Two terpene synthases in resistant Pinus massoniana contribute to defense against Bursaphelenchus xylophilus

Pine wood nematode (PWN; Bursaphelenchus xylophilus), a destructive pest of Pinus massoniana, is causing a severe epidemic of pine wilt disease in China. When invaded by PWN, resistant P. massoniana secrete an abundance of oleoresin terpenoids as a defensive strategy. However, regulatory mechanisms of this defense in resistant P. massoniana have yet to be elucidated. Here, we characterized two terpene synthase genes, α-pinene synthase (PmTPS4) and longifolene synthase (PmTPS21) identified in resistant P. massoniana and investigate the contribution of these genes to the oleoresin defense strategy in resistant masson pines. Upregulation of these two genes in the stem supported their involvement in terpene biosynthesis as part of the defense against PWN. Recombinant protein expression revealed catalytic activity for the two PmTPSs, with PmTPS4 primarily producing α-pinene, while PmTPS21 produced α-pinene and longifolene simultaneously. The major enzymatic products of the two terpene synthases had inhibitory effects on PWN in vitro. We demonstrated that PmTPS4 and PmTPS21 played positive roles in terpene-defense mechanisms against PWN infestation. The major products of these terpene synthases could directly inhibit the survival rate of PWN in vitro. We revealed that PmTPS21 was a novel bifunctional enzyme capable of simultaneous production of both monoterpene and sesquiterpene. This article is protected by copyright. All rights reserved.

Keywords: Bifunctional enzyme; Defense mechanism; Pine wood nematode; Pinus massoniana; Resistance; Signal-specific expression; Terpene synthases.