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    Agrobacterium-mediated transformation and insertional mutagenesis in colletotrichum acutatum for investigating varied pathogenicity lifestyles

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    Authors
    Talhinhas, Pedro
    Muthumeenakshi, S.
    Neves-Martins, João
    Oliveira, Helena
    Sreenivasaprasad, Surapareddy
    Affiliation
    Technical University of Lisbon
    University of Warwick
    Issue Date
    2008
    Subjects
    pathogenicity mutants
    colletotrichum acutatum
    agrobacterium tumefaciens-mediated transformation
    green fluorescent protein expression
    insertional mutagenesis
    hygromycin B resistance
    
    Metadata
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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 10(4) 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.
    Citation
    Talhinhas, P., Muthumeenakshi, S., Neves-Martins, J., Oliveira, H. and Sreenivasaprasad, S. (2008) 'Agrobacterium-Mediated Transformation and Insertional Mutagenesis in Colletotrichum acutatum for Investigating Varied Pathogenicity Lifestyles, Molecular Biotechnology, 39(1),pp.57-67
    Publisher
    Humana Press
    Journal
    Molecular Biotechnology
    URI
    http://hdl.handle.net/10547/302152
    DOI
    10.1007/s12033-007-9028-1
    Additional Links
    http://link.springer.com/10.1007/s12033-007-9028-1
    Type
    Article
    Language
    en
    ISSN
    1073-6085
    1559-0305
    ae974a485f413a2113503eed53cd6c53
    10.1007/s12033-007-9028-1
    Scopus Count
    Collections
    Environmental Monitoring Research Group

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