A reverse genetic approach for generating gene replacement mutants in Ustilago maydis

Area of Science:

• Molecular Biology
• Mycology
• Plant Pathology

Background:

• Ustilago maydis is a significant phytopathogenic fungus impacting agriculture.
• Efficient genetic engineering tools are crucial for studying fungal pathogenicity and developing control strategies.
• Existing methods for gene replacement in U. maydis may lack versatility for complex genetic manipulations.

Purpose of the Study:

• To establish a versatile strategy for generating gene replacement mutants in Ustilago maydis.
• To provide a system with multiple options for genetic engineering, including gene disruption and reporter gene insertion.
• To demonstrate the utility of the system by creating specific gene replacement mutants.

Main Methods:

• Development of a gene replacement system utilizing 32 distinct insertion cassettes.
• Employing PCR-amplified flanking sequences ligated to insertion cassettes via SfiI sites for homologous recombination.
• Generation of replacement mutants where the mfa1 promoter drives Green Fluorescent Protein (GFP) expression, regulated by heterologous promoters (crg1 or nar1).

Main Results:

• Successful generation of two gene replacement mutants in Ustilago maydis.
• Demonstrated pheromone-inducible expression of GFP driven by the endogenous mfa1 promoter.
• Confirmed that mfa1 expression, when controlled by heterologous promoters (crg1, nar1), was only detected under specific nutrient conditions.

Conclusions:

• The described versatile strategy provides a robust platform for genetic engineering in Ustilago maydis.
• This system facilitates sophisticated genetic manipulations, including the study of gene regulation and promoter function.
• The generated mutants serve as valuable tools for investigating fungal biology and gene expression in U. maydis.