Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-031-15-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2016
End Date: Dec 31, 2019
The University of Florida's overall goal of this project is to develop effective and durable disease resistance for Sclerotinia stem rot of canola/rapeseed (Brassica napus) through transgenic and/or cisgenic engineering of the host. This goal is being pursued with a multi-year strategy that builds on results from previous funding and new data concerning the HSS1 gene that holds the potential to block disease when over-expressed in canola. The HSS1 gene was first identified in Arabidopsis thaliana as a locus conferring extreme susceptibility to Sclerotinia infection when it is mutated to loss of function. Now, in its third year of funding, the University of Florida has successfully fine-mapped the A. thaliana HSS1, identified the specific mutation conferring the hss1 phenotype, overexpressed the HSS1 gene in Arabidopsis, identified a B. napus ortholog of HSS1 (BnHss1) and are in the process of testing the function of BnHSS1. Following mapping, cloning and genetic complementation, the University of Florida determined HHS1 encodes a member of the Mediator protein complex known to function in the regulation of basal resistance to pathogen infection. The Univeristy of Florida has recently discovered that S. sclerotiorum has the ability to specifically process and degrade the HSS1 protein during infection. In this current year’s funding, the University of Florida is seeking to build upon this finding to identify proteolysis-resistant forms of HSS1 and test their functions in enhancing basal resistance to S. sclerotiorium. They are focusing solely on the HSS1 gene due to its dramatic susceptibility phenotype when mutated. In the previous three years they have also included a parallel focus on the oxalate decarboxylase gene (ODC2). A lack of demonstrated disease resistance efficacy when ODC2 was overexpressed in A. thaliana however has dictated that this strategy be dropped in order to focus on the progress with HSS1. The specific objectives outlined in the University of Florida's proposal for the project’s fourth year are to: (1) Express BnHSS1 in the Arabidopsis hss1 mutant for functional complementation assays; (2) Generate B. napus transgenic lines overexpressing HSS1; and (3) Screen for proteolysis-resistant forms of the HSS1 protein identified from nonhost plants and from S. sclerotiorum.
The University of Florida's approach to accomplish the overall project goals is being carried out over the course of five years. Thus far in the first three years they completed the fine-scale genetic mapping of the hss1 mutation, verified the function of the HSS1 gene in Arabidopsis, developed plants that over-express the HSS1 and ODC2 genes, and screened over-expressing plants for disease resistance. The goals for year four are to test the function of BnHSS1, to engineer resistance in canola using HSS1 and to screen for proteolysis-resistant HSS1 proteins. This project has direct relevance to the goals and priority research needs of the Sclerotinia Initiative. The proposed studies will make significant contributions towards understanding the genetics of host resistance and directly improving crop germplasm for Sclerotinia resistance. Although the first and second year objectives have been concerned primarily with the non-crop plant A. thaliana, this has been integral to their success in mapping the HSS1 locus and their overall project goal of producing resistant canola lines. In the current funding request for year four, the University of Florida proposes to identify the functional BnHSS1 gene and verify its activity by complementation of the Arabidopsis hss1 mutant. Meanwhile, they will generate B. napus lines overexpressing HSS1 and assay transiently expressed HSS1 proteins for resistance to Sclerotinia-mediated degradation. The University of Florida has chosen to initially focus on canola as it is a crucifer with a close relationship to A. thaliana and also due to their experience with transgenic manipulation of canola. In the long term, they anticipate that the results produced here will be applicable to increasing resistance in other susceptible crops including soybean, dry bean, sunflower and pulse crops.