Submitted to: Proceedings of the International Sclerotinia Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 5/30/2005
Publication Date: 6/12/2005
Citation: Calla, B., Zhang, Y., Hubbard, S., Davidson, A., Rioux, S., Simmonds, D., Clough, S.J. 2005. Gene expression profiling of oxalate oxidase transgenic soybean challenged with sclerotinia sclerotiorum. Proceedings of the International Sclerotinia Workshop. June 12-16, 2005 pg.46
Technical Abstract: Oxalate is a major virulence factor of Sclerotinia sclerotiorum. Studies have shown that the virulence of S. sclerotiorum is substantially reduced if oxalate is removed from the interaction. We are utilizing a transgenic soybean plant that constitutively produces the oxalate-degrading enzyme, oxalate oxidase (OxO), to study how soybean plants respond to oxalate and S. sclerotiorum. Plants of transgenic line 80(30)-1 and its parent AC Colibri were inoculated behind the first lateral vein using infected flowers. Three pooled samples were obtained for each of the soybean lines: a control leaflet at time zero, a time point 1 sample (early appressorial formation, 10-12 hour post inoculation) and a time point 2 sample (early vascular entry, 18-20 hour post inoculation). The lower third of mid leaflet was removed from the plant by cutting the petiole close to the main stem and then collected and frozen in liquid nitrogen within 30 seconds. RNA was isolated from the samples using TRIzol reagent. RNA integrity was verified using Agilent 2100 BioAnalyzer instrument with RNA 6000 nanochips. RNA was reverse transcribed using aminoallyl incorporation for indirect labeling. Cy3 and Cy5 were assigned as labels according to a loop design for the experiment. Samples were hybridized on soybean cDNA microarrays and images were obtained for each of the 24 slides. A response measurement in log ratio intensity was obtained. Preliminary analyses on selected genes for the first biological replication were conducted. Several genes known to respond to infection, such as the CHS gene family and PAL, were up in abundance in both soybean lines at T12 and T18 suggesting that a defensive response was underway during the study. Significance of these results and identification of other differentially expressed genes will be assessed using various statistical tools such as MAANOVA for R and/or ANOVA in SAS after conducting additional biological replications for the experiment. This project promises to advance our understanding of the basic biology behind soybean response to this serious pathogen and the genetic/physiological effect of oxalate oxidase on resistance to Sclerotinia.