Periodic Reporting for period 1 - NECROFUNGI (The role of necrotrophic effectors in the ability of Botrytis and Monilinia species to infect host plants) Reporting period: 2018-03-01 to 2020-02-29 Summary of the context and overall objectives of the project 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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Main scientific and technical resultsGenome sequence and annotation of the stone fruit pathogen M. fructicola using PacBio technology. The annotation was manually curated in order to discard pseudogenes and repetitive regions. Moreover, we also obtained the group of proteins that encompass the secretome and the effectorome.Development of a reproducible infection assay for M. fructicola in stone fruit leaves to avoid heterogenous results due to fruit ripening.Discovery of two M. fructicola effector genes that induce cell death in host and non-host plants using different methodologies. Discovery of three B. cinerea effectors that are able to suppress the plant cell death induced by Avr4 protein. 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 use of CRISPR-Cas9 technique to generate transformants in B. cinerea and M. fructicola. The use of TRV virus to overexpress proteins in plants that possess difficulties to be infiltrated as a high throughput methodology to screen cell death inducing effectors. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Progress beyond the state of the artThe first main contribution to the state of the art was to sequence the genome of M. fructicola and to describe the transcriptome from M. fructicola-infected stone fruit leaves. Using both data we found the group of proteins that are part of the secretome and effectorome. To our knowledge there are no studies about this for M. fructicola neither from other postharvest pathogens (except B. cinerea). This data offers new research opportunities to explore the activity of those effectors in virulence of M. fructicola. Moreover, we found 3 effector proteins that induce cell death in both host and non-host plants. These effectors can influence M. fructicola virulence and can be used to screen stone fruit germplasm to find resistant/susceptible varieties in early stages of development. These results are very innovative and strongly contribute to the state of the art in the study of virulence factors in M. fructicola and can be extrapolated to the use of other postharvest diseases.We demonstrated that B. cinerea has a short early biotrophic phase before switching to necrosis. We have 3 effectors that are able to suppress plant cell death during the early infection (8-12 hpi). There are several studies about effectors that induce cell death in B. cinerea and its relation with virulence however, this is the first evidence that a necrotrophic pathogen can have a biotrophic lifestyle during the early phases of the infection. This theory will reconsider the lifestyle of B. cinerea and will open new research approaches.
