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

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

Structural and mechanistic insights into the biosynthesis of CDP-archaeol in membranes

The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg(2+)-bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg(2+) enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.Cell Research advance online publication 29 September 2017; doi:10.1038/cr.2017.122

Correction:Structural and Functional Insights into an Archaeal Lipid Synthase

(Cell Reports 33, 108294-1–9.e1–e4; October 20, 2020) In the originally published version of this article, the supplemental information file containing Figures S1–S7 and Table S1 was inadvertently removed. The complete supplemental information file is now included with the paper online. The production team regrets this error

Structural and Functional Insights into an Archaeal Lipid Synthase

The UbiA superfamily of intramembrane prenyltransferases catalyzes an isoprenyl transfer reaction in the biosynthesis of lipophilic compounds involved in cellular physiological processes. Digeranylgeranylglyceryl phosphate (DGGGP) synthase (DGGGPase) generates unique membrane core lipids for the formation of the ether bond between the glycerol moiety and the alkyl chains in archaea and has been confirmed to be a member of the UbiA superfamily. Here, the crystal structure is reported to exhibit nine transmembrane helices along with a large lateral opening covered by a cytosolic cap domain and a unique substrate-binding central cavity. Notably, the lipid-bound states of this enzyme demonstrate that the putative substrate-binding pocket is occupied by the lipidic molecules used for crystallization, indicating the binding mode of hydrophobic substrates. Collectively, these structural and functional studies provide not only an understanding of lipid biosynthesis by substrate-specific lipid-modifying enzymes but also insights into the mechanisms of lipid membrane remodeling and adaptation

Overexpression of McHB7 Transcription Factor from Mesembryanthemum crystallinum Improves Plant Salt Tolerance

Mesembryanthemum crystallinum (common ice plant) is one of the facultative halophyte plants, and it serves as a model for investigating the molecular mechanisms underlying its salt stress response and tolerance. Here we cloned one of the homeobox transcription factor (TF) genes, McHB7, from the ice plant, which has 60% similarity with the Arabidopsis AtHB7. Overexpression of the McHB7 in Arabidopsis (OE) showed that the plants had significantly elevated relative water content (RWC), chlorophyll content, superoxide dismutase (SOD), and peroxidase (POD) activities after salt stress treatment. Our proteomic analysis identified 145 proteins to be significantly changed in abundance, and 66 were exclusively increased in the OE plants compared to the wild type (WT). After salt treatment, 979 and 959 metabolites were significantly increased and decreased, respectively, in the OE plants compared to the WT. The results demonstrate that the McHB7 can improve photosynthesis, increase the leaf chlorophyll content, and affect the TCA cycle by regulating metabolites (e.g., pyruvate) and proteins (e.g., citrate synthase). Moreover, McHB7 modulates the expression of stress-related proteins (e.g., superoxide dismutase, dehydroascorbate reductase, and pyrroline-5-carboxylate synthase B) to scavenge reactive oxygen species and enhance plant salt tolerance

Salinity-Induced Palmella Formation Mechanism in Halotolerant Algae Dunaliella salina Revealed by Quantitative Proteomics and Phosphoproteomics

Palmella stage is critical for some unicellular algae to survive in extreme environments. The halotolerant algae Dunaliella salina is a good single-cell model for studying plant adaptation to high salinity. To investigate the molecular adaptation mechanism in salinity shock-induced palmella formation, we performed a comprehensive physiological, proteomics and phosphoproteomics study upon palmella formation of D. salina using dimethyl labeling and Ti4+-immobilized metal ion affinity chromatography (IMAC) proteomic approaches. We found that 151 salinity-responsive proteins and 35 salinity-responsive phosphoproteins were involved in multiple signaling and metabolic pathways upon palmella formation. Taken together with photosynthetic parameters and enzyme activity analyses, the patterns of protein accumulation and phosphorylation level exhibited the mechanisms upon palmella formation, including dynamics of cytoskeleton and cell membrane curvature, accumulation and transport of exopolysaccharides, photosynthesis and energy supplying (i.e., photosystem II stability and activity, cyclic electron transport, and C4 pathway), nuclear/chloroplastic gene expression regulation and protein processing, reactive oxygen species homeostasis, and salt signaling transduction. The salinity-responsive protein-protein interaction (PPI) networks implied that signaling and protein synthesis and fate are crucial for modulation of these processes. Importantly, the 3D structure of phosphoprotein clearly indicated that the phosphorylation sites of eight proteins were localized in the region of function domain

Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination

Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the Arabidopsis brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12-and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a uniquemechanism of E3 ligase-substrate association regulated by phosphorylation

CPKs phosphorylate WRKYs and RBOHs.

<p>(<b>A</b>) Phosphorylation of WRKYs by CPK5 <i>in vitro</i>. MBP-WRKY fusion proteins were used as the substrates for GST-CPK5 in an <i>in vitro</i> kinase assay in the presence of 1 mM Ca²⁺. Phosphorylation was analyzed by autoradiography (top panel), and the protein loading was shown by Coomassie blue staining (CBS) (bottom panel). 5 m is a kinase-dead mutant of CPK5. (<b>B</b>) Phosphorylation of WRKYs by CPK11 <i>in vitro</i>. 11 m is a kinase-dead mutant of CPK11. (<b>C</b>) Phosphorylation of WRKY DNA binding domains by different CPKs <i>in vitro</i>. (<b>D</b>) T248 is required for WRKY48 DNA binding domain phosphorylation by CPKs <i>in vitro</i>. (<b>E</b>) WRKY48 T248 is phosphorylated by CPKs with MS analysis. Sequencing of a doubly charged peptide ion at m/z 531.22 that matches to CTpTVGCGVK of WRKY48. The confident b2 and b3 ions as well as y7 ion provide strong evidence for phosphorylation of the third Thr residue. (<b>F</b>) CPKacs phosphorylated RBOHD and RBOHF with an immunocomplex kinase assay. The FLAG-tagged CPKacs or the kinase-dead mutants (m) were expressed in protoplasts, and immunoprecipitated with an α-FLAG antibody for an <i>in vitro</i> kinase assay using GST-RBOHD or GST-RBOHF as a substrate. The proteins of RBOHD and RBOHF were shown, and the expression of individual CPKacs was detected by Western blot (bottom panel). (<b>G</b>) S148 is an essential phosphorylation site of RBOHD by CPKs <i>in vitro</i>. * indicates phosphorylated RBOHD. The numbers below indicate the relative phosphorylation level compared to WT RBOHD (set as 1) as quantified by Image J. The above experiments were repeated three times with similar results. The MS analysis was repeated twice.</p

The compromised immune responses in <i>cpk</i> mutants.

<p>(<b>A</b>) Effector-induced WRKY28 phosphorylation was abolished in <i>cpk5,6</i> mutant protoplasts. An in-gel kinase assay using fusion protein of MBP-WRKY28 DNA binding domain as a substrate was performed with protoplasts transfected with <i>AvrRpm1</i> or a control vector. The equal protein loading was shown by CBS. (<b>B</b>) The <i>cpk5,6</i> mutant plants were compromised in effector-mediated disease resistance. Plant leaves were hand-inoculated with <i>Pst avrRpm1</i> or <i>avrRpt2</i> at 5×10⁵ cfu/ml. The bacterial growth was measured 4 dpi. The data are shown as mean ± SE of three repeats, and the asterisk (*) indicates a significant difference with p<0.05 when compared with data from WT plants. (<b>C</b>) <i>Pst avrRpm1</i>-induced electrolyte leakage in plants. Plant leaves were hand-inoculated with <i>Pst avrRpm1</i> at 1×10⁸ cfu/ml, and leaf discs were excised at the indicated time points. The data are shown as the mean ± SE (n = 3) and the asterisk (*) indicates a significant difference between <i>cpk1,2,5,6</i> and WT (p<0.05). (<b>D</b>) Effector-induced <i>WRKY46</i> expression was reduced in <i>cpk</i> mutant plants. <i>WRKY46</i> expression was detected in plants 6 hr after hand-inoculation with bacteria at 1×10⁷ cfu/ml. The expression of <i>WRKY46</i> was normalized to the expression of <i>UBQ10</i>. The data are shown as the mean ± SE from three independent biological replicates. * indicates a significant difference with p<0.05 when compared with data from WT plants. (<b>E</b>) Effector-induced <i>SID2</i> expression was reduced in <i>cpk</i> mutant plants. (<b>F</b>) H₂O₂ production was compromised in the <i>cpk1,2</i> mutant plants. The leaves were hand-inoculated with H₂O, <i>Pst</i>, <i>Pst avrRpm1</i> and <i>avrRpt2</i> at 5×10⁷ cfu/ml, and excised at 24 hpi for DAB staining to detect H₂O₂ production. The above experiments were repeated three times with similar results.</p