Bifurcation of Arabidopsis NLR Immune Signaling via Ca2+-Dependent Protein Kinases

Abstract

Nucleotide-binding domain leucine-rich repeat (NLR) protein complexes sense infections and trigger robust immune responses in plants and humans. Activation of plant NLR resistance (R) proteins by pathogen effectors launches convergent immune responses, including programmed cell death (PCD), reactive oxygen species (ROS) production and transcriptional reprogramming with elusive mechanisms. Functional genomic and biochemical genetic screens identified six closely related Arabidopsis Ca2+-dependent protein kinases (CPKs) in mediating bifurcate immune responses activated by NLR proteins, RPS2 and RPM1. The dynamics of differential CPK1/2 activation by pathogen effectors controls the onset of cell death. Sustained CPK4/5/6/11 activation directly phosphorylates a specific subgroup of WRKY transcription factors, WRKY8/28/48, to synergistically regulate transcriptional reprogramming crucial for NLR-dependent restriction of pathogen growth, whereas CPK1/2/4/11 phosphorylate plasma membrane-resident NADPH oxidases for ROS production. Our studies delineate bifurcation of complex signaling mechanisms downstream of NLR immune sensors mediated by the myriad action of CPKs with distinct substrate specificity and subcellular dynamics.

The Pseudomonas syringae Type III Effector AvrRpt2 Promotes Pathogen Virulence via Stimulating Arabidopsis Auxin/Indole Acetic Acid Protein Turnover

“…We coexpressed epitopetagged TIR1 or AXR with AvrRpt2 under the control of the cauliflower mosaic virus 35S promoter in Arabidopsis wild-type Columbia (Col-0) protoplasts. Protein expression was detected 6 h after transfection, prior to the occurrence of AvrRpt2-induced cell death in wildtype protoplasts (Gao et al, 2013). As shown in Figure 1A, the protein level of AXR2 was significantly reduced upon treatment with 1 mM 1-naphthalacetic acid (NAA), a synthetic auxin, which is consistent with the previous finding that perception of auxin leads to the degradation of AXR2 (Gray et al, 2001).…”

“…7B). Elicitation of AvrRpt2-RPS2 signaling is often accompanied by the transcriptional activation of an array of defense-associated genes, including WRKY46 (Gao et al, 2013). Consistently, expression of either AXR2 or axr2-1 in protoplasts did not alter AvrRpt2-mediated pWRKY46::LUC reporter activity.…”

“…The AvrRpt2 in planta expression vector construct, pIAA5::LUC, and pGH3:: LUC and pWRKY46::LUC reporter constructs were reported previously (Chen et al, 2007;Gao et al, 2013). AXR2, AXR3, or TIR1 was amplified from complementary DNA of wild-type Col-0 with the following primers (the restriction enzyme sites are underlined, and the start codon is italicized) to clone into the protoplast expression vectors: AXR2-F-BamH1, 59-CGGGATCCAT-GATCGGCCAACTTATGAA-39; AXR2-R-Stu1, 59-GAAGGCCTAGATCTGT-TCTTGCAGTACT-39; AXR3-F-BamH1, 59-CGGGATCCATGATGGGCAGTG-TCGAG-39; AXR3-R-Stu1, 59-GAAGGCCTAGCTCTGCTCTTGCACT-39; TIR1-BamH1-F, 59-CGGGATCCATGCAGAAGCGAATAGCCTT-39; TIR1-Stu1-R, 59-GAAGGCCTTAATCCGTTAGTAGTAATGA-39.…”

Genome-wide analysis and expression profiling of calcium-dependent protein kinases in potato (Solanum tuberosum)

“…Thus, tomato LeCDPK2 regulates ethylene biosynthesis in response to wound signalling (Kamiyoshihara et al 2010); Arabidopsis AtCPK1/2/4/5/11 phosphorylate and thereby activate NADPH oxidase to promote reactive oxygen species (ROS) production in response to abiotic and biotic stimuli (Gao et al 2013); rice OsCPK4 participates in rice tolerance to salt and drought stress by protecting cellular membranes from lipid peroxidation (Campo et al 2014), grapevine gene VaCPK29 confers tolerance to heat and osmotic stresses (Dubrovina et al 2017), whereas ZmCPK11 is involved in touch-and wound-induced pathways in maize (Szczegielniak et al 2012). CDPKs are widely involved in various types of disease resistance (Boudsocq et al 2010;Boudsocq and Sheen 2013;Romeis and Herde 2014;Wang et al 2015).…”

PBL13 is a serine/threonine protein kinase that negatively regulates Arabidopsis immune responses.

“…In light of our finding that PBL13 associates with RBOHD in N. benthamiana, we hypothesize that, in the absence of pathogen stimuli, PBL13 may associate with RBOHD and inhibit ROS production. Treatment with flg22 disrupts this complex, potentially enabling BIK1, PBL1, and calcium-dependent protein kinases to positively regulate RBOHD activity (Kobayashi et al, 2007;Dubiella et al, 2013;Gao et al, 2013;Kadota et al, 2014;Li et al, 2014). The requirement of these sequential events for RBOHD activation could ensure elegant control of the timing and amplitude of the extracellular ROS burst.…”