Differential innate immune signalling via Ca(2+) sensor protein kinases

Abstract

Innate immunity represents the first line of inducible defence against microbial infection in plants and animals. In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively), such as flagellin, initiates convergent signalling pathways involving mitogen-activated protein kinase (MAPK) cascades and global transcriptional changes to boost immunity. Although Ca(2+) has long been recognized as an essential and conserved primary mediator in plant defence responses, how Ca(2+) signals are sensed and relayed into early MAMP signalling is unknown. Using a functional genomic screen and genome-wide gene expression profiling, here we show that four calcium-dependent protein kinases (CDPKs) are Ca(2+)-sensor protein kinases critical for transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in the synthesis of defence peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by the 22-amino-acid peptide flg22, as well as compromised pathogen defence. In contrast to negative roles of calmodulin and a calmodulin-activated transcription factor in plant defence, the present study reveals Ca(2+) signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.

Figure 1. Functional genomic screen for CDPKs in early flg22 signalling

a, Flg22 stimulates endogenous CDPK activities. Calcium-dependent histone phosphorylation, abolished by EGTA, is stimulated by flg22 at 15 min. b, NHL10 is an early flg22 responsive gene. c, Flg22 activation of NHL10-LUC is dependent on the FLS2 receptor. The fls2 mutant is complemented by FLS2-HA. Error bars, s.d. (n=8). d, Flg22 signalling requires calcium and PK activity. Inhibitors: 1 mM LaCl₃ (La), 0.5 mM GdCl₃ (Gd), 2 μM K252a (K). Error bars, s.d. (n=6). e, Specific CPKac can activate NHL10-LUC. Error bars, s.d. (n=6). Mesophyll protoplasts (a–d) were treated with 100 nM flg22. The expression level of FLS2-HA and CPKac-FLAG was monitored by immunoblot.

Figure 2. Transcriptome profiling of CPK5ac and CPK11ac target genes in flg22, multiple MAMP and microbial signalling

a, Hierarchical clustering analysis of CPK5ac and CPK11ac target genes and early flg22 responsive genes. The genes were identified from protoplasts transiently expressing constitutively active CPK5 or CPK11, protoplasts (P30 and P60), leaves (L60) and seedlings (sd30 and sd60) treated with flg22 for 30 and 60 min. b, Diverse MAMPs activate CPK5ac and CPK11ac target genes. ABA treatment was used as a control. c, Different microbes activate CPK5ac and CPK11ac target genes. Psp: Pseudomonas syringae pv. phaseolicola, Pi: Phytophthora infestans, Bc: Botrytis cinerea. Genes are clustered into 3 groups: shared by CPK5 and CPK11 (upper panel), CPK5-specific (middle panel) and CPK11-specific (lower panel). The number of genes co-regulated by CPK5ac and/or CPK11ac is indicated at the bottom of each panel.

Figure 3. Interplay between CDPK and MAPK cascades in flg22 signalling

CDPKs and MAPKs differentially regulate four flg22-responsive gene programs. The induction of endogenous genes was monitored by qRT-PCR in protoplasts expressing individual kinases (a) or co-expressing CPKac and MKKa (b). CPKac and MKK4a: constitutively active kinases, CPK5acKM: inactive kinase mutant. Error bars, s.d. (n=6). Marker gene details are shown in the Supplementary Information.

Figure 4. CDPKs are critical positive regulators in flg22 signalling

a, Flg22 induction of gene expression is diminished in cpk mutants. Gene expression was analyzed by qRT-PCR in protoplasts treated with 2 nM flg22 for 30 min. Error bars, s.d. (n=8). b, The cpk mutants exhibit impaired flg22-induced oxidative burst. RLU: relative light units. Error bars, s.d. (n=8). c, The cpk mutants are impaired in flg22-induced resistance to the bacterial pathogen Pst DC3000. Seedlings pre-treated with flg22 were inoculated with Pst DC3000 3 days before bacterial count. Error bars, s.d. (n=6). * Significantly different from WT +flg22 (p<0.001). d, Model for convergent activation of specific CDPKs and MAPK cascades in initial MAMP signalling.