The function of effector proteins of Pseudozyma flocculosa in a tritrophic interaction plant-pathogen-biocontrol agent

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Practical application of biological control of plant pathogens presupposes a thorough understanding of the genetic, molecular and ecological events underlying such phenomena. This challenge has been the focal point of the research activities carried out in the applicant's laboratory in the context of his Discovery program. The last installment has been particularly rewarding since our team has made some major and unexpected strides toward elucidating the complex and elusive factors governing the biocontrol properties of Pseudozyma flocculosa, a natural antagonist of powdery mildew fungi. These findings have pushed our group to amalgamate the intricacies of evolutive biology, tritrophic interactions, fungal ecology, fungal genomics, and CRISPR-Cas transformation to redefine completely a dogma that had persisted for many years. Of particular significance, we reported the following: 1) the unprecedented simultaneous transcriptomic analysis of the three protagonists involved in a tritrophic interaction; 2) the discovery of new factors dictating the specificity of the interaction; 3) a new hypothesis suggesting that effector proteins released by a biocontrol agent (BCA) were involved in its activity against another fungus: 4) the development of a pipeline to identify effector genes in fungal genomes; and 5) the first application of a CRISPR-Cas knockout system in a BCA to provide evidence against the role of antibiosis, long thought to be the main mode of action of the BCA. This latter achievement was instrumental in streamlining an unforeseen phenomenon never previously described within the realm of modes of action of BCAs. In the context of this application and in direct link with our recent progress, the objectives are: 1) to apply CRISPR-Cas editing of three genes coding for effectors unique to P. flocculosa, and specifically expressed during the tritrophic interaction P. flocculosa-Blumeria graminis-barley; 2) to conduct functional analyses of the genes and quantify their impact on the biocontrol activity of the BCA; 3) to identify the target(s) of the effectors by immunolocalization; 4) to determine if the BCA effectors interact with the pathogen (powdery mildews), the plant or at their interface i.e. the extrahaustorial matrix; 5) to provide mechanistic evidence that some BCAs rely on effectors to exert their biocontrol activity. Fulfillment of these objectives will generate novel and distinctive information with regards to: the molecular determinants that regulate the biocontrol activity of P. flocculosa and how expression (or repression) of these determinants influence alter powdery mildew fungi or the plant through disturbance of the fragile biotrophic interaction linking them. These results will have a far reaching impact into the field of plant pathology as our model will provide unique data that could completely redefine the concepts by which BCAs antagonize their host.