Fig 1.
RPS4 auto-immunity is repressed by RRS1 and RRS1 increases RPS4 protein accumulation.
(A) Transient overexpression of RPS4-HA results in auto-immunity in tobacco leaves but not RRS1-R-HF. The P-loop mutant (RPS4K242A) and the TIR-domain dimerization mutant (RPS4SH/AA) abolish RPS4-dependent auto-immunity. Co-expression of RPS4-HA with RRS1-R-HF blocks HR induction in tobacco leaves. (B) Stunting and dwarf phenotype of Arabidopsis transgenic line stably overexpressing RPS4 is attenuated by crossing with RRS1-R transgenic Arabidopsis line. The 35S::RPS4-HS / 35S::RRS1-R-HF transgenic line was generated by crossing the line expressing the 35S::RPS4-HS with the transgenic line with the 35S::RRS1-R-HF. Images were taken with 4-week-old plants grown in short-day conditions at 22°C. Scale bar = 1.0 cm. (C) Quantification of rosette diameters at 4-week-old of the lines in (B). The leaf diameter was calculated from the plant rosette area measured in ImageJ. One-way ANOVA was used to calculate the statistical significance between genotypes, as indicated by different capital letters (P < 0.001). Bars represent mean ± SD (n = 40). (D) Fractionation of protein extracts show that RPS4 is stabilized by RRS1 in nucleus and cytoplasm. RPS4-Myc and RPS4-HA were transiently co-expressed in the presence or absence of RRS1-HF in N. benthamiana leaves. At 2 dpi, samples were harvested and then fractionated by the percoll-sucrose gradient method. Western blot analysis was performed with anti-FLAG, anti-Myc and anti-HA antibodies. Anti-PEPC was used as a cytosolic marker and anti-histone H3 was used as a nuclear marker. (E) RPS4-HA protein accumulation is increased by RRS1-HF expression. RPS4-HA and GUS-HF or RPS4-HA and RRS1-HF constructs in Agrobacterium tumefaciens were infiltrated into N. benthamiana leaves. A. tumefaciens cells were adjusted to the OD600 of 0.5 for RPS4-HA or 0.1 for RRS1-HF and GUS-HF constructs. After 2 dpi, samples were harvested and Western blots were performed using anti-FLAG and anti-HA antibodies. All the experiments were repeated three times with similar results.
Fig 2.
RPS4 homodimerization is dependent on RRS1.
(A) BiFC assays using nVenus- and cCFP-tagged RPS4 reveal that RPS4 self-association in the nucleus is RRS1-dependent. The nVenus-RPS4, cCFP-RPS4, and mCherry were transiently co-expressed in the presence of RRS1-HF or GUS-HF in N. benthamiana leaves. At 2 dpi, the reconstruction YFP signal is observed with confocal microscope (Leica SP5). mCherry was used as a nuclear and cytoplasmic marker. Scale bar = 10 μm. (B) Co-immunoprecipitation (co-IP) assays reveal that RPS4 self-associates only in the presence of RRS1. Agrobacterium-mediated transient co-expression of RRS1-GFP/RPS4-HF/RPS4-HA or GFP/ RPS4-HF/RPS4-HA was performed in N. benthamiana leaves. Anti-FLAG co-IPs were performed with total protein extracts and probed with anti-GFP, -FLAG, and -HA antibodies. (C) Co-IPs show that RRS1 self-associates and forms a heteromeric complex with RPS4. Transient co-expression assays of RRS1-GFP/RRS1-HF, RRS1-GFP/RPS4-HF or GFP/RRS1-HF were performed in N. benthamiana leaves. Immunoblots show the presence of proteins in total extracts (input) and after immunoprecipitation with anti-GFP beads (IP-GFP). All the experiments were repeated at least three times with similar results.
Fig 3.
RRS1 promotes association of RPS4 and EDS1 in the nucleus.
(A) In the presence of RRS1, the RPS4/EDS1 are predominantly localized to the nucleus. BiFC assays with the co-expression of nVenus-RPS4/cCFP-EDS1/GUS-HF/mCherry reveal reconstruction of YFP signal in the cytoplasmic aggregations and in the nucleus (arrows). In the presence of RRS1-HF, nVenus-RPS4/cCFP-EDS1 association revealed a YFP signal in the nucleus. Scale bar = 10 μm. (B) EDS1 associates with RPS4/RRS1. Upon transient co-delivery of RPS4-HA and RRS1-HF with GFP-EDS1 or GFP in N. benthamiana leaves, samples were harvested at 2 dpi and total extracts were immunoprecipitated with anti-GFP beads. Specific protein-protein interactions were detected by immunoblotting with the indicated antibodies. All the experiments were repeated at least three times with similar results.
Fig 4.
AvrRps4 and PopP2 do not disrupt the EDS1/PAD4/RPS4/RRS1 complex.
(A) Anti-FLAG immunoprecipitation of RRS1-HF, RPS4, EDS1 and PAD4 in the presence and absence of AvrRps4 or PopP2. Samples were prepared from transiently co-expressed RRS1-HF, RPS4-HA, EDS1-V5 and PAD4-HA in the presence of AvrRps4-GFP, PopP2-GFP or GFP in N. benthamiana. (B) Both AvrRps4 and PopP2 associate with RPS4/RRS1/EDS1/PAD4. To confirm effector protein association with a putative RPS4/RRS1/EDS1/PAD4 complex, samples were prepared from N. benthamiana leaves transiently co-expressing RRS1-HF, RPS4-Myc, EDS1-V5 and PAD4-HA in presence of AvrRps4-GFP, PopP2-GFP or GFP. Total extracts were immunoprecipitated with anti-GFP beads followed by immunoblotting with the indicated antibodies. AvrRps4C represents processed AvrRps4 C-terminus. All the experiments were repeated at least three times with similar results.
Fig 5.
PAD4 attenuates EDS1/AvrRps4 association.
(A) The EDS1/PAD4 complex strongly reduced EDS1/AvrRps4 co-immunoprecipitation in planta. EDS1-Myc or EDS1-Myc/PAD4-HA were transiently co-expressed with AvrRps4-GFP, AvrRps4E187A-GFP or GFP in N. benthamiana leaves. Immunoprecipitations were performed using anti-Myc agarose beads and then analyzed by immunoblot with the indicated antibodies. AvrRps4C represents processed AvrRps4 C-terminus. (B) The EDS1/SAG101 complex can associate with AvrRps4. HA-EDS1 or HA-EDS1 with SAG101-Myc were transiently co-expressed with AvrRps4-GFP or GFP in N. benthamiana leaves. Immunoprecipitation was performed using anti-HA agarose beads and then analyzed by immunoblot with the indicated antibodies. (C) BiFC analysis reveals that EDS1/AvrRps4 interaction is reduced in the presence of PAD4-HA but not of SAG101. Cytoplasmic aggregations are reduced in the presence of PAD4. BiFC assays were performed by co-expression of the indicated proteins in N. benthamiana. Images were obtained at 2 dpi. The experiment was repeated three times with similar results. Scale bar = 10 μm.