ZaCAD Positively Regulates Chinese pepper (Zanthoxylum armatum DC.) Resistance to Rust

Chinese pepper rust, mainly caused by a living saprophytic fungus Coleosporium zanthoxyli , can seriously reduce the yield and quality of Chinese pepper. The complex mechanism underlying Chinese pepper resistance to rust remains largely unknown. In our previous study, we performed a tranomic and metabolomics analysis of leaves of resistant and susceptible Chinese pepper varieties in response to C. zanthoxyli , and found a gene related to lignin synthesis in Z. armatum , namely cinnamyl alcohol dehydrogenase (ZaCAD) . RNAi and overexpression vectors were constructed through in vitro recombination, and silencing and overexpression transformants of both were obtained through Agrobacterium mediated transformation. The results from Real time fluorescence quantitative PCR (qRT-PCR) demonstrated that knocking down ZaCAD led to a marked decrease in both lignin and total flavonoid levels in Z. armatum . Conversely, its overexpression resulted in a heightened accumulation of these metabolites. This suggests that ZaCAD plays a crucial role in the biosynthesis of secondary metabolites in plants. In addition, six related genes of secondary metabolic pathways were selected for expression detection, which showed that the relative expression levels of ZaCAD and the six related genes were downregulated in silenced transgenic plants and upregulated in overexpressed transgenic plants. This further indicates that ZaCAD regulates the biosynthesis process of secondary metabolites by affecting the expression of secondary metabolic pathway genes. In order to verify the disease resistance level of transformants, after inoculation of pathogen spore suspension, the incidence rate and disease index of ZaCAD -RNAi transformants increased significantly, while the incidence rate and disease index of ZaCAD -overexpression transformants decreased. Subcellular localization shows that ZaCAD is located in the nucleus and cytoplasm. Finally, Y2H technology was used to demonstrate that ZaCAD interacts with GAPDH and MDH . These results strongly support that ZaCAD activates lignin production to positively regulate the resistance of plants against C. zanthoxyli .