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Updated: Feb 22, 2026
Author Spotlight: Optimized Transformation Protocol for Chlorella vulgaris Using Agrobacterium tumefaciens
Published on: October 27, 2023
Agrobacterium tumefaciens-mediated transformation: an efficient tool for targeted gene disruption in Talaromyces
Xing Xiao1,2,3, Jiao Feng1,3, Yu Li3,4
1Department of Dermatology and Venerology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.
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.
Insights
Optimizing Agrobacterium tumefaciens-mediated transformation (ATMT) allows for efficient gene disruption in Talaromyces marneffei. This advancement aids understanding of the fungus causing life-threatening infections in immunocompromised individuals.
Area of Science:
- Mycology
- Medical Mycology
- Molecular Biology
Background:
- Talaromyces marneffei infections pose significant risks to immunocompromised individuals.
- Understanding T. marneffei requires effective genetic manipulation tools.
- Thermally dimorphic fungi present unique challenges in genetic studies.
Purpose of the Study:
- To optimize Agrobacterium tumefaciens-mediated transformation (ATMT) for targeted gene disruption in Talaromyces marneffei.
- To establish an efficient method for genetic manipulation of T. marneffei.
- To facilitate further research into T. marneffei pathogenesis and host interactions.
Main Methods:
- Optimization of ATMT protocols for T. marneffei, including strain selection (EHA105 vs. AGL-1) and co-cultivation methods (solid vs. liquid).
- Utilized plasmid pDHt/acuD::pyrG for targeted gene disruption.
- Molecular analysis of transformants using Polymerase Chain Reaction (PCR), Southern blotting, and semi-quantitative Reverse Transcription PCR (RT-PCR).
Main Results:
- Agrobacterium tumefaciens strain EHA105 demonstrated higher efficiency in ATMT via solid co-cultivation compared to strain AGL-1.
- A liquid co-cultivation ratio of 1000:1 (A. tumefaciens:T. marneffei) yielded high transformation efficiency (90 transformants per 10^6 yeast cells).
- Successful disruption of the acuD gene and integration of the pyrG gene were confirmed by PCR and Southern blot; pyrG expression was verified by RT-PCR.
Conclusions:
- ATMT is a viable and efficient platform for targeted gene disruption in Talaromyces marneffei.
- Liquid co-cultivation presents a promising approach for enhancing transformation efficiency.
- These optimized methods pave the way for deeper understanding and potential therapeutic development for T. marneffei infections.
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