Finding novel virulence factors by an epigenetics approach

Host-pathogen interactions constitute fine-tuned interplays where each of the participating organism has evolved efficient strategies to win over the other. Here, epigenetic variations of pathogenesis (pathogen) and resistance (host) genes likely contribute to the phenotypic diversity and pathogen/host adaptation, and thus, may explain how both organisms rapidly and permanently adapt to these challenging conditions. Epigenetic mechanisms include histone posttranscriptional modifications (HPTMs) that do not alter the DNA sequence, but affect the read out thereof. Depending on the kind of HPTM the underlying genes are either turned on or off, suggesting that the fungal epigenetic landscape is a crucial determinant for successful host invasion. Only a limited number of studies have so far looked into this aspect of fungal-host interactions and they have found that many epigenetic mutants are affected in virulence. Study of these strains has proven to be a powerful tool to reveal novel virulence-associated genes In the proposed research project, entitled Finding novel virulence factors by using an epigenetics approach, we will focus on the so far not well characterised interaction of Fusarium fujikuroi with its host plant rice (Oryza sativa). F. fujikuroi is the causal agent of bakanae disease (chlorosis and hyperelongation) on rice resulting in huge crop losses. Some - but not all - symptoms of the disease are related to the production of the plant hormone gibberellic acid (GA) by the pathogen. Not much is known, however, regarding GA-independent virulence factors in this pathosystem. F. fujikuroi has at least 46 additional natural products, suggesting that others might also contribute to the fungal virulence. Comparison of mutant strains with an altered HPTM profile as well as affected in virulence and non-mutated strains will likely reveal genes that normally respond to the epigenetic change conveyed by the respective HPTM during the infection. Genome-wide analysis of gene expression in mutants affected in virulence will be performed during the infection and compared to the wild type. Genes associated with the infection are likely to be among the set of differentially regulated genes, and thus, will likely enable identification of virulence-associated genes. To directly correlate the changes in the gene regulation with the chromatin status we will employ Chromatin immunoprecipitation (ChIP) of the fungal chromatin during the pathogenic growth (in planta ChIP), thereby shedding light on the epigenetic mechanisms that take place during the fungal-plant interaction. A small set of target genes that show this significant correlation between changes in transcriptional activity and epigenetic marks will be chosen for a more detailed functional characterisation. This project will reveal new basic information on the epigenetic regulatory level of the fungal pathogen interaction with a host plant.

The majority of plant pathogens are fungi and many of these adversely affect food security. Decrypting what controls pathogenic development and mycotoxin production is key towards knowledge-driven strategies to combat plant infections and minimize mycotoxin contaminations. Plant-pathogen interactions constitute fine-tuned interplays where each of the participating organism has evolved efficient strategies to win over the other. Both organisms are in need to adapt to these challenging conditions quickly. Here, regulation of gene expression by chromatin-based mechanisms (e.g. posttranslational modifications (PTMs) of histones) enables a swift induction of virulence-associated genes during infection. Histone PTMs do not alter the DNA sequence, but affect the read out thereof. Depending on the histone PTM, the underlying genes are either turned on or off. This suggests that the fungal chromatin landscape (genome-wide distribution of histone PTMs) is a crucial determinant for a successful infection. Not surprisingly, fungal strains defective in writing, reading or erasing certain histone PTMs ("chromatin mutants") are often defective in fungal development, mycotoxin production and virulence. Characterization of such "chromatin mutants" constitutes a powerful tool to reveal gene networks associated with mycotoxin production and/or virulence. The overall goal of EPIVIRULENCE was to characterize the relevance of histone PTMs on the interaction between Fusarium fujikuroi and rice (Oryza sativa). Plants infected with this fungus show hyperelongated, slender and chlorotic internodes ("bakanae" disease) resulting in huge annual economic losses. The symptoms are related to gibberellins (GA), phytohormones produced by the fungus during the infection. Not much is known regarding GA-independent virulence factors in this pathosystem. To determine histone PTMs crucial for the infection, overall 26 "chromatin mutants" were characterized and the contribution to fungal virulence was evaluated. The most pronounced phenotypes were detected in strains that have lost histone acetylation and histone 3 lysine 4 methylation (H3K4me). Strains with lost H3K4me were defective in fungal development, showed an altered chemical profile and were strongly attenuated in virulence. Notably, the latter is only partially due to decreased biosynthesis of GA. To identify, GA-unrelated virulence factors, transcriptomes of strains with low and high H3K4me levels were recorded during the infection and compared to the wild-type infection. Surprisingly, only a small subset of genes was regulated antagonistically in these strains. For a selection of them the contribution to virulence was investigated within the frame of this project. As human populations increase while arable land area decreases limited food resources will become a global challenge in the near future. Finding sustainable ways to limit fungal infections and food spoilage due to mycotoxins remains a key challenge. The identification of histone PTMs as crucial determinants for infection and/or mycotoxin production, and finding additional virulence-associated genes may have a high impact on integrative management strategies in this struggle.