The MdERF1B-miR482c- <i>MdCCR4-MdTLP1b</i> module regulates apple resistance to <i>Colletotrichum gloeosporioides</i>

Abstract Glomerella leaf spot (GLS), incited by Colletotrichum gloeosporioides, poses a significant threat to apple (Malus domestica) production. microRNAs (miRNAs) are critical regulators of plant-pathogen crosstalk. However, the underlying mechanism through which miR482 regulates apple resistance to GLS remains unclear. Here, we show that overexpression of miR482c compromises resistance against C. gloeosporioides, while silencing miR482c confers resistance to C. gloeosporioides. 5’RACE assays in apple and co-expression assays in Nicotiana benthamiana confirmed that miR482c targets and cleaves the MdCCR4 mRNA. Subsequent research revealed that overexpression of MdCCR4 significantly enhances apple resistance to C. gloeosporioides. Notably, MdCCR4 physically interacts with the thaumatin-like protein MdTLP1b, which also positively regulates resistance to C. gloeosporioides. Additionally, MdERF1B was identified as an upstream regulator of miR482c through bioinformatics and Y1H screening, and was further confirmed to negatively regulate miR482c transcription by directly binding to its promoter. Meanwhile, MdERF1B overexpression enhances resistance to C. gloeosporioides, while silencing MdERF1B compromises resistance to C. gloeosporioides. Collectively, our findings indicate that the MdERF1B-miR482c-MdCCR4-MdTLP1b regulatory network is pivotal for apple defense against C. gloeosporioides, highlighting promising candidates for the genetic improvement of disease resistance.