Abstract: Cucurbitacin B (CuB), a key compound responsible for the bitterness of melon fruit, significantly reduces fruit quality and consumer acceptance. Therefore, understanding the molecular mechanism underlying CuB biosynthesis is essential for improving melon fruit quality. In this research, we investigated CuB levels in the cmwrky13-knockout mutant following treatment with 20 mg L^-1 CPPU (20CPPU) and hand pollination. The CuB content decreased by approximately 30% in the cmwrky13-knockout mutant compared with wild type under 20CPPU treatment. Yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays demonstrated that CmWRKY13 activates the promoter of CmBr, a key regulator of bitterness, and physically interacts with CmBr. Moreover, the nuclear-localized transcription factor CmMYB6 was specifically induced by 20CPPU treatment. Interestingly, CmMYB6 binds to the CmWRKY13 promoter and interacts with its protein, synergistically enhancing CuB gene expression. The cmmyb6-knockout mutants confirmed that CmMYB6 positively regulates CuB accumulation in melon fruit. Furthermore, CRISPR/Cas9-generated cmmyb6 cmwrky13 double mutants revealed that the CmMYB6-CmWRKY13 module controls CmBr expression and regulates CuB accumulation. Collectively, these findings elucidate the transcriptional cascade of CuB accumulation mediated by the CmMYB6-CmWRKY13-CmBr module in melon fruit, providing new insights into the molecular basis of bitterness formation and offering theoretical guidance for the breeding of non-bitter melon varieties.

Key words: bitterness, CmBr, CmMYB6, CmWRKY13, oriental melon