From metagenome to gene : Identification of the first Synchytrium endobioticum effector through comparative genomics

Summary

Plant pathogens can have great social and economic impact, and are a continuous threat to food security. This is clearly the case for Synchytrium endobioticum, the species causing potato wart disease. S. endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota (chytrids), which is a basal lineage in the fungal kingdom. The lack of chemical control agents, the impact of the pathogen (complete loss of tuber yields from infected field have been reported), and the production of resting spores that remain viable and infectious in infested soils for decades, led to a quarantine status for S. endobioticum in most countries world-wide. Potato wart disease has been reported from all continents where potato is cultivated, and strict phytosanitary control measures are enforced to prevent the introduction and spread of the pathogen. The use of resistant potato varieties has proven successful in achieving these goals. In chapter 1, over a century of potato wart research is reviewed to place our current knowledge in historic perspective. Observations from light microscopy and electron microscopy studies of S. endobioticum performed in the twentieth century are combined with recent molecular studies. Based on our current knowledge on molecular plant-pathogen interactions, a model is presented to describe the interaction between S. endobioticum and its host in particular with respect to plant resistance. Chapter 2 describes the independent sequencing, assembly and functionally annotation of two S. endobioticum genomes. A comparative genomics approach, in which knowledge acquired from other fungal taxa is exploited, is used to gain insights into genomic features underlying the obligate biotrophic and pathogenic lifestyle of the pathogen. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, and reflects biological differences between the phyla. Moreover, it highlights the surprising commonalities between plant pathogenic fungi that are so evolutionary distinct. In chapter 3, the within-species diversity is determined using mitochondrial genomes, and four major mitochondrial lineages are identified. Furthermore, mitochondrial genomic variation shows that S. endobioticum has likely been introduced into Europe multiple times, and that several pathotypes emerged multiple times. We also demonstrate that isolates represent communities of different genotypes, and that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype. This shift is associated with 266 increased virulence and is likely the result of disruptive selection in the community. Our analysis reveals diversity of S. endobioticum isolates, which is undetected with the currently used bioassays. In chapter 4, by means of an alternative “aboveground” bioassay, it is demonstrated that potato resistance genes are expressed in green aboveground plant organs similar to etiolated “belowground” sprouts. As potato wart resistance is active in both belowground and aboveground organs, the alternative bioassay can potentially speed up screening for S. endobioticum resistance in potato breeding programs as it omits the requirement for tuber formation. In addition, possibilities arise to express S. endobioticum effectors in potato leaves through agroinfiltration, thereby providing additional phenotyping tools for research and breeding. Chapter 5 describes the identification of the S. endobioticum effector AvrSen1, which represents the first effector gene identified in Chytridiomycota. A single dominant gene (Sen1) governs pathotype 1(D1) resistance and we hypothesized that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. Expression of AvrSen1 in Sen1 plants showed a hypersensitive response which co-segregated with Sen1 in potato populations. In non-pathotypes 1(D1) isolates, five different variants resulting in the loss of recognition by the plant were observed suggesting that AvrSen1 is under strong selective pressure. Chapter 6 is a summarizing discussion in which the chapters are integrated, and several aspects linked to pathogenicity and pathotype diversity are further discussed. The impact of the research described in chapters 2 to 5 is specified. Finally, opportunities and challenges for future research on S. endobioticum are presented with a central role for an improved Avr-R gene based pathotyping system, which will contribute to the improvement of durable potato wart resistance breeding and phytosanitary control.