Both the Extracellular Leucine-Rich Repeat Domain and the Kinase Activity of FLS2 Are Required for Flagellin Binding and Signaling in Arabidopsis

Abstract

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17 , which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli . This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex. INTRODUCTIONMany biological recognition phenomena involve ligands and receptors. For example, the critical steps of pathogen recognition in plants appear to involve interactions of plant receptors with microbial ligands, the so-called elicitors, and the induction of a phosphorylation cascade that leads to defense responses (Boller, 1995;Yang et al., 1997). One of the most active bacterially derived elicitor molecules in plant cells is flagellin (Felix et al., 1999), which induces rapid defense responses (Gómez-Gómez et al., 1999) and triggers the activation of a phosphorylation cascade (Nühse et al., 2000) in Arabidopsis plants. We recently identified a gene involved in flagellin perception, FLS2 , by positional cloning (Gómez-Gómez and Boller, 2000). FLS2 encodes a receptor kinase with an extracellular domain composed of 28 tandem leucine-rich repeats (LRRs) and an intracellular serine/threonine protein kinase domain. Interestingly, the encoded receptor kinase is homologous with the rice resistance gene Xa21, which provides resistance to Xanthomonas oryzae (Song et al., 1995), and the extracellular LRR domain of FLS2 resembles resistance genes from the tomato Cf gene family that provide resistance to various strains of Cladosporium fulvum (Jones et al., 1994; Dixon et al., 1996), suggesting that the recognition pathway for general elicitors such as flagellin may resemble the pathway involved in the highly specific resistance defined by gene-for-gene interactions (Jones and Jones, 1996). In insects and mammals, members of the family of TOL...

PhosphoThr Peptide Binding Globally Rigidifies Much of the FHA Domain from Arabidopsis Receptor Kinase-Associated Protein Phosphatase,

“…The prominence and mobility of the 8/9 loop may be important in KAPP's recognition of several RLK targets in plants. Arabidopsis KAPP interacts in a phosphorylationdependent manner with the following receptor-like kinases critically important to plant development and defense against infection: CLV1 (81), HAESA (82), WAK1 (18), BAK1 (20), SERK1 (21), and FLS2 (19). KAPP being promiscuous enough to attenuate multiple RLK-dependent signaling pathways in plants is supported moreover by KAPP being a single gene product without any known paralog of overlapping function (17).…”

“…KAPP has a C-terminal protein phosphatase 2C (PP2C) domain that dephosphorylates RLK partners to attenuate the activation of the receptor complex (15). KAPP was shown to interact with these RLKs from Arabidopsis that include HAESA (formerly RLK5) (14), CLAVATA1 (16), RLK4 (17), TMK1 (17), WAK1 (18), FLS2 (19), BAK1 (20), and SERK1 (21). KAPP uses its kinase-interacting FHA domain (KI-FHA) to bind epitopes of RLKs activated by phosphorylation of serine or threonine residues.…”

A Cysteine-Rich Extracellular Protein, LAT52, Interacts with the Extracellular Domain of the Pollen Receptor Kinase LePRK2[W]

“…A brassinosteroid is the ligand for the LRR kinase BRI1 (He et al, 2000). Flagellin, a bacterial elicitor, is the ligand for the LRR kinase FLS2 (Gomez-Gomez et al, 2001). In pollen, the best characterized LRR kinases have an extracellular domain composed of five or six LRRs, a transmembrane domain, a variable domain, and a cytoplasmic kinase domain (Mu et al, 1994;Muschietti et al, 1998).…”

Deciphering the Responses of Root Border-Like Cells of Arabidopsis and Flax to Pathogen-Derived Elicitors

“…However, to our knowledge, this is the first report of the detection of 1 O 2 in Arabidopsis root cells in response to a MAMP. To check for the specificity of the response, we have analyzed ROS production in Arabidopsis flagellinsensitive2 (fls2) and brassinosteroid insenstive1-associated kinase (bak1)-3 mutants, lacking a functional flg22 receptor, FLS2, and an associated receptor kinase, BAK1, respectively (Gómez-Gómez et al, 2001;Chinchilla et al, 2007). The response of root border-like cells to flg22 was completely abolished in the fls2 mutant (Supplemental Fig.…”