Tomato wall-associated kinase SlWak1 acts in an Fls2- and Fls3-dependent manner to promote apoplastic immune responses toPseudomonas syringae

Abstract

Wall-associated kinases (Waks) are known to be important components of plant immunity against various pathogens including Pseudomonas syringae pv. tomato (Pst) although their molecular mechanisms are largely unknown. In tomato, SlWak1 has been implicated in immunity because its transcript abundance increases significantly in leaves after treatment with the flagellin-derived peptides flg22 and flgII-28, which activate the receptors Fls2 and Fls3, respectively. We generated two SlWak1 tomato mutants (Dwak1) using CRISPR/Cas9 and investigated the role of SlWak1 in tomato-Pst interactions. PTI activated in the apoplast by flg22 or flgII-28 was 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 the ability to generate reactive oxygen species and activate MAPKs in response to flg22 and flgII-28. The 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/fls2.2/fls3 mutant plants, although both plants ultimately were similarly susceptible. SlWak1 co-immunoprecipitated with both Fls2 and Fls3 independently of flg22/flgII-28 or Bak1. These observations suggest that SlWak1 acts in a complex with Fls2/Fls3 and plays an important role at later stages of the PTI in the apoplast. We thank Robyn Roberts for comments on the manuscript and for sharing seeds of the Dfls2.1/fls2.2/fls3 line, Holly M. Roberts for assistance with vacuum infiltration and sequencing of the F2 populations, Brian Bell and Jay Miller for plant care, Joyce Van Eck for tomato transformation and Diana Lauff for assisting with experiments involving microscopy. References Anderson CM, Wagner TA, Perret M, He ZH, He D, Kohorn BD. 2001. WAKs: cell wallassociated kinases linking the cytoplasm to the extracellular matrix. Plant Mol Biol 47: 197-206. Bacete L, Melida H, Miedes E, Molina A. 2018. Plant cell wall-mediated immunity: cell wall changes trigger disease resistance responses. Plant J 93: 614-636. Bae S, Park J, Kim JS. 2014. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30: 1473-1475. Brinkman EK, Chen T, Amendola M, van Steensel B. 2014. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42: e168. Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G. 2010. A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci USA 107: 9452-9457. Chandra S, Martin GB, Low PS. 1996. The Pto kinase mediates a signaling pathway leading to the oxidative burst in tomato. Proc Natl Acad Sci USA 93: 13393-13397. Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nurnberger T, Jones JD, Felix G, Boller T. 2007. A flagellin-ind...

Receptor Kinases: A Sophisticate Protein Network to Maintain Plant life Continuity. An Update

“…In another study, it was demonstrated that wall associated kinase 1 in Solanum lycopersicum (SlWak1) plays an important role in pattern recognition receptors-triggered immunity (PTI) through transcription factor interference [32]. Recently in transcriptomic analysis, it has been found that in RP-type immune receptors regulation, EDS1, a conserved lipase like categorized as distinct subfamily known as receptor like cytoplasmic kinases (RLCKs) [4,6].…”