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Agrobacterium-Mediated Transformation and Insertional Mutagenesis in Colletotrichum acutatum for Investigating Varied Pathogenicity Lifestyles

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Abstract

Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 104 conidia, with >70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles.

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Acknowledgements

This work was partly funded by FCT POCI 2010-FEDER (POCI/AGR/56321/2004 and SFRH/BPD/7161/2001, Portugal) and BBSRC (UK). The authors thank Dr. S. Kang (Pennsylvania State University, USA) for the pBHt2 plasmid and Dr. M. P. Challen, Dr. A. Soares and Ms. V. Odon (Warwick HRI) for providing bacterial strains, plasmids and protocols.

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Authors and Affiliations

  1. Instituto Superior de Agronomia – Technical University of Lisbon, Tapada da Ajuda, Lisboa, 1349-017, Portugal

    Pedro Talhinhas, João Neves-Martins & Helena Oliveira

  2. Warwick HRI, University of Warwick, Wellesbourne, Warwickshire, CV35 9EF, UK

    Pedro Talhinhas, S. Muthumeenakshi & S. Sreenivasaprasad

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  1. Pedro Talhinhas
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  2. S. Muthumeenakshi
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  5. S. Sreenivasaprasad
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Correspondence to Pedro Talhinhas.

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Talhinhas, P., Muthumeenakshi, S., Neves-Martins, J. et al. Agrobacterium-Mediated Transformation and Insertional Mutagenesis in Colletotrichum acutatum for Investigating Varied Pathogenicity Lifestyles. Mol Biotechnol 39, 57–67 (2008). https://doi.org/10.1007/s12033-007-9028-1

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