HOME :: CHAPTER 13 :: Essay 13.3
Alkaloid-Making Fungal Symbionts
Christopher L. Schardl, Jimmy D. Blankenship, Caroline Machado, & Martin J. Spiering, Department of Plant Pathology, University of Kentucky, Lexington
Not all chemicals that plants use to defend themselves are actually made by that particular plant. Some are made by mutualistic symbionts called endophytes. For example, many temperate grasses are systemically "infected" with seed-transmissible fungal endophytes belonging to genera Epichloë and Neotyphodium. These endophytes enhance growth characteristics of the plants, increase resistance to various stresses, and act as "defensive mutualists" against herbivores. They produce an alphabet soup of secondary metabolites—particularly alkaloids—some of which protect mainly against vertebrate herbivores such as cattle, and others against invertebrate herbivores such as insects. The Epichloë and Neotyphodium endophytes are particularly common in ryegrasses and fescues, and are important worldwide both in temperate ecosystems and for human uses such as livestock forage, turf in landscapes and athletic fields, and ground cover for soil conservation. The popularity of these grasses for such uses stems from their fitness and adaptability in a wide variety of habitats, characteristics that are partially attributable to the effects of their endophytes and endophyte-derived alkaloids.
Alkaloids are chemicals with one or more nitrogen atoms bound to carbon, hydrogen, or combinations of carbon and hydrogen atoms (the term refers to the alkaline nature of most compounds fitting this description). Plant alkaloids commonly act as neurotoxins. By attacking the vital yet unique nervous systems of vertebrate or invertebrate animals, plants protect themselves from herbivores while remaining immune to their own toxins. Considering the roles of biogenic amines (such as dopamine and serotonin) in animal nervous systems, it is unsurprising that plants most often employ alkaloids as bioprotective neurotoxins. Likewise, the endophyte alkaloids attack the nervous systems of vertebrate or invertebrate animals.
Among the alkaloids produced by grass endophytes, the best known are ergot alkaloids because they are also produced by related fungi known for major poisoning events throughout history. The ergot alkaloids are so named because they are made by ergot fungi; that is, Claviceps species. "Ergots" are hard, dense resting structures of these fungi that typically replace the reproductive structures of Claviceps-infected grass florets. Ergots mimic the size, shape, and density of the seeds, so with traditional winnowing they tend to be retained in the grain rather than being removed with the chaff. In Europe up until the 20th century, ergots on rye were a particular problem for the poor; the bread of the aristocracy was mainly made from wheat, which is far less prone to ergot infestation. Symptoms included tingling in the extremities ("St. Anthony's fire"), hallucinations, abortions, and sometimes gangrene and death. The danger posed by ergots largely passed with the advent of modern grain processing techniques to remove them.
The best-known toxins in ergots, collectively called ergot alkaloids, are built upon prenylated tryptophan, and include clavines, lysergic acid, and a series of simple and.complex derivatives of lysergic acid. Perhaps the most famous ergot alkaloid is lysergic acid diethylamide (LSD), a psychedelic drug ("acid") strongly associated with counterculture in the 1970s. More complex ergopeptine alkaloids, also based on lysergic acid, are primarily responsible for acute symptoms including severe vasoconstriction leading to gangrene. After the introduction into much of the U.S. of tall fescue as a pasture and forage grass, mild symptoms of ergot alkaloid poisoning, and occasionally the more severe symptoms, were noted in cattle and other grazing livestock. Eventually it was determined that the tall fescue endophyte, Neotyphodium coenophialum, produced the ergopeptine alkaloid, ergovaline. Based on these findings, it is strongly suspected that ergovaline is responsible for tall fescue toxicosis.
The ergot alkaloids have long held interest to researchers because some, taken in low doses, are beneficial pharmaceuticals. Strains of Claviceps species have been selected for industrial production of these compounds, and the biosynthetic pathways have largely been worked out (Figure 1). Precursors of the lysergic acid portion are tryptophan, isoprene (in the form of dimethylallyl diphosphate) and methionine (donates a methyl group). Synthesis proceeds via the clavine alkaloids to lysergic acid, and amino acids or other substituents are linked to lysergic acid to make the more complex ergot alkaloids. Genes that code for enzymes in the pathway have begun to be cloned from the ergot fungi and endophytes. The first to be cloned was the dmaW gene for the first step in the pathway: namely, prenylation of tryptophan. Later, a peptide synthetase gene was identified near dmaW in Claviceps purpurea. This peptide synthetase is thought to be part of the complex that converts lysergic acid and three amino acids (alanine, phenylalanine and proline) to an ergopeptide lactam. This is the penultimate step in the.pathway, and the final cyclization step (gene not yet identified) converts the lactam to the potent neurotoxin and vasoconstrictor, ergotamine.
|Figure 1 Steps in ergovaline synthesis for which genes—dmaW and lpsA—have been cloned and characterized. (Click image to enlarge.)|
The lysergyl peptide synthetases determine some of the natural variation in ergopeptine structures. For example, the endophyte alkaloid, ergovaline, differs from ergotamine only in having a valine in place of the phenylalanine substituent. As predicted, the endophyte gene responsible for ergovaline lactam production was closely related to the previously identified C. purpurea gene. This lysergyl peptide synthetase (lpsA) gene was cloned from a ryegrass endophyte and a critical experiment was conducted that confirmed its function. Specifically, marker exchange mutagenesis was used to replace part of the gene with an antibiotic resistance gene (commonly used because it can be tracked in the mutant fungus), eliminating the function of lpsA. The result was a mutant Neotyphodium species that was incapable of producing ergovaline, but still produced simple clavines. As expected, eliminating dmaW also prevented ergovaline production, but in this case the production of clavines and simpler ergot alkaloids was also prevented.
Another family of endophyte alkaloids, the indolediterpenes, is also known for activity in animals. In perennial ryegrass, the endophyte Neotyphodium lolii produces indolediterpenes called lolitrems (Figure 2), which are potent tremorgens. The malady, ryegrass staggers, affects sheep that graze perennial ryegrass with N. lolii, and is particularly problematic in Australia and New Zealand. Though not lethal or permanently damaging, ryegrass staggers can cause accidental injury or death to the affected animal. The principle pharmacological activity that has been observed is inhibition of high-conductance potassium ion (K+) channels in smooth muscle, though it is unclear if this.completely accounts for the staggers symptoms. In South America, animals that eat certain endophyte-infected native grasses can suffer staggers syndromes and even death. For example, Neotyphodium tembladerae, endophytic in a South American bluegrass called Poa huecu, is believed to produce indolediterpenes that are partly or wholly responsible for such toxic effects.
|Figure 2 Representatives of three additional classes of endophyte-derived protective alkaloids. (Click image to enlarge.)|
Tremorgenic alkaloids produced by other fungi include paspaline and paspalitrems from the paspalum ergot fungus (Claviceps paspalx). These cause staggers in animals that graze the parasitized seed heads of Paspalum species grasses. A distantly related fungus, Penicillium paxilli, produces large amounts of paxilline when grown in the laboratory. A cluster of paxilline synthesis genes (pax) has been cloned from P. paxilli and sequenced, and the gene functions have been confirmed by marker exchange mutagenesis. Both paspaline and paxilline are found in ryegrass symbiotic with N. lolii, and potential intermediates in the pathways between these simpler alkaloids and the more complex lolitrems have also been characterized. Likely counterparts to several of the pax genes have been cloned from N. lolii.
Two other classes of endophyte alkaloids are known to be highly active against insects, yet have little or no activity against mammals. Peramine deters feeding by insects, and is thought to be very important in protecting perennial ryegrass from Argentine stem weevil in New Zealand. This plant-herbivore system is entirely introduced into New Zealand, since the grass was purposefully imported from Britain and, later, the weevil was inadvertently introduced from South America. Were it not for the endophyte, the weevil would have devastated the perennial-ryegrass pastures. This is clear because endophyte-cured pastures began to be established in New Zealand once the.connection had been revealed between ryegrass staggers and the endophyte, but within two years the endophyte-free pastures were wiped out mainly by Argentine stem weevils. What ecological role is played by peramine in more natural circumstances is unknown, but it should be important considering peramine was associated to about half of the endophytes surveyed so far, making it the most widespread of the endophyte alkaloids. A genetic analysis of Epichloë typhina indicates that a single locus probably determines whether peramine is expressed. There is no further information on that locus; however, based on what is known about the other alkaloid biosynthesis genes, it would most likely contain a biosynthesis gene cluster or a regulatory gene that controls expression of the biosynthesis genes.
A few endophytes express a more potent class of insecticidal alkaloids, the lolines. Loline alkaloids have an interesting multi-cyclic structure comprised of two 5-membered rings sharing adjacent carbon and nitrogen atoms, plus an oxygen atom bridging those rings. Based on the nitrogen-containing ring system, these substances are classified as pyrrolizidines, a class that also includes plant alkaloids known for their insecticidal activity and liver toxicity. However, chemical substituents responsible for the liver toxicity of plant pyrrolizidines are absent from lolines. Also, lolines are synthesized from amino acids, such as ornithine and aspartic acid, whereas plant pyrrolizidines are synthesized from the polyamines putrescine and spermidine. Lolines are neurotoxic to a broad range of insects, and when produced by endophytes in plants they have been shown to defend the plants from aphids. Since aphids carry many plant viruses, anti-aphid activity might also reduce viral infection, and indeed a loline-producing endophyte (N. coenophialum) has been found to reduce the proportion of tall fescue plants infected by barley yellow dwarf virus.
Genetic analysis of Epichloë festucae, a mutualist of many Festuca and Lolium grasses, indicates a single locus controlling expression of lolines. The locus contains a cluster of at least nine genes, which are also present in other loline-alkaloid producing endophytes. The cluster has been well characterized in E. festucae and Neotyphodium uncinatum, a meadow fescue endophyte. Seven of the nine genes are related to genes for known biosynthetic enzymes in fungi or other organisms: three to enzymes that use pyridoxal phosphate as a cofactor and are involved in amino acid metabolism, three others to enzymes that add molecular oxygen to substrates (mono- and dioxygenases), and one to oxidoreductases. This is likely to be the majority, but not all, of the loline alkaloid biosynthesis genes. Still to be identified are a methyltransferase responsible for N-methylation to yield loline, and one or more genes that might regulate the expression of the biosynthesis genes.
The life cycles of Neotyphodium and Epichloë species make them ideal bioprotective agents in ryegrass and fescue cultivars. These endophytes are transmitted in seeds with extremely high efficiency. When a mother plant contains an endophyte strain, the endophyte is clonally propagated in the seeds that develop on that mother plant, and ultimately resides in all of the seed progeny. Natural endophyte strains have already been identified with alkaloid profiles more suitable for grazing livestock than was typical of those in the pasture grass varieties that were originally introduced into North America, Australia, and New Zealand. Popular cultivars have been cured of their resident endophytes, and the newly identified strains have been introduced by artificial means into.those varieties to make new grass breeding stock. Conceivably, the same strategy can be also employed with endophytes in which genes for ergot or lolitrem alkaloids have been deleted, or genes for peramine or loline alkaloids have been introduced to maximize biological protection.
Panaccione, D. G., Johnson, R. D., Wang, J., Young, C. A., Scott, B., and Schardl, C. L. (2001) Elimination of ergovaline from a grass-Neotyphodium endophyte symbiosis by genetic modification of the endophyte. Proceedings of the National Academy of Sciences of U.S.A 98: 12820–12825.
Schardl, C. L. (2000) Symbiotic parasites and mutualistic pathogens: clavicipitaceous symbionts of grasses. In Fungal Pathology, J. W. Kronstad, ed. Dordrecht, The Netherlands, Kluwer Academic Publishers, pp. 307–345.
Scott, B. (2001) Epichloë endophytes: Fungal symbionts of grasses. Current Opinion in Microbiology 4: 393–398.
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: in press.
Tudzynski, P., Correia, T., and Keller, U. (2001) Biotechnology and genetics of ergot alkaloids. Applied Microbiology and Biotechnology 57: 593–605.
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