Project Number: WVA00360
CRIS Number: 0160597
MANIPULATION OF ERGOT ALKALOID PRODUCTION IN THE ENDOPHYTE OF TALL FESCUE
Investigators: Panaccione, D.
Performing Department: Plant & Soil Sciences -- 1825
Start Date: 10/23/1997
Termination Date: 09/30/2002
Reporting period: 01/01/2001 to 12/31/2001
Progress Report:
Research toward the primary objective (manipulation of ergovaline content of grass-endophyte associations by peptide synthetase gene knockout) yielded positive results. Data summarized under another report (WVA00141) demonstrate that knockout of lysergyl peptide synthetase or dimethylallyltryptophan synthase genes in Neotyphodium sp. isolate Lp1 blocks the ergot alkaloid biosynthesis pathway and eliminates ergovaline from perennial ryegrass symbiota containing these modified endophytes. Based on the success of the lysergyl peptide synthetase knockout in Neotyphodium sp. Lp1, a strategy was designed for knocking out the homologous gene in the economically important tall fescue endophyte, Neotyphodium coenophialum. The end product of this strategy will be an ergovaline-deficient strain of N. coenophialum that contains no foreign DNA. Based on DNA sequence data obtained from the Neotyphodium lolii version of the ergovaline-associated lysergyl peptide synthetase gene, oligonucleotides were designed to prime amplification of critical internal regions of the peptide synthetase gene and to introduced translation termination codons. The primers were used to successfully amplify modified internal fragments from the N. coenophialum lysergyl peptide synthetase gene. The specifically modified N. coenophialum fragments are being introduced in to a transformation construct containing selectable markers and a reporter gene that will be important in the genetic engineering process but that will be deleted by a designed recombination event, resulting in the removal of all foreign DNA from the fungus. The second objective is to investigate the feasibility of reducing or eliminating ergovaline from grass-endophyte associations by an alternate strategy -- the introduction of a gene for ergopeptine metabolism into endophytes. Progress described previously indicated that spores of a particular imperfect fungus eliminated the ergopeptine ergotamine from solution. Our results showed that ergovaline is bound by spores of the fungus rather than metabolized. Surprisingly, binding is not prevented by prior treatment of the spores with high temperatures or proteinase K. In fact, the only treatment among several tested that prevented binding was incubation with the strong cationic detergent sodium dodecylsulfate. These findings indicate that there is something beyond a simple spore-surface protein of this particular species that binds the ergopeptine and, thus, it is unlikely that the binding agent is a primary gene product. Since this trait is likely to be controlled by more than one gene, it is not a good candidate for transfer to ergopeptine-producing endophytes by transformation. Based on these findings and the success of the lysergyl peptide synthetase knockout strategy, future emphases will be placed on the knockout strategy as a means to reduce or eliminate ergovaline from grass-endophyte symbiota.
Publications:
Panaccione, D.G., Johnson, R.D., Wang, J., Young, C.A., Damrongkool, P., Scott, B. and Schardl. C.L. 2001. Elimination of ergovaline from a grass-Neotyphodium endophyte symbiosis by genetic modification of the endophyte. Proceeding of the National Academy of Science, USA 98:12820-12825.
Panaccione, D.G., and Annis, S.L. 2001. Significance of fungal peptide secondary metabolites in the agri-food industry. In Applied Biology and Biotechnology: Food and Fungi, vol. I., Khachatourians, G.G., and Arora, D.K. (Eds.) Elsevier, The Netherlands, pp. 115-143.
Panaccione, D.G., Sheets, N.L., Miller, S.P., and Cumming, J.R. 2001. Diversity of Cenococcum geophilum populations from serpentine and non-serpentine soil. Mycologia 93:645-652.
Cumming, J.R., Swiger, T.D., Kurnik, B.S., and Panaccione, D.G. 2001. Organic acid exudation by Laccaria bicolor and Pisolithus tinctorius exposed to aluminum in vitro. Canadian Journal of Forest Research 31:703-710.
Johnson, R.D., Damrongkool, P., Schardl. C.L., Panaccione, D.G. 2001. Analysis of an ergovaline-deficient Neotyphodium endophyte resulting from a peptide synthetase gene knockout. Fungal Genetics Newsletter 48A:#473.
Johnson, L.J., Johnson, R.D., Schardl, C.L., and Panaccione, D.G. 2001. Identification of differentially expressed genes involved in the symbiosis between Neotyphodium coenophialum and tall fescue by suppression subtractive hybridization analysis. Fungal Genetics Newsletter 48A:#279.
Impact:
This research is leading to the identification and characterization of mechanisms for eliminating ergovaline from economically important grass-endophyte associations. Elimination of ergovaline in forage grasses is hypothesized to improve livestock performance.