Recent studies have identified key genes that control the symbiotic interaction between Epichloë festucae and Lolium perenne. Here we report on the identification of specific E. festucae genes that control host infection. Deletion of setB, which encodes a homologue of the H3K36 histone methyltransferase Set2/KMT3, reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae ΔclrD mutant, which lacks the gene encoding the homologue of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect L. perenne. Alleles that complement the culture and plant phenotypes of both mutants also complement the histone methylation defects. Coinoculation of either ΔsetB or ΔclrD with the wildtype strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae.

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network, and also grows as an epiphyte. However, little is known about the cell wall remodelling mechanisms required to avoid host defence and maintain intercalary growth within the host. Here we use a suite of molecular probes to show that the E. festucae cell wall is remodelled by conversion of chitin to chitosan during infection of L. perenne seedlings as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth the cell wall is remodelled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalysed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption of fungal intercalary growth in the intercellular spaces of plants infected with these mutants. These results establish that these two genes are required for maintenance of the mutualistic symbiotic interaction between E. festucae and L. perenne.

Recent studies have identified key genes in Epichloë festucae that control the symbiotic interaction of this filamentous fungus with its grass host. Here we report on the identification of specific fungal genes that determine its ability to infect and colonize the host. Deletion of setB, which encodes a homolog of the H3K36 histone methyltransferase Set2/KMT3, specifically reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae ΔclrD mutant, which lacks the gene encoding the homolog of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect the host grass, and mutational studies of key residues in the catalytic SET domains from these proteins show that these phenotypes are dependent on the methyltransferase activities of SetB and ClrD. A comparison of the differences in the host transcriptome between seedlings inoculated with wild-type versus mutants suggests that the inability of these mutants to infect the host was not due to an aberrant host defense response. Co-inoculation of either ΔsetB or ΔclrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild-type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae.Author SummaryThe filamentous fungus Epichloë festucae is an endophyte that forms highly regulated symbiotic interactions with the perennial ryegrass. Proper maintenance of such interactions is known to involve several signalling pathways, but much less is understood about the infection capability of this fungus in the host. In this study, we uncovered two epigenetic marks and their respective histone methyltransferases that are required for E. festucae to infect perennial ryegrass. Null mutants of the histone H3 lysine 9 and lysine 36 methyltransferases are completely defective in colonizing the host intercellular space, and these defects are dependent on the methyltransferase activities of these enzymes. Importantly, we observed no evidence for increased host defense response to these mutants that can account for their non-infection. Rather, these infection defects can be rescued by the wild-type strain in co-inoculation experiments, suggesting that failure of the mutants to infect is due to altered expression of genes encoding infection factors that are under the control of the above epigenetic marks that can be supplied by the wild-type strain. Among genes differentially expressed in the mutants at the early infection stage is a putative small-secreted protein with a carbohydrate binding function, which deletion in E. festucae severely reduced infection efficiency.