Evolution of a subtilisin-like protease gene family in the grass endophytic fungus Epichloë festucae.

BACKGROUND: Subtilisin-like proteases (SLPs) form a superfamily of enzymes that act to degrade protein substrates. In fungi, SLPs can play either a general nutritive role, or may play specific roles in cell metabolism, or as pathogenicity or virulence factors. RESULTS: Fifteen different genes encoding SLPs were identified in the genome of the grass endophytic fungus Epichloë festucae. Phylogenetic analysis indicated that these SLPs belong to four different subtilisin families: proteinase K, kexin, pyrolysin and subtilisin. The pattern of intron loss and gain is consistent with this phylogeny. E. festucae is exceptional in that it contains two kexin-like genes. Phylogenetic analysis in Hypocreales fungi revealed an extensive history of gene loss and duplication. CONCLUSION: This study provides new insights into the evolution of the SLP superfamily in filamentous fungi.

Patterns of expression of a lolitrem biosynthetic gene in the Epichloë festucae-perennial ryegrass symbiosis.

Lolitrem B is synthesized by Epichloë festucae in associations with Pooid grasses. A complex cluster of at least 10 genes (ltm genes) is required for its synthesis. An early step in this pathway is catalyzed by ltmM, a symbiosis-expressed gene. PltmM-gusA reporter gene analysis was used to monitor ltmM gene expression patterns in planta. The minimum promoter length required for high-level gusA expression in infected seedlings is in the range of 480 to 782 bp. gusA was expressed by the endophyte in all infected vegetative plant tissues and in epiphyllous hyphae. Spikelets from reproductive tillers were analyzed at different developmental stages. During pre-anthesis, gusA expression was observed in all infected floral organs except the immature gynoecium. In post-anthesis florets, gene expression occurred almost exclusively in the gynoecium. Expression of gusA by the endophyte was observed in germinating seeds 24 h postimbibition and seedlings older than 6 days postimbibition in hyphae from the mesocotyl to the tip of the emerging first leaf. This work provides a detailed analysis of the spatial and temporal expression patterns of a symbiosis-expressed gene in planta.

Functional analysis of a beta-1,6-glucanase gene from the grass endophytic fungus Epichloë festucae.

beta-1,6-glucanases degrade the polysaccharide beta-1,6-glucan, a cell wall component in some filamentous fungi. A single copy of a beta-1,6-glucanase gene, designated gcnA, was identified in each of the grass endophytic fungi Neotyphodium lolii and Epichloë festucae. Phylogenetic analysis indicates that the GcnA protein is a member of glycosyl hydrolase family 5, and is closely related to fungal beta-1,6-glucanases implicated in mycoparasitism. The E. festucae gcnA gene was expressed in mycelium grown in culture and in both vegetative and reproductive tissues of perennial ryegrass. A gcnA replacement mutant had reduced beta-1,6-glucanase activity when grown in media containing pustulan as the major carbon source. beta-1,6-glucanase activity was restored in the replacement mutant by introducing multiple copies of the gcnA gene. Growth of DeltagcnA and gcnA-overexpressing strains in vegetative grass tissues was indistinguishable from wild type strains.