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Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

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Abstract

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

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Acknowledgments

This work was funded by the FAPESP/BIOTA (Grant 04/13910-6 and fellowship 06/57060-1 awarded to PTL) and CNPq (fellowship awarded to F.L.S.S.). We thank Dr. K. M. Plummer (La Trobe University, Australia) for providing the pFAT-gfp plasmid to L.C.L.F, and Dr. M.L.R. Aguiar-Perecin and Dr. A. Ferreira for their help with fluorescence microscopy analysis.

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  1. Department of Genetics, Escola Superior de Agricultura “Luiz de Queiroz”, University of São Paulo, Av. Pádua Dias 11, PO Box 83, Piracicaba, SP, 13400-970, Brazil

    Fernanda L. S. Sebastianes, Maria B. C. Rodrigues, João L. Azevedo & Aline A. Pizzirani-Kleiner

  2. Institute of Natural Sciences, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 700, Alfenas, MG, 37130-000, Brazil

    Paulo T. Lacava

  3. Embrapa Agroenergia, Brazilian Agricultural Research Corporation, Brasilia, DF, 70770-90, Brazil

    Léia C. L. Fávaro

  4. Department of Microbiology, Biomedical Sciences Institute, University of São Paulo, São Paulo, SP, 05508-900, Brazil

    Welington L. Araújo

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  1. Fernanda L. S. Sebastianes
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Correspondence to Paulo T. Lacava.

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Sebastianes, F.L.S., Lacava, P.T., Fávaro, L.C.L. et al. Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens . Curr Genet 58, 21–33 (2012). https://doi.org/10.1007/s00294-011-0362-2

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