfalfa, beans, chick pea (Cicer arietinum), other Vigna species and peanut / Legumes (e.g. peanut, beans and chick pea) / About 50% seed transmission through embryo / [S18-S21]
11 / Pea seed-borne mosaic virus (PSbMV) / N. benthamiana and [pea] / Peanut / – / About 30% seed transmission through embryo / [S19,S20,S22,S23]
12 / Poplar mosaic virus (PopMV) / N. benthamiana / – / Poplar species / No report of seed transmission / [S24]
13 / Plum pox virus (PPV) / N. benthamiana / – / Plum, peach and apricots / About 10% transmission through seed coat / [S25]
14 / Pepino mosaic virus (PepMV) / N. benthamiana / – / Tomato, pepino and other Solanaceae members / About 10% transmission through seed coat / [S26,S27]
15 / Sweet potato feathery mottle poty virus (SPFMV) / N. benthamiana / – / Sweet potato / No report of seed transmission / [S28,S29]
16 / Satellite of tobacco mosaic virus (STMV) / Tobacco / – / Pepper and tomato / No report of seed transmission / [S30]
17 / Sunn-hemp mosaic virus (SHMV) / Tobacco and [barrel medic] / Other legumes (e.g. alfalfa) / Cowpea and Bengal bean / 4%–20% transmission through seed coat / [S31,S32]
18 / Tobacco mosaic virus (TMV) / N. benthamiana / – / Arabidopsis, potato, tomato, muskmelon, cucumber, squash and spinach / Less than 10% through seed coat transmission / [S33-S36]
19 / TTO1A vector (TMV strain U1 and ToMV strain F) / N. benthamiana / – / – / TMV and ToMV can be transmitted through seed coat / [S37]
20 / Tomato bushy stunt virus (TBSV) / N. benthamiana / – / Tomato / Low level of seed transmission / [S38]
21 / *Tobacco rattle virus (TRV) / [N. benthamiana, Arabidopsis, cotton (Gossypium spp.),Aquilegia, opium or California poppy, Jatropha curcas, tomato,
Pepper (Capsicum spp.), peach] and several other Solanaceae species / Other Gossypium species, Jatropha species and other Solanaceae plants / Onion, cucumber, spinach, beet, beans, castor, rubber plant, cassava and other Solanaceae plants / Up to 40% seed transmission / [S39-S46]
22 / Turnip yellow mosaic virus (TYMV) / N. benthamiana and Arabidopsis / – / Cabbage and turnip / Less than 30% seed transmission (through embryo and seed coat) / [S35,S47]
DNA viruses
23 / Abutilon mosaic virus (AbMV) / N. benthamiana / –- / Cotton and beans / No report of seed transmission / [S48]
24 / African cassava mosaic virus (ACMV) / N. benthamiana and [cassava (Manihot esculenta)] / Other Manihot species / Arabidopsis, tobacco and tomato / No report of seed transmission / [S49,S50]
25 / Beet curly top virus (BCTV) / Spinach (Spinaciaspp.)and tomato / – / Pepper / No report of seed transmission / [S51,S52]
26 / Cabbage leaf curl virus (CaLCuV) / N. benthamiana, Arabidopsis, cabbage (Brassicaspp.) and cauliflower (Brassicaspp.) / – / No report of seed transmission / [S53,S54]
27 / Cotton leaf crumple virus (CLCV) / [N. benthamiana and cotton] / Bean and other Gossypium species / Bean, tomato and pepper / No report of seed transmission / [S55]
28 / Grapevine virus A (GVA) / Grapevine (Vitis vinifera) / – / – / No report of seed transmission / [S56]
29 / Pepper huasteco yellow vein virus (PHYVV) / [pepper(Capsicum spp.), tobacco and tomato] / Other Solanaceae plants / – / No report of seed transmission / [S57]
30 / Rice tungro bacilliform virus
(RTBV) / Rice / – / Indian goosegrass / No report of seed transmission / [S8,S58]
31 / Tomato golden mosaic virus (TGMV) / N. benthamiana / – / Tomato / No report of seed transmission / [S59]
32 / Tobacco curly shoot virus (TCSV) with or without Geminivirus satellite vector (2mDNA1) / Tobacco and tomato / – / Pepper / No report of seed transmission / [S60]
33 / Tomato yellow leaf curl China virus (TYLCCNV) / N. benthamiana and [tomato] / Other Solanaceae plants / Kidney bean / No report of seed transmission / [S61]
34 / Tomato leaf curl virus (ToLCV) / N. benthamiana and tomato / – / – / No report of seed transmission / [S62,S63]
Viruses (DNA or RNA) potentially suitable for VIGS vector development and their host
35 / Clover yellow vein virus (CIYVV) / – / – / Soybean / – / [S2]
36 / Cucumber necrosis virus (CNV) / – / – / N. benthamiana / – / [S64]
37 / Pepper ringspot virus (PepRSV) / – / – / Pepper and tomato / – / [S65]
38 / Soybean mosaic virus (SMV) / – / – / Soybean / – / [S2]
39 / Tobacco yellow dwarf virus (TYDV) / – / – / Petunia, pea and beans / – / [S66,S67]
40 / Tobacco streak virus (TSV) / – / – / Soybean / – / [S68]
41 / Other vectors and resources / – / – / – / – / [S5,S69]

*Transcriptional gene silencing (and also post-transcriptional gene silencing) has been reported for these viruses (all other unmarked virus vectors induce PTGS).

+Values given are based on a report for any one species listed in column C

This table describes the use of VIGS to identify (forward genetic screen) and characterize (reverse genetic screen) gene function in crop plants (listed in column D) that currently do not have a VIGS vector. Although efforts should be made to develop a VIGS vector specific for the target species, this process would take several years. Hence, this table should help researchers to study the relevance of genes in species that are recalcitrant to VIGS.

Column C:Plant species underlined (and in parenthesis) are VIGS-model plants that are phylogenetically close to crop plants mentioned in column D. To understand the functional relevance of a gene for a species mentioned in column D, a corresponding homolog of that gene can be identified from the species given in column C and silenced in the same plant. Information from VIGS-amenable model plants can be used to infer gene function in closely related crop plants [S70]. Thus, target gene functions of crop species listed in column D can be identified. Based on the comparative genomic information presented earlier [S71] and our sequence analysis, we selected closely related VIGS-amenable model plants for each potential crop plant that is currently recalcitrant to VIGS.

Column D:Heterologous VIGS: genes from currently recalcitrant crop species (that do not have specific VIGS vector) can also be characterized by VIGS. This can be achieved by cloning the gene fragments of interest from this recalcitrant plant (column D) in to a VIGS vector (respective row of column B) and silencing them in a closely related VIGS-amenable plant [S72]. Closely related model plants are listed in column C. By using siRNA prediction software [S73] and NCBI BLAST analysis, 21 nucleotide similarities were checked for at least 10 randomly selected genes from each species. Model plants (column C) that shared more than 21 nt for the selected genes are considered closely related to crop species (listed under column D).

Column E:Based on the host range for the specific virus, we predicted (based on literature information showing infection, spread of virus and use of the viral vector for gene expression studies) the possibility of VIGS protocol standardization using already available VIGS vectors (column B).

Column F: Extent of the virus's seed transmission ability was derived based on available information in the literature.

Note: widely used VIGS vectors are highlighted in mustard yellow.

Table S2. List of major crop and model plants that can be currently benefitted by use of virus-induced gene silencing vectors
Sl. No. / Plant species/family / Name of VIGS vector / VIGS protocol – currently standardized method of inoculation / Refs
Monocotyledonous plants
Barley / BMV / Rub-inoculation of invitro synthesized transcripts / [S74]
BSMV b / Rub-inoculation of invitro synthesized transcripts
BSMV binary VIGS constructs delivered through particle bombardment
Sap inoculation / [S75-S77]
Brachypodium distachyon / BSMV / Rub-inoculation of invitro synthesized transcripts / [S9,S78]
Ginger / BSMV / Sap inoculation / [S79]
Foxtail millet / BMV / Agro-inoculation
Sap inoculation
Rub-inoculation of invitro synthesized transcripts / Dr Rick Nelson, personal communication
Maize / BMV / Rub-inoculation of invitro synthesized transcripts / [S74]
Oat / BSMV / Rub-inoculation of invitro synthesized transcripts / [S9]
Orchids (Phalaenopsisspp.) / CymMV / Rub-inoculation of invitro synthesized transcripts / [S12]
Rice / BMV / Agro-inoculation
Sap inoculation
Rub-inoculation of invitro synthesized transcripts / [S80]
RTBV / Agro-inoculation / [S58]
Sorghum / BMV / Agro-inoculation
Sap inoculation / Dr Rick Nelson, personal communication
Wheat / BSMV / Rub-inoculation of invitro synthesized transcripts / [S75,S81,S82]
Dicotyledonous plants
Apple / ALSV / Total RNA extracted from VIGS construct inoculated host plant leaves delivered to apple seedlings through particle bombardment / [S1,S3]
Thale cress(Arabidopsis thaliana) / ALSV / Mechanical inoculation of virus particles (or sap) obtained from Chenopodium quinoa, a host plant used to multiply ASLV construct / [S1]
CalCuV / Particle bombardment of constructs / [S53]
TYMV / Rub-inoculation of in vitro RNA transcripts / [S47]
TRV a, b / Agro-inoculation
Sap inoculation / [S83-S85]
Barrel medic / PEBV / Agro-inoculation / [S18]
Cotton / CLCV / Particle bombardment of constructs / [S55]
TRV / Agro-inoculation / [S39]
Cowpea / ALSV / Mechanical inoculation of virus particles (or sap) obtained from Chenopodium quinoa, a host plant used to multiply ASLV construct / [S1]
Cucumis sativus(and other cucurbits) / ALSV / Mechanical inoculation of virus particles (or sap) obtained from C. quinoa, a host plant used to multiply ASLV construct / [S1]
Grapevine / GVA / Agro-inoculation / [S56]
Jatropha / TRV / Agro-inoculation / [S43]
Lotus / PEBV / Agro-inoculation / [S18]
N. benthamianac / PVXa / Agro-inoculation
Agro-drench / [S40]
TRV / Agro-inoculation
Pricking leaves using took pick carrying Agrobacterium
Agro-drench / [S40,S86]
Pea / PEBV / Agro-inoculation / [S87]
Pepper / PHYVV / Particle bombardment of construct / [S57]
TRV / Agro-inoculation / [S40,S86]
Petunia / TRV / Agro-inoculation / [S88]
Pear / ASLV / Total RNA extracted from VIGS construct inoculated C. quinoa leaves delivered to young seedlings through particle bombardment / [S3]
Poppy (Papaver somniferumand Eschscholzia californica) / TRV / Agro-inoculation (vacuum used) / [S89,S90]
Potato / PVX / Agro-inoculation / [S15]
Soybean / ALSV / Mechanical inoculation of virus particles (or sap) obtained from C. quinoa, a host plant used to multiply ASLV construct
Total RNA extracted from VIGS construct inoculated host plant leaves delivered to cotyledons of young seedlings through particle bombardment / [S1,S91]
BPMV / Rub-inoculation of in vitro synthesized RNA transcripts
Inoculation by direct DNA-rubbing of BPMV constructs
Biolistic bombardment / [S10,S92]
CMV / Sap inoculation / [S11]
Spinach / BCTV / Biolistic bombardment / [S51]
Tapioca (cassava) / ACMV / Biolistic bombardment / [S49]
Tobacco (and several Nicotiana species) / ALSV / Mechanical inoculation of virus particles (or sap) obtained from C. quinoa, a host plant used to multiply ASLV construct / [S1]
STMV / Rub-inoculation of invitro synthesized transcripts
Agro-inoculation / [S30,S93]
TCSV / Agro-inoculation / [S60]
TRV / Agro-inoculation / [S86]
Tomato (and many Solanum spp.) / ALSV / Mechanical inoculation of virus particles (or sap) obtained from C. quinoa, a host plant used to multiply ASLV construct / [S1]
BCTV / Agro-inoculation / [S51]
PVX / Direct rub-inoculation of plasmid DNA of VIGS construct / [S94]
TRV / Agro-inoculation
Agro-drench / [S41,S86]
ToLCV / Agro-inoculation / [S62]
TYLCCNV / Agro-inoculation / [S95]
TCSV / Agro-inoculation / [S60]

avectors suitable for high throughput cloning using gateway cloning technology

b vectors suitable for ligation free cloning [S9,S96]

c Almost all VIGS vectors shown in Table S1 are known to infect the VIGS-model plant, N. benthamiana. Only two widely used vectors are listed here for this species

Notes: (1) Only important crop species and model plant species are mentioned in this table. (2) A detailed list of plant species and VIGS constructs suitable for them are available in previous publications [S5,S40,S71]. (3) Agro-inoculation is a method to deliver binary VIGS vectors into plants through Agrobacterium-mediated T-DNA transfer [S97]. (4) Sap inoculation method is described in the main text and in previous literatures [S42,S85]. (5) In vitro transcripts can be produced through transcription of linearised plasmids containing respective VIGS vector genomes. The transcripts are capped during the reactions and the products mixed with buffer prior to rub-inoculation on to plants [S80]. (6) For abbreviations of VIGS vectors and scientific names of plant species listed in this table refer to Table S1. (7) Model plants listed in this table are highlighted (mustard yellow).

Table S3. Comparison of non-integration-based stable PTGS and non-transgenic stable TGS techniques with other available gene functional analysis techniques
Mechanism / Characteristics / Advantages over mutagenesis / Advantages over stable RNAi / Advantages over conventional VIGS
Non-integration-based stable PTGS
(proposed as an alternative to RNAi plants) / Translation inhibition
RNA silencing by mRNA cleavage (siRNA targeted to mRNA)
Mediated by VIGS vector carrying a fragment of the gene of interest homologous to the endogenous target plant gene / No genomic DNA modification
Seed transmission of virus vector required for seed-only propagated plants. However, vegetative and tissue culture-mediated regeneration is possible for amenable plant species
Silencing trigger (virus or dsRNA) should always be present in parent and progeny plants
siRNA spread contributes to gene silencing
Silencing persists for several generations / Genomic DNA is not altered
Genes causing embryo-lethal or severely malformed phenotype upon mutation can be studied
Identification of genes that confer a particular phenotype is much faster in forward genetics screens
No segregation in progeny plants
Can be combined with stable mutants to knock down and knock out multiple genes. This will be faster than generating double mutants
Can be applied on only vegetatively propagated plants
Silencing can be reverted (virus-free plants can be made). This way the same plant can be used for other applications. e.g. reducing flowering time during breeding or increasing Agrobacterium-mediated transformation efficiency in recalcitrant plants
Targeted gene silencing is possible
Reverse genetics can be faster than using stable mutants for the same / Can be used on transformation- recalcitrant crop plants
Exact vector control plants can be developed (in RNAiintegrating vector control at the same site of the genome is difficult)
Less time consumingto develop gene-silenced plants
Plants that are recalcitrant to Agrobacterium-mediated transformation can be silenced using virus-sap inoculation.
Even embryo lethal genes can be studied
Can be easily combined with stable RNAi plants to silence multiple genes
Can be applied on only vegetatively propagated plants / Gene silencing in seeds (can help to elucidate molecular and physiological responses during seed development, maturation and nutrient storage in endosperm
Genes involved in seed germination and early seedling vigor can be silenced
Gene silencing throughout plant life cycle (temporal and spatial) from seed to seed possible
Non-transgenic stable TGS
(proposed as alternative to mutant plants) / DNA (e.g. promoter) methylation (dsRNA or siRNA targeted to DNA)
Histone-lysine methylation
Mediated by viral vector carrying fragment of gene that can generate dsRNA homologous to DNA (e.g. promoter region); this is transported to cell nucleus to cause the above said modifications / Genomic DNA or histone methylated, but no alteration in genome structure
Silencing trigger (virus or dsRNA) should be present in reproductive tissues in parent (P0). Virus or dsRNA or virus-derived siRNA contributes to methylation
Presence of silencing trigger (virus or dsRNA) or siRNA is not required in progeny plants (if seed-transmitted virus is used as a trigger, virus-free plants can be developed)
Progeny plants are non-transgenic (after removal of recombinant virus vector)
Silencing persists for several generations
Can be applied on seed-only and tissue-culture-propagated plants. However, this cannot be applied on plants with only vegetatively propagated (and recalcitrant to tissue culture-based regeneration) / Non transgenic
Applicable to transformation- recalcitrant plants
Targeted gene silencing possible
Easy to develop silenced plants
Less time
No alteration in genomic DNA structure
Can be applied to vegetatively propagated plants / Can be applied to elucidate genes involved in PTGS pathway and other epigenetic gene regulation
Advantages mentioned under PTGS-VIGS / Virus-free plants can be developed (a silencing trigger is not required to maintain TGS)
Can be applied to elucidate genes involved in PTGS pathway and other epigenetic gene regulation

Note: In both cases, initial virus infection shall be performed by direct virus inoculation using a non-binary VIGS vector. Agrobacterium and binary vectors has to be avoided to prevent any possible integration of virus vector within the genome.