Functional characterization of PCRK1, a putative protein kinase with a role in immunity

Abstract

In Arabidopsis, defense signaling is triggered by the perception of conserved molecular patterns by pattern recognition receptors (PRRs). Signal transduction from the PRRs requires members of a family of Receptor-Like Cytoplasmic Kinases (RLCKs). Previously, we described one such RLCK, PTI Compromised Receptor-Like Cytoplasmic Kinase 1 (PCRK1) that is important for immunity induced by Microbe Associated Molecular Patterns (MAMPs) as well as Damage Associated Molecular Patterns (DAMPs). In this study, we measured the growth of Pma ES4326 in double mutants carrying pcrk1 together with the salicylic acid (SA) biosynthesis mutation sid2–2 or the jasmonic acid (JA) receptor mutation coi1–1, showing that the function of PCRK1 is SA independent but may be partially dependent on JA. Mutation of phosphorylated serine residues S232, S233 and S237 compromised the immune signaling function of PCRK1.

Plants recognize evolutionarily conserved molecular signatures called Microbe Associated Molecular Patterns (MAMPs) and endogenous signals generated as a consequence of pathogen activity called Damage Associated Molecular Patterns (DAMPs). Perception of MAMPs or DAMPs by pattern recognition receptors (PRRs) triggers several physiological changes that collectively induce immunity called Pattern Triggered Immunity (PTI). PRRs are cell surface receptors belonging to the Receptor-Like Kinase family (RLKs). The RLK family includes the Receptor-Like Cytoplasmic Kinases (RLCKs), of which several members, including BIK1, PBS1, PBL1, PBL27 and RIPK, play important roles in immune signal transduction.^1-6

Previously, we demonstrated that PCRK1, a member of the RLCK subfamily VII, is important for defense against Pseudomonas syringae and that it plays an important role in PTI elicited by multiple MAMPs.⁷ Mutation of a conserved lysine that forms part of the ATP binding pocket in other RLCKs resulted in failure to complement a pcrk1–1 null allele. Here, we show that certain phosphorylated serine (Ser) residues in PCRK1 are important for its function in immunity. We also show that the function of PCRK1 is independent of SA signaling but may be partially dependent on JA signaling.

Phosphorylation of Specific Residues of PCRK1 is Important for its Function

Other RLCKs have been shown to be phosphorylated and the phosphorylation of certain residues is important for their function in defense responses.⁸ A Phosphoproteomics study identified phosphorylated PCRK1(S385) peptide from a membrane fraction of cells after flg22 and xylanase treatment.⁹ PCRK1 was also shown to be autophosphorylated in a study using a wheat germ cell-free translation system to profile Arabidopsis proteins.¹⁰ Serine residues S373 and S377 were predicted to be phosphorylated in addition to S385 (PhosphAt database http://phosphat.mpimp-golm.mpg.de/).¹¹ PCRK1 was also identified in a screen of phosphorylated membrane proteins. In this study, S373, S377 and S385 were found using tandem mass spectrometry (MS-MS) to be phosphorylated (MH and BM, unpublished results).

Since multiple lines of evidence suggested that PCRK1 is phosphorylated, we tested if phosphorylation of PCRK1 is important for its function in defense signaling. We made a phosphomimetic version of PCRK1 by substituting the 3 serines at positions 373, 377 and 385 with aspartate residues (S373D, S377D, and S385D) and a non-phosphorylatable version by substituting the serines with alanine (S373A, S377A and S385A). We then monitored the growth of Pma ES4326 in transgenic lines expressing the phosphomimetic or the non-phosphorylatable versions of PCRK1 expressed from its native promoter in a pcrk1–1 background. Transgenic lines carrying the phosphomimetic version of PCRK1 fully complemented pcrk1–1 for bacterial growth while the lines carrying the non-phosphorylatable version of PCRK1 failed to complement the pcrk1–1 phenotype suggesting that phosphorylation of serine at positions 373, 377 or 385 is important for the function of PCRK1 in immunity (Fig. 1).

Figure 1.

Phosphorylation of specific residues of PCRK1 is important for its function. Growth of Pma ES4326 was measured in independent transgenic lines expressing the phosphomimetic (PM) or the non-phosphorylatable version of PCRK1 (NP) expressed from the native promoter in a pcrk1–1 background. Four weeks old plants were inoculated with Pma ES4326 (OD600 = 0.0001) and the bacterial titer was determined at 0 and 3 d post infection (dpi). Data were obtained from 2 independent experiments with 4 or 12 biological replicates for each genotype at 0 or 3 dpi respectively. Bars represent means and standard error calculated using a mixed linear model. Different letters indicate significant differences, p < 0.01. The experiment was conducted as described previously.⁷

In RLCKs such as BIK1, several phosphorylated serine residues within the kinase domain are known to be important for function.^2,4,8 The serine residues important for PCRK1 functions are however from the C-terminal end of the protein, outside the predicted kinase domain. Unlike the kinase domains, the C-terminal regions of RLKs are highly variable.¹² In RLKs such as BRASSINOSTEROID-INSENSITIVE1 (BRI1), substitution of Ser and threonine (Thr) residues from the C-terminal end resulted in reduced substrate phosphorylation without affecting the autophosphorylation activity of the protein.¹² Thus phosphorylation of residues in the C-terminal end of the protein may affect a PCRK1 specific signaling activity.

PCRK1 function is independent of SA but partially dependent on JA

Many mutants with enhanced susceptibility to bacterial pathogens have reduced salicylic acid (SA)-dependent signaling.¹³ However, SA levels in pcrk1 mutants are similar to wild type plants.⁷ The enhanced bacterial growth in pcrk1 mutants might be due to changes in SA signaling, and not SA levels per se. To test this possibility, we made homozygous double mutants carrying pcrk1 and sid2–2, a mutation blocking SA biosynthesis.¹⁴ Since jasmonic acid (JA) is also an important immune signaling hormone, we decided to test for a role of PCRK1 in JA signaling by making homozygous double mutants carrying pcrk1 and coi1–1, a mutation in the JA receptor.^15,16 Growth of Pma ES4326 in each genotype was determined 3 d after inoculation. The effects of pcrk1 and sid2–2 were additive, suggesting that PCRK1 functions in an SA-independent manner. The pcrk1 coi1–1 double mutants were significantly more susceptible to Pma ES4326 than coi1–1, but the increase in susceptibility conferred by pcrk1 in the coi1–1 background was significantly less than in the wild type background, suggesting that the contribution of PCRK1 to immunity is partially JA-dependent (Fig. 2).Thus, it is possible that PCRK1 promotes immunity partly through an effect on JA signaling.

Figure 2.

Bacterial growth assay in pcrk1 mutant lines in comparison with an SA biosynthetic mutant (sid2–2), a JA receptor mutant (coi1–1) and double mutants of pcrk1 with sid2–2 and coi1–1. Four weeks old plants were inoculated with Pma ES4326 (OD600 = 0.0001) and the bacterial titer was determined at 0 and 3 dpi. Each bar represents data from at least 3 independent experiments each containing at least 4 and 12 replicates for 0 and 3 dpi, respectively. Means and standard error were estimated by a mixed linear model. Different letters indicate significant differences (p < 0.01). The experiment was conducted as described previously.⁷

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Greg Barrett-Wilt, Mass Spectrometry Facility, Biotechnology Center, University of Wisconsin-Madison, for mass spectrometric instrumentation, and Michael R. Sussman of the Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 for support of the mass spectrometry study. We thank Fumiaki Katagiri of the Department of Plant Biology, University of Minnesota, for statistical analysis.

Funding

This work was supported by grants IOS-0925375 and MCB-0929395 from the National Science Foundation to J.G. and Michael R. Sussman, respectively. B.B.M. was supported by NHGRI training grant 5T32HG002760 to the Genomic Science Training Program.