Can Wang, 
Xiaolong Hao, 
Yao Wang, 
Itay Maoz, 
Wei Zhou, 
Zhigang Zhou, 
Guoyin Kai 

Camptothecin (CPT) is a chemotherapeutic drug widely used to treat various cancers. Ophiorrhiza pumila is an ideal model for a camptothecin-producing plant with various advantages for studying camptothecin biosynthesis and regulation. The DNA binding protein WRKY transcription factors have a key regulatory role in secondary metabolite biosynthesis in plants. However, their involvement in camptothecin biosynthesis in O. pumila is still in its infancy. We identified 46 OpWRKY genes unevenly distributed within 11 chromosomes of O. pumila. Phylogenetic and multiple sequence alignment analyses divided the OpWRKY proteins into three subfamilies. Based on spatial and co-expression, we targeted a candidate gene, OpWRKY6. Overexpression of OpWRKY6 significantly reduced the accumulation of camptothecin compared to the control. Conversely, compared to the control, camptothecin accumulation increased in OpWRKY6 knock-out lines. Further biochemical assays showed that OpWRKY6 negatively regulates camptothecin biosynthesis, from both iridoid and shikimate pathways, by directly down-regulating the gene expression of OpGES, Op10HGO, Op7DLH, and OpTDC. Our data provide direct evidence to the involvement of WRKYs in the regulation of camptothecin biosynthesis and offer valuable information for the enrichment production of camptothecin in plant systems.