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Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles
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Orientador(es)
Resumo(s)
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.
Descrição
Research
Palavras-chave
colletotrichum acutatum agrobacterium tumefaciens-mediated transformation hygromycin B resistance green fluorescent protein expression insertional mutagenesis pathogenicity mutants
Contexto Educativo
Citação
"Molecular Biotechnology". ISSN 1073-6085. 39:1 (2008) 57-67