Surrogate transformation of perennial ryegrass,Lolium perenne, using genetically modifiedAcremonium endophyte

  • Fiona R. Murray
  • Garrick C. M. Latch
  • D. Barry Scott


Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/μg DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The β-glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.

Key words

Acremonium sp. Transformation Chromosomal karyotype Surrogate grass transformation uidA andhph genes 


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  1. Brimble MA, Rowan DD (1988) Synthesis of peramine, an insect feeding deterrent mycotoxin fromAcremonium lolii. J Chem Soc Chem Commun 978–979Google Scholar
  2. Brody H, Carbon J (1989) Electrophoretic karyotype ofAspergillus nidulans. Proc Natl Acad Sci USA 86:6260–6263Google Scholar
  3. Case ME, Schweizer M, Kushner SR, Giles NH (1979) Efficient transformation ofNeurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci USA 76:5259–5263Google Scholar
  4. Chakraborty BN, Kapoor M (1990) Transformation of filamentous fungi by electroporation. Nucleic Acids Res 18:6737Google Scholar
  5. Christensen MJ, Latch GCM, Tapper BA (1991) Variation within isolates ofAcremonium endophytes from perennial rye-grasses. Mycological Res, 95:918–923Google Scholar
  6. Chu G, Vollrath D, Davis RW (1986) Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234:1582–1585Google Scholar
  7. Clay K (1990) Fungal endophytes of grasses. Annu Rev Ecol Syst 21:275–297Google Scholar
  8. Cooley RN, Shaw RK, Franklin FCH, Caten CE (1988) Transformation of the phytopathogenic fungusSeptoria nodorum to hygromycin B resistance. Curr Genet 13:383–389Google Scholar
  9. Debets AJM, Holub EF, Swart K, van den Brock HWJ, Bos CJ (1990) An electrophoretic karyotype ofAspergillus niger. Mol Gen Genet 224:264–268Google Scholar
  10. Dhawale SS, Marzluf GA (1985) Transformation ofNeurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA. Curr Genet 10:205–212Google Scholar
  11. Dumas DJ (1988) Total synthesis of peramine. J Org Chem 53:4650–4653Google Scholar
  12. Fan J-B, Chikashige Y, Smith CL, Niwa O, Yanagida M, Cantor CR (1989) Construction of aNotI restriction map of the fission yeastSchizosaccharomyces pombe genome. Nucleic Acids Res 17:2801–2818Google Scholar
  13. Fincham JRS (1989) Transformation in Fungi. Microbiol Rev 53:148–170Google Scholar
  14. Fletcher LR, Harvey IC (1981) An association of aLolium endophyte with ryegrass staggers. NZ Vet J 29:185–186Google Scholar
  15. 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 29:189–190Google Scholar
  16. Gallagher RT, Campbell AG, Hawkes AD, Holland PT, McGaveston DA, Pansier EA, Harvey IC (1982) Ryegrass staggers: the presence of lolitrem neurotoxins in perennial ryegrass seed. NZ Vet J 30:183–184Google Scholar
  17. 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 614–616Google Scholar
  18. Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2998Google Scholar
  19. Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5:387–405Google Scholar
  20. Latch GCM, Christensen MJ (1985) Artificial infection of grasses with endophytes. Ann Appl Biol 107:17–24Google Scholar
  21. Latch GCM, Christensen MJ, Samuels GJ (1984) Five Endophytes ofLolium andFestuca in New Zealand. Mycotaxon 20:535–550Google Scholar
  22. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  23. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  24. Mortimer RK, Schild D (1985) Genetic map ofSaccharomyces cerevisiae, edn 9. Microbiol Rev 49:181–213Google Scholar
  25. Oliver RP, Roberts IN, Harling R, Kenyon L, Punt PJ, Dingemanse MA, van den Hondel CAMJJ (1987) Transformation ofFulvia fulva, a fungal pathogen of tomato, to hygromycin B resistance. Curr Genet 12:231–233Google Scholar
  26. Orbach MJ, Vollrath D, Davis RW, Yanofsky C (1988) An electrophoretic karyotype ofNeurospora crassa. Mol Cell Biol 8:1469–1473Google Scholar
  27. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: A model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358Google Scholar
  28. Prestige RA, Pottinger RP, Barker GM (1982) An association ofLolium endophyte with ryegrass resistance to Argentine stem weevil. Proc 35th NZ Weed and Pest Control Conf 35:119–122Google Scholar
  29. Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CAMJJ (1987) Transformation ofAspergillus based on the hygromycin B resistance marker fromEscherichia coli. Gene 56:117–124Google Scholar
  30. Rambosek JA, Leach J (1987) Recombinant DNA in filamentous fungi: progress and prospects. Rev Biotechnol 6:357–373Google Scholar
  31. Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ (1988) Genetically transformed maize plants from protoplasts. Science 240:204–207Google Scholar
  32. Roberts IN, Oliver RP, Punt PJ, van den Hondel CAMJJ (1989) Expression of theEscherichia coli β-glucuronidase gene in industrial and phytopathogenic filamentous fungi. Curr Genet 15:177–180Google Scholar
  33. Rowan DD, Gaynor DL (1986) Isolation of feeding deterrents against argentine stem weevil from ryegrass infected with the endophyteAcremonium loliae. J Chem Ecology 12:647–658Google Scholar
  34. Rowan DD, Hunt MB, Gaynor DL (1986) Peramine, a Novel Insect Feeding Deterrent from Ryegrass infected with EndophyteAcremonium loliae. J Chem Soc Chem Commun 935–936Google Scholar
  35. Shimamoto K, Terada R, Izawa T, Fujimoto H (1989) Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274–276Google Scholar
  36. Siegel MR, Latch GCM, Johnson MC (1987) Fungal endophytes of grasses. Annu Rev Phytopathol 25:293–315Google Scholar
  37. Skatrud PL, Queener SW (1989) An electrophoretic molecular kayotype for an industrial strain ofCephalosporium acremonium. Gene 78:331–338Google Scholar
  38. Skatrud PL, Queener SW, Carr LG, Fisher DL (1987) Efficient integrative transformation ofCephalosporium acremonium. Curr Genet 12:337–348Google Scholar
  39. Smith CL, Matsumoto T, Niwa O, Klco S, Fan J-B, Yanagida M, Cantor CR (1987) An electrophoretic karyotype forSchizosaccharomyces pombe by pulsed field gel electrophoresis. Nucleic Acids Res 15:4481–4489Google Scholar
  40. Southern EM (1975) Detection of specific sequences among fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  41. Unkles SE, Campbell El, Carrez D, Grieve C, Contreras R, Fiers W, Van den Hondel CAMJJ, Kinghorn JR (1989) Transformation ofAspergillus niger with the homologous nitrate reductase gene. Gene 78:157–166Google Scholar
  42. Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes ofNeurospora crassa. Proc Natl Acad Sci USA 83:4869–4873Google Scholar
  43. Wang J, Holden DW, Leong SA (1988) Gene transfer system for the phytopathogenic fungusUstilago maydis. Proc Natl Acad Sci USA 85:865–869Google Scholar
  44. Weedon CM, Mantle PG (1987) Paxilline biosynthesis byAcremonium loliae; a step towards defining the origin of lolitrem neurotoxins. Phytochemistry 26:969–971Google Scholar
  45. Whitfeld PL, Seeburg PH, Shine J (1982) The human pro-opio-melanocortin gene: organisation, sequence, and interspersion with repetitive DNA. DNA 1:133–143Google Scholar
  46. Wnendt S, Jacobs M, Stahl U (1990) Transformation ofAspergillus giganteus to hygromycin B resistance. Curr Genet 17:21–24Google Scholar
  47. Yelton MM, Hamer JE, Timberlake WE (1984) Transformation ofAspergillus nidulans by using atrpC plasmid. Proc Natl Acad Sci USA 81:1470–1474Google Scholar
  48. Yoder OC (1988)Cochliobolus heterostrophus, cause of southern corn leaf blight. Adv Plant Pathol 6:93–112Google Scholar
  49. Zhang W, Wu R (1988) Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants. Theor Appl Genet 76:835–840Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Fiona R. Murray
    • 1
  • Garrick C. M. Latch
    • 2
  • D. Barry Scott
    • 1
  1. 1.Molecular Genetics Unit, Department of Microbiology and GeneticsMassey UniversityPalmerston NorthNew Zealand
  2. 2.DSIR Plant ProtectionMassey UniversityPalmerston NorthNew Zealand

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