Characterization of plant-fungal interactions involving necrotrophic effector-producing plant pathogens.

Recently, great strides have been made in the area of host-pathogen interactions involving necrotrophic fungi. In this article we describe a method to identify, produce, and characterize effectors that are important in host-necrotrophic fungal pathogen interactions, and to genetically characterize the interactions. The main strength of this method is the combined use of pathogen inoculation, a pathogen culture filtrate bioassay, and genetic analysis of susceptibility and sensitivity in segregating host-mapping populations. These methods have been successfully used to identify several Stagonospora nodorum necrotrophic effectors and to characterize the genetic and phenotypic effects of individual host-effector interactions in the wheat-S. nodorum system. S. nodorum isolates that induce a differential response on two lines are used to produce culture filtrates that contain necrotrophic effectors while the wheat lines differing in reaction to the pathogen are used to develop a mapping population. The wheat population is used to develop DNA marker-based genetic linkage maps and culture filtrates are infiltrated across the mapping population. Linkage and quantitative trait loci (QTL) analysis is used to identify regions of the wheat genome harboring genes that govern sensitivity to necrotrophic effectors. The same populations are inoculated with the effector-producing isolate to determine the significance and proportion of disease explained by individual host gene-effector interactions. Additionally, from this information, differential lines that are sensitive to single effectors are developed for further purification and characterization of the effectors, eventually resulting in the identification, molecular cloning, and characterization of the effector genes.