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Agrobacterium tumefaciens-mediated transformation: an efficient tool for targeted gene disruption in Talaromyces marneffei

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Abstract

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.

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Acknowledgements

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).

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Author notes

  1. Xing Xiao and Jiao Feng have contributed equally to this work.

Authors and Affiliations

  1. Department of Dermatology and Venerology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China

    Xing Xiao, Jiao Feng, Zhiwen Chen, Minglan Shi, Liyan Xi & Junmin Zhang

  2. Department of Dermatology and Venerology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China

    Xing Xiao

  3. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China

    Xing Xiao, Jiao Feng, Yu Li, Zhiwen Chen, Minglan Shi, Liyan Xi & Junmin Zhang

  4. Medical Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China

    Yu Li

  5. Division of Infectious Diseases, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, USA

    Eleftherios Mylonakis

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  1. Xing Xiao
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  2. Jiao Feng
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  3. Yu Li
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  7. Eleftherios Mylonakis
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  8. Junmin Zhang
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Correspondence to Junmin Zhang.

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Xiao, X., Feng, J., Li, Y. et al. Agrobacterium tumefaciens-mediated transformation: an efficient tool for targeted gene disruption in Talaromyces marneffei . World J Microbiol Biotechnol 33, 183 (2017). https://doi.org/10.1007/s11274-017-2352-0

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