Project Number: WVA00141
CRIS Number: 0181275
TOXIN BIOSYNTHESIS GENES IN ERGOPEPTINE-PRODUCING FUNGI
Investigators: Panaccione, D. G., Schardl, C. L.
Performing Department: Plant & Soil Sciences -- 1825
Start Date: 11/15/1998
Termination Date: 11/30/2001
Reporting period: 11/15/1998 to 11/30/2001
Termination Report:
We have fully met our stated objectives of (1) cloning and characterizing putative ergot alkaloid biosynthesis genes from related fungi in the Clavicipitaceae, including Claviceps and Neotyphodium species, and (2) demonstrating the functions of the putative ergot alkaloid biosynthesis genes. In fulfilling these objectives we have generated stable grass-endophyte associations (symbiota) that are free of the important ergopeptine mycotoxin, ergovaline, and that will be important tools for future applied and basic research. Peptide synthetase genes involved in ergopeptine production (lysergyl peptide synthetases or lpsA) were cloned from Claviceps purpurea (2 genes) and Neotyphodium lolii. Functional analysis was conducted in the N. lolii derivative, Neotyphodium sp. Lp1. Through gene knockout experiments we demonstrated that role of this gene in ergopeptine biosynthesis, as symbiota containing the lpsA knockout strain did not produce ergovaline, but did produce simpler ergot alkaloids, as predicted. The establishment of stable symbioses by the ergovaline-deficient knockout strain was determined qualitatively, by immunoblotting, and quantitatively, by competitive PCR. The data showed that the ergovaline-deficient lpsA knockout strain colonized plants with the same frequency (assessed by artificial inoculation and transmission through tillers) and to the same relative extent (assessed by relative mass fungal DNA/total symbiotum DNA). The data demonstrate that ergovaline is not important in endophyte colonization and in maintenance of the symbiosis (in the absence of applied stress). Characterization of the N. lolii versus C. purpurea lysergyl peptide synthetase genes showed that although they are clearly homologous (based on sequence identity and intron placement), there are important differences near their 5' ends that have significant functional implications. Sequences homologous to lpsA were found in all known ergopeptine producing fungi but not in related fungi that lack the ability to produce ergopeptines. We have cloned dimethylallyltryptophan synthase (dmaW) genes from C. purpurea (2 genes), Claviceps fusiformis, Balansia obtecta, Neotyphodium sp. Lp1, and N. coenophialum (2 genes). The function of C. purpurea, C. fusiformis, and B. obtecta genes was demonstrated by expression in yeast. The function of the Neotyphodium sp. Lp1 dmaW was demonstrated by gene knockout and subsequent introduction into perennial ryegrass to produce ergovaline-free symbiota. DNA sequence analysis showed that the dmaW genes were homologues and had no significant similarity to other prenyl transferases, indicating that dmaW represents a new family of prenyl transferases. In Southern hybridization analyses, only members of the Clavicipitaceae that produce ergot alkaloids contained homologous sequences. Our results confirm the identity of the ergot alkaloid biosynthesis genes and establish that ergovaline can be eliminated from a Neotyphodium-grass symbiosis by genetic modification of the endophyte at two different steps in the pathway.
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.
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.
Panaccione, D.G., and Schardl, C.L. 200X (in press). Molecular genetics of ergot alkaloid biosynthesis. In The Clavicipitalean Fungi: Evolutionary Biology, Chemistry, Biocontrol, and Cultural Impacts, Bacon, C., Hywel-Jones, N., Spatafora, J., and White, J.F., Jr., (eds.). Marcel-Dekker, NY.
Panaccione, D.G., Johnson, R.D., Rasmussen, J.B., and Friesen, T.L. 200X (in press). Fungal phytotoxins. In The Mycota, vol. XI, Kempken, F. (Ed.), Springer-Verlag, Berlin-Heidelburg.
Impact:
Neotyphodium spp. that grow endophytically in grasses provide their plant hosts with important fitness enhancements. However, the animal toxicoses associated with these endophytes is a substantial agricultural problem in the U.S., and is thought primarily to be due to the mycotoxin, ergovaline. We have generated ergovaline-deficient endophyte strains and grass-endophyte associations that may be used in direct tests of the role of ergovaline in mammalian toxicosis and in several endophyte-associated beneficial traits. If these tests indicate that ergovaline is the main agent of livestock toxicosis but is unnecessary for important fitness enhancements, such genetically modified endophytes may be useful for the improvement of forage grasses.