A virulent <scp>milRNA</scp> inhibits host immunity by silencing a host receptor‐like kinase <scp>MaLYK3</scp> and facilitates infection by <i>Fusarium oxysporum</i> f. sp. <i>cubense</i>

MicroRNA-like RNAs (milRNAs) play a significant role in the infection process by plant-pathogenic fungi. However, the specific functions and regulatory mechanisms of fungal milRNAs remain insufficiently elucidated. This study investigated the function of Foc-milR138, an infection-induced milRNA secreted by Fusarium oxysporum f. sp. cubense (Foc), which is the causal agent of Fusarium wilt of banana. Initially, through precursor gene knockout and phenotypic assessments, we confirmed that Foc-milR138 acts as a virulent milRNA prominently upregulated during the early stages of Foc infection. Subsequent bioinformatic analyses and transient expression assays in Nicotiana benthamiana leaves identified a host receptor-like kinase gene, MaLYK3, as the direct target of Foc-milR138. Functional investigations of MaLYK3 revealed its pivotal role in triggering immune responses of N. benthamiana by upregulating a suite of resistance genes, bolstering reactive oxygen species (ROS) accumulation and callose deposition, thereby fortifying disease resistance. This response was markedly subdued upon co-expression with Foc-milR138. Expression pattern analysis further verified the specific suppression of MaLYK3 by Foc-milR138 during the early root infection by Foc. In conclusion, Foc secretes a virulent milRNA (Foc-milR138) to enter the host banana cells and inhibit the expression of the plant surface receptor-like kinase MaLYK3, subverting the disease resistance activated by MaLYK3, and ultimately facilitating pathogen invasion. These findings shed light on the roles of fungal milRNAs and their targets in resistance and pathogenicity, offering promising avenues for the development of disease-resistant banana cultivars.