Identification of the WAK/WAKL gene family in melon and expression analysis in response to Phytophthora capsici infection

【Objective】As a crucial class of receptor-like protein kinases, genes in the WAK/WAKL family play a vital role in plant growth and development, disease resistance, and stress responses. The aim of this study was to identify the MeWAK/WAKL family in melon, analyze their expression patterns in resistant and susceptible materials under Phytophthora capsici infection, and explore the relationship between this gene family and melon's resistance to Phytophthora blight.【Methods】The melon ZQK9 and E31 are high- resistant and high-susceptible resources to P. capsici, respectively, and they were identified through disease resistance screening in our laboratory. These materials were used to analyze geneexpression patterns after inoculation with P. capsici. The pathogen was isolated from diseased melon plants collected in Sanya, Hainan, China. The melon genome data were referenced from Melon (DHL92) v4.0. Based on the conserved domain features, the melon WAK/WAKL family members were identified through genome-wide screening. Subsequent analyses including gene structure, protein physicochemical properties, chromosome distribution, subcellular localization prediction, promoter cis- acting elements, and phylogenetic relationships were conducted by a serious of softwares and visualized using TBtools software. Quantitative real-time PCR (qRT-PCR) was employed to investigate the expression patterns of family members in resistant (ZQK9) and susceptible (E31) samples at 2, 24 and 72 hours post-inoculation (hpi) with P. capsici, aiming to elucidate their correlation with disease resistance in melon.【Results】The WAK/WAKL family in melon comprised 20 genes, including 4 WAK genes and 16 WAKL genes, which were distributed across 9 melon chromosomes. Among them, 7 genes were clustered on chromosome 3, suggesting that these genes might function through coordinated expression. The results of subcellular localization prediction indicated that the WAK/WAKL family genes in melon were mainly localized in the extracellular or plasma membrane, consistent with reports in other crops. The phylogenetic tree divided 113 WAK/WAKL family genes from melon and model plants such as Nicotiana benthamiana, Arabidopsis thaliana, and Solanum lycopersicum into 6 groups. The MeWAK/WAKL family genes in melon were mainly distributed in group I and VI. Group I was independently composed of 7 MeWAKL genes, indicating that the members of the MeWAK/WAKL family in melon might have undergone strong natural selection (such as extreme environments) during the evolutionary process, showing a phenomenon of rapid evolution. In addition, 6 of these 7 genes were clustered on chromosome 3. This clustering might lead to functional redundancy, but it could also reduce the negative impacts caused by structural variations such as chromosome breakage in individuals under strong external pressure, thus being retained in evolution. Domain analysis showed that the WAK/WAKL family genes in melon contained 1 to 7 exons. Among the 20 family genes, 17 genes contained the GUB-WAK bind (wall-associated receptor kinase galacturonan-binding) domain. Seven genes contained the GUB-WAK bind, EGF (epidermal growth factor), and serine-threonine kinase domains simultaneously. Seven genes only contained the GUB-WAK bind domain, and 2 genes contained both EGF domain and the kinase domain without GUB-WAK bind domain. Analysis of promoter cis-acting elements revealed that the promoters of the WAK/WAKL family genes in melon contained light-responsive elements (LTR), regulatory element related to meristem expression (MEE), response elements for various hormones (MeJA, SA, ABA, Auxin, and GA), as well as elements related to drought-inducibility and defense and stress responsiveness. This suggested that the WAK/WAKL family genes in melon might play important roles in disease resistance, stress tolerance, and growth and development of melon. Two genes, MeWAKL4 and MeWAKL16, which were specifically up- regulated after inoculation in resistant melon ZQK9, were screened through qRT- PCR analysis. MeWAKL4 was a transmembrane protein containing the GUBWAK bind domain, WAK_assoc domain, and kinase domain, might play an important role in recognizing pathogen infection signals and transmitting them into the cell. The MeWAKL16 protein had only one GUB-WAK bind domain, was localized on the plasma membrane, and had a transmembrane structure. Based on the resistance mechanism of the maize gray leaf spot resistance gene ZmWAKL, we speculated that MeWAKL16 might play a role in recognizing pathogen infection signals and transmitting the signals into the cell by forming a complex with other kinase(s) on the plasma membrane. The detail regulatory mechanism required further experimental research.【Conclusion】In this study, 20 MeWAK/ WAKL family genes in melon were identified. Through gene domain and promoter cis-acting element analyses, it could be speculated that MeWAKs/WAKLs could respond to various hormone or stress signals and regulate the plant's disease resistance, stress tolerance, and growth and development processes. Two genes, MeWAKL4 and MeWAKL16, were screened via qRT-PCR and showed specific up- regulation at 24 and 72 hpi in resistant melon ZQK9, while their expression levels exhibited no significant differences in susceptible E31. They might be involved in the signal pathway of melon's resistance to Phytophthora blight. This study laid a foundation for further research on the specific relationship between melon MeWAK/WAKL family genes and the resistance response to Phytophthora blight.