Overexpression of Anthocyanidin Reductase Increases Flavonoids Content to Combat Fusarium Wilt in the Root Xylem of <i>Vernicia montana</i>

Tung wilt disease, caused by Fusarium oxysporum f. sp. fordiis (Fof-1), poses a serious threat to tung oil tree (Vernicia fordii) production. Fortunately, another native Vernicia species in China, V. montana (woody tung oil tree) exhibits high resistance ability to the pathogen. The resistant and susceptible tung trees provide material for the investigation on the mechanism underlying the resistance to Fusarium wilt disease. Root xylem extracts of resistant V. montana significantly inhibited Fof-1 growth compared with that of susceptible V. fordii. Metabolomic analysis of V. montana root xylem revealed that 13 types of flavonoids increased after Fof-1 infection. Of the 13 flavonoids, antimicrobial assays showed that catechin, (-)-epicatechin and (-)-epigallocatechin exhibited an obvious inhibitory effect on Fof-1 growth. Transcriptomic analysis revealed that several genes with up-regulated expression patterns were also enriched in the flavonoid biosynthesis pathway after Fof-1 infection in V. montana. Among them, the anthocyanidin reductase (ANR) gene is directly involved in the biosynthesis of antimicrobial (-)-epicatechin and (-)-epigallocatechin. Moreover, transgenic V. montana lines overexpressing the VmANR gene elevated eight types of flavonoid concentrations, and silencing VmANR resulted in a substantial reduction in the levels of catechin and myricitrin. The enzyme activity assay in vitro further verified that VmANR catalysed the formation of (-)-epigallocatechin from the substrate cyanidin. This study identifies VmANR as a critical gene to promote biosynthesis of antimicrobial flavonoids in shaping resistance to Fof-1 infection, and offers an effective strategy for breeding Fusarium-resistant tung oil trees.