Identification and characterization of two P450 enzymes from involved in TMTT and DMNT biosyntheses and Asian citrus psyllid defense.

The homoterpenes (3)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (,)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) are the major herbivore-induced plant volatiles that help in defense directly by acting as repellants and indirectly by recruiting insects' natural enemies. In this study, DMNT and TMTT were confirmed to be emitted from citrus () leaves infested with Asian citrus psyllid ( Kuwayama; ACP), and two cytochrome P450 (CYP) genes ( and ) were newly identified and characterized. Understanding the functions of these genes in citrus defense will help plan strategies to manage huanglongbing caused by Liberibacter asiaticus (Las) and spread by ACP. Quantitative real-time PCR (qPCR) analysis showed that and were significantly upregulated in citrus leaves after ACP infestation. Yeast recombinant expression and enzyme assays indicated that CsCYP82L1 and CsCYP82L2 convert ()-nerolidol to DMNT and ()-geranyllinalool to TMTT. However, citrus calluses stably overexpressing generated only DMNT, whereas those overexpressing produced DMNT and TMTT. Furthermore, ACPs preferred wild-type lemon () over the -overexpressing line in dual-choice feeding assays and mineral oil over TMTT or DMNT in behavioral bioassays. Finally, yeast one-hybrid, electrophoretic mobility shift, and dual luciferase assays demonstrated that CsERF017, an AP2/ERF transcription factor, directly bound to the CCGAC motif and activated . Moreover, the transient overexpression of in lemon leaves upregulated in the absence and presence of ACP infestation. These results provide novel insights into homoterpene biosynthesis in and demonstrate the effect of homoterpenes on ACP behavior, laying a foundation to genetically manipulate homoterpene biosynthesis for application in huanglongbing and ACP control.