Botrytis cinerea and Monilinia fructicola are both devastating pathogens that can cause decay in a broad range of hosts during pre and postharvest handling. Despite the current use of chemical fungicides, both fungal pathogens are still responsible of important economic losses worldwide. The knowledge of pathogen virulence mechanisms is an important step to guide the search for new control strategies alternative to the fungicides currently in use. Crucially, the genome sequences of both B. cinerea and M. fructicola can be exploited to identify genes and proteins that determine their ability to infect host plants. By applying molecular and bioinformatic analyses, NECROFUNGI aimed to identify genes that encode effector proteins which can modulate cell death processes in host plants, by either inducing or suppressing cell death. The identification of genes responsible for disease will lead the way towards new strategies to control plant diseases based on the development of new classes of chemicals that are not directly toxic to fungi and to design more efficient breeding strategies. In the case of M. fructicola, the genome was sequenced with PacBio and the de novo assembly resulted in a genome size of 42.95 Mb. After a manual curation supported by RNA-Seq libraries, 10.086 predicted genes were annotated. The genome was examined for the presence of genes that encode secreted proteins and more specifically for effector proteins. A set of 134 putative effectors was identified, which were subject of functional studies. We developed a reproducible infection assay for M. fructicola in nectarine leaves and selected different time points for gene expression analysis. Several candidate effector genes were cloned into Agrobacterium tumefaciens for transient expression in Nicotiana benthamiana plants and some tested candidates triggered necrosis. Two of these effector proteins were produced using Pichia pastoris and also triggered necrosis in N. benthamiana and in tomato leaves. In stone fruit leaves, those effectors were overexpressed using Agrobacterium infiltration carrying TRV virus and both also induced necrosis. In conclusion, we found two effectors that induce plant cell death which can be exploited in the future in effector-based selection of (partially) resistant stone fruit germplasm. In the case of B. cinerea, a transcriptome analysis of the early infection time points of B. cinerea-tomato leaf infection was analysed to determine which genes were highly expressed. The B. cinerea genome revealed an abundance of genes coding for secreted proteins of which 135 could serve as effectors. From this group we selected candidate effectors with high transcript levels in early infection stages, without a known protein domain and without enzymatic activity. By using these criteria a group of 51 effector candidates was obtained. Several of these candidates were tested in a transient expression system for cell death suppressing activity in N. benthamiana (expressing Cf4) in which PCD was triggered by A. tumefaciens containing the Cladosporium fulvum Avr4 gene. From all tested candidates, three were able to suppress the plant cell death induced by Avr4 protein. The role of one of these effector proteins in virulence was tested using a knock-out mutant. Infection assays in tomato leaf indicated that the knockout-mutant was less virulent in some assays, but equally virulent in others. In conclusion, these results support the hypothesis that B. cinerea has a brief biotrophic phase prior to switching to the induction of necrosis but we still do not know the implications in the infection process. The multidisciplinary perspective adopted, combining fruit pathology, comparative genomics, molecular biology and bioinformatics built the necessary information to help in the design of new control approaches for these two devastating pathogens by effector-based technologies that would provide a great economic benefit for EU agriculture.
