The wall-associated kinase (WAK) and wall-associated kinase like (WAKL) is a subfamily of receptor-like kinases associated with the cell wall, which have been suggested as sensors of the extracellular environment and triggers of intracellular signals. However, these proteins have not yet been comprehensively analyzed in tomato (Solanum lycopersicum L.). In this study, 11 SlWAK and 18 SlWAKL genes were identified in an uneven distribution in 9 of 12 chromosomes. GUB-WAK-bind (wall-associated receptor kinase galacturonan-binding) and epidermal growth factor (EGF) domains appear more often in SlWAK proteins. However, more SlWAKLs (wall-associated kinase like) have a WAK-assoc (wall-associated receptor kinase C-terminal) domain. Based on their phylogenetic relationships, 29 SlWAK-RLKs (wall associated kinase-receptor like kinases) were clustered into three distinct categories analogous to those in Arabidopsis thaliana. High similarities were found in conserved motifs of the genes within each group. Cis-elements in the promoter region of these 29 genes were found mainly in response to methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), anaerobic, light, wound, and MYB transcription factors. Public tomato genome RNA-seq data indicates that multiple SlWAK-RLKs showed different expression patterns under developmental and ripening stages of fruits, such as SlWAK4, SlWAKL11, SlWAKL9, SlWAKL15, SlWAKL14, and SlWAKL1, their RPKM (Reads Per Kilo bases per Million reads) value constantly increases during the fruit expansion period, and decreases as the fruit matures. In tomato leaves, our RNA-seq data showed that nine SlWAK-RLKs transcripts (SlWAK3, SlWAK4, SlWAK10,SlWAKL1, SlWAKL2, SlWAKL3, SlWAKL5, SlWAKL14, and SlWAKL18) were significantly induced (p < 0.001), and three transcripts (SlWAK2, SlWAK5, and SlWAKL15) were significantly inhibited (p < 0.001) under mechanical wounding. The qRT-PCR (Quantitative reverse transcription polymerase chain reaction) of SlWAKL1 and SlWAKL6 verify these results.

Jasmonates accumulate rapidly and act as key regulators in response to mechanical wounding, however few studies link receptor-like cytoplasmic kinases (RLCKs) to wound-induced JA signaling cascades. Here, we identified a novel wounding induced RLCK-XII-2 subfamily member in tomato (SlZRK1) that was closely related to Arabidopsis HOPZ-ETI-DEFICIENT 1 (ZED1)-related kinases 1 based on the phylogenetic analysis. SlZRK1 was targeted to the plasma membrane (PM) of tobacco mesophyll protoplasts as determined by transient co-expressing with PM marker mCherry-H +-ATPase. Catalytic residue sequence analysis and in vitro kinase assay indicated that SlZRK1 may act as a pseudokinase. To further analyze the function of SlZRK1, we developed two stable knock-out mutants by CRISPR/Cas9. Loss of SlZRK1 significantly altered expression of genes involved in jasmonic acid (JA) biosynthesis, salicylic acid (SA) biosynthesis, and ethylene response. Furthermore, after mechanical wounding treatment, slzrk1 mutants increased transcription of early wound inducible genes involved in JA biosynthesis and signaling. In addition, JA accumulation after wounding and plant resistance to herbivorous insects also were enhanced. Our findings expand plant regulatory networks in the wound-induced JA production by adding RLCKs as a new component in wound signal transduction pathway.