Plant MAPK cascade performs a critical role in the regulation of plant immunity and disease resistance. Although the function of MAPK cascade in immunity regulation is partially conserved between different species, the mechanism varies in different host and pathogen combinations. To date, the MAPK cascade function of woody plants in the regulation of disease resistance has seldom been reported. Here, we present evidence to show that apple MdMAPKKK1 performed an important role in the regulation of apple resistance to Botryosphaeria dothidea, the causal agent of apple ring rot. B. dothidea infection leads to enhanced MdMAPKKK1 expression and MAPK cascade activation, indicating that the MAPK cascade is involved in the defense against B. dothidea. MdMAPKKK1 overexpression-induced pathogen-independent cell death. MdMAPKKK1 silencing decreases the resistance of apple calli and fruits to B. dothidea. Further analysis indicates that MdMAPKKK1 can bind MdBSK1 and is likely phosphorylated by it. The MdBSK1-mediated phosphorylation of MdMAPKKK1 is important for resistance to B. dothidea. These results collectively indicate that apple resistance to B. dothidea is regulated by the interaction between MAPKKK1 and MdBSK1.

Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is one of the most devastating apple diseases. Our previous study reported that the GLS resistance locus was defined on the chromosome 15 region. Here, we further found a single-nucleotide polymorphism (SNP) site (SNP_7309212) in the GLS resistance that was able to distinguish resistant cultivars (lines) from susceptible ones. On the basis of the SNP site, we cloned a TNL gene from the GLS resistant locus and named it MdTNL1 (NCBI Accession Number: ON402514). This gene contains a toll/interleukin-1 receptor transmembrane domain (TIR), nucleotide-binding sites (NBS), and leucine-rich repeat (LRR) domain. Subcellular location indicated that MdTNL1 was expressed in the nucleus and cell membrane. Ectopic overexpression of MdTNL1 in Nicotiana benthamiana caused cell death. We further demonstrated allelic polymorphisms in MdTNL1. It is noteworthy that NBS and LRR domains of the MdTNL1 protein serve as the repository for generating allelic diversity. Quantitative real-time PCR (qRT-PCR) assay revealed that MdTNL1 was highly expressed in resistant apple cultivar ‘Fuji’ after inoculation with C. fructicola, whereas susceptible cultivar ‘Golden Delicious’ exhibited low expression after inoculation. Over-expression of MdTNL1-1 in susceptible apple fruits and leaves improved disease resistance, while in ‘Orin’ calli, silencing the MdTNL1-1 gene conversely decreased GLS resistance. In conclusion, we identified a GLS associated with SNP_7309212 and demonstrated that a TIR-NBS-LRR gene MdTNL1-1 positively regulates GLS resistance in apple.