2013 Annual Report
1a.Objectives (from AD-416):
To identify new necrotrophic effectors that are important in Stagonospora nodorum leaf blotch.
1b.Approach (from AD-416):
Stagonospora nodorum blotch (SNB) has long been a problem for both wheat growers and breeders alike. For growers, this disease is a destructive foliar and glume disease that causes major yield and quality losses. In the Northern Great Plains, consistent yield losses of 5-10% occur with similar losses in other wheat growing regions of the world. For breeders, SNB has been a difficult problem to overcome due to the lack of understanding of the underlying basis of the disease. Rarely have major resistance genes been identified and instead, numerous minor quantitative trait loci (QTL) have been identified on most chromosomes spread throughout the A, B, and D genomes. We have identified, cloned and characterized three necrotrophic effectors including SnTox1, SnTox3, and SnToxA. Each of these NEs have been shown to contribute significantly to SNB disease.
In this project, multiple approaches will be used to identify and validate additional necrotrophic effectors from S. nodorum. Personnel on this project will use.
1)an NE purification approach and.
2)an NE candidate gene approach to isolate NEs involved in SNB disease.
Several chromatography methods have been worked out using culture filtrates containing NEs. So far, the most robust method across all NEs has been ion exchange. Ion exchange matrixes that have been most successful as initial concentration steps are SP Sepharose™ Fast Flow and SP Sepharose™ XL. Additionally, ion exchange matrixes including Q Sepharose™ Fast Flow and CM Sepharose™ Fast Flow (GE Healthcare) have been useful in downstream steps. Size exclusion chromatography has been used in final purification steps before separation of proteins on a gel for use in mass spectrometry (MS) analysis. HPLC size exclusion columns have been used effectively both in purification and size estimation. Size estimation is critical for prioritizing candidate gene lists for each NE. Using specific active fractions, we have generated a list of candidate genes based on MS analysis of protein fractions with specific necrosis activity. These genes have been prioritized and transformation constructs will be developed for expressing these genes in P. pastoris and in avirulent isolates. This process of identifying and verifying candidates is highly effective and was used in the identification and cloning of SnTox3.
Candidate gene approach:
Using the bioinformatics data which we have accumulated, we are using a scoring approach to identify the top candidate genes based on several weighted criteria. A database of ~16,000 genes was ranked based on criteria including observations by MS in necrosis producing fractions, observations by MS in untested fractions, absence in the avirulent isolate Sn79-1087, predicted signal sequence, expression profiles similar to SnToxA, SnTox3, and SnTox1, no significant blast hits, and a predicted molecular weight <50 kDa. The top 100 genes will be evaluated and expressed in P. pastoris. This process of identifying and verifying candidates has also proven effective and was used in the identification and cloning of SnTox3.
Several necrotrophic effector (NE) candidate genes have been identified using criteria that we would expect in necrotrophic effectors including small size, secretion prediction, expression early in disease, absence in avirulent isolates, etc. Three of these genes, after being expressed in Pichia pastoris cultures, have been shown to induce necrosis on susceptible wheat lines in a host specific manner, indicating that these genes are responsible for production of NE proteins that are important in disease. Currently we are developing and evaluating host populations to characterize the importance of these proteins in disease development.
A necrotrophic effector purification approach is also being used to identify new NEs. We have worked out a method to produce highly concentrated NEs in small amounts of media using a spore germination fluid approach. Preliminary data has shown that this technique is highly efficient in producing NEs.