Functional Characterization of Fungal Effectors in the Phytopathogen Fusarium graminearum

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Summary Fungi are the main cause of diseases in crops. Fusarium head blight (FHB) is a devastating disease of wheat, maize, and other small grain crops and is caused by several related Fusarium species. The disease reduces grain quality due to mycotoxin deposition and the contaminated grains with deoxynivalenol (DON) mycotoxin can restrict trade and impact the livelihood of producers. To maintain high-quality grain production and reduce DON accumulation, research efforts have produced partially resistant cereal crops and identified many quantitative trait loci (QTL) in wheat. However, the underlying mechanisms involved in partial or complete FHB resistance remain unclear. Colonization of plants by pathogenic fungi is governed by the secretion of effector molecules into the host cell. Effectors can be defined as molecules that alter host cell structure and function, facilitating infection and/or triggering defense responses; the molecules include proteins, peptides, and secondary metabolites (toxins). Our overarching goal is to delineate the mechanisms that allow the fungal effectors to promote virulence or induce defense response in the plant. Although great strides have been made in the identification and characterization of effectors in bacteria and oomycetes, a similar statement cannot be said with respect to studying phytopathogenic fungal effectors. My lab has been studying mechanisms underlying the pathogenesis of F. graminearum. We use genetics (CRISPR/Cas9 editing), and Omics platforms (genomics, transcriptomics, proteomics, and metabolomics) to unravel signalling pathways that regulate the biosynthesis of the mycotoxin DON in the pathogen and understand the infection processes in wheat. This proposal will use datasets from various Omics platforms to prioritize candidate effector proteins (CEPs) in F. graminearum that will be used for functional characterization studies. Prioritization will be based on machine learning algorithms, phenotypic studies, and expression patterns in axenic cultures and infected wheat tissues. Next, the selected CEPs (~ 40) will be monitored for secretion into plant cells using the self-assembling split super-folder fluorescent protein system in Arabidopsis. Localization of CEPs will be assessed based on the split Green fluorescent protein marker in various organelles in Arabidopsis. In parallel, the CEPs function will be assessed based on phenotype (defense or disease) using the well-developed type III AvrRpt2/RPS2 translocation/recognition system. Additionally, CEPs will also be functionally validated in their native host (wheat) using the Fusarium-wheat pathosystem, well-established in my lab. The CEPs will be rendered non-functional either individually or in combination using the CRISPR/Cas9 technique and monitored for disease symptoms on wheat. Finally, those CEPs that display phenotype will be used as baits to identify host targets using the proximal-dependent biotin identification (BioID) system.