Skip to main content

Advertisement

SpringerLink
Log in

Virus-induced gene silencing (VIGS) of genes expressed in root, leaf, and meiotic tissues of wheat

  • Original Paper
  • Published:
Functional & Integrative Genomics Aims and scope Submit manuscript

Abstract

Barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in wheat leaves, but its utility to transiently express genes, and silencing in other tissues including root, flower, and developing grains, has not been demonstrated in monocots. We monitored green fluorescent protein (GFP) expression to demonstrate the utility of BSMV as a transient expression vector and silenced genes in various wheat tissues to expand VIGS utility to characterize tissue-specific genes. An antisense construct designed for coronatine insensitive1 (COI1) showed an 85% decrease in COI1 transcript level in roots accompanied by a 26% reduction in root length. Similarly, silencing of seed-specific granule-bound starch synthase by antisense and hairpin constructs resulted in up to 82% reduction in amylose content of the developing grains. VIGS of meiosis-specific genes demonstrated by silencing wheat homologue of disrupted meiosis cDNA1 (DMC1) by an antisense construct resulted in a 75–80% reduction in DMC1 transcript level accompanied by an average of 37.2 univalents at metaphase I. The virus-based transient GFP expression was observed in the leaf, phloem, and root cortex at 10–17 days post-inoculation. A novel observation was made that 8–11% of the first selfed generation progeny showed VIGS inheritance and that this proportion increased to 53–72% in the second and to 90–100% in the third generations. No viral symptoms were observed in the progeny, making it possible to study agronomic traits by VIGS. VIGS inheritance is particularly useful to study genes expressing during seed germination or other stages of early plant growth.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Bennett CW (1969) Seed transmission of plant viruses. Adv Virus Res 14:221–261

    Article  PubMed  CAS  Google Scholar 

  • Bennypaul HS (2008) Genetic analysis and functional genomic tool development to characterize resistance gene candidates in wheat (Triticum aestivum L.). PhD thesis, Washington State University, USA

  • Bruun-Rasmussen M, Madsen CT, Jessing S, Albrechtsen M (2007) Stability of Barley stripe mosaic virus-induced gene silencing in barley. Mol Plant Microbe Interact 20:1323–1331

    Article  PubMed  CAS  Google Scholar 

  • Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746

    Article  PubMed  CAS  Google Scholar 

  • Burch-Smith TM, Schiff M, Liu Y, Dinesh-Kumar SP (2006) Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol 142:21–27

    Article  PubMed  CAS  Google Scholar 

  • Cai X-Z, Xu Q-F, Wang C-C, Zheng Z (2006) Development of a virus-induced gene-silencing system for functional analysis of the RPS2-dependent resistance signaling pathways in Arabidopsis. Plant Mol Biol 62:223–232

    Article  PubMed  CAS  Google Scholar 

  • Cakir C, Scofield S (2008) Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes. Cereal Res Commun 36:217–222

    Article  CAS  Google Scholar 

  • Cakir C, Tör M (2010) Factors influencing barley stripe mosaic virus-mediated gene silencing in wheat. Physiol Mol Plant Pathol 74:246–253

    Article  CAS  Google Scholar 

  • Camborde L, Tournier V, Noizet M, Jupin I (2007) A Turnip yellow mosaic virus infection system in Arabidopsis suspension cell culture. FEBS Lett 581:337–341

    Article  PubMed  CAS  Google Scholar 

  • Carroll TW, Mayhew DE (1976a) Anther and pollen infection in relation to the pollen and seed transmissibility of two strains of barley stripe mosaic virus in barley. Botany 54:1604–1621

    Article  Google Scholar 

  • Carroll TW, Mayhew DE (1976b) Occurrence of virions in developing ovules and embryo sacs of barley in relation to the seed transmissibility of barley stripe mosaic virus. Botany 54:2497–2512

    Article  Google Scholar 

  • Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980

    PubMed  CAS  Google Scholar 

  • Constantin GD, Krath BN, MacFarlane SA, Nicolaisen M, Johansen IE, Lund OS (2004) Virus-induced gene silencing as a tool for functional genomics in a legume species. Plant J 40:622–631

    Article  PubMed  CAS  Google Scholar 

  • Couteau F, Belzile F, Horlow C, Grandjean O, Vezon D, Doutriaux MP (1999) Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis. Plant Cell 11:1623–1634

    Article  PubMed  CAS  Google Scholar 

  • Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, Beachy RN, Fauquet C (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33

    Article  CAS  Google Scholar 

  • Deng ZY, Wang T (2007) OsDMC1 is required for homologous pairing in Oryza sativa. Plant Mol Biol 65:31–42

    Article  PubMed  CAS  Google Scholar 

  • Elmer S, Rogers SG (1990) Selection for wild type size derivatives of tomato golden mosaic virus during systemic infection. Nucleic Acids Res 18:2001–2006

    Article  PubMed  CAS  Google Scholar 

  • Etessami P, Watts J, Stanley J (1989) Size reversion of African cassava mosaic virus coat protein gene deletion mutants during infection of Nicotiana benthamiana. J Gen Virol 70:277–289

    Article  PubMed  CAS  Google Scholar 

  • Fu DQ, Zhu BZ, Zhu HL, Jiang WB, Luo YB (2005) Virus-induced gene silencing in tomato fruit. Plant J 43:299–308

    Article  PubMed  CAS  Google Scholar 

  • Fu D, Uauy C, Blechl A, Dubcovsky J (2007) RNA interference for wheat functional gene analysis. Transgenic Res 16:689–701

    Article  PubMed  CAS  Google Scholar 

  • Gilbertson RL, Sudarshana M, Jiang H, Rojas MR, Lucas WJ (2003) Limitations on geminivirus genome size imposed by plasmodesmata and virus-encoded movement protein:insights into DNA trafficking. Plant Cell 15:2578–2591

    Article  PubMed  CAS  Google Scholar 

  • Hammond J (1999) Overview: the many uses and applications of transgenic plants. Curr Top Microbiol Immunol 240:1–19

    PubMed  CAS  Google Scholar 

  • Haupt S, Duncan GH, Holzberg S, Oparka KJ (2001) Evidence for symplastic phloem unloading in sink leaves of barley. Plant Physiol 125:209–218

    Article  PubMed  CAS  Google Scholar 

  • Hayes RJ, Coutts RH, Buck KW (1989) Stability and expression of bacterial genes in replicating geminivirus vectors in plants. Nucleic Acids Res 17:2391–2403

    Article  PubMed  CAS  Google Scholar 

  • Holzberg S, Brosio P, Gross C, Pogue GP (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J 30:315–327

    Article  PubMed  CAS  Google Scholar 

  • Joshi RL, Joshi V, Ow DW (1990) BSMV genome mediated expression of a foreign gene in dicot and monocot plant cells. EMBO J 9:2663–2669

    PubMed  CAS  Google Scholar 

  • Lacomme C, Hrubikova K, Hein I (2003) Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. Plant J 34:543–553

    Article  PubMed  CAS  Google Scholar 

  • Lawrence DM, Jackson AO (2001) Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus. J Virol 75:8712–8723

    Article  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • McNeal FH, Berg MA, Carroll TW (1976) Barley stripe mosaic virus data from six infected spring wheat cultivars. Plant Dis Report 60:730–733

    Google Scholar 

  • Mok YG, Uzawa R, Lee J, Weiner GM, Eichman BF, Fisher RL, Huh JH (2010) Domain structure of the DEMETER 5-methylcytosine DNA glycosylase. Proc Natl Acad Sci USA 107:19225–19230

    Article  PubMed  CAS  Google Scholar 

  • Okubara PA, Bonsall RF (2008) Accumulation of Pseudomonas-derived 2,4-diacetylphloroglucinol on wheat seedling roots is influenced by host cultivar. Biol Control 46:322–331

    Article  CAS  Google Scholar 

  • Pflieger S, Blanchet S, Camborde L, Drugeon G, Rousseau A, Noizet M, Planchais S, Jupin I (2008) Efficient virus-induced gene silencing in Arabidopsis using a ‘one-step’ TYMV-derived vector. Plant J 56:678–690

    Article  PubMed  CAS  Google Scholar 

  • Pogue GP, Lindbo JA, Dawson WO, Turpen TH (1998) Tobamovirus transient expression vectors: tools for plant biology and high-level expression of foreign proteins in plants. In: Gelvin SB, Schilperoot RA (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp 1–27

    Google Scholar 

  • Scofield SR, Huang L, Brandt AS, Gill BS (2005) Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol 138:2165–2173

    Article  PubMed  CAS  Google Scholar 

  • Scofield SR, Nelson RS (2009) Resources for virus-induced gene silencing in the grasses. Plant Physiol 149:152–157

    Article  PubMed  CAS  Google Scholar 

  • Sharma AK, Sharma A (1980) Chromosome techniques: theory and practice. Butterworths, London

    Google Scholar 

  • Smith KM, Gee L, Bode HR (2000) HyAlx, an aristaless-related gene, is involved in tentacle formation in hydra. Development 127:4743–4752

    PubMed  CAS  Google Scholar 

  • Tai YS, Bragg J (2007) Dual applications of a virus vector for studies of wheat–fungal interactions. Biotechnology 6:288–291

    Article  Google Scholar 

  • Thomas CL, Jones L, Baulcombe DC, Maule AJ (2001) Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector. Plant J 25:417–425

    Article  PubMed  CAS  Google Scholar 

  • Valentine T, Shaw J, Blok VC, Phillips MS, Oparka KJ, Lacomme C (2004) Efficient virus-induced gene silencing in roots using a modified tobacco rattle virus vector. Plant Physiol 136:3999–4009

    Article  PubMed  CAS  Google Scholar 

  • Vance V, Vaucheret H (2001) RNA silencing in plants—defense and counter defense. Science 292:2277–2280

    Article  PubMed  CAS  Google Scholar 

  • Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590

    Article  PubMed  CAS  Google Scholar 

  • Zhang C, Ghabrial SA (2006) Development of Bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean. Virology 344:401–411

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Gregory Pogue (Large Scale Biology Corporation, CA, USA) for providing VIGS vectors and Dr. Simon Chuong (Franchesci Electron Microscopy Center, Washington State University) for expert assistance with confocal microscopy. This work was supported by Washington Wheat Commission, The Vogel Endowment funds, and USDA ARS Project no. 5248-22000-012-00D (P.O.).

Author information

Author notes

  1. Harvinder S. Bennypaul

    Present address: Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PE, Canada, C1E 2J5

  2. Jasdeep S. Mutti

    Present address: Pioneer Hi-Bred International, Inc., PO Box 1000, Johnston, IA, 50131-0184, USA

Authors and Affiliations

  1. Department of Crop and Soil Sciences, Washington State University, Pullman, WA, 99164, USA

    Harvinder S. Bennypaul, Jasdeep S. Mutti, Sachin Rustgi, Neeraj Kumar & Kulvinder S. Gill

  2. USDA ARS, Root Disease and Biological Control Research Unit, Pullman, WA, 99164, USA

    Patricia A. Okubara

Authors
  1. Harvinder S. Bennypaul
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jasdeep S. Mutti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sachin Rustgi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Neeraj Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Patricia A. Okubara
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kulvinder S. Gill
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kulvinder S. Gill.

Additional information

H.S. Bennypaul and J.S. Mutti contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1

Genomic organization of BSMV (strain ND-18)-based VIGS vector. RNA α (a); β.Δβa, a coat protein deletion mutant of BSMV RNA β (b); pγ.wWxhp, RNA γ modified to express hairpin WAXY fragment (c); pγ.wWxas, RNA γ modified to express antisense WAXY fragment (d); pγ.TaCOI1, RNA γ modified to express antisense TaCOI1 fragment (e); pγ.TaDMC1, RNA γ modified to express antisense TaDMC1 fragment (f); pγ.MCS, RNA γ modified to express sense multiple cloning site (MCS) fragment of pBluescript (g). Sub-genomic promoters are indicated by arrows and stop codons by ST. The positions of selected restriction enzyme sites are indicated (PPT 144 kb)

Fig. S2

Pictorial representations of Oligo-based technique for making hairpin-encoding and antisense constructs for virus-induced gene silencing (PPT 252 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bennypaul, H.S., Mutti, J.S., Rustgi, S. et al. Virus-induced gene silencing (VIGS) of genes expressed in root, leaf, and meiotic tissues of wheat. Funct Integr Genomics 12, 143–156 (2012). https://doi.org/10.1007/s10142-011-0245-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10142-011-0245-0

Keywords

Search

Navigation