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First published online July 15, 2005; 10.1104/pp.105.061861

Plant Physiology 138:2165-2173 (2005)
© 2005 American Society of Plant Biologists

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PLANTS INTERACTING WITH OTHER ORGANISMS

Development of a Virus-Induced Gene-Silencing System for Hexaploid Wheat and Its Use in Functional Analysis of the Lr21-Mediated Leaf Rust Resistance Pathway1

Steven R. Scofield2,*, Li Huang2, Amanda S. Brandt and Bikram S. Gill

United States Department of Agriculture, Agricultural Research Service, Crop Production and Pest Control Research Unit, West Lafayette, Indiana 47907 (S.R.S., A.S.B.); Department of Agronomy, Purdue University, West Lafayette, Indiana 47907 (S.R.S., A.S.B.); and Department of Plant Pathology, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, Kansas 66506 (L.H., B.S.G.)

Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. In VIGS, viruses engineered to carry sequences derived from plant gene transcripts activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus contains transcribed plant sequence, homologous host mRNAs are also targeted for destruction. While routinely used in some dicots, no VIGS system was known for monocot plants until the recent report of silencing in barley (Hordeum vulgare) by barley stripe mosaic virus (BSMV). Here, we report development of protocols for use of BSMV to efficiently silence genes in hexaploid wheat (Triticum aestivum). The VIGS system was first optimized in studies silencing phytoene desaturase expression. Next, we used it to assay genes functioning in leaf rust resistance mediated by Lr21, which encodes a nucleotide binding site-leucine-rich repeat class resistance gene product. We demonstrated that infection with BSMV constructs carrying a 150-bp fragment of Lr21 caused conversion of incompatible interactions to compatible, whereas infection with a control construct or one that silences phytoene desaturase had no effect on resistance or susceptibility. Additionally, silencing the RAR1, SGT1, and HSP90 genes, known to be required in many but not all nucleotide binding site-leucine-rich repeat resistance pathways in diverse plant species, resulted in conversion to compatibility, indicating that these genes are essential in Lr21-mediated resistance. These studies indicate that BSMV-VIGS is a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.

1 This work was supported by the U.S. Department of Agriculture, Agricultural Research Service Current Research Information System (project 3602–21220–008–00D; S.R.S. and A.S.B.), and by the Plant Biotechnology Center at Kansas State University and the Kansas Wheat Commission (funds to L.H. and B.S.G.).

2 These authors contributed equally to the paper.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.061861.

* Corresponding author; e-mail scofield{at}purdue.edu; fax 765–496–2926.

Received February 23, 2005; returned for revision May 12, 2005; accepted May 27, 2005.




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