Abstract
We have developed an improved Agrobacterium-mediated transformation (AMT) system for the functional genetic analysis of Penicillium marneffei, a thermally dimorphic, human pathogenic fungus. Our AMT protocol included the use of conidia or pre-germinated conidia of P. marneffei as the host recipient for T-DNA from Agrobacterium tumefaciens and co-cultivation at 28°C for 36 hours. Bleomycin-resistant transformants were selected as yeast-like colonies following incubation at 37°C. The efficiency of transformation was approximately 123 ± 3.27 and 239 ± 13.12 transformants per plate when using 5 × 104 conidia and pre-germinated conidia as starting materials, respectively. Southern blot analysis demonstrated that 95% of transformants contained single copies of T-DNA. Inverse PCR was employed for identifying flanking sequences at the T-DNA insertion sites. Analysis of these sequences indicated that integration occurred as random recombination events. Among the mutants isolated were previously described stuA and gasC defective strains. These AMT-derived mutants possessed single T-DNA integrations within their particular coding sequences. In addition, other morphological and pigmentation mutants possessing a variety of gene-specific defects were isolated, including two mutants having T-DNA integrations within putative promoter regions. One of the latter integration events was accompanied by the deletion of the entire corresponding gene. Collectively, these results indicated that AMT could be used for large-scale, functional genetic analyses in P. marneffei. Such analyses can potentially facilitate the identification of those genetic elements related to morphogenesis, as well as pathogenesis in this medically important fungus.
Acknowledgements
This work was supported by a Royal Golden Jubilee PhD research assistant fellowship to AK from the Thailand Research Fund, and the Faculty of Medicine, Chiang Mai University. The University Research Council and the Office of Student Affairs at Youngstown State University provided additional support. We also thank Dr Rosa Elena Cardoza and Dr Santiago Gutiérrez for kindly providing A. tumefaciens AGL1-pUPRSO and technical advice, as well as Ms Ashley Whitfield and Ms Karen Stangl for their technical assistance.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
This paper was first published online on Early Online on 14 May 2010.