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BY-NC-ND 3.0 license Open Access Published by De Gruyter December 31, 2011

A new BSMV-based vector with modified β molecule allows simultaneous and stable silencing of two genes

  • Adam Kawalek EMAIL logo , Marta Dmochowska-Boguta , Anna Nadolska-Orczyk and Waclaw Orczyk


Virus-induced gene silencing is an important tool for functional gene analysis and the vector based on Barley stripe mosaic virus (BSMV) is widely used for the purpose in monocots. Of the tripartite BSMV genome, currently the BSMV:γMCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:β molecule was engineered with a unique BamHI site between the open reading frame of βc (ORF βc) and poly(A). The mixture of RNA particles α, βBamHI and γMCS was fully infectious. Barley phytoene desaturase and wheat phospholipase Dα fragments were cloned to βBamHI and γMCS. Delivery of the target gene fragment in γMCS induced stronger silencing, while delivery in βBamHI yielded more stable transcript reduction. A quantitative analysis (qRT-PCR) of the transcripts showed that the silencing induced with a fragment carried in both particles was stronger and more stable than that from a fragment placed in one particle. The modification of β enables simultaneous silencing of two genes. Quantifying the β and γ particles in virus-inoculated plants revealed a 2.5-fold higher level of γ than β, while the stability of the insert was higher in β compared with γ. The possible influence of the relative quantity of β and γ particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.

[1] Kumagai, M.H., Donson, J., Della-Cioppa, G., Harvey, D., Hanley, K. and Grill, L.K. Cytoplasmic inhibition of carotenoid biosynthesis with virusderived RNA. Proc. Nat. Acad. Sci. USA 92 (1995) 1679–1683. in Google Scholar PubMed PubMed Central

[2] Ratcliff, F., Harrison, B.D. and Baulcombe, D.C. A similarity between viral defense and gene silencing in plants. Science 276 (1997) 1558–1560. in Google Scholar PubMed

[3] Ratcliff, F., Martin-Hernandez, A.M. and Baulcombe D.C. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25 (2001) 237–245. in Google Scholar PubMed

[4] Liu, Y., Schiff, M. and Dinesh-Kumar, S.P. Virus-induced gene silencing in tomato. Plant J. 31 (2002) 777–786. in Google Scholar

[5] Ding, X., Schneider, W.L., Chaluvadi, S.G., Mian, M.A.R. and Nelson R.S. Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts. Mol. Plant Microbe Interact. 19 (2006) 1229–1239. in Google Scholar PubMed

[6] Pacak, A., Strozycki, P.M., Barciszewska-Pacak, M., Alejska, M., Lacomme, C., Jarmolowski, A., Szweykowska-Kulinska, Z. and Figlerowicz, M. The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence. Arch. Virol. 155 (2010b) 169–179. in Google Scholar PubMed

[7] Van der Linde, K.., Kastner, C., Kumlehn, J., Kahmann, R. and Doehlemann, G. Systemic virus-induced gene silencing allows functional characterization of maize genes during biotrophic interaction with Ustilago maydis. New Phytol. 189 (2010) 471–483. in Google Scholar PubMed

[8] Holzberg, S., Brosio, P., Gross, C. and Pogue, G.P. Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J. 30 (2002) 315–327. in Google Scholar PubMed

[9] Lacomme, C., Hrubikova, K. and Hein, I. Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. Plant J. 34 (2003) 543–553. in Google Scholar PubMed

[10] Oikawa, A., Rahman, A., Yamashita, T., Taira, H. and Idou, S. Virusinduced gene silencing of P23k in barley leaf reveals morphological changes involved in secondary wall formation. J. Exp. Bot. 58 (2007) 2617–2625. in Google Scholar PubMed

[11] Hein, I., Barciszewska-Pacak, M., Hrubikova, K., Williamson, S., Dinesen, M., Soenderby, I. E., Sundar, S., Jarmolowski, A., Shirasu, K. and Lacomme, C. Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley. Plant Physiol. 138 (2005) 2155–2164. in Google Scholar PubMed PubMed Central

[12] Pacak, A., Geisler, K., Jørgensen, B., Barciszewska-Pacak, M., Nilsson, L., Nielsen, T.H., Johansen, E., Grønlund, M., Jakobsen I. and Albrechtsen M. Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat. Plant Methods 6 (2010a) 26. in Google Scholar PubMed PubMed Central

[13] Scofield, S.R., Huang, L., Brandt, A.S. and Gill, B.S. 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 (2005) 2165–2173. in Google Scholar PubMed PubMed Central

[14] Tai, Y-S., Bragg, J. and Edwards, M.C. Virus vector for gene silencing in wheat. Biotechniques 39 (2005) 310–314. in Google Scholar PubMed

[15] Van Eck, L., Schultz, T., Leach, J.E., Scofield, S.R., Peairs, F.B., Botha, A.M. and Lapitan, N.L.V. Virus-induced gene silencing of WRKY53 and an inducible phenylalanine ammonia-lyase in wheat reduces aphid resistance. Plant Biotechnol. J. 8 (2010) 1023–1032. in Google Scholar PubMed

[16] Demircan, T. and Akkaya, M.S. Virus induced gene silencing in Brachypodium distachyon, a model organism for cereals. Plant Cell Tiss. Organ. Cult. 100 (2010) 91–96. in Google Scholar

[17] Scofield, S.R. and Nelson, R.S. Resources for virus-induced gene silencing in the grasses. Plant Physiol. 149 (2009) 152–157. in Google Scholar PubMed PubMed Central

[18] Petty, I.T., Hunter, B.G., Wei, N. and Jackson, A.O. Infectious barley stripe mosaic virus RNA transcribed in vitro from full-length genomic cDNA clones. Virology 171 (1989) 342–349. in Google Scholar PubMed

[19] Bruun-Rasmussen, M., Madsen, C.T., Jessing, S. and Albrechtsen, M. Stability of Barley stripe mosaic virus-induced gene silencing in barley. Mol. Plant Microbe Interact. 20 (2007) 1323–1331. in Google Scholar PubMed

[20] Avesani, L., Marconi, G., Morandini, F., Albertini, E., Bruschetta, M., Bortesi, L., Pezzotti, M. and Porceddu, A. Stability of Potato virus X expression vectors is related to insert size: Implications for replication models and risk assessment. Transgenic Res. 16 (2007) 587–597. in Google Scholar PubMed

[21] Zhong, X., Hou, H. and Qiu, W. Integrity of nonviral fragments in recombinant Tomato bushy stunt virus and defective interfering RNA is influenced by silencing and the type of inserts. Mol. Plant Microbe Interact. 18 (2005) 800–807. in Google Scholar PubMed

[22] Pogue, G.P., Lindbo, J.A., Dawson, W.O. and Turpen T.H. (Eds.). Tobamovirus transient expression vectors: tools for plant biology and high-level expression of foreign proteins in plants. Kluwer Academic Publishers, Dordrecht, The Netherlands, 1998. 10.1007/978-94-011-5242-6_5Search in Google Scholar

[23] Orczyk, W., Dmochowska-Boguta, M., Czembor, H.J. and Nadolska-Orczyk, A. Spatiotemporal patterns of oxidative burst and micronecrosis in resistance of wheat to brown rust infection. Plant Pathol. 59 (2010) 567–575. in Google Scholar

[24] Cakir, C. and Tör, M. Factors influencing Barley stripe mosaic virusmediated gene silencing in wheat. Physiol. Mol. Plant Pathol. 74 (2010) 246–253. in Google Scholar

[25] Cakir, C. and Scofield, S.R. Evaluating the ability of the Barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes. Cereal Res. Commun. 36 (2008) 217–222. in Google Scholar

[26] Wang, X., Devaiah, S.P., Zhang, W. and Welti, R. Signaling functions of phosphatidic acid. Prog. Lipid Res. 45 (2006) 250–278. in Google Scholar PubMed

Published Online: 2011-12-31
Published in Print: 2012-3-1

© 2011 University of Wrocław, Poland

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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