Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2005
Publication Date: 7/15/2005
Citation: Scofield, S.R., Huang, L., Brandt, A.S., Gill, B.S. 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 Physiology. 10.1104:105.061861 Interpretive Summary: Wheat is one of the most important crops in the world's food supply. Unfortunately, the essential steps in genetic improvement of wheat by application of molecular biology to identify and analyze the function of critical genes are difficult and slow. In this paper, we describe an effective method to determine gene function in hexaploid wheat and show that it is applicable in a range of wheat lines. Instead of creating transgenic wheat plants, the process, termed virus-induced gene silencing (VIGS), relies on infecting wheat with a plant virus containing a fragment of the wheat gene to be tested. Development of a convenient tool for gene silencing in wheat is highly significant for all investigators performing genetic analyses in wheat. VIGS provides a novel and rapid strategy for gene identification and functional analysis that overcomes obstacles that currently confound research in wheat genetics. As demonstrated in this paper, the technique is well suited for identifying new genes that are involved in diseases resistance, which is essential to enhance productivity of wheat.
Technical Abstract: Virus-induced gene silencing (VIGS) is a proven tool for the analysis of gene function in several dicotyledonous plant species. VIGS strategies utilize viruses engineered to carry sequences derived from plant gene transcripts to activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus also contains transcribed plant sequence, the homologous host mRNAs are also targeted for destruction. No VIGS system was known for monocot plants until the recent report of silencing in 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. After the wheat VIGS system was optimized in studies silencing phytoene desaturase (PDS) expression, we employed it to assay genes functioning in leaf rust resistance mediated by Lr21, that encodes a nucleotide binding site - leucine-rich repeat class R-gene product (NB-LRR). It was demonstrated that infection with BSMV constructs carrying a 150bp fragment of Lr21 caused conversion of incompatible interactions to compatible, while infection with a control construct, or one that silences PDS had no effect on resistance or susceptibility. Additionally, silencing the RAR1, SGT1 and HSP90 genes, known to be required in many, but not all NB-LRR resistance pathways in diverse plant species, also resulted in conversion to compatibility indicating that these genes are essential in Lr21-mediated resistance. These studies indicate that BSMV-VIGS is a powerful new tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.