P-170 - Genetic manipulation of the fungus Moniliophthora perniciosa, the causal agent of witches’ broom disease in cacao

Witches' broom disease (WBD), caused by the basidiomycete fungus Moniliophthora perniciosa, poses a significant threat to cacao production in the Americas. Despite advances in understanding the disease, the molecular mechanisms underlying M. perniciosa pathogenicity remain elusive, largely due to challenges in genetically manipulating the fungus. To overcome this barrier, we developed an efficient protoplast-based transformation protocol for M. perniciosa, now routinely used in our laboratory. Using this approach, we successfully introduced a hygromycin resistance marker into multiple M. perniciosa isolates and confirmed stable transformation by recovering fruiting bodies (mushrooms) and spores carrying the marker. Furthermore, we generated transformants expressing the mClover3 fluorescent reporter gene, facilitating live-cell imaging. To enable targeted gene disruption, we constructed a CRISPR/Cas9 plasmid carrying guide RNAs for genome editing. Additionally, we developed a ribonucleoprotein (RNP)-based approach for transgene-free mutagenesis. Our next goal is to generate the first M. perniciosa mutant strains to dissect key virulence determinants and advance our understanding of WBD pathogenesis. This work establishes a robust genetic toolkit for M. perniciosa, opening new avenues for functional genomics in a complex and agriculturally significant non-model pathosystem.