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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 mutants do not exhibit increased interference with AvrRpt2-mediated cleavage of RIN4.Pfo expressing wild type AvrRpt2 and either wild type or AvrRpm1 missense mutations in trans was infiltrated into leaves of 4-week-old rpm1 rps2 plants at 108 cfu/mL. Samples were collected over a time course (as indicated) and probed for the presence of RIN4 as an output of AvrRpt2 function.
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pone-0042633-g004: AvrRpm1 mutants do not exhibit increased interference with AvrRpt2-mediated cleavage of RIN4.Pfo expressing wild type AvrRpt2 and either wild type or AvrRpm1 missense mutations in trans was infiltrated into leaves of 4-week-old rpm1 rps2 plants at 108 cfu/mL. Samples were collected over a time course (as indicated) and probed for the presence of RIN4 as an output of AvrRpt2 function.

Mentions: The type III effector AvrRpt2 functions as a cysteine protease that directly interacts with RIN4, cleaving it at N- and C-terminal RCS (RIN4 cleavage sites) resulting in rapid degradation of the remaining RIN4 fragments [17], [18], [64]. One hypothesis to explain the results reported above is that AvrRpm1 prevents binding and cleavage of RIN4, or a RIN4-like substrate, by AvrRpt2. In this model, this blockade of the proposed substrate's ability to interact appropriately with RPS2 would lead to ectopic RPS2 activation in the same manner that RIN4 is genetically required to negatively regulate an otherwise lethal activation of RPS2 [6], [15]. Thus, AvrRpm1 and its missense alleles were tested for their ability to directly inhibit the ability of AvrRpt2 to cleave RIN4. Each AvrRpm1 missense mutation was expressed in trans together with AvrRpt2 in Pseudomonas fluorescens, a non-pathogen engineered to carry a competent type III secretion apparatus [45]. The ability of AvrRpt2 to cleave RIN4 leading to the overall disappearance of RIN4 over time was assayed via western blot analysis using native RIN4 antisera (Figure S4). Neither wild type AvrRpm1 nor the missense alleles consistently inhibited the cleavage and clearance of RIN4 by AvrRpt2 by 6 hours post-infection (Figure 4), consistent with the lack of effect of RIN4 on the growth suppression phenotype displayed by the AvrRpm1 missense alleles. However, we reproducibly detected attenuation of cleavage and clearance of RIN4 by AvrRpt2 in the presence of wild type AvrRpm1, mislocalized AvrRpm1G2A or the putative non-functional AvrRpm1D185A. The double mutant AvrRpm1G2A D185A reproducibly lacked this ability. We speculate that this attenuation is due to weak inhibitory activity of mislocalized, but functional, AvrRpm1G2A on RIN4 [11], and a similar inhibitory activity of properly localized, but non-functional, AvrRpm1D185A on RIN4. The lack of attenuation of RIN4 cleavage by AvrRpt2 observed for AvrRpm1G2A D185A can be explained by the combined loss of function of the two single mutants. In sum these data do not eliminate the possibility that the AvrRpm1 missense alleles exhibit their novel phenotype via binding to another RIN4-related target more tightly than does the wild type AvrRpm1, leading to a previously undefined activation of RPS2 and subsequent pathogen growth suppression.


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 mutants do not exhibit increased interference with AvrRpt2-mediated cleavage of RIN4.Pfo expressing wild type AvrRpt2 and either wild type or AvrRpm1 missense mutations in trans was infiltrated into leaves of 4-week-old rpm1 rps2 plants at 108 cfu/mL. Samples were collected over a time course (as indicated) and probed for the presence of RIN4 as an output of AvrRpt2 function.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412798&req=5

pone-0042633-g004: AvrRpm1 mutants do not exhibit increased interference with AvrRpt2-mediated cleavage of RIN4.Pfo expressing wild type AvrRpt2 and either wild type or AvrRpm1 missense mutations in trans was infiltrated into leaves of 4-week-old rpm1 rps2 plants at 108 cfu/mL. Samples were collected over a time course (as indicated) and probed for the presence of RIN4 as an output of AvrRpt2 function.
Mentions: The type III effector AvrRpt2 functions as a cysteine protease that directly interacts with RIN4, cleaving it at N- and C-terminal RCS (RIN4 cleavage sites) resulting in rapid degradation of the remaining RIN4 fragments [17], [18], [64]. One hypothesis to explain the results reported above is that AvrRpm1 prevents binding and cleavage of RIN4, or a RIN4-like substrate, by AvrRpt2. In this model, this blockade of the proposed substrate's ability to interact appropriately with RPS2 would lead to ectopic RPS2 activation in the same manner that RIN4 is genetically required to negatively regulate an otherwise lethal activation of RPS2 [6], [15]. Thus, AvrRpm1 and its missense alleles were tested for their ability to directly inhibit the ability of AvrRpt2 to cleave RIN4. Each AvrRpm1 missense mutation was expressed in trans together with AvrRpt2 in Pseudomonas fluorescens, a non-pathogen engineered to carry a competent type III secretion apparatus [45]. The ability of AvrRpt2 to cleave RIN4 leading to the overall disappearance of RIN4 over time was assayed via western blot analysis using native RIN4 antisera (Figure S4). Neither wild type AvrRpm1 nor the missense alleles consistently inhibited the cleavage and clearance of RIN4 by AvrRpt2 by 6 hours post-infection (Figure 4), consistent with the lack of effect of RIN4 on the growth suppression phenotype displayed by the AvrRpm1 missense alleles. However, we reproducibly detected attenuation of cleavage and clearance of RIN4 by AvrRpt2 in the presence of wild type AvrRpm1, mislocalized AvrRpm1G2A or the putative non-functional AvrRpm1D185A. The double mutant AvrRpm1G2A D185A reproducibly lacked this ability. We speculate that this attenuation is due to weak inhibitory activity of mislocalized, but functional, AvrRpm1G2A on RIN4 [11], and a similar inhibitory activity of properly localized, but non-functional, AvrRpm1D185A on RIN4. The lack of attenuation of RIN4 cleavage by AvrRpt2 observed for AvrRpm1G2A D185A can be explained by the combined loss of function of the two single mutants. In sum these data do not eliminate the possibility that the AvrRpm1 missense alleles exhibit their novel phenotype via binding to another RIN4-related target more tightly than does the wild type AvrRpm1, leading to a previously undefined activation of RPS2 and subsequent pathogen growth suppression.

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