Tomato Wall-Associated Kinase SlWak1 Depends on Fls2/Fls3 to Promote Apoplastic Immune Responses to Pseudomonas syringae

Abstract

Wall-associated kinases (Waks) are important components of plant immunity against various pathogens, including the bacterium Pseudomonas syringae pv. tomato (Pst). However, the molecular mechanisms of their role(s) in plant immunity are largely unknown. In tomato (Solanum lycopersicum), wall-associated kinase 1 (SlWak1), has been implicated in pattern recognition receptor (PRR)-triggered immunity (PTI) because its transcript abundance increases significantly after treatment with the flagellin-derived, microbe-associated molecular patterns flg22 and flgII-28, which activate the PRRs Fls2 and Fls3, respectively. We generated two SlWak1 tomato mutants (Dwak1) using CRISPR/Cas9 gene editing technology and investigated the role of SlWak1 in tomato-Pst interactions. Late PTI responses activated in the apoplast by flg22 or flgII-28 were compromised in Dwak1 plants, but PTI at the leaf surface was unaffected. The Dwak1 plants developed fewer callose deposits than wild-type plants, but retained early PTI responses such as generation of reactive oxygen species and activation of mitogen-activated protein kinases upon exposure to flg22 and flgII-28. Induction of Wak1 gene expression by flg22 and flgII-28 was greatly reduced in a tomato mutant lacking Fls2 and Fls3, but induction of Fls3 gene expression by flgII-28 was unaffected in Dwak1 plants. After Pst inoculation, Dwak1 plants developed disease symptoms more slowly than Dfls2.1/2.2/3 mutant plants, although ultimately, both plants were similarly susceptible. SlWak1 coimmunoprecipitated with both Fls2 and Fls3, independently of flg22/flgII-28 or of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1. These observations suggest that SlWak1 acts in a complex with Fls2/Fls3 and is important at later stages of PTI in the apoplast.

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2

“…The presumed orthologs of BAK1 in tomato are Serk3A/Serk3B, but this has not been experimentally investigated. Additionally, Fls2 and Fls3 both coimmunoprecipitate with Wak1 independently of flg22/ flgII-28 or Arabidopsis BAK1 (Zhang et al, 2020a). It is possible that Fls3 and Fls2 may interact differently with the Serks, and that will be a focus of future studies.…”

“…Fls2 and Fls3 appear to contribute equally to PTI, which suggests that they may use some similar mechanisms and/or signaling pathways. In fact, we reported recently that activation of the Fls2 or Fls3 pathways induces the expression of the wall-associated kinase Wak1 gene, and both PRRs coimmunoprecipitate with the Wak1 protein, which appears to act independently of ROS production and at a later stage of PTI (Zhang et al, 2020a). However, the differences in several readouts from the Fls2 and Fls3 pathways also suggest underlying differences in some aspect of their functions.…”

Genome-Wide Identification, Classification, Characterization, and Expression Analysis of the Wall-Associated Kinase Family during Fruit Development and under Wound Stress in Tomato (Solanum lycopersicum L.)

“…Wounding induced WAK-RLKs maybe as an injury signal receptor quickly participated in plant defense response paralleled or combined with other RLKs pathway. Recently, Zhang et al found tomato SlWAK1 (named SlWAK4 in our study) coimmunoprecipitated with both pattern recognition receptors Fls2 and Fls3, acting in a complex and playing an important role during later stages of pattern-triggered immunity in the apoplast [ 19 ].…”

“…For example, SlWAK5 , SlWAK6, SlWAK7, and SlWAK8; SlWAKL6, SlWAKL7, SlWAKL8, SlWAKL9, SlWAKL10, SlWAKL11, and SlWAK12 have the same motifs respectively. This phenomenon indicates that genes from the same cluster may share extremely similar functions [ 19 ].…”

“…In another study, a reporter construct containing the OsWAK11 promoter fused to GUS is activated in response to wounding [ 18 ]. In tomato, Zhang et al generated two homozygous wak1 mutant lines using CRISPR/Cas9, and found that Wak1 protein acted as an important positive regulator in later stages of flagellin-mediated PTI (PAMP-triggered immunity) response in the apoplast and formed a complex with Fls2 and Fls3 to trigger immune signaling [ 19 ].Genome sequences can provide valuable information for genome-based investigation, such as gene cloning and genome-wide analysis of a target gene family [ 20 ]. WAK-RLKs had been investigated using global identification techniques and genome analysis in some plants.…”