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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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Missense mutants of AvrRpm1 do not elicit an RPM1-mediated hypersensitive response, but can be translocated.(A) Four week old Col-0 plants were hand inoculated with 5×107 cfu/mL Pto DC3000 carrying either an empty vector or avrRpm1 with missense mutations eliminating localization to the membrane (G2A) [11], to the putative catalytic triad (H63A, Y122A, and D185A) and a double mutant (G2A D185A) and assayed for the ability to promote electrolyte leakage via RPM1-mediated hypersensitive response (HR) (see Methods). Error bars represent 2× SEM. (B) Five week old rpm1 RPS2 plants were infiltrated with 5×107 cfu/mL Pto DC3000 carrying missense mutations of avrRpm1 cloned to produce fusion proteins with Δ79avrRpt2. The ability to elicit an RPS2-mediated hypersensitive response was assayed at 20 hours post inoculation (HPI). Leaf counts (HR positive/total inoculated) are displayed under representative leaves.
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pone-0042633-g002: Missense mutants of AvrRpm1 do not elicit an RPM1-mediated hypersensitive response, but can be translocated.(A) Four week old Col-0 plants were hand inoculated with 5×107 cfu/mL Pto DC3000 carrying either an empty vector or avrRpm1 with missense mutations eliminating localization to the membrane (G2A) [11], to the putative catalytic triad (H63A, Y122A, and D185A) and a double mutant (G2A D185A) and assayed for the ability to promote electrolyte leakage via RPM1-mediated hypersensitive response (HR) (see Methods). Error bars represent 2× SEM. (B) Five week old rpm1 RPS2 plants were infiltrated with 5×107 cfu/mL Pto DC3000 carrying missense mutations of avrRpm1 cloned to produce fusion proteins with Δ79avrRpt2. The ability to elicit an RPS2-mediated hypersensitive response was assayed at 20 hours post inoculation (HPI). Leaf counts (HR positive/total inoculated) are displayed under representative leaves.

Mentions: Identification of a putative catalytic triad (H63-Y122-D185; Figure 1A) via homology modeling guided our introduction of missense mutations and subsequent functional tests following conjugation of mutant genes into Pto DC3000 (Methods). We assayed each of the three missense mutations (H63A, Y122A, D185A) for their ability to elicit AvrRpm1-dependent activation of RPM1 as measured by cellular electrolyte leakage, a proxy for HR cell death (Figure 2A). We found that each of the missense mutations was compromised in their ability to trigger RPM1-mediated HR, comparable to a previously characterized loss of function, mislocalization mutant G2A [11]. We also assayed for the ability of the missense mutations to trigger RPM1-dependent growth restriction of Pto DC3000 in wild-type plants (Col-0) [63]. We found that Pto DC3000 carrying the missense mutations were, surprisingly, unable to grow (Figure S2). One interpretation of this result is that these missense alleles retain the ability to initiate RPM1-dependent growth restriction, but not HR. However, data subsequently presented complicate this overly simple conclusion, and offer a clearer interpretation. To ensure that the AvrRpm1 missense alleles were not merely compromised in their ability to traverse the type III secretion system, we cloned each loss of function mutant as a fusion protein to truncated AvrRpt2 effector protein lacking the N-terminal 79 amino acids required for its own translocation [44]. These constructs were conjugated into Pto DC3000 and infiltrated into leaves of plants lacking RPM1, but expressing functional RPS2. These fusion effector proteins thus rely on the native type III secretion signals from AvrRpm1 for delivery into the host cell, but on the activity of Δ79AvrRpt2 to initiate RPS2-dependent HR. Each of the missense mutations was translocated via the type III secretion system (Figure 2B), an indication that the proteins are both expressed and stably accumulate to levels necessary for delivery into the host.


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)

Missense mutants of AvrRpm1 do not elicit an RPM1-mediated hypersensitive response, but can be translocated.(A) Four week old Col-0 plants were hand inoculated with 5×107 cfu/mL Pto DC3000 carrying either an empty vector or avrRpm1 with missense mutations eliminating localization to the membrane (G2A) [11], to the putative catalytic triad (H63A, Y122A, and D185A) and a double mutant (G2A D185A) and assayed for the ability to promote electrolyte leakage via RPM1-mediated hypersensitive response (HR) (see Methods). Error bars represent 2× SEM. (B) Five week old rpm1 RPS2 plants were infiltrated with 5×107 cfu/mL Pto DC3000 carrying missense mutations of avrRpm1 cloned to produce fusion proteins with Δ79avrRpt2. The ability to elicit an RPS2-mediated hypersensitive response was assayed at 20 hours post inoculation (HPI). Leaf counts (HR positive/total inoculated) are displayed under representative leaves.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412798&req=5

pone-0042633-g002: Missense mutants of AvrRpm1 do not elicit an RPM1-mediated hypersensitive response, but can be translocated.(A) Four week old Col-0 plants were hand inoculated with 5×107 cfu/mL Pto DC3000 carrying either an empty vector or avrRpm1 with missense mutations eliminating localization to the membrane (G2A) [11], to the putative catalytic triad (H63A, Y122A, and D185A) and a double mutant (G2A D185A) and assayed for the ability to promote electrolyte leakage via RPM1-mediated hypersensitive response (HR) (see Methods). Error bars represent 2× SEM. (B) Five week old rpm1 RPS2 plants were infiltrated with 5×107 cfu/mL Pto DC3000 carrying missense mutations of avrRpm1 cloned to produce fusion proteins with Δ79avrRpt2. The ability to elicit an RPS2-mediated hypersensitive response was assayed at 20 hours post inoculation (HPI). Leaf counts (HR positive/total inoculated) are displayed under representative leaves.
Mentions: Identification of a putative catalytic triad (H63-Y122-D185; Figure 1A) via homology modeling guided our introduction of missense mutations and subsequent functional tests following conjugation of mutant genes into Pto DC3000 (Methods). We assayed each of the three missense mutations (H63A, Y122A, D185A) for their ability to elicit AvrRpm1-dependent activation of RPM1 as measured by cellular electrolyte leakage, a proxy for HR cell death (Figure 2A). We found that each of the missense mutations was compromised in their ability to trigger RPM1-mediated HR, comparable to a previously characterized loss of function, mislocalization mutant G2A [11]. We also assayed for the ability of the missense mutations to trigger RPM1-dependent growth restriction of Pto DC3000 in wild-type plants (Col-0) [63]. We found that Pto DC3000 carrying the missense mutations were, surprisingly, unable to grow (Figure S2). One interpretation of this result is that these missense alleles retain the ability to initiate RPM1-dependent growth restriction, but not HR. However, data subsequently presented complicate this overly simple conclusion, and offer a clearer interpretation. To ensure that the AvrRpm1 missense alleles were not merely compromised in their ability to traverse the type III secretion system, we cloned each loss of function mutant as a fusion protein to truncated AvrRpt2 effector protein lacking the N-terminal 79 amino acids required for its own translocation [44]. These constructs were conjugated into Pto DC3000 and infiltrated into leaves of plants lacking RPM1, but expressing functional RPS2. These fusion effector proteins thus rely on the native type III secretion signals from AvrRpm1 for delivery into the host cell, but on the activity of Δ79AvrRpt2 to initiate RPS2-dependent HR. Each of the missense mutations was translocated via the type III secretion system (Figure 2B), an indication that the proteins are both expressed and stably accumulate to levels necessary for delivery into the host.

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