Abstract
<jats:p> <jats:italic>Pseudocercospora fijiensis</jats:italic> is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of <jats:italic>P. fijiensis</jats:italic> and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for <jats:italic>P. fijiensis</jats:italic> . We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR/Cas9 to knockout the polyketide synthase gene <jats:italic>PKS10-1</jats:italic> , which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene <jats:italic>Fus3</jats:italic> . Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector <jats:italic>Avr4</jats:italic> in <jats:italic>P. fijiensis</jats:italic> . The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and <jats:italic>Avr4</jats:italic> knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that <jats:italic>Avr4</jats:italic> does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana- <jats:italic>P. fijiensis</jats:italic> interaction, thereby supporting the discovery of resistance genes against BLSD. </jats:p>