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, 7 (8), e42633

AvrRpm1 Missense Mutations Weakly Activate RPS2-mediated Immune Response in Arabidopsis Thaliana

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AvrRpm1 Missense Mutations Weakly Activate RPS2-mediated Immune Response in Arabidopsis Thaliana

Karen A Cherkis et al. PLoS One.

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. AvrRpm1 exhibits structural homology to the catalytic domain of Poly-ADP-ribosyl polymerase (PARP).
(A) Sequence alignment of DT family ADP-ribosylating proteins 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).
Figure 2
Figure 2. 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) , 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.
Figure 3
Figure 3. 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 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).
Figure 4
Figure 4. 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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References

    1. Cunnac S, Lindeberg M, Collmer A (2009) Pseudomonas syringae type III secretion system effectors: repertoires in search of functions. Curr Opin Microbiol 12: 53–60. - PubMed
    1. Galan JE, Collmer A (1999) Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284: 1322–1328. - PubMed
    1. Lindeberg M, Cunnac S, Collmer A (2009) The evolution of Pseudomonas syringae host specificity and type III effector repertoires. Mol Plant Pathol 10: 767–775. - PMC - PubMed
    1. Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411: 826–833. - PubMed
    1. Belkhadir Y, Subramaniam R, Dangl JL (2004) Plant disease resistance protein signaling: NBS-LRR proteins and their partners. Curr Opin Plant Biol 7: 391–399. - PubMed

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