Elimination of ergovaline from a grass-Neotyphodium endophyte symbiosis bygenetic modification of the endophyte

The fungal endophytes Neotyphodium lolii and Neotyphodium sp. Lp1 from pere nnial ryegrass (Lolium perenne), and related endophytes in other grasses, p roduce the ergopeptine toxin ergovaline, among other alkaloids, while also increasing plant fitness and resistance to biotic and abiotic stress. In th e related fungus, Claviceps purpurea, the biosynthesis of ergopeptines requ ires the activities of two peptide synthetases, LPS1 and LPS2. A peptide sy nthetase gene hypothesized to be important for ergopeptine biosynthesis was identified in C purpurea by its clustering with another ergot alkaloid bio synthetic gene, dmaW. Sequence analysis conducted independently of the rese arch presented here indicates that this gene encodes LPS1 [Tudzynski, P., H olter, K., Correia, T., Arntz, C., Grammel, N. & Keller, U. (1999) Mol Gen. Genet. 261, 133-141]. We have cloned a similar peptide synthetase gene fro m Neotyphodium lolii and inactivated it by gene knockout in Neotyphodium sp . Lp1. The resulting strain retained full compatibility with its perennial ryegrass host plant as assessed by immunoblotting of tillers and quantitati ve PCR. However, grass-endophyte associations containing the knockout strai n did not produce detectable quantities of ergovaline as analyzed by HPLC w ith fluorescence detection. Disruption of this gene provides a means to man ipulate the accumulation of ergovaline in endophyte-infected grasses for th e purpose of determining the roles of ergovaline in endophyte-associated tr aits and, potentially, for ameliorating toxicoses in livestock.