Journal of Zhejiang University SCIENCE B

, Volume 9, Issue 10, pp 823–828 | Cite as

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

Article

Abstract

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.

Key words

Penicillium digitatum Agrobacterium tumefaciens-mediated transformation (ATMT) Hygromycin B resistance gene Insertional mutagenesis 

CLC number

Q93 Q3 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. An, G., Ebert, P.R., Mitra, A., Ha, S.B., 1988. Binary Vectors. In: Gelvin, S.B., Schilperoort, R.A. (Eds.), Plant Molecular Biology Manual. Kluwer Academic Publishers, Great Britain, p.1–19.Google Scholar
  2. Bowyer, P., 2001. DNA-mediated Transformation of Fungi. In: Talbot, N. (Ed.), Molecular and Cellular Biology of Filamentous Fungi. Oxford Univ. Press, Oxford, p.33–46.Google Scholar
  3. Bundock, P., Dulk-Ras, A., Beijersbergen, A.G.M., Hooykaas, P.J.J., 1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Ssaccharomyces cerevisiae. EMBO J., 14(13):3206–3214.PubMedGoogle Scholar
  4. Bush, D.A., Codner, R.C., 1968. The nature of macerating factor of Penicillium digitatum saccardo. Phytochemistry, 7(5):863–869. [doi:10.1016/S0031-9422(00)84844-X]CrossRefGoogle Scholar
  5. Hamamoto, H., Nawata, O., Hasegawa, K., Nakaune, R., Lee Y.J., Makizumi, Y., Akutsu, K., Hibi, T., 2001. The role of the ABC transporter gene PMR1 in demethylation inhibitor resistance in Penicillium digitatum. Pest. Biochem. Physiol., 70(1):19–26. [doi:10.1006/pest.2001.2530]CrossRefGoogle Scholar
  6. Holmes, G.J., Eckert, J.W., 1999. Sensitivity of Penicilium digitatum and P. italicum to postharvest citrus fungicides in California. Phytopathology, 89(9):716–721. [doi:10.1094/PHYTO.1999.89.9.716]CrossRefGoogle Scholar
  7. Hooykaas, P.J.J., Roobol, C., Schilperoort, R.A., 1979. Regulation of the transfer of Ti-plasmids of Agrobacterium tumefaciens. J. Gen. Microbiol., 110(1): 99–109.Google Scholar
  8. Irie, T., Honda, Y., Watanabe, T., Kuwahara, M., 2001. Efficient transformation of the filamentous fungus Pleurotus ostreatus using single-strand carrier DNA. Appl. Microbiol. Biotechnol., 55(5):563–565. [doi:10.1007/s002530000535]PubMedCrossRefGoogle Scholar
  9. Malonek, S., Meinhardt, F., 2001. Agrobacterium tumefaciens mediated genetic transformation of the phytopathogenic ascomycete Calonectria morganii. Curr. Genet., 40(2): 152–155. [doi:10.1007/s002940100236]PubMedCrossRefGoogle Scholar
  10. Morioka, L.R.I., Furlaneto, M.C., Bogas, A.C., Pompermayer, P., Duarte, R.T.D., Vieira, M.L.C., Watanabe, M.A.E., Fungaro, M.H.P., 2006. Efficient genetic transformation system for the ochratoxigenic fungus Aspergillus carbonarius. Curr. Microbiol., 52(6):469–472. [doi:10.1007/s00284-005-0402-6]PubMedCrossRefGoogle Scholar
  11. Mullins, E.D., Chen, X., Romaine, P., Raina, R., Geiser, D.M., Kang, S., 2001. Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 91(2):173–180. [doi:10.1094/PHYTO.2001.91.2.173]CrossRefGoogle Scholar
  12. Nakaune, R., Adachi, K., Nawata, O., Tomiyama, M., Akutsu, K., Hibi, T., 1998. A novel ATP-binding cassette transporter involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum. Appl. Environ. Microbiol., 64(10):3983–3988.PubMedGoogle Scholar
  13. Nakaune, R., Hamamoto, H., Imada, J., Akutsu, K., Hibi, T., 2002. A novel ABC transporter gene, PMR5, is involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum. Mol. Genet. Genomics, 267(2): 179–185. [doi:10.1007/s00438-002-0649-6]PubMedCrossRefGoogle Scholar
  14. Raeder, U., Broda, P., 1985. Rapid prepraration of DNA from filamentous fungi. Lett. Appl. Microbiol., 1(1):17–20. [doi:10.1111/j.1472-765X.1985.tb01479.x]CrossRefGoogle Scholar
  15. Rooney, P.J., Sullivan, T.D., Klein, B.S., 2001. Selective expression of the virulence factor BAD1 upon morphogenesis to the pathogenic yeast form of Blastomyces dermatitidis: evidence for transcriptional regulation by a conserved mechanism. Mol. Microbiol., 39(4):875–889. [doi:10.1046/j.1365-2958.2001.02300.x]PubMedCrossRefGoogle Scholar
  16. Sambrock, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  17. Smilanick, J.L., Mansour, M.F., Sorensen, D., 2006. Pre-and postharvest treatments to control green mold of citrus fruit during ethylene degreening. Plant Dis., 90(1):89–96. [doi:10.1094/PD-90-0089]CrossRefGoogle Scholar
  18. Song, A.H., Li, H.Y., Liu, X.H., 2004. Isolation and regeneration of protoplasts from Penicillium digitatum. Chin. J. Agric. Biotechnol., 1(3):197–202. [doi:10.1079/CJB200442]CrossRefGoogle Scholar
  19. Zhang, J.X., 2007. The potential of a new fungicide fludioxonil for stem-end rot and green mold control on Florida citrus fruit. Postharvest Biol. Technol., 46(3): 262–270. [doi:10.1016/j.postharvbio.2007.05.016]CrossRefGoogle Scholar
  20. Zhu, J.W., Xie, Q.Y., Li, H.Y., 2006. Occurrence of imazalil-resistant biotype of Penicillium digitatum in China and the resistant molecular mechanism. J. Zhejiang Univ. Sci. A, 7(2):362–365. [doi:10.1631/jzus.2006.AS0362]CrossRefGoogle Scholar

Copyright information

© Zhejiang University and Springer-Verlag GmbH 2008

Authors and Affiliations

  1. 1.Institute of BiotechnologyZhejiang UniversityHangzhouChina

Personalised recommendations