Melatonin Enhances Panax vietnamensis Resistance to Leaf Blight Pathogen Neofusicoccum ribis via the PvWRKY40-PvCOMT2 Module-Driven Lignin Biosynthesis

Abstract

Panax vietnamensis, a medicinally valuable perennial herb, is highly susceptible to leaf blight under cultivation; however, the molecular mechanisms underlying this disease remain poorly understood. In this study, we identified Neofusicoccum ribis as the causal agent of P. vietnamensis leaf blight through pathogen isolation and fulfilment of Koch's postulates. Transcriptomic analysis revealed activation of phytohormone signalling (salicylic acid, jasmonic acid, and melatonin [MT]) and phenylpropanoid metabolism during infection. Among these, MT exhibited superior efficacy in inducing lignin biosynthesis compared to other hormones, with exogenous application of MT significantly enhancing lignin accumulation and improving disease resistance by 8 days post-inoculation. Further, we identified PvWRKY40 as a negative regulator of lignin synthesis, which directly binds to the W-box motif in the PvCOMT2 promoter to suppress its expression. MT counteracted this repression by downregulating PvWRKY40. Heterologous overexpression of PvCOMT2 in Nicotiana benthamiana increased lignin content and conferred enhanced resistance to Fusarium oxysporum. This study reveals a novel MT-PvWRKY40-PvCOMT2 regulatory axis governing lignin-mediated defence in P. vietnamensis, providing critical insights for combating leaf blight in cultivated ginseng.

Keywords: Neofusicoccum ribis; Panax vietnamensis; leaf blight; lignin biosynthesis; melatonin.

FIGURE 1

Neofusicoccum ribis is the pathogenic agent of Panax vietnamensis leaf blight. (a) Colony morphology of N. ribis on potato dextrose agar. (b) Scanning electron micrographs of N. ribis conidia and hyphal structures. (c) Phylogenetic identification of N. ribis. (d) Disease progression in P. vietnamensis leaves inoculated with N. ribis at 0, 2, 4, 6, and 8 days post‐inoculation (dpi). Scale bar: 1 cm.

FIGURE 2

Transcriptomic profiling of Panax vietnamensis during Neofusicoccum ribis infection. (a) Number of differentially expressed genes (DEGs) identified at 0, 2, 4, 6, and 8 days post‐inoculation (dpi). (b) Venn diagram analysis of DEGs across infection time points. (c) Gene Ontology (GO) enrichment analysis of DEGs. (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs.

FIGURE 3

Lignin and phytohormone accumulation in Panax vietnamensis during Neofusicoccum ribis infection. (a) Transcriptional profiles of lignin biosynthesis‐related genes, values: log₂(fold change). (b) Reverse transcription‐quantitative PCR (RT‐qPCR) validation of lignin biosynthesis gene expression in P. vietnamensis . (c) Transcriptional profiles of salicylic acid (SA), jasmonic acid (JA), and melatonin (MT) biosynthesis genes, value: log₂(fold change). (d) RT‐qPCR validation of SA, JA, and MT biosynthesis gene expression. (e) Temporal changes in lignin content in P. vietnamensis leaves at 0, 2, 4, 6, and 8 days post‐inoculation (dpi), S (syringyl lignin), G (guaiacyl lignin), H (para‐hydroxy‐phenyl lignin), and temporal changes in hormone content: MT (f), SA (g) and JA (h) at 0, 2, 4, 6, and 8 dpi.

FIGURE 4

Melatonin enhances lignin biosynthesis to improve Neofusicoccum ribis resistance in Panax vietnamensis. (a) Expression of lignin biosynthesis genes under salicylic acid (SA) treatment 0–12 h. (b) Expression of lignin biosynthesis genes under jasmonic acid (JA) treatment 0–12 h. (c) Expression of lignin biosynthesis genes under melatonin (MT) treatments 0–12 h. (d) The expression levels of lignin synthesis genes after 0–8 days of MT treatment. (e) Lignin accumulation dynamics after MT‐, JA‐, SA‐treated leaves. (f) Disease resistance assay of MT‐treated plants at 8 days, left: control, right: MT treatment. (g) Lignin content in MT‐treated calli. (h) The expression levels of lignin biosynthesis gene in MT‐treated calli.

FIGURE 5

PvWRKY40 is a potential regulator of melatonin (MT)‐mediated lignin biosynthesis in Panax vietnamensis. (a) Schematic of promoter cis‐elements in lignin biosynthesis genes. (b) Quantification of cis‐element types and numbers. (c) Transcriptional profiles of differentially expressed WRKY transcription factors (TFs) in the transcriptome of P. vietnamensis leaves after Neofusicoccum ribis infection, log₂ (fold change). (d) Reverse transcription‐quantitative PCR validation of WRKY TF expression during N. ribis infection. (e) Temporal expression of WRKY TFs after MT treatment (0–12 h). (f) PvWRKY40 expression dynamics under MT treatment (0–8 days post‐inoculation [dpi]). (g) PvWRKY40 expression in MT‐treated calli.

FIGURE 6

PvWRKY40 represses PvCOMT2 expression by binding W‐box motifs in its promoter. (a) Dual‐luciferase reporter assay schematic. (b) PvWRKY40 suppresses PvCOMT2 (but not PvCOMT1) expression. (c) Melatonin (MT) alleviates PvWRKY40‐mediated repression of PvCOMT2. (d) Probe design and sequences for electrophoretic mobility shift assay. (e) PvWRKY40 specifically binds W‐box motifs in the PvCOMT2 promoter.

FIGURE 7

PvCOMT2 enhances lignin biosynthesis and disease resistance in transgenic Nicotiana benthamiana. (a) Semiquantitative analysis of PvCOMT2 expression in transgenic lines. (b) Protein accumulation of PvCOMT2 in transgenic plants. (c) Lignin content in PvCOMT2‐overexpressing plants. (d) Disease resistance assay of PvCOMT2 transgenic plants.

FIGURE 8

The disease‐resistance mechanism diagram of Panax vietnamensis. After infection Neofusicoccum ribis, P. vietnamensis activates the synthesis of melatonin (MT). The elevation in the endogenous melatonin content inhibits the expression level of PvWRKY40, thereby relieving the inhibition of PvWRKY40 on PvCOMT2. Ultimately, this results in an increase in lignin accumulation level and enhances the physical resistance of P. vietnamensis to N. ribis.