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Functional & Integrative Genomics

, Volume 12, Issue 1, pp 143–156 | Cite as

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

  • Harvinder S. Bennypaul
  • Jasdeep S. Mutti
  • Sachin Rustgi
  • Neeraj Kumar
  • Patricia A. Okubara
  • Kulvinder S. GillEmail author
Original Paper

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.

Keywords

VIGS Wheat BSMV Gene silencing VIGS inheritance Transient gene expression 

Notes

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.).

Supplementary material

10142_2011_245_MOESM1_ESM.ppt (144 kb)
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)
10142_2011_245_MOESM2_ESM.ppt (252 kb)
Fig. S2 Pictorial representations of Oligo-based technique for making hairpin-encoding and antisense constructs for virus-induced gene silencing (PPT 252 kb)

References

  1. Bennett CW (1969) Seed transmission of plant viruses. Adv Virus Res 14:221–261PubMedCrossRefGoogle Scholar
  2. 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, USAGoogle Scholar
  3. 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 20:1323–1331PubMedCrossRefGoogle Scholar
  4. 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 39:734–746PubMedCrossRefGoogle Scholar
  5. Burch-Smith TM, Schiff M, Liu Y, Dinesh-Kumar SP (2006) Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol 142:21–27PubMedCrossRefGoogle Scholar
  6. 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 62:223–232PubMedCrossRefGoogle Scholar
  7. 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 36:217–222CrossRefGoogle Scholar
  8. Cakir C, Tör M (2010) Factors influencing barley stripe mosaic virus-mediated gene silencing in wheat. Physiol Mol Plant Pathol 74:246–253CrossRefGoogle Scholar
  9. Camborde L, Tournier V, Noizet M, Jupin I (2007) A Turnip yellow mosaic virus infection system in Arabidopsis suspension cell culture. FEBS Lett 581:337–341PubMedCrossRefGoogle Scholar
  10. 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 54:1604–1621CrossRefGoogle Scholar
  11. 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 54:2497–2512CrossRefGoogle Scholar
  12. 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 115:971–980PubMedGoogle Scholar
  13. 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 40:622–631PubMedCrossRefGoogle Scholar
  14. 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 11:1623–1634PubMedCrossRefGoogle Scholar
  15. 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 7:25–33CrossRefGoogle Scholar
  16. Deng ZY, Wang T (2007) OsDMC1 is required for homologous pairing in Oryza sativa. Plant Mol Biol 65:31–42PubMedCrossRefGoogle Scholar
  17. Elmer S, Rogers SG (1990) Selection for wild type size derivatives of tomato golden mosaic virus during systemic infection. Nucleic Acids Res 18:2001–2006PubMedCrossRefGoogle Scholar
  18. 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 70:277–289PubMedCrossRefGoogle Scholar
  19. Fu DQ, Zhu BZ, Zhu HL, Jiang WB, Luo YB (2005) Virus-induced gene silencing in tomato fruit. Plant J 43:299–308PubMedCrossRefGoogle Scholar
  20. Fu D, Uauy C, Blechl A, Dubcovsky J (2007) RNA interference for wheat functional gene analysis. Transgenic Res 16:689–701PubMedCrossRefGoogle Scholar
  21. 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 15:2578–2591PubMedCrossRefGoogle Scholar
  22. Hammond J (1999) Overview: the many uses and applications of transgenic plants. Curr Top Microbiol Immunol 240:1–19PubMedGoogle Scholar
  23. Haupt S, Duncan GH, Holzberg S, Oparka KJ (2001) Evidence for symplastic phloem unloading in sink leaves of barley. Plant Physiol 125:209–218PubMedCrossRefGoogle Scholar
  24. Hayes RJ, Coutts RH, Buck KW (1989) Stability and expression of bacterial genes in replicating geminivirus vectors in plants. Nucleic Acids Res 17:2391–2403PubMedCrossRefGoogle Scholar
  25. Holzberg S, Brosio P, Gross C, Pogue GP (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J 30:315–327PubMedCrossRefGoogle Scholar
  26. Joshi RL, Joshi V, Ow DW (1990) BSMV genome mediated expression of a foreign gene in dicot and monocot plant cells. EMBO J 9:2663–2669PubMedGoogle Scholar
  27. Lacomme C, Hrubikova K, Hein I (2003) Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. Plant J 34:543–553PubMedCrossRefGoogle Scholar
  28. Lawrence DM, Jackson AO (2001) Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus. J Virol 75:8712–8723PubMedCrossRefGoogle Scholar
  29. 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 25:402–408PubMedCrossRefGoogle Scholar
  30. McNeal FH, Berg MA, Carroll TW (1976) Barley stripe mosaic virus data from six infected spring wheat cultivars. Plant Dis Report 60:730–733Google Scholar
  31. 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 107:19225–19230PubMedCrossRefGoogle Scholar
  32. Okubara PA, Bonsall RF (2008) Accumulation of Pseudomonas-derived 2,4-diacetylphloroglucinol on wheat seedling roots is influenced by host cultivar. Biol Control 46:322–331CrossRefGoogle Scholar
  33. 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 56:678–690PubMedCrossRefGoogle Scholar
  34. 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 1–27Google Scholar
  35. 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 138:2165–2173PubMedCrossRefGoogle Scholar
  36. Scofield SR, Nelson RS (2009) Resources for virus-induced gene silencing in the grasses. Plant Physiol 149:152–157PubMedCrossRefGoogle Scholar
  37. Sharma AK, Sharma A (1980) Chromosome techniques: theory and practice. Butterworths, LondonGoogle Scholar
  38. Smith KM, Gee L, Bode HR (2000) HyAlx, an aristaless-related gene, is involved in tentacle formation in hydra. Development 127:4743–4752PubMedGoogle Scholar
  39. Tai YS, Bragg J (2007) Dual applications of a virus vector for studies of wheat–fungal interactions. Biotechnology 6:288–291CrossRefGoogle Scholar
  40. 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 25:417–425PubMedCrossRefGoogle Scholar
  41. 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 136:3999–4009PubMedCrossRefGoogle Scholar
  42. Vance V, Vaucheret H (2001) RNA silencing in plants—defense and counter defense. Science 292:2277–2280PubMedCrossRefGoogle Scholar
  43. 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 27:581–590PubMedCrossRefGoogle Scholar
  44. 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 344:401–411PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Harvinder S. Bennypaul
    • 1
    • 2
  • Jasdeep S. Mutti
    • 1
    • 3
  • Sachin Rustgi
    • 1
  • Neeraj Kumar
    • 1
  • Patricia A. Okubara
    • 4
  • Kulvinder S. Gill
    • 1
    Email author
  1. 1.Department of Crop and Soil SciencesWashington State UniversityPullmanUSA
  2. 2.Canadian Food Inspection AgencyCharlottetownCanada
  3. 3.Pioneer Hi-Bred International, Inc.JohnstonUSA
  4. 4.USDA ARSRoot Disease and Biological Control Research UnitPullmanUSA

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