, Volume 19, Issue 7, pp 501–513 | Cite as

Isolation and sequence analysis of a β-tubulin gene from arbuscular mycorrhizal fungi

  • Zola Msiska
  • Joseph B. Morton
Original Paper


A full-length β-tubulin gene has been cloned and sequenced from Gigaspora gigantea and Glomus clarum, two arbuscular mycorrhizal fungi (AMF) species in the phylum Glomeromyota. The gene in both species is organized into five exons and four introns. Both genes are 94.9% similar and encode a 447 amino acid protein. In comparison with other fungal groups, the amino acid sequence is most similar to that of fungi in the Chytridiomycota. The codon usage of the gene in both AMF species is broad and biased in favor of an A or a T in the third position. The four introns varied in length from 87 to 168 bp for G. gigantea and from 90 to 136 bp for G. clarum. Of all fungi in which full-length sequences have been published, only AMF do not have an intron before codon 174. The introns positioned at codons 174 and 257 in AMF match the position of different introns in β-tubulin genes of some Zygomycete, Basidiomycete, and Ascomycete fungi. The 5′ and 3′ splice site consensus sequences are similar to those found in introns of most fungi. Sequence analysis from single-strand conformation polymorphism analysis confirmed the presence of two β-tubulin gene copies in G. clarum, but only one copy was evident in G. gigantea based on Southern hybridization analysis.


Glomeromycota Inverse PCR Beta-tubulin Arbuscular mycorrhizal fungi 



The authors wish to thank Bill Wheeler for helping with the manipulation and processing of AMF cultures. We also thank Dr. Jed Doelling for reviewing the manuscript. We are grateful to one reviewer for providing an additional source of information for full-length β-tubulin sequences of the Chytridiomycota and Zygomycota. Funding was provided by a Fulbright scholarship to Zola Msiska and National Science Foundation grants DBI0650735 and DEB0649341 to Joseph Morton.


  1. Aono T, Maldonado-Mendoza TE, Dewbre GR, Harrison MJ, Saito M (2004) Expression of alkaline phophatase genes in arbuscular mycorrhizas. New Phytol 162:525–534. doi: 10.1111/j.1469-8137.2004.01041.x CrossRefGoogle Scholar
  2. Ayliffe MA, Dodds PN, Lawrence GJ (2001) Characterisation of a beta-tubulin gene from Melampsora lini and comparison of fungal beta-tubulin genes. Mycol Res 105:818–826. doi: 10.1017/S0953756201004245 CrossRefGoogle Scholar
  3. Bago B, Zipfel W, Williams RM, Jun J, Arreola R, Lammers PJ, Pfeffer PE, Shachar-hill Y (2002) Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis. Plant Physiol 128:108–124. doi: 10.1104/pp.010466 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Breuninger M, Trujillo CG, Serrano E, Fischer R, Requena N (2004) Different nitrogen sources modulate activity but not expression of glutamine synthetase in arbuscular mycorrhizal fungi. Fungal Genet Biol 41:542–552. doi: 10.1016/j.fgb.2004.01.003 CrossRefPubMedGoogle Scholar
  5. Buhr TL, Dickman MB (1994) Isolation, characterization, and expression of a second β-tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene. Appl Environ Microbiol 60:4155–4159PubMedPubMedCentralGoogle Scholar
  6. Burleigh SH, Harrison MJ (1998) A cDNA from the arbuscular mycorrhizal fungus Glomus versiforme with homology to a cruciform DNA-binding protein from Ustilago maydis. Mycorrhiza 7:301–306. doi: 10.1007/s005720050196 CrossRefGoogle Scholar
  7. Cappellazzo G, Lanfranco L, Fitz M, Wipf D, Bonfante P (2008) Characterization of an amino acid permease from the endomycorrhizal fungus Glomus mosseae. Plant Physiol 147:429–437. doi: 10.1104/pp.108.117820 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chiocchio V, Venedikian N, Martinez AE, Menendez A, Ocampo JA, Godeas A (2000) Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae. Int Microbiol 3:173–175PubMedGoogle Scholar
  9. Cleveland DW, Theodorakis NG (1994) Regulation of tubulin synthesis. In: Hyams JS, Lloyd CW (eds) Microtubules. Wiley-Liss, New York, pp 47–58Google Scholar
  10. Comai L (2005) The advantages and disadvantages of being polyploid. Nat Rev Genet 6:836–846. doi: 10.1038/nrg1711 CrossRefPubMedGoogle Scholar
  11. Corradi N, Sanders IR (2006) Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices. BMC Evol Biol 6:21. doi: 10.1186/1471-2148-6-21 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Corradi N, Kuhn G, Sanders IR (2004a) Monophyly of b-tubulin and H+-ATPase gene variants in Glomus intraradices: consequences for molecular evolutionary studies of AM fungal genes. Fungal Genet Biol 41:262–273. doi: 10.1016/j.fgb.2003.11.001 CrossRefPubMedGoogle Scholar
  13. Corradi N, Hijri M, Fumagalli L, Sanders IR (2004b) Arbuscular mycorrhizal fungi (Glomeromycota) harbour ancient fungal tubulin genes that resemble those of the chytrids (Chytridiomycota). Fungal Genet Biol 41:1037–1045. doi: 10.1016/j.fgb.2004.08.005 CrossRefPubMedGoogle Scholar
  14. Cross D, Farias G, Dominguez J, Avila J, Maccioni RB (1994) Carboxyl terminal sequences of the beta-tubulin involved in the interaction of HMW-MAPs. Studies using site specific antibodies. Mol Cell Biochem 16:81–90. doi: 10.1007/BF00925677 CrossRefGoogle Scholar
  15. Cruz MC, Edlind T (1997) Beta-tubulin genes and the basis for benzimidazole sensitivity of the opportunistic fungus Cryptococcus neoformans. Microbiology 143:2003–2008CrossRefPubMedGoogle Scholar
  16. Farr GW, Sternlicht H (1992) Site-directed mutagenesis of the GTP-binding domain of beta-tubulin. J Mol Biol 227:307–321. doi: 10.1016/0022-2836(92)90700-T CrossRefPubMedGoogle Scholar
  17. Fitter AH, Nichols R (1988) The use of benomyl to control infection by vesicular–arbuscular mycorrhizal fungi. New Phytol 110:201–206. doi: 10.1111/j.1469-8137.1988.tb00253.x CrossRefGoogle Scholar
  18. Franken P, Requena N (2001) Analysis of gene expression in arbuscular mycorrhizas: new approaches and challenges. New Phytol 150:517–523CrossRefGoogle Scholar
  19. Gadkar V, Rillig MC (2006) The arbuscular mycorrhizal fungal protein glomalin is a putative homolog of heat shock protein 60. FEMS Microbiol Lett 263:93–101. doi: 10.1111/j.1574-6968.2006.00412.x CrossRefPubMedGoogle Scholar
  20. Gerdemann JW, Nicolson TH (1963) Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans Br Mycol Soc 46:235–244CrossRefGoogle Scholar
  21. Gilbert SF (ed) (2006) Developmental biology, 8th edn. Sinauer, MassachussettsGoogle Scholar
  22. Goldman GH, Temmerman W, Jacobs D, Contreras R, van Montagu M, Herrera-Estrella A (1993) A nucleotide substitution in one of the beta-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate. Mol Gen Genet 240:73–80. doi: 10.1007/BF00276886 CrossRefPubMedGoogle Scholar
  23. Harrier LA (2001) The arbuscular mycorrhizal symbiosis: a molecular review of the fungal dimension. J Exp Bot 52:469–478CrossRefPubMedGoogle Scholar
  24. Harrier LA, Wright F, Hooker JE (1998) Isolation of the 3-phosphoglycerate kinase gene of the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe. Curr Genet 34:386–392. doi: 10.1007/s002940050411 CrossRefPubMedGoogle Scholar
  25. Harrison MJ, van Buuren ML (1995) A phosphate transporter from the mycorrhizal fungus Glomus versiforme. Nature 378:626–629. doi: 10.1038/378626a0 CrossRefPubMedGoogle Scholar
  26. Helgason T, Watson IJ, Young JP (2003) Phylogeny of the Glomerales and Diversiporales (Fungi: Glomeromycota) from actin and eleongation factor 1-alpha sequences. FEMS Microbiol Lett 229:127–132. doi: 10.1016/S0378-1097(03)00802-4 CrossRefPubMedGoogle Scholar
  27. Hosny M, Debarros JPP, Gianinazzi-Pearson V, Dulieu H (1997) Base composition of DNA from Glomalean fungi—high amounts of methylated cytosine. Fungal Genet Biol 22:103–111. doi: 10.1006/fgbi.1997.1008 CrossRefPubMedGoogle Scholar
  28. Huang S-H, Chen SHM, Jong AY (2003) Use of inverse PCR to clone cDNA ends. In: Ying S-Y (ed) Methods in molecular biology vol. 221: generation of cDNA libraries: methods and protocols. Humana, Totowa, NJ, pp 51–58Google Scholar
  29. Jung MK, Wilder IB, Oakley BR (1992) Amino acid alterations in the benA (beta-tubulin) gene of Aspergillus nidulans that confer benomyl resistance. Cell Motil Cytoskelet 22:170–174. doi: 10.1002/cm.970220304 CrossRefGoogle Scholar
  30. Juuti JT, Jokela S, Tarkka MT, Paulin L, Lahdensalo J (2005) Two phylogenetically highly distinct beta-tubulin genes of the basidiomycete Suillus bovinus. Curr Genet 47:253–263. doi: 10.1007/s00294-005-0564-6 CrossRefPubMedGoogle Scholar
  31. Keeling PJ, Luker MA, Palmer JD (2000) Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi. Mol Biol Evol 17:23–31CrossRefPubMedGoogle Scholar
  32. Koonin EV (2006) The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate? Biol Direct 1:1–22. doi: 10.1186/1745-6150-1-1 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lanfranco L, Garnero L, Bonfante P (1999) Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme: Full sequence of a gene encoding a class IV chitin synthase. FEMS Microbiol Lett 170:59–67. doi: 10.1111/j.1574-6968.1999.tb13355.x CrossRefPubMedGoogle Scholar
  34. Li M, Yang Q (2007) Isolation and characterization of a beta-tubulin gene from Trichoderma harzianum. Biochem Genet 45:529–534. doi: 10.1007/s10528-007-9094-x CrossRefPubMedGoogle Scholar
  35. Ma Z, Yoshima MA, Michailides TJ (2005) PCR-based assays for detection of benzimidazole resistant isolates of Monilinia laxa in California. Pest Manag Sci 61:449–457. doi: 10.1002/ps.982 CrossRefPubMedGoogle Scholar
  36. May GS (1989) The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable. J Cell Biol 109:2267–2274. doi: 10.1083/jcb.109.5.2267 CrossRefPubMedGoogle Scholar
  37. May GS, Tsang MLS, Smith H, Fidel S, Morris NR (1987) Aspergillus nidulans beta-tubulin genes are unusually divergent. Gene 55:231–243. doi: 10.1016/0378-1119(87)90283-6 CrossRefPubMedGoogle Scholar
  38. McKean PG, Vaughan S, Gull K (2001) The extended tubulin superfamily. J Cell Sci 114:2723–2733PubMedGoogle Scholar
  39. Msiska Z, Morton JB (2009) Phylogenetic analysis of the Glomeromycota by partial beta-tubulin gene sequences. Mycorrhiza 19:247–254. doi: 10.1007/s00572-008-0216-z CrossRefPubMedGoogle Scholar
  40. Mukherjee M, Hadar R, Mukherjee PK, Horwitz BA (2003) Homologous expression of a mutated beta-tubulin gene does not confer benomyl resistance on Trichoderma virens. J Appl Microbiol 95:861–867. doi: 10.1046/j.1365-2672.2003.02061.x CrossRefPubMedGoogle Scholar
  41. Neff NF, Thomas JH, Grisafi P, Botstein D (1983) Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell 33:211–219. doi: 10.1016/0092-8674(83)90350-1 CrossRefPubMedGoogle Scholar
  42. Nogales E, Downing KH, Amos LA, Lowe J (1998) Tubulin and FtsZ form a distinct family of GTPases. Nat Struct Mol Biol 5:451–458. doi: 10.1038/nsb0698-451 CrossRefGoogle Scholar
  43. Ochman H, Gerber S, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120:621–623PubMedPubMedCentralGoogle Scholar
  44. Ocon A, Hampp R, Requena N (2007) Trehalose turnover during abiotic stress in arbuscular mycorrhizal fungi. New Phytol 174:879–891. doi: 10.1111/j.1469-8137.2007.02048.x CrossRefPubMedGoogle Scholar
  45. Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for beta-tubulin from a benomyl resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461CrossRefPubMedPubMedCentralGoogle Scholar
  46. Panaccione DG, Hanau RM (1990) Characterization of two divergent beta-tubulin genes from Colletotrichum graminicola. Gene 86:163–170. doi: 10.1016/0378-1119(90)90275-V CrossRefPubMedGoogle Scholar
  47. Panaccione DG, McKiernan M, Hanau RM (1988) Colletotrichum graminicola transformed with homologous and heterologous benomyl-resistant genes retains expected pathogenicity to corn. Mol Plant Microbe Interact 1:113–120CrossRefGoogle Scholar
  48. Redecker D, Raab P (2006) Phylogeny of the Glomeromycota (arbuscular mycorrhizal fungi): recent developments and new gene markers. Mycologia 98:885–895. doi: 10.3852/mycologia.98.6.885 CrossRefPubMedGoogle Scholar
  49. Requena N, Fuller P, Franken P (1999) Molecular characterization of GmFOX2, an evolutionarily highly conserved gene from the mycorrhizal fungus Glomus mosseae, down regulated during interaction with rhizobacteria. Mol Plant Microbe Interact 12:934–942. doi: 10.1094/MPMI.1999.12.10.934 CrossRefPubMedGoogle Scholar
  50. Requena N, Mann P, Franken P (2000) A homologue of the cell cycle check point TOR2 from Saccharomyces cerevisiae exists in the arbuscular mycorrhizal fungus Glomus mosseae. Protoplasma 212:89–98. doi: 10.1007/BF01279350 CrossRefGoogle Scholar
  51. Requena N, Breuninger M, Franken P, Ocon A (2003) Symbiotic status, phosphate, and sucrose regulate the expression of two plasma membrane H+-ATPase genes from the myccorrhizal fungus Glomus mosseae. Plant Physiol 132:1540–1549. doi: 10.1104/pp.102.019042 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Spokes JR, MacDonald RM, Hayman DS (2006) Effects of plant protection chemicals on vesicular–arbuscular mycorrhizas. Pest Manag Sci 12:346–350CrossRefGoogle Scholar
  53. Weatherbee JA, May GS, Gambino J, Morris NR (1985) Involvement of a particular species of tubulin (beta3) in conidial development in Aspergillus nidulans. J Cell Biol 101:706–711. doi: 10.1083/jcb.101.3.706 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.West Virginia UniversityMorgantownUSA

Personalised recommendations