Abstract

Cross-kingdom RNA interference has emerged as an important mechanism in plant–pathogen interactions, yet how fungal small RNAs coordinate host immune suppression remains unclear. Here, we demonstrate that Alternaria alternata f. sp. mali , the causal agent of apple leaf spot, suggests microRNA-like RNAs (milRNAs) into host tissues in association with extracellular vesicles (EVs). Small RNA sequencing of fungal EVs identified multiple milRNAs, among which AamilR251 , AamilR292 and AamilR004 were the three most abundant species. These AamilRNAs target three apple ( Malus domestica ) immune-related kinases ( MdMAPKKK1 , MdRLK2 and MdMAPKK6 ). Molecular and genetic analyses revealed that these proteins form an interconnected signalling module and a linear phosphorylation cascade (MdRLK2-MdCRK10-MdMAPKKK1-MdMAPKK6) required for effective defence activation. Deletion of individual AamilRNAs significantly attenuated fungal virulence and restored expression of their respective host targets. Conversely, overexpression of MdMAPKKK1 , MdRLK2 or MdMAPKK6 enhanced resistance to infection. Pull-down assays identified MdCRK10 as an interacting partner of MdRLK2, forming a complex with MAPKs that activates defence signalling. MdRLK2 phosphorylates MdCRK10 at S144 and S363, and mutation of these sites disrupts signal transduction and compromises disease resistance. These findings reveal a central immune signalling network in apple and identify key components for improving resistance against fungal pathogens.