Location: Cereal Crops Research
Project Number: 3060-22000-048-28-N
Project Type: Nonfunded Cooperative Agreement
Start Date: Aug 1, 2013
End Date: Jul 31, 2016
The main goal of this project is to provide the tools and knowledge for more precise and effective breeding of resistance to Stagonospora nodorum blotch in wheat and net blotch in barley. Subgoals include the characterization of the virulence profiles and genetic structure of the local pathogen populations of Stagonospora nodorum and Pyrenophora teres and to have determined the role of necrotrophic effectors (NEs) in the two pathosystems. This will allow the identification of the most important genetic factors for resistance/susceptibility in Norwegian breeding material of wheat and barley. The end result will be the development of a breeder’s toolbox with optimized testing methods based on seedling tests in the greenhouse and field testing of adult plant resistance and validated marker assays for use in the wheat and barley breeding programs.
Collected isolates of the pathogens will be screened for specific reactions against differential sets of host lines at the seedling stage following established methodology. For SNB in wheat and barley net blotch, this will include commonly used differential lines for the different supplemented with a small subset of Norwegian lines with varying levels of field resistance. Parents of potential mapping populations will be included to facilitate selection of isolates for genetic studies of host resistance. Culture filtrates will be produced from a selection of fungal isolates causing differential reactions in the seedling tests and infiltrated onto seedling leaves of the same lines following our established methodology for SNB and barley net blotch. For SNB, these tests will also include extracts of the known NEs SnToxA, SnTox1 and SnTox3 produced in Pichia pastoris. This will determine to what degree seedling reactions to pathogen isolates can be explained by sensitivity to known and unknown soluble compounds released by the isolates in culture. Additionally, intercellular wash fluids can be extracted following an established protocol used for identification of NEs produced in planta. Protease treatment of culture filtrates will determine whether these putative NEs are proteinaceous in nature. Seedlings of mapping populations will be infiltrated with culture filtrates from candidate isolates that are expressing potentially novel NEs in order to map the corresponding sensitivity loci. Strong interactions shown to be important for adult plant resistance in the field will be investigated further following established protocols, which briefly include fractionation of culture filtrates by HPLC, mass spectrometry of active fractions and searches against expected proteins encoded by the S. nodorum genome sequence. Resulting candidate genes will be cloned and expressed in P. pastoris and the culture filtrates infiltrated onto wheat seedlings to verify that they cause the expected sensitivity reactions.