Abstract

Cucumber is an important horticultural vegetable crop with considerable potential for proanthocyanidin extraction. Flavonoids play crucial roles in plant defense and growth regulation, with MYB transcription factors and miRNAs being key regulators of their biosynthesis. In this study, we identified a differentially expressed miRNA , Cs-miR858 , in cucumber cultivars with contrasting flavonoid contents and root rot resistance. Cs-miR858 was found to negatively regulate the accumulation of Proanthocyanidin (PA) by targeting CsMYB60 , a key regulator of flavonoid biosynthesis. Overexpression of Cs-miR858 in cucumber via transient and stable transformation reduced PA content by downregulating structural genes such as CsLAR and increased susceptibility to Fusarium solani ( F. solani) . Conversely, silencing Cs-miR858 promoted PA biosynthesis and enhanced resistance. Molecular validation demonstrated that Cs-miR858 directly targets the coding sequence of CsMYB60 . Additionally, heterologous expression of Cs-miR858 in Arabidopsis inhibited anthocyanin and PA accumulation by targeting CsMYB60 . Furthermore, we found that CsBBX22 bound to the Cs-miR858 promoter and induced its expression, reducing PA accumulation in cucumber. Our findings unveil a miRNA-MYB regulatory module in which Cs-miR858 fine-tunes flavonoid metabolism by suppressing CsMYB60 , thereby modulating cucumber's resistance to F. solani . This study provides a theoretical basis for breeding new cucumber cultivars with high flavonoid content, strong disease resistance, and pronounced medicinal value.