Construction of a Normalized Library and Screening of Transcriptional Regulators of the cas5 Gene in Corynespora cassiicola

In tropical rubber-growing regions, Corynespora leaf fall disease stands as a predominant and economically significant threat to rubber trees. The toxin protein encoded by the cas5 gene is the main pathogenic factor of Corynespora cassiicola. To identify transcription factors capable of binding with the cas5 gene promoter sequence of C. cassiicola, the promoter of the cas5 gene was predicted by bioinformatics, and the 1000 bp promoter of the cas5 gene was isolated to construct a yeast one-hybrid bait vector for self-activation detection. A yeast one-hybrid cDNA expression library of C. cassiicola was constructed to screen for potential transcriptional regulators interacting with the 1000 bp promoter of the cas5 gene. The transcriptional regulators interacting with the cas5 gene were determined by the yeast one-hybrid (Y1H) point-to-point verification experiment. Y1H results showed that the bait vector did not have self-activation. The cDNA library had a titer of 2.516 × 108 cfu/mL and a total clone count of 5.032 × 108. Screening identified 30 candidate transcriptional regulators. Through point-to-point yeast one-hybrid verification, only one of the 30 candidate transcription factors (named CcbZIP3629) showed interaction with cas5. Molecular docking was performed using the AlphaFold3-predicted structure of CcbZIP3629, which revealed its binding to two ACGT core motifs within the promoter. These findings provide the groundwork for elucidating the regulatory mechanism of the cas5 gene, particularly by which CcbZIP3629 mediates the expression of the Cc-Cas5 toxin.