Location: Cereal Crops Research2010 Annual Report
1a. Objectives (from AD-416)
1) The identification of HST-host gene interactions using purified toxins and wheat mapping populations; 2) Identification of proteinaceous toxin genes using purified toxins in conjunction with mass spec analysis to identify candidate genes for further evaluation; 3) Verification of candidate genes using heterologous expression, transformation, and site directed gene disruption; and 4) Mode of actions studies to identify the molecular and biochemical mechanism whereby the toxin is instrumental in causing disease, including protein-protein interaction studies, inhibitor studies, and protein localization studies.
1b. Approach (from AD-416)
Stagonospora nodorum will be used to produce extracts of the proteinaceous host-selective toxins (HSTs) that we have identified. HSTs will be purified and active fractions will be used to identify protein sequences using mass-spectrometry. Each of the presently characterized toxins has been shown to be a protein that interacts with a corresponding host sensitivity gene in an inverse gene-for-gene manner. Candidate genes will be revealed using the S. nodorum genome sequence. Additionally, bioinformatics will be used to scan the S. nodorum genome to identify candidate genes based on signal sequence domains and predicted protein size in addition to other relevant criteria. We have isolated a nonpathogenic strain of S. nodorum that appears to secrete no toxins. We will use this strain to verify toxin gene candidates. Candidate genes will initially be expressed in this isolate to verify toxin production and pathogenicity changes. Lastly, we will continue to identify new toxins using an international S. nodorum collection. This proposal will enhance economic opportunities for agricultural producers. This will be accomplished by providing valuable information to scientists including breeders for the improvement of wheat as a food crop especially as it relates to providing durable resistance sources to growers.
3. Progress Report
Stagonospora nodorum blotch (SNB) is a destructive disease of wheat worldwide. We have shown that the causal agent, S. nodorum, produces a suite of necrotrophic effectors that each interact with specific host sensitivity gene products to induce disease on wheat. New interactions that we have identified include host sensitivity genes on wheat chromosomes 4B and 3D. Wheat chromosome 4B has been shown to harbor a sensitivity gene, Snn5, that accounts for up to 53% of the disease phenotype. Wheat chromosome 5D has been shown to harbor a sensitivity gene, Snn3D1, which accounts for up to 100% of the disease phenotype and has also been shown to interact with the previously reported necrotrophic effector, SnTox3. Snn3D1 has also been shown to be homeologous to Snn3B1 (formerly Snn3), which was originally shown to interact with SnTox3. Additionally, using a bioinformatically generated candidate gene list, we have identified and cloned the gene responsible for the expression of SnTox1, the first HST /necrotrophic effector identified in this system. Currently we are characterizing the gene SnTox1 and the corresponding protein SnTox1. We anticipate that the work outlined in this proposal will lead to a better understanding of SNB of wheat. Specifically we aim to characterize the necrotrophic effector-host gene interactions in order to provide basic knowledge about necrotrophic plant-pathogen interactions that can be used to better characterize other similar systems.