Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass


Lolitrems are potent tremorgenic mycotoxins that are synthesised by clavicipitaceous fungal endophytes of the Epichloë/Neotyphodium group in association with grasses. These indole–diterpenes confer major ecological benefits on the grass–endophyte symbiotum. A molecular signature for diterpene biosynthesis is the presence of two geranylgeranyl diphosphate (GGPP) synthases. Using degenerate primers for conserved domains of fungal GGPP synthases, we cloned two such genes, ltmG and ggsA, from Neotyphodium lolii. Adjacent to ltmG are two genes, ltmM and ltmK, that are predicted to encode an FAD-dependent monooxygenase and a cytochrome P450 monooxygenase, respectively. The cluster of ltm genes is flanked by AT-rich retrotransposon DNA that appears to have undergone extensive repeat induced point (RIP) mutation. Epichloë festucae, the sexual ancestor of N. lolii, contains an identical ltm gene cluster, but lacks the retrotransposon “platform’‘ on the right flank. Associations established between perennial ryegrass and an E. festucae mutant deleted for ltmM lack detectable levels of lolitrems. A wild-type copy of ltmM complemented this phenotype, as did paxM from Penicillium paxilli. Northern hybridization and RT-PCR analysis showed that all three genes are weakly expressed in culture but strongly induced in planta. The relative endophyte biomass in these associations was estimated by real-time PCR to be between 0.3 and 1.9%. Taking this difference into account, the steady-state levels of the ltm transcripts are about 100-fold greater than the levels of the endogenous ryegrass β-tubulin (β -Tub1) and actin (Act1) RNAs. Based on these results we propose that ltmG, ltmM and ltmK are members of a set of genes required for lolitrem biosynthesis in E. festucae and N. lolii.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


  1. Acklin W, Weibel F, Arigoni D (1977) Zur Biosynthese von Paspalin und verwandten Metaboliten aus Claviceps paspali. Chimia 31:63

  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

  3. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

  4. Basse CW, Kolb S, Kahmann R (2002) A maize-specifically expressed gene cluster in Ustilago maydis. Mol Microbiol 43:75–93

  5. Bennett JW, Lasure LL (1985) Conventions for gene symbols. In: Bennett JW, Lasure LL (eds) Gene manipulation in fungi. Academic, London, pp 537–544

  6. Bhatnagar D, Ehrlich KC, Cleveland TE (2003) Molecular genetic analysis and regulation of aflatoxin biosynthesis. Appl Microbiol Biotechnol 61:83–93

  7. Bohnert HU, Fudal I, Dioh W, Tharreau D, Notteghem JL, Lebrun MH (2004) A putative polyketide synthase/peptide synthetase from Magnaporthe grisea signals pathogen attack to resistant rice. Plant Cell 16:2499–2513

  8. Bradshaw RE, Bhatnagar D, Ganley RJ, Gillman CJ, Monahan BJ, Seconi JM (2002) Dothistroma pini, a forest pathogen, contains homologs of aflatoxin biosynthetic pathway genes. Appl Environ Microbiol 68:2885–2892

  9. Brown DW, McCormick SP, Alexander NJ, Proctor RH, Desjardins AE (2001) A genetic and biochemical approach to study trichothecene diversity in Fusarium sporotrichioides and Fusarium graminearum. Fungal Genet Biol 32:121–133

  10. Bruchez JJP, Eberle J, Russo VEA (1993) Regulatory sequences involved in the translation of Neurospora crassa mRNA: Kozak sequences and stop codons. Fungal Genet Newslett 40:85–88

  11. Bullock WO, Fernandez JM, Short JM (1987) XL1-Blue: a high efficiency plasmid transforming recAEscherichia coli strain with beta-galactosidase selection. Biotechniques 5:376–378

  12. Bush LP, Wilkinson HH, Schardl CL (1997) Bioprotective alkaloids of grass-fungal endophyte symbioses. Plant Physiol 114:1–7

  13. Byrd AD, Schardl CL, Songlin PJ, Mogen KL, Siegel MR (1990) The β-tubulin gene of Epichloë typhina from perennial ryegrass (Lolium perenne). Curr Genet 18:347–354

  14. Byrne KM, Smith SK, Ondeyka JG (2002) Biosynthesis of nodulisporic acid A: precursor studies. J Am Chem Soc 124:7055–7060

  15. Cambareri EB, Jensen BC, Schabtach E, Selker EU (1989) Repeat-induced G-C to A-T mutations in Neurospora. Science 244:1571–1575

  16. Carroll AM, Sweigard JA, Valent B (1994) Improved vectors for selecting resistance to hygromycin. Fungal Genet Newslett 22:

  17. Chen AP, Kroon PA, Poulter CD (1994) Isoprenyl diphosphate synthases: protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure. Protein Sci 3:600–607

  18. Christensen MJ, Leuchtmann A, Rowan DD, Tapper BA (1993) Taxonomy of Acremonium endophytes of tall fescue (Festuca arundinacea), meadow fescue (F. pratensis) and perennial rye-grass (Lolium perenne). Mycol Res 97:1083–1092

  19. Clay K (1990) Fungal endophytes of grasses. Annu Rev Ecol Syst 21:275–297

  20. Clay K, Schardl C (2002) Evolutionary origins and ecological consequences of endophyte symbiosis with grasses. Am Naturalist 160:S99–S127

  21. Clergeot PH, Gourgues M, Cots J, Laurans F, Latorse MP, Pepin R, Tharreau D, Notteghem JL, Lebrun MH (2001) PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea. Proc Natl Acad Sci USA 98:6963–6968

  22. Cole RJ, Dorner JW, Lansden JA, Cox RH, Pape C, Cunfer BM, Nicholson SS, Bendell DM (1977) Paspalum staggers: isolation and identification of tremorgenic metabolites from sclerotia of Claviceps paspali. J Agric Food Chem 25:1197–1201

  23. Cox GB, Stout RW (1987) Study of the retention mechanisms for basic compounds on silica under “pseudo-reversed phase” conditions. J Chromatogr 384:315–336

  24. de Jesus AE, Gorst-Allman CP, Steyn PS, van Heerden FR, Vleggar R, Wessels PL, Hull WE (1983) Tremorgenic mycotoxins from Penicillium crustosum. Biosynthesis of Penitrem A. J Chem Soc Perkin Trans 1863–1868

  25. Eggink G, Engel H, Vriend G, Terpstra P, Witholt B (1990) Rubredoxin reductase of Pseudomonas oleovorans: structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints. J Mol Biol 212:135–142

  26. Fletcher LR, Harvey IC (1981) An association of a Lolium endophyte with ryegrass staggers. NZ Vet J 29:185–186

  27. Frischauf AM, Lehrach H, Poustka A, Murray N (1983) Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170:827–842

  28. Gallagher RT, White EP, Mortimer PH (1981) Ryegrass staggers: isolation of potent neurotoxins lolitrem A and lolitrem B from staggers-producing pastures. NZ Vet J 29:189–190

  29. Gallagher RT, Campbell AG, Hawkes AD, Holland PT, McGaveston DA, Pansier EA (1982) Ryegrass staggers: the presence of lolitrem neurotoxins in perennial ryegrass seed. NZ Vet J 30:183–184

  30. Gallagher RT, Hawkes AD, Steyn PS, Vleggaar R (1984) Tremorgenic neurotoxins from perennial ryegrass causing ryegrass staggers disorder of livestock: structure elucidation of lolitrem B. J Chem Soc Chem Commun 614–616

  31. Gallagher RT, Hawkes AD, Stewart JM (1985) Rapid determination of neurotoxin lolitrem B in perennial ryegrass by high-performance liquid chromatography with fluorescence detection. J Chromatogr 321:217–226

  32. Gardiner DM, Cozijnsen AJ, Wilson LM, Pedras MS, Howlett BJ (2004) The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans. Mol Microbiol 53:1307–1318

  33. Graham-Lorence SE, Peterson JA (1996) Structural alignments of P450s and extrapolations to the unknown. Methods Enzymol 272:315–326

  34. Gwinn KD, Collins-Shepard MH, Reddick BB (1991) Tissue print-immunoblot, an accurate method for the detection of Acremonium coenophialum in tall fescue. Phytopathology 81:747–748

  35. Idnurm A, Howlett BJ (2003) Analysis of loss of pathogenicity mutants reveals that repeat-induced point mutations can occur in the Dothideomycete Leptosphaeria maculans. Fungal Genet Biol 39:31–37

  36. Itoh Y, Johnson R, Scott B (1994) Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli. Curr Genet 25:508–513

  37. Keller NP, Hohn TM (1997) Metabolic pathway gene clusters in filamentous fungi. Fungal Genet Biol 21:17–29

  38. Kempken F, Kück U (1998) Transposons in filamentous fungi-facts and perspectives. BioEssays 20:652–659

  39. Latch GCM, Christensen MJ (1985) Artificial infection of grasses with endophytes. Ann Appl Biol 107:17–24

  40. Mantle PG, Weedon CM (1994) Biosynthesis and transformation of tremorgenic indole-diterpenoids by Penicillium paxilli and Acremonium lolii. Phytochemistry 36:1209–1217

  41. McLeay LM, Munday-Finch SC, Smith BL (1999) Tremorgenic mycotoxins paxilline, penitrem and lolitrem B, the non-tremorgenic 31-epilolitrem B and electromyographic activity of the reticulum and rumen of sheep. Res Vet Sci 66:119–127

  42. McMillan LK et al (2003) Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli and effects of paxilline intermediates on mammalian maxi-K ion channels. Mol Genet Genomics 270:9–23

  43. Miles CO, Munday SC, Wilkins AL, Ede RM, Towers NR (1994) Large-scale isolation of lolitrem B and structure determination of lolitrem E. J Agric Food Chem 42:1488–1492

  44. Möller EM, Bahnweg G, Sandermann H, Geiger HH (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Res 20:6115–6116

  45. Moon CD, Tapper BA, Scott B (1999) Identification of Epichloë endophytes in planta by a microsatellite-based PCR fingerprinting assay with automated analysis. Appl Environ Microbiol 65:1268–1279

  46. Moon CD, Scott B, Schardl CL, Christensen MJ (2000) The evolutionary origins of Epichloë endophytes from annual ryegrasses. Mycologia 92:1103–1118

  47. Munday-Finch SC, Miles CO, Wilkins AL, Hawkes AD (1995) Isolation and structure elucidation of lolitrem A, a tremorgenic mycotoxin from perennial ryegrass infected with Acremonium lolii. J Agric Food Chem 43:1283–1288

  48. Munday-Finch SC, Wilkins AL, Miles CO (1996) Isolation of paspaline B, an indole-diterpenoid from Penicillium paxilli. Phytochemisty 41:327–332

  49. Namiki F, Matsunaga M, Okuda M, Inoue I, Nishi K, Fujita Y, Tsuge T (2001) Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis. Mol Plant Microbe Interact 14:580–584

  50. Panaccione DG, Tapper BA, Lane GA, Davies E, Fraser K (2003) Biochemical outcome of blocking the ergot alkaloid pathway of a grass endophyte. J Agric Food Chem 51:6429–6437

  51. Parker EJ, Scott DB (2004) Indole–diterpene biosynthesis in ascomycetous fungi. In: An Z (ed) Handbook of industrial mycology. Marcel Dekker, New York, pp 405–426

  52. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448

  53. Penn J, Garthwaite I, Christensen MJ, Johnson CM, Towers NR (1993) The importance of paxilline in screening for potentially tremorgenic Acremonium isolates. In: Easton HS (ed) Proceedings of the Second International Symposium on Acremonium/Grass Interactions. AgResearch Grasslands Research Centre, Palmerston North, New Zealand, pp 88–92

  54. Proctor RH, Brown DW, Plattner RD, Desjardins AE (2003) Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis. Fungal Genet Biol 38:237–249

  55. Reinholz J, Paul VH (2001) Toxin-free Neotyphodium-isolates achieved without genetic engineering—a possible strategy to avoid “ryegrass staggers”. In: Dapprich PD (ed) Fourth International Neotyphodium/grass Interactions Symposium. University of Paderborn-Soest, Germany, pp 261–271

  56. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY

  57. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

  58. Schardl CL (1996) Interactions of grasses with endophytic Epichlö e species and hybrids. In: Keen N (ed) Plant–microbe interactions. Chapman and Hall, New York, pp 107–140

  59. Schardl CL, Leuchtmann A, Tsai H-F, Collett MA, Watt DM, Scott DB (1994) Origin of a fungal symbiont of perennial ryegrass by interspecific hybridization of a mutualist with the ryegrass choke pathogen, Epichloë typhina. Genetics 136:1307–1317

  60. Schmid J, Spiering MJ, Christensen MJ (2000) Metabolic activity, distribution, and propagation of grass endophytes in planta: investigations using the GUS reporter gene system. In: White JFJ (ed) Microbial endophytes. Marcel Dekker, New York, pp 295–322

  61. Selala MI, Laekeman GM, Loenders B, Masuka A, Herman AG, Schepens P (1991) In vitro effects of tremorgenic mycotoxins. J Nat Prod 54:207–212

  62. Selker EU, Cambareri EB, Jensen BC, Haack KR (1987) Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 51:741–752

  63. Siegel MR, Latch GCM, Bush LP, Fannin FF, Rowan DD, Tapper BA, Bacon CW, Johnson MC (1990) Fungal endophyte-infected grasses: alkaloid accumulation and aphid response. J Chem Ecol 16:3301–3315

  64. Smith BL, McLeay LM, Embling PP (1997) Effects of the mycotoxins penitrem, paxilline and lolitrem B on the electromyographic activity of skeletal and gastrointestinal smooth muscle of sheep. Res Vet Sci 62:11–116

  65. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

  66. Spiering MJ, Davies E, Tapper BA, Schmid J, Lane GA (2002) Simplified extraction of ergovaline and peramine for analysis of tissue distribution in endophyte-infected grass tillers. J Agric Food Chem 50:5856–5862

  67. Springer JP, Clardy J, Wells JM, Cole RJ, Kirksey JW (1975) The structure of paxilline, a tremorgenic metabolite of Penicillium paxilli Bainier. Tetrahedron Lett 30:2531–2534

  68. Toyomasu T, Nakaminami K, Toshima H, Mie T, Watanabe K, Ito H, Matsui H, Mitsuhashi W, Sassa T, Oikawa H (2004) Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha. Biosci Biotechnol Biochem 68:146–152

  69. Trinci APJ (1978) The duplication cycle and branching in fungi. In: Trinci APJ (ed) Symposium of the British Mycological Society, Queen Elizabeth College, London, pp 319–357

  70. Tudzynski B, Hölter K (1998) Gibberellin biosynthetic pathway in Gibberella fujikuroi: evidence for a gene cluster. Fungal Genet Biol 25:157–170

  71. Vallon O (2000) New sequence motifs in flavoproteins: evidence for common ancestry and tools to predict structure. Protein Struct Funct Genet 38:95–114

  72. Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci USA 83:4869–4873

  73. Wierenga RK, Terpstra P, Hol WGJ (1986) Predictions of the occurrence of the ADP-binding bab-fold in proteins using an amino acid sequence fingerprint. J Mol Biol 187:101–107

  74. Wu R, Hirai A, Mundy J, Nelson R, Rodriguez R (1991) Guidelines for nomenclature of cloned genes or DNA fragments in rice. Rice Genet Newslett 8:51–53

  75. Yoder OC (1988) Cochliobolus heterostrophus, cause of southern corn leaf blight. Adv Plant Pathol 6:93–112

  76. Young C, Itoh Y, Johnson R, Garthwaite I, Miles CO, Munday-Finch SC, Scott B (1998) Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration. Curr Genet 33:368–377

  77. Young CA, McMillan L, Telfer E, Scott B (2001) Molecular cloning and genetic analysis of an indole–diterpene gene cluster from Penicillium paxilli. Mol Microbiol 39:754–764

  78. Zhang S, Monahan BJ, Tkacz JS, Scott B (2004) An indole–diterpene gene cluster from Aspergillus flavus. Appl Environ Microbiol 70:6875–6883

Download references


This research was supported by grants MAU-X0127 and C10X0203 from the New Zealand Foundation for Research, Science and Technology (FRST), and a grant (MAU103) from the Royal Society of New Zealand Marsden Fund. The authors thank Andrea Bryant (Massey) for technical assistance, Joanne Dobson for constructing the Lp19 genomic library, Wayne Simpson and Elizabeth Davies (AgResearch) for technical assistance and advice, and Emily Parker (Massey) for discussions on the chemistry of lolitrem B biosynthesis. Carolyn Young was a recipient of a FRST Bright Futures Scholarship.

Author information

Correspondence to B. Scott.

Additional information

Communicated by P.Punt

Electronic Supplementary Material

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Young, C.A., Bryant, M.K., Christensen, M.J. et al. Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass. Mol Genet Genomics 274, 13–29 (2005).

Download citation


  • Neotyphodium lolii
  • Epichloë festucae
  • Lolitrem B
  • Endophyte
  • Retrotransposons