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First published online March 18, 2005; 10.1104/pp.104.057257 Plant Physiology 137:1345-1353 (2005) © 2005 American Society of Plant Biologists RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae1Donald Danforth Plant Science Center, St. Louis, Missouri 63132 (S.S., O.Y.); and Department of Plant Pathology, Plant Molecular Biology and Biotechnology Program, Ohio State University and Ohio Agricultural Research and Development Center, Columbus, Ohio 43210 (M.Y.G., T.L.G.)
Isoflavones are thought to play diverse roles in plant-microbe interactions and are also potentially important to human nutrition and medicine. Isoflavone synthase (IFS) is a key enzyme for the formation of the isoflavones. Here, we examined the consequences of RNAi silencing of genes for this enzyme in soybean (Glycine max). Soybean cotyledon tissues were transformed with Agrobacterium rhizogenes carrying an RNAi silencing construct designed to silence expression of both copies of IFS genes. Approximately 50% of emerging roots were transformed with the RNAi construct, and most transformed roots exhibited >95% silencing of isoflavone accumulation. Silencing of IFS was also demonstrated throughout the entire cotyledon (in tissues distal to the transformation site) both by high-performance liquid chromatography analysis of isoflavones and by real-time reverse transcription-PCR. This distal silencing led to a nearly complete suppression of mRNA accumulation for both the IFS1 and IFS2 genes and of isoflavone accumulations induced by wounding or treatment with the cell wall glucan elicitor from Phytophthora sojae. Preformed isoflavone conjugates were not reduced in distal tissues, suggesting little turnover of these stored isoflavone pools. Distal silencing was established within just 5 d of transformation and was highly efficient for a 3- to 4-d period, after which it was no longer apparent in most experiments. Silencing of IFS was effective in at least two genotypes and led to enhanced susceptibility to P. sojae, disrupting both R gene-mediated resistance in roots and nonrace-specific resistance in cotyledon tissues. The soybean cotyledon system, already a model system for defense signal-response and cell-to-cell signaling, may provide a convenient and effective system for functional analysis of plant genes through gene silencing.
1 This work was supported by the Ohio Plant Biotechnology Consortium (to M.Y.G.), by the Ohio Agricultural Research and Development Center, the Ohio State University (to T.L.G.), and by the Illinois-Missouri Biotechnology Alliance and Missouri Soybean Merchandising Council (to O.Y.). Salary and additional research support was also provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.057257. * Corresponding author; e-mail graham.1{at}osu.edu; fax 614–292–4455. Received November 26, 2004; returned for revision January 30, 2005; accepted January 30, 2005. Related articles in Plant Physiol.:
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