RRS1 promotes association of RPS4 and EDS1 in the nucleus.

<p>(A) In the presence of RRS1, the RPS4/EDS1 are predominantly localized to the nucleus. BiFC assays with the co-expression of <i>nVenus-RPS4</i>/<i>cCFP-EDS1</i>/<i>GUS-HF/mCherry</i> 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 <i>RPS4-HA</i> and <i>RRS1-HF</i> with <i>GFP-EDS1</i> or <i>GFP</i> in <i>N</i>. <i>benthamiana</i> 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.</p

RPS4 homodimerization is dependent on RRS1.

<p>(A) BiFC assays using nVenus- and cCFP-tagged RPS4 reveal that RPS4 self-association in the nucleus is RRS1-dependent. The <i>nVenus-RPS4</i>, <i>cCFP-RPS4</i>, and <i>mCherry</i> were transiently co-expressed in the presence of <i>RRS1-HF</i> or <i>GUS-HF</i> in <i>N</i>. <i>benthamiana</i> leaves. At 2 dpi, the reconstruction YFP signal is observed with confocal microscope (Leica SP5). <i>mCherry</i> 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. <i>Agrobacterium</i>-mediated transient co-expression of <i>RRS1-GFP</i>/<i>RPS4-HF</i>/<i>RPS4-HA</i> or <i>GFP</i>/ <i>RPS4-HF</i>/<i>RPS4-HA</i> was performed in <i>N</i>. <i>benthamiana</i> 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 <i>RRS1-GFP</i>/<i>RRS1-HF</i>, <i>RRS1-GFP</i>/<i>RPS4-HF</i> or <i>GFP</i>/<i>RRS1-HF</i> were performed in <i>N</i>. <i>benthamiana</i> 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.</p

AvrRps4 and PopP2 do not disrupt the EDS1/PAD4/RPS4/RRS1 complex.

<p>(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 <i>RRS1-HF</i>, <i>RPS4-HA</i>, <i>EDS1-V5</i> and <i>PAD4-HA</i> in the presence of <i>AvrRps4-GFP</i>, <i>PopP2-GFP</i> or <i>GFP</i> in <i>N</i>. <i>benthamiana</i>. (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 <i>N</i>. <i>benthamiana</i> leaves transiently co-expressing <i>RRS1-HF</i>, <i>RPS4-Myc</i>, <i>EDS1-V5</i> and <i>PAD4-HA</i> in presence of <i>AvrRps4-GFP</i>, <i>PopP2-GFP</i> or <i>GFP</i>. Total extracts were immunoprecipitated with anti-GFP beads followed by immunoblotting with the indicated antibodies. AvrRps4^C represents processed AvrRps4 C-terminus. All the experiments were repeated at least three times with similar results.</p