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.