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AvrRpm1 missense mutations weakly activate RPS2-mediated immune response in Arabidopsis thaliana.

Cherkis KA, Temple BR, Chung EH, Sondek J, Dangl JL - PLoS ONE (2012)

Bottom Line: Plants recognize microbes via specific pattern recognition receptors that are activated by microbe-associated molecular patterns (MAMPs), resulting in MAMP-triggered immunity (MTI).AvrRpm1 is a P. syringae type III effector.Site-directed mutagenesis of each residue in the putative catalytic triad, His63-Tyr122-Asp185 of AvrRpm1, results in loss of both AvrRpm1-dependent virulence and AvrRpm1-mediated activation of RPM1, but, surprisingly, causes a gain of function: the ability to activate the RPS2 nucleotide binding leucine-rich repeat sensor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Plants recognize microbes via specific pattern recognition receptors that are activated by microbe-associated molecular patterns (MAMPs), resulting in MAMP-triggered immunity (MTI). Successful pathogens bypass MTI in genetically diverse hosts via deployment of effectors (virulence factors) that inhibit MTI responses, leading to pathogen proliferation. Plant pathogenic bacteria like Pseudomonas syringae utilize a type III secretion system to deliver effectors into cells. These effectors can contribute to pathogen virulence or elicit disease resistance, depending upon the host plant genotype. In disease resistant genotypes, intracellular immune receptors, typically belonging to the nucleotide binding leucine-rich repeat family of proteins, perceive bacterial effector(s) and initiate downstream defense responses (effector triggered immunity) that include the hypersensitive response, and transcriptional re-programming leading to various cellular outputs that collectively halt pathogen growth. Nucleotide binding leucine-rich repeat sensors can be indirectly activated via perturbation of a host protein acting as an effector target. AvrRpm1 is a P. syringae type III effector. Upon secretion into the host cell, AvrRpm1 is acylated by host enzymes and directed to the plasma membrane, where it contributes to virulence. This is correlated with phosphorylation of Arabidopsis RIN4 in vivo. RIN4 is a negative regulator of MAMP-triggered immunity, and its modification in the presence of four diverse type III effectors, including AvrRpm1, likely enhances this RIN4 regulatory function. The RPM1 nucleotide binding leucine-rich repeat sensor perceives RIN4 perturbation in disease resistant plants, leading to a successful immune response. Here, demonstrate that AvrRpm1 has a fold homologous to the catalytic domain of poly(ADP-ribosyl) polymerase. Site-directed mutagenesis of each residue in the putative catalytic triad, His63-Tyr122-Asp185 of AvrRpm1, results in loss of both AvrRpm1-dependent virulence and AvrRpm1-mediated activation of RPM1, but, surprisingly, causes a gain of function: the ability to activate the RPS2 nucleotide binding leucine-rich repeat sensor.

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AvrRpm1 exhibits structural homology to the catalytic domain of Poly-ADP-ribosyl polymerase (PARP).(A) Sequence alignment of DT family ADP-ribosylating proteins [35] and the four AvrRpm1 family proteins illustrating key regions of conservation. Secondary structure for each region is shown above. Highly conserved residues are highlighted in blue. Red carets denote the catalytic triad of PARP. (B) Homology model of the AvrRpm1 reference allele (copper) from P. syringae pv. maculicola M6 (Psm M6) with the catalytic domain of Poly-ADP-ribosyl polymerase 1 (PARP-1; PDB ID: 3GJW) (silver). The side chains for residues highlighted in (A) are denoted by dark blue (AvrRpm1) and light blue (PARP-1). Residues in the catalytic triad are labeled according to AvrRpm1. “N” and “C” represent the amino- and carboxy-terminus of the protein respectively. Independent homology models for the remaining three AvrRpm1 family members from (B) P. syringae pvs. syringae B728a (Psy B728a), (C), pisi race 6 (Ppi race 6) (D), and phaseolicola 2708 (Psp 2708).
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pone-0042633-g001: AvrRpm1 exhibits structural homology to the catalytic domain of Poly-ADP-ribosyl polymerase (PARP).(A) Sequence alignment of DT family ADP-ribosylating proteins [35] and the four AvrRpm1 family proteins illustrating key regions of conservation. Secondary structure for each region is shown above. Highly conserved residues are highlighted in blue. Red carets denote the catalytic triad of PARP. (B) Homology model of the AvrRpm1 reference allele (copper) from P. syringae pv. maculicola M6 (Psm M6) with the catalytic domain of Poly-ADP-ribosyl polymerase 1 (PARP-1; PDB ID: 3GJW) (silver). The side chains for residues highlighted in (A) are denoted by dark blue (AvrRpm1) and light blue (PARP-1). Residues in the catalytic triad are labeled according to AvrRpm1. “N” and “C” represent the amino- and carboxy-terminus of the protein respectively. Independent homology models for the remaining three AvrRpm1 family members from (B) P. syringae pvs. syringae B728a (Psy B728a), (C), pisi race 6 (Ppi race 6) (D), and phaseolicola 2708 (Psp 2708).

Mentions: We generated a computational homology model for AvrRpm1 to identify conserved structural domains shared with proteins of known function. After removing the first 30 residues, which are predicted to be disordered, we input the remaining AvrRpm1 amino acid sequence into the BioInfoBank Institute's metaserver [51]. The highest-ranking outputs for predicted homologous folds from the aggregated databases were to various catalytic domains of poly(ADP-ribosyl)polymerase (PARP) [26], [52]. PARP is a member of the larger family of Diphtheria toxin-like ADP-ribosyl transferases [35], [53], [54]. The catalytic domain of these proteins can be broken down into three regions (Figure 1A). The N-terminal region 1 is a span of primarily conserved residues highlighted by an aromatic residue (Figure 1A, denoted with Φ) followed by the first catalytic triad member H63 (in AvrRpm1; all residues noted refer to the allele from Psm M6, GEN BANK ID AF359557.1 unless stated otherwise) and a glycine (G64). We also noted the presence of a conserved leucine (L62) preceding this region and a serine or threonine (T64) at its end in the majority of the Diphtheria toxin-like ADP-ribosyl transferase proteins. The centrally located region 2 is denoted by a pair of tyrosine residues Y111 and Y122 separated by ten amino acids, where Y122 corresponds to the second member of the catalytic triad. The C-terminal region 3 contains the third catalytic triad residue, glutamate, or in the case of AvrRpm1 (Psm M6) aspartate (D185). Mutation of the glutamate residue to an aspartate did not abolish PARP-1 activity, but rather altered the in vitro kinetics [55]. The overall sequence identity between the catalytic domain of PARP-1 and AvrRpm1 is relatively low, however these regions and the relative spacing between them are conserved.


AvrRpm1 missense mutations weakly activate RPS2-mediated immune response in Arabidopsis thaliana.

Cherkis KA, Temple BR, Chung EH, Sondek J, Dangl JL - PLoS ONE (2012)

AvrRpm1 exhibits structural homology to the catalytic domain of Poly-ADP-ribosyl polymerase (PARP).(A) Sequence alignment of DT family ADP-ribosylating proteins [35] and the four AvrRpm1 family proteins illustrating key regions of conservation. Secondary structure for each region is shown above. Highly conserved residues are highlighted in blue. Red carets denote the catalytic triad of PARP. (B) Homology model of the AvrRpm1 reference allele (copper) from P. syringae pv. maculicola M6 (Psm M6) with the catalytic domain of Poly-ADP-ribosyl polymerase 1 (PARP-1; PDB ID: 3GJW) (silver). The side chains for residues highlighted in (A) are denoted by dark blue (AvrRpm1) and light blue (PARP-1). Residues in the catalytic triad are labeled according to AvrRpm1. “N” and “C” represent the amino- and carboxy-terminus of the protein respectively. Independent homology models for the remaining three AvrRpm1 family members from (B) P. syringae pvs. syringae B728a (Psy B728a), (C), pisi race 6 (Ppi race 6) (D), and phaseolicola 2708 (Psp 2708).
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getmorefigures.php?uid=PMC3412798&req=5

pone-0042633-g001: AvrRpm1 exhibits structural homology to the catalytic domain of Poly-ADP-ribosyl polymerase (PARP).(A) Sequence alignment of DT family ADP-ribosylating proteins [35] and the four AvrRpm1 family proteins illustrating key regions of conservation. Secondary structure for each region is shown above. Highly conserved residues are highlighted in blue. Red carets denote the catalytic triad of PARP. (B) Homology model of the AvrRpm1 reference allele (copper) from P. syringae pv. maculicola M6 (Psm M6) with the catalytic domain of Poly-ADP-ribosyl polymerase 1 (PARP-1; PDB ID: 3GJW) (silver). The side chains for residues highlighted in (A) are denoted by dark blue (AvrRpm1) and light blue (PARP-1). Residues in the catalytic triad are labeled according to AvrRpm1. “N” and “C” represent the amino- and carboxy-terminus of the protein respectively. Independent homology models for the remaining three AvrRpm1 family members from (B) P. syringae pvs. syringae B728a (Psy B728a), (C), pisi race 6 (Ppi race 6) (D), and phaseolicola 2708 (Psp 2708).
Mentions: We generated a computational homology model for AvrRpm1 to identify conserved structural domains shared with proteins of known function. After removing the first 30 residues, which are predicted to be disordered, we input the remaining AvrRpm1 amino acid sequence into the BioInfoBank Institute's metaserver [51]. The highest-ranking outputs for predicted homologous folds from the aggregated databases were to various catalytic domains of poly(ADP-ribosyl)polymerase (PARP) [26], [52]. PARP is a member of the larger family of Diphtheria toxin-like ADP-ribosyl transferases [35], [53], [54]. The catalytic domain of these proteins can be broken down into three regions (Figure 1A). The N-terminal region 1 is a span of primarily conserved residues highlighted by an aromatic residue (Figure 1A, denoted with Φ) followed by the first catalytic triad member H63 (in AvrRpm1; all residues noted refer to the allele from Psm M6, GEN BANK ID AF359557.1 unless stated otherwise) and a glycine (G64). We also noted the presence of a conserved leucine (L62) preceding this region and a serine or threonine (T64) at its end in the majority of the Diphtheria toxin-like ADP-ribosyl transferase proteins. The centrally located region 2 is denoted by a pair of tyrosine residues Y111 and Y122 separated by ten amino acids, where Y122 corresponds to the second member of the catalytic triad. The C-terminal region 3 contains the third catalytic triad residue, glutamate, or in the case of AvrRpm1 (Psm M6) aspartate (D185). Mutation of the glutamate residue to an aspartate did not abolish PARP-1 activity, but rather altered the in vitro kinetics [55]. The overall sequence identity between the catalytic domain of PARP-1 and AvrRpm1 is relatively low, however these regions and the relative spacing between them are conserved.

Bottom Line: Plants recognize microbes via specific pattern recognition receptors that are activated by microbe-associated molecular patterns (MAMPs), resulting in MAMP-triggered immunity (MTI).AvrRpm1 is a P. syringae type III effector.Site-directed mutagenesis of each residue in the putative catalytic triad, His63-Tyr122-Asp185 of AvrRpm1, results in loss of both AvrRpm1-dependent virulence and AvrRpm1-mediated activation of RPM1, but, surprisingly, causes a gain of function: the ability to activate the RPS2 nucleotide binding leucine-rich repeat sensor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Plants recognize microbes via specific pattern recognition receptors that are activated by microbe-associated molecular patterns (MAMPs), resulting in MAMP-triggered immunity (MTI). Successful pathogens bypass MTI in genetically diverse hosts via deployment of effectors (virulence factors) that inhibit MTI responses, leading to pathogen proliferation. Plant pathogenic bacteria like Pseudomonas syringae utilize a type III secretion system to deliver effectors into cells. These effectors can contribute to pathogen virulence or elicit disease resistance, depending upon the host plant genotype. In disease resistant genotypes, intracellular immune receptors, typically belonging to the nucleotide binding leucine-rich repeat family of proteins, perceive bacterial effector(s) and initiate downstream defense responses (effector triggered immunity) that include the hypersensitive response, and transcriptional re-programming leading to various cellular outputs that collectively halt pathogen growth. Nucleotide binding leucine-rich repeat sensors can be indirectly activated via perturbation of a host protein acting as an effector target. AvrRpm1 is a P. syringae type III effector. Upon secretion into the host cell, AvrRpm1 is acylated by host enzymes and directed to the plasma membrane, where it contributes to virulence. This is correlated with phosphorylation of Arabidopsis RIN4 in vivo. RIN4 is a negative regulator of MAMP-triggered immunity, and its modification in the presence of four diverse type III effectors, including AvrRpm1, likely enhances this RIN4 regulatory function. The RPM1 nucleotide binding leucine-rich repeat sensor perceives RIN4 perturbation in disease resistant plants, leading to a successful immune response. Here, demonstrate that AvrRpm1 has a fold homologous to the catalytic domain of poly(ADP-ribosyl) polymerase. Site-directed mutagenesis of each residue in the putative catalytic triad, His63-Tyr122-Asp185 of AvrRpm1, results in loss of both AvrRpm1-dependent virulence and AvrRpm1-mediated activation of RPM1, but, surprisingly, causes a gain of function: the ability to activate the RPS2 nucleotide binding leucine-rich repeat sensor.

Show MeSH
Related in: MedlinePlus