Deconstruction of the phytotoxic secretome to decipher the necrotrophic pathogenesis of Botrytis cinerea

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Botrytis cinerea is one of the economically most important plant pathogens worldwide, causing pre- and postharvest grey rot on numerous crops. During infection, the fungus secretes a complex mixture of lytic enzymes and phytotoxic proteins and metabolites, thereby inducing rapid necrosis of host plant cells. To better understand the key infection mechanism of this broad host range necrotroph, we are using a subtractive approach to serially eliminate all plant cell death-inducing proteins (CDIPs) and metabolites, until complete loss of necrosis induction of the resulting mutants. To this end, we have established a highly efficient CRISPR/Cas editing method to generate multiple mutants within short time. Our preliminary work has shown that each of the known CDIPs secreted by B. cinerea contributes only to a minor extent to infection. To fully address the high redundancy of phytotoxic compounds, we want to identify the as yet unknown number of remaining CDIPs and analyse their roles in different stages of necrotrophic infection, by investigating the phenotypes of single and multiple mutants. We are further studying the interaction of CDIPs with their plant receptors or targets. Several fungal CDIPs have been shown to activate pattern recognitions receptors (PRR) at the cell surface, which upon activation induce a basic plant defence called PTI (pattern triggered immunity). Notably, PTI-activation by Botrytis often results eventually in plant cell death, a rather unusual feature of this basic plant defence. Therefore, we want to investigate how Botrytis is using CDIPs to exploit PTI for its own benefit without being stopped by plant defence mechanisms. To identify plant CDIP receptors, screenings will be performed with Arabidopsis PRR mutants and with Arabidopsis and tomato ecotypes that vary in their genetic PRR equipment. Identification of receptor-ligand pairs will allow functional insight into plant cell death induction and might lead to a better understanding of the wide host range of Botrytis. We believe that our proposed project will shed light on a central aspect of microbial pathogenesis, and will pave the way towards a comprehensive understanding of the necrotrophic lifestyle of B. cinerea.