| Ergot alkaloids produced by fungi of family Clavicipitaceae can cause poisoning in humans and livestock. Toxicosis often occurs when livestock graze Neotyphodium spp.-infected grasses. A promising solution to prevent livestock toxicosis is to generate ergot alkaloid non-producing Neotyphodium endophytes by disrupting a gene involved in ergot alkaloid biosynthesis. The biosynthesis of ergot alkaloids involves multiple steps. In the first pathway-specific step, dimethylallyltryptophan synthase (EC: 2.5.1.34) catalyzes the formation of dimethylallyltryptophan from dimethylallyl diphosphate and tryptophan. In a terminal step, a peptide synthetase is involved in ergopeptine production. The gene (dmaW) encoding dimethylallyltryptophan synthase was cloned from Claviceps purpurea (ATCC 20102) by screening a genomic cosmid library using the Claviceps fusiformis dmaW as probe. The coding sequence of C. purpurea dmaW was expressed in yeast, to confirm the identity of this gene by enzyme activity assay. Another gene, encoding a putative peptide synthetase was also cloned from C. purpurea, and was located in the same cosmid clones as the dmaW.; Segments of dmaW homologous sequences were identified by PCR from four endophytic fungal species, Neotyphodium coenophialum , Neotyphodium sp. Lp1 (a hybrid of Neotyphodium lolii and Epichloë typhina), Balansia obtecta, and an endophyte of the grass Achnatherum inebrians . The full-length sequences of dmaW from N. coenophialum, Lp1 and B. obtecta were obtained by cassette-mediated PCR or by screening a cosmid library. The deduced amino acid sequences of these genes shared 60% to 70% identities to those of dmaW from C. purpurea and C. fusiformis . The presence of cDNA in the library from N. coenophialum -infected tall fescue but not in that of N. coenophialum culture suggests that dmaW is specifically expressed in the interaction with its host grass. This is consistent with the ergot alkaloid production profiles whereby N. coenophialum produces alkaloids in planta, but not in culture unless special medium is used. Homologous sequences were also identified by Southern blot and PCR analysis in various, but not all grass endophytes. The existence of dmaW homology in all ergot alkaloid producers supports the hypothesis that dimethylallyltryptophan synthase encoded by dmaW is associated with the biosynthesis of ergot alkaloids. |
