Transcriptomic, metabonomic and proteomic analyses reveal that terpenoids and flavonoids are required for Pinus koraiensis early defence against Bursaphelenchus xylophilus infection.

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, threatens Pinus seriously. Pinus koraiensis is one of the most important pine species in China and is the host for PWN. However, our understanding of the defence-regulating process following infection by B. xylophilus at the molecular level remains limited. To understand the mechanisms that P. koraiensis responds to B. xylophilus invasion, P. koraiensis was inoculated with B. xylophilus solutions and observed no obvious symptoms during the early stage; symptoms began to appear at 5 dpi. Therefore, we conducted comparative transcriptomic, metabonomic and proteomic analyses between P. koraiensis 5dpi and 0 dpi. In infected plants, 1574 genes were significantly up-regulated, including 17 terpenoid-, 41 phenylpropanoid- and 22 flavonoid-related genes. According to GO and KEGG enrichment analyses of significantly up-regulated genes, 86 GO terms and 16 KEGG pathways were significantly enriched. Most terms and pathways were associated with terpenoid-, phenylpropanoid-, flavonoid- and carbohydrate-related events. Similarly, the abundance of 36 and 30 metabolites, significantly increased in positive and negative polarity modes, respectively. Among them, naringenin and 3-methyl-2-oxovaleric acid exhibited significant toxic effects on B. xylophilus. According to functional analysis of significantly up-regulated metabolites, most terms were enriched in above pathways, in addition to alkaloid biosynthesis. Although the abundance of few proteins changed, response to stress term was significantly enriched in significant up-regulated proteins. Furthermore, plant receptor-like serine/threonine kinases, pectin methylation modulators, pinosylvin O-methyltransferase and arabinogalactan/proline-rich proteins were significantly up-regulated in the infected P. koraiensis compared to healthy plants. These proteins were not abundant in the healthy plant. Overall, these results indicate that P. koraiensis can actively response to PWN via various defense strategies, including events related to terpenoids, flavonoids, phenylpropanoids, lipids and alkaloids. Particularly, terpenoids and flavonoids are required for the early defence of P. koraiensis against B. xylophilus infection.