The <scp>miR159a</scp> ‐ <scp> <i>MYB33</i> </scp> module induces male sterility by regulating polygalacturonase‐mediated anther dehiscence in watermelon

Heterosis, characterized by enhanced resistance and yield, has been widely utilized in watermelon breeding. However, our understanding of the regulatory mechanisms underlying male-sterile phenotypes in watermelon remains limited. Here, we determined that the miR159a targets ClMYB33 to regulate anther dehiscence, leading to male sterility in watermelon. Both overexpression of Cl-miR159a (OE-miR159a) and knockout of ClMYB33 (clmyb33) in watermelon suppressed the degradation of septum and stomium tissues, thereby impairing anther dehiscence and preventing successful pollen release. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, ClPG1 and ClQRT2 were identified as downstream target genes of ClMYB33; both were positively regulated by ClMYB33. Both ClPG1 and ClQRT2 exhibited polygalacturonase (PG) activity in vivo. The knockout of ClQRT2 led to reduced PG activity and a failure in anther dehiscence. Furthermore, the GST-ClQRT2 fusion protein was capable of rescuing the indehiscent anther phenotype observed in both OE-miR159a and clmyb33 plants. Our results reveal a new mechanism by which the miR159a-ClMYB33 module regulates anther dehiscence by mediating PG activity, and provide a new molecular tool to create male sterility in watermelon.