RPG1-B-derived resistance to AvrB-expressing Pseudomonas syringae requires RIN4-like proteins in soybean

Abstract

Soybean (Glycine max) RPG1-B (for resistance to Pseudomonas syringae pv glycinea) mediates species-specific resistance to P. syringae expressing the avirulence protein AvrB, similar to the nonorthologous RPM1 in Arabidopsis (Arabidopsis thaliana). RPM1-derived signaling is presumably induced upon AvrB-derived modification of the RPM1-interacting protein, RIN4 (for RPM1-interacting 4). We show that, similar to RPM1, RPG1-B does not directly interact with AvrB but associates with RIN4-like proteins from soybean. Unlike Arabidopsis, soybean contains at least four RIN4-like proteins (GmRIN4a to GmRIN4d). GmRIN4b, but not GmRIN4a, complements the Arabidopsis rin4 mutation. Both GmRIN4a and GmRIN4b bind AvrB, but only GmRIN4b binds RPG1-B. Silencing either GmRIN4a or GmRIN4b abrogates RPG1-B-derived resistance to P. syringae expressing AvrB. Binding studies show that GmRIN4b interacts with GmRIN4a as well as with two other AvrB/RPG1-B-interacting isoforms, GmRIN4c and GmRIN4d. The lack of functional redundancy among GmRIN4a and GmRIN4b and their abilities to interact with each other suggest that the two proteins might function as a heteromeric complex in mediating RPG1-B-derived resistance. Silencing GmRIN4a or GmRIN4b in rpg1-b plants enhances basal resistance to virulent strains of P. syringae and the oomycete Phytophthora sojae. Interestingly, GmRIN4a- or GmRIN4b-silenced rpg1-b plants respond differently to AvrB-expressing bacteria. Although both GmRIN4a and GmRIN4b function to monitor AvrB in the presence of RPG1-B, GmRIN4a, but not GmRIN4b, negatively regulates AvrB virulence activity in the absence of RPG1-B.

Figure 1.

Sequence conservation and phylogenetic analysis of Arabidopsis (At; At3g25070), lettuce (Ls; GQ497774 and GQ497780), soybean (Gm), and tomato (Sl; TC174419) RIN4 proteins. A, Sequence conservation among the various RIN4 proteins. Numbers on the left denote amino acid positions. Identical residues are shaded in gray. The two AvrRPT2 cleavage sites (RCS1 and RCS2) are boxed. The AvrB-binding region is underlined by a dotted line. Residues that contact and/or form hydrogen bonds with AvrB are indicated by asterisks. A solid line underlines the palmitoylation site essential for plasma membrane localization. Sequence alignment was carried out using ClustalW in the Megalign program of the DNASTAR package. B, Phylogenetic analysis of the various RIN4 proteins. The tree was constructed using the program PAUP*, version 4b10.

Figure 2.

GmRIN4 (GmRIN4a to GmRIN4d = 4a to 4d) proteins interact with AvrB. A, Y2H assay showing interactions between AvrB and 4a, 4b, 4c, or 4d. Growth on His⁻Trp⁻Leu⁻ plates is shown. Numbers indicate β-galactosidase activity with sd (n = 3). B, Pull-down assays showing in vitro protein binding. Immobilized 4a/4b (Ni-NTA) and 4c/4d (glutathione) were incubated with increasing amounts (0.05–0.5 μg) of AvrB (His tag cleaved), and bound proteins were visualized using anti-AvrB antibodies. Purified AvrB was loaded as a positive control (+) or incubated with empty Ni-NTA/glutathione agarose as a negative control (−) on respective gels. C, BiFC assay showing in planta interactions. CFP and YFP overlay images (40× magnification) of micrographs at 48 h post infiltration from leaves coexpressing the indicated proteins are shown. Images are representative of three separate infiltrations from two independent experiments for each interaction. Bars = 10 μm. D, Western-blot analysis showing expression and localization of 4a/4b/4c/4d. Ten micrograms of protein each of the total (T), soluble (S), and membrane (M) fractions from CFP-H2B-expressing transgenic N. benthamiana leaves infiltrated with nEYFP-fused GmRIN4 proteins were subjected to western-blot analysis using anti-GFP antibodies.

Figure 3.

GmRIN4 (GmRIN4a to GmRIN4d = 4a to 4d) proteins interact with RPG1-B. A, Y2H assay between 4a, 4b, 4c, or 4d and RPG1-B (N-terminal 187 amino acids) or RPG1-B lacking 16 amino acids of the CC domain (N-terminal 171 amino acids; RPG1-BΔCC₁₆). Growth on His⁻Trp⁻Leu⁻ plates is shown. Numbers indicate β-galactosidase activity with sd (n = 3). B, Pull-down assays showing in vitro protein binding. RPG1-B was immobilized on Ni-NTA incubated with increasing amounts of AvrB (0.05–0.5 μg) or 4b, 4c, or 4d (0.1–1 μg), and bound proteins were visualized using anti-AvrB, anti-Myc (4b), or anti-GST (4c/4d) antibodies. Purified AvrB, 4b, 4c, or 4d was loaded as a positive control (+) or incubated with empty Ni-NTA as a negative control (−) on respective gels. C, BiFC assay showing in planta interactions. CFP and YFP overlay images (40× magnification) of micrographs at 48 h post infiltration from leaves coexpressing the indicated proteins are shown. Images are representative of three separate infiltrations from two independent experiments for each interaction. Bars = 10 μm. D, Western-blot analysis showing expression and localization of 4a/4b/4c/4d in 10 μg each of membrane (M) or soluble (S) fractions of N. benthamiana leaves coexpressing the four nEYFP-fused GmRIN4 isoforms and cEYFP-fused RPG1-B. Proteins were detected using anti-GFP antibodies that cross-react with nEYFP alone.

Figure 4.

GmRIN4b rescues the rin4 mutation in Arabidopsis. A, Northern-blot analysis showing expression of GmRIN4a or GmRIN4b and PR-1 in wild-type (Col-0), rin4 rps2, 35S-GmRIN4a (35S-4a), and 35S-GmRIN4b (35S-4b) plants. Ethidium bromide staining of rRNA was used as a loading control. B, Western-blot analysis of protein extracts from the indicated genotypes. GmRIN4a and GmRIN4b proteins in the respective transgenic lines were visualized using GmRIN4a-specific antibodies. Ponceau-S staining of the membrane was used as a loading control. C to E, Response to AvrB (C), AvrRpm1 (D), or DC3000 (E) strains of P. syringae in the wild type (Col-0; black bars), a mutant (rin4 rps2; red bars), and two transgenic lines each expressing GmRIN4a (green and yellow bars) or GmRIN4b (orange and blue bars). Bacterial numbers at 0, 2, or 4 d post inoculation (dpi) are presented as log₁₀ values of colony-forming units (cfu) per unit of leaf area. Error bars indicate sd (n = 5). Statistical significance was determined using Student's t test. Asterisks denote data significantly different from the wild type (Col-0), where P < 0.001 (n = 5). F, BiFC assay showing in planta interactions. CFP and YFP overlay images (40× magnification) of micrographs at 48 h post infiltration from leaves coexpressing the indicated proteins are shown. Images are representative of three separate infiltrations from two independent experiments for each interaction. Bars = 10 μm.

Figure 5.

GmRIN4a and GmRIN4b are required for RPG1-B-mediated resistance signaling. A, RT-PCR analysis showing expression of the GmRIN4 isoforms in mock/vector (M/V)-inoculated and GmRIN4a (S_4a)- or GmRIN4b (S_4b)-silenced plants. β-Tubulin levels were used as an internal control for cDNA amounts. B, Northern-blot analysis showing expression of PR1a in V, S_4a, or S_4b plants or V plants inoculated with an avirulent (AvrB) strain of P. syringae pv glycinea (V-AvrB; 24 h). Ethidium bromide staining of rRNA was used as a loading control. C, Bacterial counts in RPG1-B (cv Harosoy) S_4a (gray bars) or S_4b (black bars) plants as compared with V (white bars) plants. Plants were infiltrated with AvrB bacteria. Log₁₀ values of colony-forming units (cfu) per unit of leaf area from infected leaves at 0, 2, or 4 d post inoculation (dpi) are presented. Error bars indicate sd (n = 5). Statistical significance was determined using Student's t test. Asterisks denote data significantly different from control (V), where P < 0.001.

Figure 6.

GmRIN4 isoforms (GmRIN4a to GmRIN4d = 4a to 4d) interact with each other. A, Y2H assay showing interactions between the various GmRIN4 proteins. Growth on His⁻Trp⁻Leu⁻ plates is shown. Numbers indicate β-galactosidase activity ± sd (n = 3). B, Pull-down assays showing in vitro binding between the GmRIN4 proteins. For pull-down assay between 4a and 4b, 4c, or 4d, 4a was immobilized on Ni-NTA and incubated with increasing amounts (0.1–1 μg) of 4b, 4c, or 4d. The His tag was cleaved from 4b before application to a 4a affinity column. For interactions between 4b and 4c or 4d, 4b was immobilized on Ni-NTA and incubated with increasing amounts (0.1–1 μg) of 4c or 4d. Purified 4b, 4c, or 4d was loaded as a positive control (+) and incubated with empty Ni-NTA/glutathione agarose as a negative control (−) on respective gels. Proteins were visualized by immunoblot analysis using anti-Myc (4b) or anti-GST (4c and 4d) antibodies. C, BiFC assay showing in planta interactions. CFP and YFP overlay images (40× magnification) of micrographs at 48 h post infiltration from leaves coexpressing the indicated proteins are shown. Images are representative of three separate infiltrations from two independent experiments for each interaction. Bars = 10 μm.

Figure 7.

GmRIN4a and GmRIN4b mediate basal immunity in soybean. A, Bacterial counts in rpg1-b (cv Essex) plants silenced for GmRIN4a (S_4a) or GmRIN4b (S_4b) and those inoculated with the empty silencing vector (V). Plants were infiltrated with virulent (vir) or avirulent (avrB) strains of P. syringae pv glycinea. Log₁₀ values of colony-forming units (cfu) per unit of leaf area from infected leaves at 0 d post inoculation (dpi; white bars) or 4 dpi (black bars) are presented. Error bars indicate sd (n = 5). Student's t test was used to determine statistical significance. Asterisks denote significant differences from V plants inoculated with vir bacteria as follows: ** P < 0.001, *** P < 0.0001. Significant difference between avrB-inoculated V and S_4a plants is denoted by a, where P < 0.05. Significant difference between vir and avrB-inoculated S_4b plants is denoted by b, where P < 0.0001. B, Percentage survival of V (white squares), S_4a (gray circles), or S_4b (black triangles) plants at 1, 5, 7, 8, 9, 12, and 15 d post inoculation with P. sojae. Error bars indicate sd (n = 3). Asterisks denote significant differences from V, where P < 0.001, as determined using Student's t test. Results are representative of three independent experiments.