Evolutionary conservedcis-transregulation machinery for diterpenoid phytoalexin production in Poaceae

Abstract

SummaryMomilactones and phytocassanes are diterpenoid phytoalexins involved in plant chemical defense. These metabolites, along with biosynthetic gene clusters (BGCs), are conserved in wild rice. However, the mechanisms by which phytoalexins are regulated in wild rice are unclear. Thus, we aimed to investigate the regulatory mechanisms for biosynthetic genes within the BGCs of diterpenoid phytoalexins.We conducted a transcriptome analysis of five wild rice species,Oryza rufipogon,Oryza punctata,Oryza officinalis,Oryza brachyantha, andLeersia perrieri, after CuCl2treatment.Among the CuCl2-responsive transcription factors, diterpenoid phytoalexin factor (DPF), which regulates phytoalexin production in cultivated rice (Oryza sativa), was broadly conserved in wild rice and showed phytoalexin-inducing activity when introduced into cultivated rice. Highly conserved genomic regions containing N-boxes (5′-CACGAG-3′), the potential binding motif of DPF, were found. CRISPR/Cas9 genome editing to remove these regions showed that biosynthetic gene expression and phytoalexin production were significantly attenuated after CuCl2treatment in the leaves of the edited plants. Thus, thecis-transfactor combination of DPF and N-boxes is a key determinant of regulation.DPF has evolved as a strongcis-transregulatory system for diterpenoid phytoalexin production, with N-boxes generated within the cluster region during the evolution from wild rice to cultivated rice.