Summary One of the most devastating fungal diseases of soybean in the southern USA is Cercospora leaf blight (CLB), which is caused mainly by Cercospora cf. flagellaris. Recent studies found that the fungal effector AVR4, originally identified in Cladosporium fulvum as a chitin‐binding protein, is highly conserved among other Cercospora species. We wanted to determine whether it is present in C. cf. flagellaris and, if so, whether it plays a role in the pathogen infection of soybean. We cloned the Avr4 gene and created C. cf. flagellaris ∆avr4 mutants, which produced little cercosporin and significantly reduced expression of cercosporin biosynthesis genes. The ∆avr4 mutants were also more sensitive to chitinase and showed reduced virulence on soybean compared to the wild‐type. The observed reduced virulence of C. cf. flagellaris ∆avr4 mutants on detached soybean leaves is likely due to reduced cercosporin biosynthesis. The phenotypes of reduced cercosporin production and cercosporin pathway gene expression, similar to those of the ∆avr4 mutants, were reproduced when wild‐type C. cf. flagellaris was treated with double‐stranded RNA targeting Avr4 in vitro. These two independent approaches demonstrated for the first time the direct involvement of AVR4 in the biosynthesis of cercosporin.

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