| Summary: | The RNA polymerase II (Pol II) transcription process is coordinated by the reversible phosphorylation of its largest subunit-carboxy terminal domain (CTD). Ssu72 is identified as a CTD phosphatase with specificity for phosphorylation of Ser5 and Ser7 and plays critical roles in regulation of transcription cycle in eukaryotes. However, the biofunction of Ssu72 is still unknown in <i>Aspergillus flavus</i>, which is a plant pathogenic fungus and produces one of the most toxic mycotoxins-aflatoxin. Here, we identified a putative phosphatase Ssu72 and investigated the function of Ssu72 in <i>A. flavus</i>. Deletion of <i>ssu72</i> resulted in severe defects in vegetative growth, conidiation and sclerotia formation. Additionally, we found that phosphatase Ssu72 positively regulates aflatoxin production through regulating expression of aflatoxin biosynthesis cluster genes. Notably, seeds infection assays indicated that phosphatase Ssu72 is crucial for pathogenicity of <i>A. flavus</i>. Furthermore, the Δ<i>ssu72</i> mutant exhibited more sensitivity to osmotic and oxidative stresses. Taken together, our study suggests that the putative phosphatase Ssu72 is involved in fungal development, aflatoxin production and pathogenicity in <i>A. flavus</i>, and may provide a novel strategy to prevent the contamination of this pathogenic fungus.
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