TY - JOUR
T1 - Agrobacterium tumefaciens-mediated transformation
T2 - an efficient tool for targeted gene disruption in Talaromyces marneffei
AU - Xiao, Xing
AU - Feng, Jiao
AU - Li, Yu
AU - Chen, Zhiwen
AU - Shi, Minglan
AU - Xi, Liyan
AU - Mylonakis, Eleftherios
AU - Zhang, Junmin
N1 - Funding Information:
We thank Cao Cunwei at the Guangxi Medical University for the T. marneffei △ligD pyrG strain. We also thank all of the medical workers in our hospital ward for their cooperation and clinical assistance. This study was supported by a grant from the National Natural Science Foundation of China (Grant No. 81571970). The authors declare that they have no conflict of interest.
Publisher Copyright:
© 2017, Springer Science+Business Media B.V.
PY - 2017/10/1
Y1 - 2017/10/1
N2 - Talaromyces marneffei causes life-threatening infections in immunocompromised hosts. An efficient tool for genetic manipulation of T. marneffei will allow for increased understanding of this thermally dimorphic fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) was optimized for targeted gene disruption in T. marneffei using the plasmid pDHt/acuD::pyrG. Molecular analyses of transformants were performed by PCR, Southern blot and semi-quantitative RT-PCR. A. tumefaciens strain EHA105 was more efficient at transformation than strain AGL-1 in ATMT via solid co-cultivation. An A. tumefaciens:T. marneffei ratio of 1000:1 in an ATMT liquid co-cultivation led to a relatively high transformation efficiency of 90 transformants per 106 yeast cells. Using ATMT-mediated knockout mutagenesis, we successfully deleted the acuD gene in T. marneffei. PCR and Southern blot analysis confirmed that acuD was disrupted and that the foreign pyrG gene was integrated into T. marneffei. Semi-quantitative RT-PCR analysis further confirmed that pyrG was expressed normally. These results suggest that ATMT can be a potential platform for targeted gene disruption in T. marneffei and that liquid co-cultivation may provide new opportunities to develop clinical treatments.
AB - Talaromyces marneffei causes life-threatening infections in immunocompromised hosts. An efficient tool for genetic manipulation of T. marneffei will allow for increased understanding of this thermally dimorphic fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) was optimized for targeted gene disruption in T. marneffei using the plasmid pDHt/acuD::pyrG. Molecular analyses of transformants were performed by PCR, Southern blot and semi-quantitative RT-PCR. A. tumefaciens strain EHA105 was more efficient at transformation than strain AGL-1 in ATMT via solid co-cultivation. An A. tumefaciens:T. marneffei ratio of 1000:1 in an ATMT liquid co-cultivation led to a relatively high transformation efficiency of 90 transformants per 106 yeast cells. Using ATMT-mediated knockout mutagenesis, we successfully deleted the acuD gene in T. marneffei. PCR and Southern blot analysis confirmed that acuD was disrupted and that the foreign pyrG gene was integrated into T. marneffei. Semi-quantitative RT-PCR analysis further confirmed that pyrG was expressed normally. These results suggest that ATMT can be a potential platform for targeted gene disruption in T. marneffei and that liquid co-cultivation may provide new opportunities to develop clinical treatments.
KW - Agrobacterium tumefaciens-mediated transformation
KW - Liquid co-cultivation
KW - Talaromyces marneffei
KW - Targeted gene disruption
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U2 - 10.1007/s11274-017-2352-0
DO - 10.1007/s11274-017-2352-0
M3 - Article
C2 - 28948456
AN - SCOPUS:85029878964
SN - 0959-3993
VL - 33
JO - World Journal of Microbiology and Biotechnology
JF - World Journal of Microbiology and Biotechnology
IS - 10
M1 - 183
ER -