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 [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).
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) [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.
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 [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).
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.