An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei

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...

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Main Authors: Kummasook A., Cooper Jr. C.R., Vanittanakom N.
Format: Article
Language:English
Published: 2014
Online Access:http://www.scopus.com/inward/record.url?eid=2-s2.0-77958147226&partnerID=40&md5=5ce602507ae453268973a9b9dfec1c0a
http://www.ncbi.nlm.nih.gov/pubmed/20465521
http://cmuir.cmu.ac.th/handle/6653943832/2511
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Institution: Chiang Mai University
Language: English
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spelling th-cmuir.6653943832-25112014-08-30T02:00:55Z An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei Kummasook A. Cooper Jr. C.R. Vanittanakom N. 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. © 2010 ISHAM. 2014-08-30T02:00:55Z 2014-08-30T02:00:55Z 2010 Article 13693786 10.3109/13693781003801219 20465521 MEMYF http://www.scopus.com/inward/record.url?eid=2-s2.0-77958147226&partnerID=40&md5=5ce602507ae453268973a9b9dfec1c0a http://www.ncbi.nlm.nih.gov/pubmed/20465521 http://cmuir.cmu.ac.th/handle/6653943832/2511 English
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
language English
description 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. © 2010 ISHAM.
format Article
author Kummasook A.
Cooper Jr. C.R.
Vanittanakom N.
spellingShingle Kummasook A.
Cooper Jr. C.R.
Vanittanakom N.
An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei
author_facet Kummasook A.
Cooper Jr. C.R.
Vanittanakom N.
author_sort Kummasook A.
title An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei
title_short An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei
title_full An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei
title_fullStr An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei
title_full_unstemmed An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei
title_sort improved agrobacterium-mediated transformation system for the functional genetic analysis of penicillium marneffei
publishDate 2014
url http://www.scopus.com/inward/record.url?eid=2-s2.0-77958147226&partnerID=40&md5=5ce602507ae453268973a9b9dfec1c0a
http://www.ncbi.nlm.nih.gov/pubmed/20465521
http://cmuir.cmu.ac.th/handle/6653943832/2511
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