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

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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.

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References

  • 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 

  • 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 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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]

    Article  CAS  Google Scholar 

  • 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]

    Article  CAS  Google Scholar 

  • 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]

    Article  CAS  Google Scholar 

  • 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.

    CAS  Google Scholar 

  • 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]

    Article  PubMed  CAS  Google Scholar 

  • 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]

    Article  PubMed  CAS  Google Scholar 

  • 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]

    Article  PubMed  CAS  Google Scholar 

  • 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]

    Article  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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]

    Article  PubMed  CAS  Google Scholar 

  • 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]

    Article  CAS  Google Scholar 

  • 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]

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • 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]

    Article  Google Scholar 

  • 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]

    Article  Google Scholar 

  • 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]

    Article  CAS  Google Scholar 

  • 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]

    Article  Google Scholar 

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Correspondence to Hong-ye Li.

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Project supported by the National Natural Science Foundation of China (No. 30571236), the Modern Agriculture (Citrus) Technology System (MATS) of China, and the Science and Technology Department of Zhejiang Province, China (No. 2007C22007)

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Wang, Jy., Li, Hy. Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum . J. Zhejiang Univ. Sci. B 9, 823–828 (2008). https://doi.org/10.1631/jzus.B0860006

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  • DOI: https://doi.org/10.1631/jzus.B0860006

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