Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei
Piyan Zhang1, Bin Xu, Yuezhu Wang
1Radiation Oncology Department, Thomas Jefferson University, Philadelphia, PA 19107, USA. piyan.zhang@jeffersonhospital.org
Abstract:
Penicillium marneffei is an opportunistic fungal pathogen of humans, causing respiratory, skin, and systemic mycosis in south-east Asia. Here we describe the transformation of P. marneffei with Agrobacterium tumefaciens, and the optimization of the transformation procedure. Transformations in different combinations between A. tumefaciens stains (LBA4404 and EHA105) and binary vectors (pCB309A, pBI129A, and pCaMBIA1312A) showed that EHA105/pBI129A were the most efficient partners. Southern blot analysis suggested that 87.5% of transformants obtained with this protocol displayed single hybridization bands, indicating a single insert of T-DNA in each of the transformants. Unique hybridization patterns, along with thermal asymmetric interlaced PCR (TAIL-PCR) analysis of T-DNA insertion sites, suggested that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in P. marneffei. Several mutants with altered phenotypes were obtained during the construction of the mutant library, indicating the usefulness of the approach for functional genetic analysis in this important fungal pathogen.
Insights
Agrobacterium tumefaciens-mediated transformation was optimized for Penicillium marneffei, an opportunistic fungal pathogen. This method enables insertional mutagenesis for functional genetic analysis of P. marneffei.
Area of Science:
- Mycology
- Molecular Biology
- Genetics
Background:
- Penicillium marneffei is an opportunistic fungal pathogen causing significant human mycosis in Southeast Asia.
- Understanding its genetic mechanisms is crucial for developing effective treatments.
Purpose of the Study:
- To optimize Agrobacterium tumefaciens-mediated transformation for Penicillium marneffei.
- To establish a method for insertional mutagenesis and functional genetic analysis.
Main Methods:
- Optimization of transformation using different Agrobacterium tumefaciens strains (LBA4404, EHA105) and binary vectors (pCB309A, pBI129A, pCaMBIA1312A).
- Southern blot analysis to confirm T-DNA integration.
- Thermal Asymmetric Interlaced PCR (TAIL-PCR) to analyze T-DNA insertion sites.
Main Results:
- The combination of EHA105/pBI129A demonstrated the highest transformation efficiency.
- 87.5% of transformants exhibited single hybridization bands, indicating single T-DNA inserts.
- TAIL-PCR analysis confirmed unique insertion patterns, suggesting potential for insertional mutagenesis.
Conclusions:
- Agrobacterium tumefaciens-mediated transformation is an effective tool for genetic manipulation of Penicillium marneffei.
- This optimized protocol facilitates insertional mutagenesis for functional genetic studies of this important fungal pathogen.
