GENOMICS ANALYSIS OF PLANT RESPONSES TO THE FUNGICIDE FOSETYL BY COMBINING SSH AND DNA MICROARRAYS

Texas A&M University Dept of Biology BSBE - Room 201 College-Station, TX 77843-3258

Fosetyl is a systemic fungicide which is widely used in agriculture for controlling plant pathogens. It has been shown that fosetyl can induce the expression of plant pathogenesis-related gene, PR-1, in Arabidopsis. In this study, we combined suppression subtractive hybridization (SSH) and DNA microarrays to gain insight about plant responses to fosetyl. PolyA+ RNA prepared from fosetyl treated and control plant were subjected to subtractive hybridization. PCR products from the forward and reverse subtractions were fluorescently labeled with Cy3 and Cy5 and hybridized to a DNA array containing 2,500 Arabidopsis ESTs. 17 ESTs had Cy3/Cy5 ratios greater than 2.5 and were confirmed to be fosetyl inducible (2 to 10 folds of induction) by RNA gel blots. In another approach, a DNA microarray containing clones from the subtractive library was made from the forward subtraction. This microarray was hybridized with fluorescently labeled cDNAs prepared by reverse transcription from the fosetyl-treated (Cy3) and control (Cy5) polyA+ RNA. 15 clones had high Cy3/Cy5 ratios and were confirmed to be up-regulated by fosetyl by RNA gel blot analysis. In this study, we show that 1) a combination of DNA microarray and SSH is an effective tool for gene discovery. 2) fosetyl, in addition to its fungicidal activity, triggers a broad range of responses in Arabidopsis.