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Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles
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Talhinhas, Pedro, Muthumeenakshi, S., Martins, J. M. Neves (João M. Neves), Oliveira, Helena and Sreenivasaprasad, S. (2008) Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles. Molecular Biotechnology, Vol.39 (No.1). pp. 57-67. doi:10.1007/s12033-007-9028-1 ISSN 1073-6085.
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Official URL: http://dx.doi.org/10.1007/s12033-007-9028-1
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.
| Item Type: | Journal Article | ||||
|---|---|---|---|---|---|
| Subjects: | Q Science > QP Physiology Q Science > QR Microbiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) > Warwick HRI (2004-2010) | ||||
| Library of Congress Subject Headings (LCSH): | Agrobacterium tumefaciens, Colletotrichum, Green fluorescent protein, Gene expression, Mutagenesis | ||||
| Journal or Publication Title: | Molecular Biotechnology | ||||
| Publisher: | Humana Press, Inc. | ||||
| ISSN: | 1073-6085 | ||||
| Official Date: | May 2008 | ||||
| Dates: |
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| Volume: | Vol.39 | ||||
| Number: | No.1 | ||||
| Number of Pages: | 11 | ||||
| Page Range: | pp. 57-67 | ||||
| DOI: | 10.1007/s12033-007-9028-1 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published | ||||
| Access rights to Published version: | Restricted or Subscription Access | ||||
| Funder: | Fundação para a Ciência e a Tecnologia (FCT), Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC) | ||||
| Grant number: | POCI/AGR/56321/2004 (FCT), SFRH/BPD/7161/2001 (FCT) |
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