Project Number: 5442-21220-023-49
Project Type: Specific Cooperative Agreement

Start Date: Jun 01, 2007
End Date: May 31, 2009

Objective:
Elucidation of the molecular basis of resistance to Sclerotinia sclerotiorum will enhance traditional plant breeding, genetic engineering, and disease management in the field. In a model system, Arabidopsis thaliana, we will: (1) Characterize the role of protein phosphatase 2C in resistance to S. sclerotiorum; (2) Determine the contribution of the ERECTA gene to resistance against S. sclerotiorum; and (3) Identify additional genes and pathways that are involved in resistance to S. sclerotiorum.

Approach:
Water loss will be measured using excised leaves of Arabidopsis thaliana. Three rosette leaves per plant will be inoculated with either oxalate-deficient or wild-type S. sclerotiorum. Plants will be incubated in a humid chamber for 2 to 3 days until symptoms form. Leaves will be excised form abi1 mutant and wild-type plants, incubated at ambient laboratory conditions, and weighed at 20 min intervals for 3 hours. With the help of this assay, it will be possible to determine whether differences in susceptibility and water loss are correlated. As a secondary test, we will measure the width of stomatal apertures in abi1 mutant and wild-type leaves. Oxalate-dependent effects can further be studied by analyzing the production of ROS. We will track hydrogen peroxide (H2O2) and superoxide (O2.-) production in infected tissues histochemically using 3,3-diaminobenzidine and nitro blue tetrazolium staining, respectively. Quantitative analysis of ROS production is possible using digital imaging (ImageJ, NIH). Differences in ROS accumulation during infection of abi1 and abi2 mutants are likely important because suppression of ROS production by oxalate and virulence of S. sclerotiorum are correlated. The contribution of ROS signaling to resistance against S. sclerotiorum will also be tested genetically. Lines with T-DNA insertions in the NADPH oxidase genes atrbohD and atrbohF will be used in addition to the double mutant atrbohD/F. All of these lines have been obtained from Dr. Julian Schroeder (University of California, San Diego). Hypersusceptibility of these lines to S. sclerotiorum would support the hypothesis that ROS protect against this fungal pathogen. To improve our understanding of the genetic basis of resistance to S. sclerotiorum, we have decided to acquire and screen the Ler-2 x Cvi-1 recombinant inbred line population. Susceptibility of Ler-1 and Cvi-0 to S. sclerotiorum was significantly (t-test, p = 0.0134, n = 20). Our rational is that identification of resistance QTL in Arabidopsis can be combined with information about candidate genes, which were obtained from screening for oxalate sensitivity and susceptibility to S. sclerotiorum in yeast and Arabidopsis, respectively. Major QTL can be used for back-crossing and generation of near-isogenic lines. Also, locations of resistance QTL in Arabidopsis and B. napus can be compared.