| Summary: | The endophytic fungus <i>Epichloë festucae</i> is known to produce bioactive metabolites, which consequently protect the host plants from biotic and abiotic stresses. We previously found that the overexpression of <i>vibA</i> (a gene for transcription factor) in <i>E. festucae</i> strain E437 resulted in the secretion of an unknown fungicide. In the present study, the active substance was purified and chemically identified as ε-poly-L-lysine (ε-PL), which consisted of 28−34 lysine units. The productivity was 3.7-fold compared with that of the wild type strain E437. The isolated ε-PL showed inhibitory activity against the spore germination of the plant pathogens <i>Drechslera erythrospila</i>, <i>Botrytis cinerea</i>, and <i>Phytophthora infestans</i> at 1−10 μg/mL. We also isolated the fungal gene “<i>epls</i>” encoding ε-PL synthetase Epls. Overexpression of <i>epls</i> in the wild type strain E437 resulted in the enhanced production of ε-PL by 6.7-fold. Interestingly, overexpression of <i>epls</i> in the different strain <i>E. festucae</i> Fl1 resulted in the production of shorter ε-PL with 8−20 lysine, which exhibited a comparable antifungal activity to the longer one. The results demonstrate the first example of ε-PL synthetase gene from the eukaryotic genomes and suggest the potential of enhanced expression of <i>vibA</i> or/and <i>epls</i> genes in the <i>Epichloë</i> endophyte for constructing pest-tolerant plants.
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