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The fungus Stemphylium lycopersici (S. lycopersici) is an economically important plant pathogen that causes grey leaf spot disease in tomato. However, functional genomic studies in S. lycopersici are lacking, and the factors influencing its pathogenicity remain largely unknown. Here, we present the first example of genetic transformation and targeted gene replacement in S. lycopersici. We functionally analyzed the NLP gene, which encodes a necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein (NLP). We found that targeted disrup