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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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Putative catalytic triad residues are required for AvrRpm1 virulence that is inhibited via weak activation of RPS2-mediated disease resistance.(A–C) Growth of Psm CR299, a derivative of Psm M2 that carries an insertion in avrRpm1[13] was complemented in trans with plasmids expressing wild type AvrRpm1 and missense mutations as noted. Four week old rpm1 (A), rpm1 rps2 (B) or rpm1 rps2 rin4 (C) plants were inoculated with 106 cfu/mL and samples were collected on day 0 and day 3. Error bars represent 2× SEM. An analysis of variance (ANOVA) was performed among the day 3 samples followed by Tukey's post-hoc analysis (α = 0.05) with significance groups indicated by letters on the graph. (D) Immunoblot assay for accumulation of the wild type and mutant AvrRpm1 proteins at 3 days post inoculation for strains used in (B) and (C).
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pone-0042633-g003: Putative catalytic triad residues are required for AvrRpm1 virulence that is inhibited via weak activation of RPS2-mediated disease resistance.(A–C) Growth of Psm CR299, a derivative of Psm M2 that carries an insertion in avrRpm1[13] was complemented in trans with plasmids expressing wild type AvrRpm1 and missense mutations as noted. Four week old rpm1 (A), rpm1 rps2 (B) or rpm1 rps2 rin4 (C) plants were inoculated with 106 cfu/mL and samples were collected on day 0 and day 3. Error bars represent 2× SEM. An analysis of variance (ANOVA) was performed among the day 3 samples followed by Tukey's post-hoc analysis (α = 0.05) with significance groups indicated by letters on the graph. (D) Immunoblot assay for accumulation of the wild type and mutant AvrRpm1 proteins at 3 days post inoculation for strains used in (B) and (C).

Mentions: Each AvrRpm1 missense mutation was tested for its virulence [13]. AvrRpm1 missense mutations were expressed in P. syringae pv maculicola (Psm) strain M2 CR299, which carries an insertion in avrRpm1 that disables this gene (CR299; [13]) (Figure 3A). Psm M2 CR299 carrying a wild type copy of avrRpm1 in trans grew at least ten-fold more than either Psm M2 CR299, or an isogenic strain that can deliver the mislocalized AvrRpm1 G2A missense mutant [11]. Each of the putative AvrRpm1 catalytic triad missense mutations was also compromised for virulence mediated by AvrRpm1. In fact, the expression of these putative catalytic triad mutants inhibited the growth of Psm M2 CR299 to a higher extent with respect to CR299 or to CR299 complemented with the localization AvrRpm1G2A mutant (Figure 3A). To determine if plasma membrane localization was required for this surprising phenotype, we tested the virulence activity of an AvrRpm1 double mutant in both the putative catalytic activity and localization/myristoylation (AvrRpm1G2A D185A). We found that this strain grew to levels equal to Psm M2 CR299 expressing the mislocalized missense mutation G2A (Figure S3). These data suggest that the missense mutations must be properly localized inside the host cell in order to inhibit the growth of Psm M2 CR299.


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)

Putative catalytic triad residues are required for AvrRpm1 virulence that is inhibited via weak activation of RPS2-mediated disease resistance.(A–C) Growth of Psm CR299, a derivative of Psm M2 that carries an insertion in avrRpm1[13] was complemented in trans with plasmids expressing wild type AvrRpm1 and missense mutations as noted. Four week old rpm1 (A), rpm1 rps2 (B) or rpm1 rps2 rin4 (C) plants were inoculated with 106 cfu/mL and samples were collected on day 0 and day 3. Error bars represent 2× SEM. An analysis of variance (ANOVA) was performed among the day 3 samples followed by Tukey's post-hoc analysis (α = 0.05) with significance groups indicated by letters on the graph. (D) Immunoblot assay for accumulation of the wild type and mutant AvrRpm1 proteins at 3 days post inoculation for strains used in (B) and (C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412798&req=5

pone-0042633-g003: Putative catalytic triad residues are required for AvrRpm1 virulence that is inhibited via weak activation of RPS2-mediated disease resistance.(A–C) Growth of Psm CR299, a derivative of Psm M2 that carries an insertion in avrRpm1[13] was complemented in trans with plasmids expressing wild type AvrRpm1 and missense mutations as noted. Four week old rpm1 (A), rpm1 rps2 (B) or rpm1 rps2 rin4 (C) plants were inoculated with 106 cfu/mL and samples were collected on day 0 and day 3. Error bars represent 2× SEM. An analysis of variance (ANOVA) was performed among the day 3 samples followed by Tukey's post-hoc analysis (α = 0.05) with significance groups indicated by letters on the graph. (D) Immunoblot assay for accumulation of the wild type and mutant AvrRpm1 proteins at 3 days post inoculation for strains used in (B) and (C).
Mentions: Each AvrRpm1 missense mutation was tested for its virulence [13]. AvrRpm1 missense mutations were expressed in P. syringae pv maculicola (Psm) strain M2 CR299, which carries an insertion in avrRpm1 that disables this gene (CR299; [13]) (Figure 3A). Psm M2 CR299 carrying a wild type copy of avrRpm1 in trans grew at least ten-fold more than either Psm M2 CR299, or an isogenic strain that can deliver the mislocalized AvrRpm1 G2A missense mutant [11]. Each of the putative AvrRpm1 catalytic triad missense mutations was also compromised for virulence mediated by AvrRpm1. In fact, the expression of these putative catalytic triad mutants inhibited the growth of Psm M2 CR299 to a higher extent with respect to CR299 or to CR299 complemented with the localization AvrRpm1G2A mutant (Figure 3A). To determine if plasma membrane localization was required for this surprising phenotype, we tested the virulence activity of an AvrRpm1 double mutant in both the putative catalytic activity and localization/myristoylation (AvrRpm1G2A D185A). We found that this strain grew to levels equal to Psm M2 CR299 expressing the mislocalized missense mutation G2A (Figure S3). These data suggest that the missense mutations must be properly localized inside the host cell in order to inhibit the growth of Psm M2 CR299.

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