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Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three putative knockouts were identified. Southern blot analysis of digested genomic DNA indicated that homologous gene replacement at dmaW2 locus took place while dmaW1 was still present. Chromosome separation followed by Southern-blot hybridization showed that the dmaW genes in N. coenophialum are located on different chromosomes. The aim of this study was to obtain a nontoxic endophyte free of marker genes that could be used to inoculate popular tall fescue cultivars. Therefore the Cre/loxP system developed in this study allows reusing the marker gene for sequential transformations. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum, or Epichloë festucae isolates, containing a floxed hygromycin phosphotransferase (hph) gene (loxP::hph::loxP), were transfected with a Crerecombinase expression plasmid and then cultured without selection. The marker was excised in 0.5-2% of the colonies, leaving a single loxP sequence. This strategy will help to reduce the concerns related to field release or commercialization of economically important grasses associated with manipulated fungal strains. It is expected that the technology will likely be adapted and applied in other fungal species. Manipulation of the ergot alkaloid (EA) gene cluster from C. purpurea and C. fusiformis by introducing and expressing its genes in different fungal-grass symbionts was also investigated. Heterologous expression of the ergot alkaloid cluster could result either in the synthesis of compounds similar to the ones produced by the host or in synthesis of novel compounds with new modes of action. Even though the results indicated that several EA genes were expressed in the new symbiota, none of the ergot alkaloids intermediates were detected. |
