Integrated Analysis of <scp>GC</scp> – <scp>MS</scp> ‐Based Metabolomics and Proteomics Reveals the Importance of Volatile Metabolite Stigmasterol in the Defence Response of <i>Panax notoginseng</i> Against Root Rot

Root rot disease in Panax notoginseng, primarily caused by the pathogenic fungus Fusarium solani, significantly impacts the growth and production of this medicinal herb. To elucidate the defence mechanisms of P. notoginseng against root rot, we employed proteomics and gas chromatography-mass spectrometry (GC-MS)-based metabolomics analyses. These analyses revealed significant accumulations of metabolites involved in phenylpropanoid, terpenoid and steroid biosynthesis pathways in F. solani-infected P. notoginseng roots. This accumulation correlated with the up-regulation of synthetases in these pathways as indicated by proteomics data. Focusing on stigmasterol, a representative steroid with differential accumulation levels, and its biosynthesis gene PnCYP710A, we investigated the role of stigmasterol metabolism in the defence response against root rot. Stigmasterol exhibited significant inhibitory effects on spore germination and hyphal growth of F. solani. Furthermore, PnCYP710A was up-regulated upon F. solani infection and induced by hormonal signals such as methyl jasmonate (MeJA). Overexpression of PnCYP710A in tobacco enhanced resistance to F. solani, up-regulated expression of JA biosynthesis/signalling pathway-related genes, increased accumulation of stigmasterol/lignin/callus, and maintained reactive oxygen species homeostasis during F. solani infection. Conversely, RNA interference (RNAi) of PnCYP710A in P. notoginseng yielded opposite effects. Additionally, PnWRKY4 positively regulated the transcription level of PnCYP710A by binding to its promoter. In summary, this study not only identifies volatile metabolites and proteins involved in the defence response of P. notoginseng against root rot but also discovers that PnWRKY4 activates stigmasterol biosynthesis to resist root rot pathogen infection.