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Published on: September 30, 2016
Gene Replacement by a Selectable Marker in the Filamentous FungusMagnaporthe oryzae
Nalleli Garcia1, Alexa N Farmer1, Richmond Baptiste1
1Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
Abstract:
Magnaporthe oryzaeis a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain. Key features Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.
Insights
This study details a split marker method for creating gene knockout mutants in Magnaporthe oryzae, a key fungus causing rice blast disease. This genetic manipulation technique aids in understanding plant-pathogen interactions.
Area of Science:
- Plant Pathology
- Mycology
- Molecular Biology
Background:
- Magnaporthe oryzae causes significant global rice crop losses.
- M. oryzae is a model organism for studying plant-pathogen interactions due to its genetic tractability.
- Existing gene deletion methods include CRISPR-Cas9 and homologous recombination.
Purpose of the Study:
- To provide a detailed protocol for gene replacement in M. oryzae using a split marker method.
- To generate knockout mutants of M. oryzae for further research.
Main Methods:
- The protocol involves replacing a gene's open reading frame with a selectable marker gene.
- Key steps include primer design, polyethylene glycol (PEG)-mediated protoplast transformation, and fungal DNA extraction.
- The split marker technique facilitates the identification of successful gene replacement events.
Main Results:
- Successful generation of M. oryzae knockout mutants is achieved through the described gene replacement protocol.
- The method allows for disruption of target gene transcription, leading to loss-of-function phenotypes.
- The protocol is comprehensive, covering essential molecular and genetic manipulation techniques.
Conclusions:
- The split marker method offers a reliable approach for generating gene knockout mutants in M. oryzae.
- This protocol enhances the study of M. oryzae's pathogenicity and host interactions.
- The described techniques are crucial for advancing research on the rice blast fungus.
