The molecular mechanism of miRN65-PlWAT1 in resisting Paeonia lactiflora black spot disease by increasing lignin content

Paeonia lactiflora Pall., is a perennial herbaceous plant with high ornamental value, yet its cut-flower industry is severely limited by stem bending and disease susceptibility, particularly black spot caused by Alternaria alternata . This study explored molecular improvement strategies by leveraging the precise regulatory role of miRNAs. Using multiple small RNA and degradome datasets, a de novo analysis was conducted to construct a peony miRNA database. The regulatory module miRN65– PlWAT1 was identified. Dual-luciferase assays confirmed that PlWAT1 is a direct target of miRN65 and is negatively regulated by it. qRT-PCR showed that upon A. alternata inoculation for 48 h, miRN65 expression increased significantly while PlWAT1 expression decreased markedly in leaves. Functional analysis of transgenic materials revealed that PlWAT1 overexpression in tobacco enhanced physiological indices, increased lignin content, enlarged xylem radial width and thick-walled cell layers, reduced lesion area after inoculation, and elevated SOD, POD, CAT activities. Conversely, PlWAT1 silencing in peony decreased lignin content, narrowed xylem, enlarged lesions, and lowered antioxidant enzyme activities. The phenotypes induced by miRN65 overexpression in peony were consistent with PlWAT1 silencing. Collectively, miRN65 negatively regulates lignin accumulation by post-transcriptionally suppressing PlWAT1 transcript abundance, thereby playing a key role in peony resistance to black spot disease. These findings provide new insights for genetic improvement of cut-flower quality and disease resistance. • Established P. lactiflora miRNA database via small RNA and degradome sequencing. • Identified miRN65- PlWAT1 module, validated by multiple assays. • miRN65- PlWAT1 enhances P. lactiflora black spot resistance via lignin.