MdMYB1R1 Activates MdMAPKKK18 and Additional Defense Pathways to Confer Resistance to Woody Fungal Pathogens in Apple

Trunk diseases caused by woody plant pathogens, including Valsa mali and Botryosphaeria dothidea, represent a major threat to global apple production; however, the molecular basis of host resistance remains largely elusive. In this study, we employed TMT-based quantitative proteomic profiling to compare necrotic and uninfected twig tissues of apple following V. mali inoculation, identifying 904 differentially expressed proteins (DEPs). Among these, the MYB transcription factor, MdMYB1R1, exhibited a pronounced induction in response to pathogen infection. Subcellular localization and transactivation assays revealed that MdMYB1R1 localizes to the nucleus and acts as a transcriptional activator. Transient overexpression of MdMYB1R1 in Nicotiana benthamiana potentiated reactive oxygen species (ROS) accumulation and activated MAPK signaling, correlating with enhanced resistance against Sclerotinia sclerotiorum. Stable overexpression of MdMYB1R1 in apple markedly enhanced resistance to both V. mali and B. dothidea, whereas silencing of MdMYB1R1 impaired disease tolerance. Through DNA affinity purification sequencing (DAP-seq), yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assays, we demonstrated that MdMYB1R1 binds to the promoter region of MdMAPKKK18 and activates its transcription, a gene encoding a mitogen-activated protein kinase kinase kinase involved in MAPK signaling. Genetic epistasis analysis further revealed that overexpression of MdMAPKKK18 only partially rescued the compromised resistance in MdMYB1R1-RNAi plants. Collectively, our findings identify MdMYB1R1 as a key positive regulator of apple resistance against trunk fungal diseases, acting at least in part by directly activating the MdMAPKKK18-mediated MAPK cascade and by regulating additional defense pathways, thus providing a promising candidate for breeding disease-resistant apple cultivars.