ABSTRACT
Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1-resistance complex, resulting in ETI activation.
Subject(s)
Acetyltransferases/immunology , Arabidopsis Proteins/immunology , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/immunology , Carrier Proteins/immunology , Phosphotransferases/metabolism , Pseudomonas syringae/immunology , Acetyltransferases/metabolism , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Blotting, Western , Carrier Proteins/metabolism , Chromatography, Liquid , Cloning, Molecular , Cluster Analysis , Immunoprecipitation , Phosphotransferases/genetics , Phylogeny , Pseudomonas syringae/enzymology , Surface Plasmon Resonance , Tandem Mass Spectrometry , Two-Hybrid System TechniquesABSTRACT
The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.
Subject(s)
Acetyltransferases/metabolism , Arabidopsis/microbiology , Bacterial Outer Membrane Proteins/metabolism , Cytoskeleton/metabolism , Microtubules/metabolism , Pseudomonas syringae/pathogenicity , Acetylation , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/immunology , Arabidopsis Proteins/metabolism , Cell Line , HEK293 Cells , Humans , Phytic Acid/metabolism , Plant Diseases/microbiology , Pseudomonas syringae/enzymology , Pseudomonas syringae/genetics , Tubulin/metabolismABSTRACT
Induction of intracellular and secreted acid phosphatases (APases) is a widespread response of orthophosphate (Pi)-starved (-Pi) plants. APases catalyze Pi hydrolysis from a broad range of phosphomonoesters at an acidic pH. The largest class of nonspecific plant APases is comprised of the purple APases (PAPs). Although the biochemical properties, subcellular location, and expression of several plant PAPs have been described, their physiological functions have not been fully resolved. Recent biochemical studies indicated that AtPAP26, one of 29 PAPs encoded by the Arabidopsis (Arabidopsis thaliana) genome, is the predominant intracellular APase, as well as a major secreted APase isozyme up-regulated by -Pi Arabidopsis. An atpap26 T-DNA insertion mutant lacking AtPAP26 transcripts and 55-kD immunoreactive AtPAP26 polypeptides exhibited: (1) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (2) a 40% decrease in secreted APase activity during Pi deprivation, (3) 35% and 50% reductions in free and total Pi concentration, respectively, as well as 5-fold higher anthocyanin levels in shoots of soil-grown -Pi plants, and (4) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function occurred under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. Transient expression of AtPAP26-mCherry in Arabidopsis suspension cells verified that AtPAP26 is targeted to the cell vacuole. Our results confirm that AtPAP26 is a principal contributor to Pi stress-inducible APase activity, and that it plays an important role in the Pi metabolism of -Pi Arabidopsis.
Subject(s)
Acclimatization , Acid Phosphatase/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/physiology , Glycoproteins/metabolism , Phosphates/deficiency , Acclimatization/drug effects , Acid Phosphatase/genetics , Alleles , Arabidopsis/cytology , Arabidopsis/drug effects , Arabidopsis Proteins/genetics , DNA, Bacterial/genetics , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Plant/drug effects , Glycoproteins/genetics , Intracellular Space/drug effects , Intracellular Space/enzymology , Isoenzymes/metabolism , Mutagenesis, Insertional/drug effects , Mutagenesis, Insertional/genetics , Mutation/genetics , Phosphates/pharmacology , Protein Transport/drug effects , Reproducibility of Results , Seedlings/drug effects , Seedlings/enzymology , Seedlings/growth & development , Up-Regulation/drug effects , Vacuoles/drug effects , Vacuoles/enzymologyABSTRACT
Plant purple acid phosphatases (PAPs) belong to a large multigene family whose specific functions in Pi metabolism are poorly understood. Two PAP isozymes secreted by Pi-deficient (-Pi) Arabidopsis thaliana were purified from culture filtrates of -Pi suspension cells. They correspond to an AtPAP12 (At2g27190) homodimer and AtPAP26 (At5g34850) monomer composed of glycosylated 60 and 55 kDa subunit(s), respectively. Each PAP exhibited broad pH activity profiles centred at pH 5.6, and overlapping substrate specificities. Concanavalin-A chromatography resolved a pair of secreted AtPAP26 glycoforms. AtPAP26 is dual targeted during Pi stress because it is also the principal intracellular (vacuolar) PAP up-regulated by -Pi Arabidopsis. Differential glycosylation appears to influence the subcellular targeting and substrate selectivity of AtPAP26. The significant increase in secreted acid phosphatase activity of -Pi seedlings was correlated with the appearance of immunoreactive AtPAP12 and AtPAP26 polypeptides. Analysis of atpap12 and atpap26 T-DNA mutants verified that AtPAP12 and AtPAP26 account for most of the secreted acid phosphatase activity of -Pi wild-type seedlings. Semi-quantitative RT-PCR confirmed that transcriptional controls exert little influence on the up-regulation of AtPAP26 during Pi stress, whereas AtPAP12 transcripts correlate well with relative levels of secreted AtPAP12 polypeptides. We hypothesize that AtPAP12 and AtPAP26 facilitate Pi scavenging from soil-localized organophosphates during nutritional Pi deprivation.
Subject(s)
Acid Phosphatase/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Phosphates/metabolism , Seedlings/enzymology , Acid Phosphatase/genetics , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Computational Biology , DNA, Bacterial/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Glycosylation , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutation , RNA, Plant/genetics , Sequence Alignment , Substrate SpecificityABSTRACT
PEPC [PEP(phosphoenolpyruvate) carboxylase] is a tightly controlled cytosolic enzyme situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PPCK (PEPC kinase) in plant acclimation to nutritional P(i) deprivation. However, little is known about the genetic origin or phosphorylation status of native PEPCs from -P(i) (P(i)-deficient) plants. The transfer of Arabidopsis suspension cells or seedlings to -P(i) growth media resulted in: (i) the marked transcriptional upregulation of genes encoding the PEPC isoenzyme AtPPC1 (Arabidopsis thaliana PEPC1), and PPCK isoenzymes AtPPCK1 and AtPPCK2; (ii) >2-fold increases in PEPC specific activity and in the amount of an immunoreactive 107-kDa PEPC polypeptide (p107); and (iii) In vivo p107 phosphorylation as revealed by immunoblotting of clarified extracts with phosphosite-specific antibodies to Ser-11 (which could be reversed following P(i) resupply). Approx. 1.3 mg of PEPC was purified 660-fold from -P(i) suspension cells to apparent homogeneity with a specific activity of 22.3 units x mg(-1) of protein. Gel filtration, SDS/PAGE and immunoblotting demonstrated that purified PEPC exists as a 440-kDa homotetramer composed of identical p107 subunits. Sequencing of p107 tryptic and Asp-N peptides by tandem MS established that this PEPC is encoded by AtPPC1. P(i)-affinity PAGE coupled with immunoblotting indicated stoichiometric phosphorylation of the p107 subunits of AtPPC1 at its conserved Ser-11 phosphorylation site. Phosphorylation activated AtPPC1 at pH 7.3 by lowering its Km(PEP) and its sensitivity to inhibition by L-malate and L-aspartate, while enhancing activation by glucose 6-phosphate. Our results indicate that the simultaneous induction and In vivo phosphorylation activation of AtPPC1 contribute to the metabolic adaptations of -P(i) Arabidopsis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Phosphates/deficiency , Phosphoenolpyruvate Carboxylase/metabolism , Adaptation, Physiological , Aspartic Acid/pharmacology , Glucose-6-Phosphate/pharmacology , Malates/pharmacology , Phosphorylation , Transcriptional ActivationABSTRACT
Effector-triggered immunity (ETI) was originally termed gene-for-gene resistance and dates back to fundamental observations of flax resistance to rust fungi by Harold Henry Flor in the 1940s. Since then, genetic and biochemical approaches have defined our current understanding of how plant "resistance" proteins recognize microbial effectors. More recently, proteomic approaches have expanded our view of the protein landscape during ETI and contributed significant advances to our mechanistic understanding of ETI signaling. Here we provide an overview of proteomic techniques that have been used to study plant ETI including both global and targeted approaches. We discuss the challenges associated with ETI proteomics and highlight specific examples from the literature, which demonstrate how proteomics is advancing the ETI research field.
Subject(s)
Bacterial Proteins/metabolism , Plant Diseases/immunology , Plant Immunity , Plant Proteins/metabolism , Plants/microbiology , Proteomics , Pseudomonas syringae/pathogenicity , Mutation , Plant Diseases/microbiology , Plants/immunology , Plants/metabolism , Proteomics/methods , Pseudomonas syringae/immunology , Pseudomonas syringae/metabolismABSTRACT
Pseudomonas syringae subverts plant immune signalling through injection of type III secreted effectors (T3SE) into host cells. The T3SE HopF2 can disable Arabidopsis immunity through Its ADP-ribosyltransferase activity. Proteomic analysis of HopF2 interacting proteins identified a protein complex containing ATPases required for regulating stomatal aperture, suggesting HopF2 may manipulate stomatal immunity. Here we report HopF2 can inhibit stomatal immunity independent of its ADP-ribosyltransferase activity. Transgenic expression of HopF2 in Arabidopsis inhibits stomatal closing in response to P. syringae and increases the virulence of surface inoculated P. syringae. Further, transgenic expression of HopF2 inhibits flg22 induced reactive oxygen species production. Intriguingly, ADP-ribosyltransferase activity is dispensable for inhibiting stomatal immunity and flg22 induced reactive oxygen species. Together, this implies HopF2 may be a bifunctional T3SE with ADP-ribosyltransferase activity required for inhibiting apoplastic immunity and an independent function required to inhibit stomatal immunity.