Research Accomplishment Reports 2007

Genetics and Biochemistry of Alkaloid Production by Endophytes

C.L. Schardl
Department of Plant Pathology

 

Project Description

Ergot alkaloids and loline alkaloids are produced by symbiotic fungi (endophytes) in grasses. In the common forage grass, tall fescue, ergot alkaloids cause livestock toxicoses, whereas lolines help protect the plants and improve stand persistence. The ability of the tall fescue endophyte to produce ergot alkaloids will be eliminated by engineering the inactivation of two genes. Meanwhile, the pathway and associated genes for producing loline alkaloids will be elucidated.

Objective 1. Characterize and knock out the genes for the first determinant step in ergot alkaloid production by the tall fescue endophyte, Neotyphodium coenophialum: the tall fescue endophyte, N. coenophialum, is an interspecies hybrid with two copies of DMAW, the gene for the first pathway-specific step in ergot alkaloid biosynthesis. These copies are designated DMAW1 and DMAW2. Three transformants were identified (out of 1600 analyzed) with the wild type DMAW2 replaced by a deletion mutant containing a hygromycin resistance gene (HPH) modified to allow deletion by the CRE recombinase. Expression of CRE in one of these mutants deleted the HPH marker, to permit a knockout of DMAW1 by the same strategy. A plasmid construct was designed and constructed for DMAW1 knockout, and several hundred transformants with this plasmid are currently undergoing analysis. Once DMAW1 has been eliminated, it is unlikely that the endophyte will produce ergot alkaloids. Tests of the agronomic potential of this modified endophyte will follow.

Objective 2. Elucidate the pathway and genes for biosynthesis of the exo-1-aminopyrrolizidine alkaloid, n-formylloline, an anti-insect endophyte alkaloid: a cluster of nine protein-coding genes was identified in loline-producing endophytes, but was absent in endophytes that lack lolines. Experiments in Neotyphodium uncinatum, an endophyte of meadow fescue (a close relative of tall fescue and the ryegrasses) indicated that this cluster, designated lol, is likely to be required for loline alkaloid biosynthesis. Expression of the lol genes was coordinated with production of loline alkaloids, and when expression of one of them (lolc) was reduced, loline production was reduced. Another of the lol genes, lolp, was demonstrated to encode a p450-type monooxygenase responsible for conversion of n-methylloline (nml) to the more insecticidal n-formylloline. Studies of the biosynthetic pathway indicated that the first step is condensation l-proline with l-homoserine in an unprecedented gamma-substitution reaction. This is followed by two decarboxylations and formation of the second ring to give a saturated exo-1-aminopyrrolizidine. Finally, an oxygen bridge is formed to give the core loline alkaloid structure. Decorations of the 1-amine with methyl, acetyl or formyl groups gives the various loline alkaloids found in tall fescue and meadow fescue with their respective endophytes. Thus, this study revealed several novel mechanisms and genes for natural product biosynthesis.

Impact

Forage grasses with endophytes in which ergot alkaloid production has been eliminated should be highly beneficial to livestock farmers because the endophytes enhance stand longevity and reduce maintenance costs and labor. Eliminating ergot alkaloid production is expected to eliminate tall fescue toxicosis to livestock that graze on endophyte-infected tall fescue. Even with toxic endophytes, tall fescue is an extremely popular forage and pasture grass and hay crop in a large part of the U.S., and fescue toxicosis causes close to $1 billion annual loss in livestock productivity in this country.

The expected outcome of this research is to eliminate fescue toxicosis. Although ergot alkaloids are undesirable in a pasture grass, another class of endophyte alkaloids called lolines are desirable for their specific antiinsect activities. Control of expression of loline alkaloids should further enhance the agricultural benefits of the grass endophytes. Furthermore, loline alkaloid production genes may eventually be introduced into forage, fiber, ornamental or amenity plants, for enhanced resistance to insects.

Publications

Blankenship, J.D., Houseknecht, J.B., Pal, S., Bush, L.P., Grossman, R.B. and Schardl, C.L. 2005. Biosynthetic precursors of fungal pyrrolizidines, the loline alkaloids. Chembiochem 6:1016-1022.

Clay, K. and Schardl, C. 2002. Evolutionary origins and ecological consequences of endophyte symbiosis with grasses. American Naturalist 160:S99-S127.

Craven, K.D. and Schardl, C.L. 2003. Tall fescue - systematics and morphologyin Tall fescue information system: Tall fescue book on-line (H. A. Fribourg, ed.) Oregon State University, Corvallis, Oregon.

Damrongkool, P., Sedlock, A.B., Young, C.A., Johnson, R.D., Goetz, K.E., Scott, B., Schardl, C.L. and Panaccione, D.G. 2005. Structural analysis of a peptide synthetase gene required for ergopeptine production in the endophytic fungus Neotyphodium lolii. DNA Sequence 16:379-385.

Florea, S., Machado, C., Zhang, D., Panaccione, D.G. and Schardl, C.L. 2007. Towards the elimination of ergot alkaloid biosynthesis genes in Neotyphodium coenophialum. Pages 477-479 in New Zealand Grassland Association: Endophyte Symposium (A. Popay, and E. R. Thom, eds.). New Zealand Grassland Association, Christchurch, New Zealand.

Haarmann, T., Machado, C., Lubbe, Y., Correia, T., Schardl, C.L., Panaccione, D.G. and Tudzynski, P. 2005. The ergot alkaloid gene cluster in Claviceps purpurea: Extension of the cluster sequence and intra species evolution. Phytochemistry 66:1312-1320.

Hesse, U., Maynard, P., Macmil, S., Wiley, G., Andreeva, K., Beech, W.E., Arnaoudova, E., Willey, B.T., Puram, V.-G., Wiseman, J., Webb, J., Gill, L., Farman, M.L., Jaromczyk, J.W., Roe, B.A. and Schardl, C.L. 2007. The genome and the genes of Epichloe festucae. Pages 461-465 in New Zealand Grassland Association: Endophyte Symposium (A. Popay, and E. R. Thom, eds.). New Zealand Grassland Association, Christchurch, New Zealand.

Johnson, L.J., Johnson, R.D., Schardl, C.L. and Panaccione, D.G. 2003. Identification of differentially expressed genes in the mutualistic association of tall fescue with Neotyphodium coenophialum. Physiological and Molecular Plant Pathology 63:305-317.

Kutil, B.L., Greenwald, C., Liu, G., Spiering, M.J., Schardl, C.L. and Wilkinson, H.H. 2007. Comparison of loline alkaloid gene clusters across fungal endophytes: Predicting the co-regulatory sequence motifs and the evolutionary history. Fungal Genetics and Biology 44:1002-1010.

Lorenz, N., Wilson, E.V., Machado, C., Schardl, C.L. and Tudzynski, P. 2007. Comparison of ergot alkaloid biosynthesis gene clusters in Claviceps species indicates loss of late pathway steps in evolution of C. fusiformis. Applied and Environmental Microbiology 73:7185-7191.

Moon, C.D., Craven, K.D., Leuchtmann, A., Clement, S.L. and Schardl, C.L. 2004. Prevalence of interspecific hybrids amongst asexual fungal endophytes of grasses. Molecular Ecology 13:1455-1467.

Moon, C.D., Miles, C.O., Jarlfors, U. and Schardl, C.L. 2002. The evolutionary origins of three new Neotyphodium endophyte species from grasses indigenous to the Southern Hemisphere. Mycologia 94:694-711.

Panaccione, D.G. and Schardl, C.L. 2003. Molecular genetics of ergot alkaloid biosynthesis. Pages 399-424 in Clavicipitalean fungi: evolutionary biology, chemistry, biocontrol and cultural impacts (J. F. White, Jr., C. W. Bacon, N. L. Hywel-Jones, and J. W. Spatafora, eds.). Marcel-Dekker, Inc., New York and Basel.

Potter, D.A., Stokes, J.T., Redmond, C.T., Schardl, C.L. and Panaccione, D.G. 2008. Contribution of ergot alkaloids to suppression of a grass-feeding caterpillar assessed with gene-knockout endophytes in perennial ryegrass. Entomologia Experimentalis et Applicata 126:138-147.

Schardl, C.L. 2002. Epichloe festucae and related mutualistic symbionts of grasses (vol 33, pg 69, 2001). Fungal Genetics and Biology 35:79.

Schardl, C.L., Blankenship, J.D., Machado, C. and Spiering, M.J. 2002. Web essay: alkaloid-making fungal symbiontsin Plant Physiology (L. Taiz, and E. Zeiger, eds.). Sinauer Associates, Sunderland, Massachusetts.

Schardl, C.L., Blankenship, J.D., Spiering, M.J. and Machado, C. 2004. Loline and ergot alkaloids in grass endophytes. Pages 427-448 in Handbook of Industrial Mycology (Z. An, ed.) Marcel Dekker, Inc., New York & Basel.

Schardl, C.L., Grossman, R.B., Blankenship, J.D., Spiering, M.J. and Faulkner, J.R. 2007. Biosynthesis of loline alkaloids by epichloe endophytes of grasses. Pages 482 in New Zealand Grassland Association: Endophyte Symposium (A A. Popay, and E. R. Thom, eds.). New Zealand Grassland Association, Christchurch, New Zealand.

Schardl, C.L., Grossman, R.B., Nagabhyru, P., Faulkner, J.R. and Mallik, U.P. 2007. Loline alkaloids: currencies of mutualism. Phytochemistry 68:980-996.

Schardl, C.L. and Leuchtmann, A. 2005. The epichloe endophytes of grasses and the symbiotic continuum. Pages 475-503 in The Fungal Community: its organization and role in the ecosystem (J. Dighton, J. F. White, and P. Oudemans, eds.). CRC Press, Boca Raton, Florida. Schardl, C.L., Leuchtmann, A. and Spiering, M.J. 2004. Symbioses of grasses with seedborne fungal endophytes. Annual Review of Plant Biology 55:315-340.

Schardl, C.L. and Moon, C.D. 2003. Processes of species evolution in Epichloe/ Neotyphodium endophytes of grasses. Pages 273-310 in Clavicipitalean fungi: evolutionary biology, chemistry, biocontrol and cultural impacts (J. F. White, Jr., C. W. Bacon, N. L. Hywel-Jones, and J. W. Spatafora, eds.). Marcel-Dekker, Inc., New York and Basel.

Schardl, C.L., Panaccione, D.G., Potter, D.A., Florea, S., Hesse, U., Konnova, E., Liu, M., Machado, C. and Stokes, J.T. 2007. Genetic tests of ecological roles of ergot alkaloids produced by an epichloe endophyte of ryegrass. Pages 172 in New Zealand Grassland Association: Endophyte Symposium (A. Popay, and E. R. Thom, eds.). New Zealand Grassland Association, Christchurch, New Zealand.

Schardl, C.L., Panaccione, D.G. and Tudzynski, P. 2006. Ergot alkaloids--biology and molecular biology. Alkaloids: Chemistry and Biology 63:45-86.

Spiering, M.J., Moon, C.D., Wilkinson, H.H. and Schardl, C.L. 2005. Gene clusters for insecticidal loline alkaloids in the grass-endophytic fungus Neotyphodium uncinatum. Genetics 169:1403-1414.

Spiering, M.J., Wilkinson, H.H., Blankenship, J.D. and Schardl, C.L. 2002. Expressed sequence tags and genes associated with loline alkaloid expression by the fungal endophyte Neotyphodium uncinatum. Fungal Genetics and Biology 36:242-254.

Sullivan, T.J., Rodstrom, J., Vandop, J., Librizzi, J., Graham, C., Schardl, C.L. and Bultman, T.L. 2007. Symbiont-mediated changes in Lolium arundinaceum inducible defenses: evidence from changes in gene expression and leaf composition. New Phytologist 176:673-679.

Wang, J., Machado, C., Panaccione, D.G., Tsai, H.-F. and Schardl, C.L. 2004. The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass. Fungal Genetics and Biology 41:189-198.