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

In tropical rubber-growing regions, <i>Corynespora</i> leaf fall disease stands as a predominant and economically significant threat to rubber trees. The toxin protein encoded by the <i>cas5</i> gene is the main pathogenic factor of <i>Corynespora cassiicola</i>. To identify transcription factors capable of binding with the <i>cas5</i> gene promoter sequence of <i>C. cassiicola</i>, the promoter of the <i>cas5</i> gene was predicted by bioinformatics, and the 1000 bp promoter of the <i>cas5</i> gene was isolated to construct a yeast one-hybrid bait vector for self-activation detection. A yeast one-hybrid cDNA expression library of <i>C. cassiicola</i> was constructed to screen for potential transcriptional regulators interacting with the 1000 bp promoter of the <i>cas5</i> gene. The transcriptional regulators interacting with the <i>cas5</i> 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 <i>cas5.</i> 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 <i>cas5</i> gene, particularly by which CcbZIP3629 mediates the expression of the Cc-Cas5 toxin.