2012 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.
Stagonospora nodorum blotch (SNB) caused by the necrotrophic fungal pathogen S. nodorum is a destructive disease throughout the wheat growing regions of the world. We have shown that S. nodorum produces a suite of necrotrophic effectors that each interact with specific host sensitivity gene products to induce disease on wheat. In the previous year we have continued to characterize the mode of action of SnTox1 and SnTox3 as it relates to the corresponding sensitivity genes Snn1 and Snn3 respectively, as well as continue our work on the identification of new necrotrophic effectors (NEs) using multiple approaches.
In the past year, 50 newly prioritized candidate NE genes were amplified and cloned based on criteria deemed important to effector-like molecules. Among these genes, 16 were chosen to be expressed in Pichia pastoris for verification of necrotrophic effector activity. Two candidate genes, showed obvious necrotic activities, indicating that these two genes are strong necrotrophic candidates encoding two novel NEs.
The novel interaction, SnTox6-Snn6, has been further characterized. An isolate producing SnTox6 was used to inoculate the International Triticeae Mapping Initiative hexaploid wheat mapping population which segregates for sensitivity to SnTox6 (Snn6). QTL analysis of a 7-day inoculation on the ITMI population detected a significant QTL at the Snn6 locus which corresponds to SnTox6 sensitivity. Snn6 was mapped to the distal end of the long arm of chromosome 6A. SnTox6 was purified from the isolate Sn6 through ion-exchange chromatography, low pressure size exclusion and HPLC size exclusion chromatography. The estimated size of SnTox6 is 6.5-13 kDa based on protein size standards used in size exclusion chromatography.
Previously, we showed that SnTox1 was an important S. nodorum virulence factor by transforming it into an avirulent S. nodorum isolate. To further demonstrate its function, SnTox1 was transformed with its native promoter into the barley fungal pathogen Pyrenophora teres f. teres and the sugar beet fungal pathogen Cercospora beticola, both of which are nonpathogenic to wheat. After acquiring the SnTox1 gene, both fungi were capable of producing SnTox1 in culture; however, they showed different virulence toward wheat lines carrying Snn1. P. teres f. teres transformants became highly virulent causing large necrotic lesions, while C. beticola transformants only induced small white flecks.
A Prosite motif search and manual alignment identified several sites and regions in SnTox1 that may have functional importance, including multiple potential chitin-binding domains, a lysine-rich region, and a casein kinase II phosphorylation site, each of which has been targeted for site-directed mutagenesis or deletion mutation. Fifteen mutations have been completed and characterized. Mutations produced at the casein kinase II phosphorylation site, certain lysine residues of the lysine-rich region, and several cysteine residues significantly reduced the SnTox1 activity. Mutation at one of the putative chitin-binding domains also affected the SnTox1 activity, but to a lesser degree. The deletion of 17 aa following the signal peptide did not have significant impact on SnTox1 activity; however, all other deletion mutations involving cysteine residues completely eliminated the SnTox1 activity.
A highly interesting discovery was that SnTox1 was capable of causing widespread necrotic lesions on wheat lines carrying Snn1 by directly spraying the protein on the leaf surface. The two other cloned S. nodorum necrotrophic effectors, SnTox3 and SnToxA, were not able to induce a reaction on their sensitive lines by direct spraying, indicating that SnTox1 has a different mode of action than SnToxA and SnTox3 to induce necrosis, and likely, SnTox1 interacts with a substance on the leaf surface to initiate plant cell death. We further examined the time period required for SnTox1 to be attached or recognized on the leaf surface by washing away SnTox1 using high pressure water at different time points after the initial spray application. It was found that the necrosis development caused by SnTox1 was nearly unaffected if washed off at or after 25 min following SnTox1 spraying. We also found that co-spray of SnTox1 with protein kinase inhibitors did not inhibit the development of the necrosis caused by SnTox1.
Using a fungal strain expressing an SnTox1-GFP fusion protein, we examined the location of the SnTox1 protein during fungal growth and infection. SnTox1 was observed in higher concentration on parts of several fungal structures, including the outer surface of conidia and mycelium, and hyphal tips as well as hyphal septa. This, along with the identification of the putitive chitin binding domains, suggested that SnTox1 may have a chitin-binding function to protect the fungal mycelium from host produced hydrolytic enzymes. The accumulation of SnTox1GFP is particularly obvious at hyphal regions where new hyphae are arising. In planta, SnTox1 is highly expressed in the hyphopodia where the penetration is initiated, providing further evidence that SnTox1 plays a role in penetration. Polyclonal antibodies generated against SnTox1 have been successfully generated from rabbits and is being used to determine the cellular location of SnTox1 during disease induction using immunolocalization methods. The yeast strain expressing a histidine tagged SnTox1 has been generated and provides a useful tool for purification of SnTox1.
The functional sites of the SnTox3 protein have been subjected to extensive investigation using site-directed mutagenesis or deletion mutations, including a putative C-terminal GTPase domain, which also, is predicated to contain a carbamoyl transferase signature. Deletion of the whole or partial GTPase domain led to the complete loss of SnTox3 activity. Several amino acid residues within this domain have been identified to be important through substitutions with alanine. However, mutations at other places such as a PPNP motif, a CK II phosphorylation site, and an N-glycosylation site did not affect the SnTox3 activity. Using the two different antibodies with one specifically against a pro sequence and the other against the end of the mature protein, we have demonstrated that SnTox3 contains a pro-sequence (position of 21~72 aa) that is cleaved during maturing. The change of the cysteine residue at the very end of the protein resulted in an unsuccessful cleavage of the pro-sequence, hence, eliminating the function of the protein. This suggested that the cleavage of the pro-sequence is necessary for proper protein function. We have successfully developed an SnTox3RFP fusion construct and expressed it in P. pastoris and this protein fusion has been successfully produced and shown to be fully functional. A protocol to purify SnTox3RFP from the yeast culture is being developed. The SnTox3RFP fusion protein as well as the SnTox3 antibodies provides us with a powerful tool to investigate the cellular location of SnTox3 in planta.