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Genetic Analysis of Biosynthesis and Roles of Anti-Herbivore Alkaloids Produced by Grass Endophytes

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Genetic Analysis of Biosynthesis and Roles of Anti-Herbivore Alkaloids Produced by Grass Endophytes 118 Genetic Analysis of Biosynthesis and Roles of Anti-Herbivore Alkaloids Produced by Grass Endophytes C. L. Schardl, J. Wang, H. H. Wilkinson and K.-R. Chung Abstract A characteristic of seed-borne clavicipitaceous symbionts (endophytes) that mediates their mutualisms with host grasses is production of anti-herbivore metabolites. Ergot alkaloids and indolediterpenes are potent neurotoxins in vertebrates ; saturated 1- aminopyrrolizidines (lolines) and the pyrrolopyrazine alkaloid, peramine, are active against insects. All except lolines are reported to be produced in cultures of fungal endophytes free of plant material. We identified lolines in defined-medium cultures of Neotyphodium un- cinatum. We have also developed Epichloe festucae and Epichloe typhina as Mendelian genetic models to test the effects on aphids of lolines and peramine, respectively. In each case, the phenotypic difference of expression or non-expression was apparently governed by a single locus. Genotypes of E. typhina expressing peramine caused killing of greenbug aphid (Schizaphis graminum) on the host plants. Lolines were associated with killing of both greenbug and bird-cherry oat aphid (Rhopalosiphum padi). Statistically, the anti-aphid activities of the endophytes were entirely attributable to their alkaloids. Recent progress on genetic control of ergot alkaloid and indolediterpene expression holds promise for analogous tests for roles of these alkaloids in host benefits. Mendelian segregation and molecular knockouts can be used eventually to test the ecological importance of all known endophyte alkaloids in the many established endophyte effects, including increased drought tolerance, competitiveness, resistance to nematodes, and resistance to vertebrate and insect herbivores. Genetic knockouts in endophytes of genes for anti-vertebrate alkaloids will likely become an integral part of forage cultivar development. Key words : Neotyphodium, Epichloe, symbiosis, bioprotection, genetics. Introduction Endophytes of the closely related fungal genera Epichloe and Neotyphodium are com- monly symbiotic with many cool season grasses (Poaceae subfam. Pooideae), to which they provide protection from a variety of stresses. These mutualistic associations are bio- chemically diverse, though these endophytes belong to a group of related genera within the fungal family Clavicipitaceae (order Hypocreales). The occurrence of ergot alkaloids and indolediterpenes (tremorgens) is widespread within the family, and known also among distantly related ascomycetes. Ergot alkaloids even occur in a family of green plants, the Convovulaceae. In contrast, the pyrrolopyrazine, peramine, and a subgroup of pyrrolizidine alkaloids, the lolines (saturated 1-aminopyrrolizidines with an oxygen bridge) are known Department of Plant Pathology, S-305 ASN, University of Kentucky, Lexington, KY 40546-0091, U.S.A. fax: 1-606-323-1961 119 almost exclusively from Neotyphodium and Epichloe species. All of these alkaloid groups have potent anti-herbivore activities. Ergot alkaloids and indolediterpenes are known anti-mammalian neurotoxins. Lolines are neurotoxic to insects and peramine is potent insect feeding deterrent. This paper reviews recent progress on the biochemistry and genetics of expression of lolines and peramine, and on key genes for the biosynthesis of ergot alkaloids and indolediterpenes. Lolines Saturated aminopyrrolizidines are known almost exclusively from grass-endophyte symbiota"2'. The loline alkaloids are most likely derived from either spermine or sper- midine (Fig. 1). Likely enzymatic steps from spermine to norloline are (1) polyamine oxidase, releasing 1,3-diaminobutane and aminopropylpyrroline ; (2) cyclization of the latter to yield a 1-amino nescine ring structure;(3) oxidation to form the tricyclic norloline. The more common loline alkaloids have elaborations on the primary amine of norloline (Fig. 1). The plant apoplast, the natural environment of Neotyphodium species , is thought to be poor in nutrients, particular nitrogen3'. Therefore, in preliminary tests we grew Neoty- phodium uncinatum in a rich medium to increase fungal biomass, then transferred to minimal medium still cultures4' without added nitrogen. After several weeks N-formylloline began accumulating. New cultures were set up similarly and provided either no nitrogen or various nitrogen sources. Remarkably, including the preferred nitrogen source, asparagine, Fig. 1 Possible biosynthetic pathways for saturated 1-aminopyrrolizidine (loline) alkaloids. 120 Fig. 2 N-Formylloline levels in 7-week old minimal medium still cultures with Fig. 3 Expression time course of the loline different amine sources. Open bars alkaloid, N-formylloline, in minimal indicate where poor nitrogen sources medium still cultures with no added were provided, and shaded bars indi- nitrogen, or with asparagine or cate good nitrogen sources. The glutamate as the nitrogen source. identity of N-formylloline was The identity of N-formylloline was confirmed by gas chromatography- confirmed as in Fig. 2. mass spectrometry as described by Petroski et al. resulted in the highest loline alkaloid levels (Fig. 2). In a time course with no nitrogen or with asparagine or glutamate as a nitrogen source, the fungus began producing detectable lolines after one week (Fig. 3). The fungal biomass did not increase, and loline levels peaked in 4-6 weeks. The newly described species, Epichloe festucaes' has the necessary diversity in alkaloid profiles for Mendelian genetic studies of the alkaloids' protective roles. Giant fescue (Lolium giganteum) with its common E. festucae symbiont typically possesses loline alka- loids, and this is the only association with E. festucae currently known to do so. A mating of the giant fescue symbiont with a red fescue (Festuca rubra) symbiont was conducted, and the F, progeny were introduced into plants of meadow fescue (Lolium pratense). The resulting symbiota were tested by gas chromatography (GC) for N-formylloline and N- acetylloline. The F, generation segregated for loline alkaloid expression with a ratio very close to 1 : 1. This result supported the hypothesis of a single genetic locus controlling loline alkaloid expression, since a 1 : 1 segregation ratio is expected for a single gene in a haploid organism. Subsequent tests entailed backcrosses (BCC and BC2 generations), and other crosses (F2) between loline producing and nonproducing progeny. The results consistently supported the single locus model. The Mendelian genetic system was useful for testing the hypothesis that loline alkaloids provide protection of the host plant against insect herbivores. We conducted tests with two species of aphids, greenbug (Schizaphis graminum) and bird-cherry oat aphid (Rhopalosiphum padi). These were no-choice tests with 25 juvenile aphids introduced into small cages with three tillers of each plant, and left to feed and reproduce for 3 days. The lolines-producing endophytes provided significantly greater protection from both aphid species than did their lolines-nonproducing siblings. In fact, in most tests nearly all aphids on symbiota with 121 lolines were killed, whereas symbiota without lolines had no significant effect on aphids compared to endophyte-free plants. This result further indicated a biological function of lolines in plant protection from aphids, important also because aphids are vectors of many plant viruses. Peramine Peramine, classified as a pyrrolopyrazine alkaloid, so far appears to be unique to the Epichloe and Neotyphodium endophytes. Peramine is reported to be produced in cultures', a finding we confirmed from minimal medium cultures of Epichloe typhina (unpubl. data). Thus, like lolines, peramine is a fungal alkaloid. There is no experimental information on the biosynthesis of peramine. Inspection of its structure (Fig. 4) suggests that the cyclic dipeptide, cycloprolylarginine, would require two desaturation steps, a reduction, a dehydra- tion, and an N-methylation to generate peramine. Peramine is known as an insect feeding deterrents, and its presence in symbiota is associated with activity against the greenbug aphid. The role of peramine against aphids was tested in a manner similar to that for the lolines (above). In this case, the parents were isolates of Epichloe typhina that differed in peramine production. Segregation of peramine production in F1 and BC1 generations was not significantly different from 1:1. Anti-aphid activity was tested on perennial ryegrass symbiota with BCl progeny. Greenbugs were placed on tillers from the peramine-containing and peramine-minus symbiota. Significantly more aphids died on peramine-containing than peramine-minus symbiota. Thus, once again a Mendelian test supported the postulated role of a fungal alkaloid in biological protection of the plant. Fig. 4 Possible biosynthetic scheme for peramine. 122 Ergot Alkaloids The ergot alkaloids are well known for their effects on the central and peripheral nervous systems of vertebrates, as well as their toxicity to insects. Ergot fungi, Claviceps species, are known and feared as toxic contaminants of grains. Very high levels of ergot alkaloids, indolediterpenes or both characterize the resting structures (called sclerotia or ergots) of Claviceps species. In modern times mechanical elimination of most ergot contami- nation of food grains greatly reduces the risk of poisoning. The very widely planted symbiota, red fescue with E. festucae, perennial ryegrass (Lolium perenne) with Neotyphodium lolii, and tall fescue
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