1a.Objectives (from AD-416):
Objective 1. Evaluate the importance of HST-host gene interactions identified using culture filtrates and purified toxins in conjunction with wheat mapping populations including the development of differential wheat lines for each toxin.
Objective 2. Identify candidate HSTs by mass spec analysis of purified active protein preparations and by using a bioinformatics gene selection approach.
Objective 3. Verify candidate genes using heterologous expression, transformation, and site directed gene disruption.
Objective 4. Characterize the function and mode of action of SnTox3 to identify the molecular and biochemical mechanism whereby the toxin is effective in inducing disease.
1b.Approach (from AD-416):
We have established that the Stagonospora nodorum-wheat interaction consists of pathogen produced effector proteins i.e. host selective toxins (HSTs) that interact with dominant host gene products to induce disease. This interaction is similar to a classical gene-for-gene interaction except that host recognition of the effector proteins leads to susceptibility rather than resistance and therefore acts in an inverse gene-for-gene manner. We have published or submitted for publication five of these HST – host gene interactions. Additionally, we have accumulated evidence for four more interactions. Each of these nine interactions involves a single proteinaceous HST that interacts directly or indirectly with a dominant host gene product leading to disease. In this proposal we would like to. 1)characterize the newly identified interactions,. 2)clone the associated HST encoding genes from the pathogen for further analysis of individual interactions, and. 3)do functional analysis and evaluate the mode of action of SnTox3, a recently cloned HST involved in the SnTox3-Snn3 interaction.
Characterization of SnTox1 mode of action and localization – SnTox1 has significant homology to other chitin binding proteins and we have shown that SnTox1 physically binds chitin as well other polysaccharides found in the plant cell wall. These polysaccharides include cellulose, xylan, chitosan, crab shell chitin, and shrimp shell chitin. Using an SnTox1-GFP fusion, we showed that SnTox1 localized to the outside of the fungal mycelium as well as to the plant cell wall. Using a His tag, SnTox1 was purified and treated with a fluorescent label for localization. Wheat plants harboring Snn1, the corresponding wheat sensitivity gene, were inoculated and cross sections were made in order to visualize where SnTox1 was localized to during the infection process. Preliminary data indicate that SnTox1 is able to induce necrosis in the mesophyll cells, while remaining localized to the top of the epidermal layer of the plant.
Necrotrophic effector candidate gene identification – Continued work on a necrotrophic effector candidate gene list has been useful in identifying several new NE candidates that induce necrosis on certain susceptible cultivars in a host specific manner. These genes have been expressed in Pichia pastoris (yeast) and have been shown to induce necrosis. Further work in validating these genes is in progress.