A Csi‐ miR3954b ‐ CsCE70/CsPLA1 Regulatory Module Enhances Citrus Fruit Resistance to Penicillium digitatum by Promoting Phenolic Acid and Flavonoid Biosynthesis

ABSTRACT Green mould is one of the major postharvest diseases affecting citrus. Although evidence indicates that microRNAs (miRNAs) participate in plant defence responses, their roles and regulatory mechanisms in citrus resistance to green mould remain unclear. Small RNA sequencing identified several defence‐associated miRNAs involved in citrus resistance to Penicillium digitatum . Among them, transient overexpression screening identified Csi‐miR3954b as the most effective miRNA, reducing disease incidence by 24.4% compared with the control at 5 days post‐inoculation (dpi). Subsequent transient overexpression and short tandem target mimic (STTM)‐mediated suppression assays demonstrated that Csi‐miR3954b positively regulates citrus resistance to green mould, with transient overexpression reducing disease incidence by 22.7% and STTM‐mediated suppression increasing it by 9.6% at 5 dpi. Degradome sequencing, transient assays in Nicotiana benthamiana leaves and expression analysis identified CsCE70 and CsPLA1 as the direct targets of Csi‐miR3954b. Transcriptome analysis showed that CsCE70 and CsPLA1 mainly regulated phenolic acid and flavonoid biosynthesis, respectively. Transient overexpression of CsCE70 and CsPLA1 increased disease incidence by 17.4% and 10.4%, respectively, compared with the control at 5 dpi, suggesting that both genes negatively regulated citrus resistance to P. digitatum . Silencing these genes enhanced its resistance, accompanied by the accumulation of phenolics and flavonoids in the peel. In general, the results showed that the Csi‐miR3954b– CsCE70/CsPLA1 regulatory modules enhanced citrus resistance to P. digitatum by promoting the biosynthesis of phenolic acids and flavonoids. These results provide new insights into miRNA‐mediated postharvest disease resistance and potential molecular strategies for improving citrus postharvest protection.