Volume 4, Issue 6, Pages 1168-1184 (September 2013) Double-Stranded RNA-Binding Protein 4 Is Required for Resistance Signaling against Viral and Bacterial Pathogens  Shifeng Zhu, Rae-Dong Jeong, Gah-Hyun Lim, Keshun Yu, Caixia Wang, A.C. Chandra-Shekara, Duroy Navarre, Daniel F. Klessig, Aardra Kachroo, Pradeep Kachroo  Cell Reports  Volume 4, Issue 6, Pages 1168-1184 (September 2013) DOI: 10.1016/j.celrep.2013.08.018 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure 1 RNA Silencing Suppressor Function Is Not Required for the Activation of HRT-Mediated Resistance (A) Visual phenotypes in Col-0 and Di-17 leaf at 3 days postinoculation (dpi) with WT-TCV, CPB, or CPC strains of TCV. The HR phenotype was evaluated in at least 35 plants in four separate experiments. (B) Trypan-blue-stained leaves from Di-17 and Col-0 plants inoculated with WT-TCV, or CPB and CPC mutants. Leaves were sampled at 3 dpi, and the arrows indicate isolated cell death zones corresponding to visible HR lesions. This experiment was repeated three times with similar results. (C) RNA gel blot analysis showing expression of PR-1 and TCV CP in indicated genotypes after inoculation with buffer (mock), WT-TCV, or CPB and CPC mutants. Total RNA was extracted from inoculated leaves at 1 or 3 dpi. Ethidium bromide staining of rRNA was used as the loading control. The experiment was repeated twice with similar results. (D) Typical morphological phenotypes of Di-17 and Col-0 plants inoculated with WT-TCV, CPB, or CPC viruses. Plants were photographed at 18 dpi. Approximately 30–40 plants were inoculated in three separate experiments and analyzed for disease phenotypes. (E) Western blot showing relative CP levels in Col-0 and Di-17 plants at 1 or 3 dpi with WT-TCV, CPB, or CPC viruses. Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated three times with similar results. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure 2 HRT-Mediated Resistance to TCV Is Dependent on RDR6 and DCL4 (A) HR formation in mock- or TCV-inoculated WT plants (Col-0 and Di-17) or F2 progeny derived from various crosses at 3 dpi. The HR phenotype was evaluated in at least 100 F2 progeny. (B) Trypan-blue-stained leaves showing microscopic cell death phenotype after TCV inoculation. Scale bars, 270 μm. The cell death phenotype in TCV-inoculated HRT dcl1 plants was similar to Di-17. At least five independent leaves were analyzed with similar results. (C) RNA gel blot analysis showing expression of PR-1 transcript in indicated genotypes after mock or TCV inoculation. Total RNA was extracted from inoculated leaves at 3 dpi. This experiment was repeated twice with similar results. Ethidium bromide staining of rRNA was used as the loading control. The PR-1 levels in TCV-inoculated HRT dcl1 plants were similar to Di-17. (D) Typical morphological phenotypes of TCV-inoculated HRT (Di-17), HRT rdr, HRT dcl, and hrt (Col-0) genotypes. Plants were photographed at 18 dpi. (E) Time-course analysis of TCV CP levels in the inoculated leaves of indicated genotypes at 1, 2, and 3 dpi. Enzyme-linked immunosorbent assay was used to quantify the CP levels. This experiment was repeated three times with similar results. The error bars indicate SD. (F) SA and SAG levels in mock- or TCV-inoculated plants at 3 dpi. Asterisks indicate data statistically significant from mock-inoculated plants (p < 0.05, n = 6). The error bars indicate SD. The experiment was repeated twice with similar results. See also Figure S1. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure 3 The HRT drb4 Plants Show Spreading HR and Support Increased Replication of Virus (A) HR formation in TCV-inoculated Di-17, HRT drb4, and HRT crt1 plants at 3, 6, and 10 dpi. The HR phenotype was evaluated in ∼40 plants that were analyzed in three separate experiments. (B) Trypan-blue-stained leaves showing microscopic cell death phenotype at 10 dpi with TCV. Scale bars, 270 μm. At least five independent leaves were analyzed with similar results. (C) RNA gel blot analysis showing expression of PR-1 and CP transcripts in indicated genotypes at 3 dpi after TCV inoculation. This experiment was repeated three times with similar results. Ethidium bromide staining of rRNA was used as the loading control. (D) Western blot showing relative CP levels in TCV-inoculated genotypes shown in (C). Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated three times with similar results. (E) Western blot showing relative CP levels in Di-17 and HRT drb4 plants inoculated with CPB, CPC, R8A, and WT TCV. Leaves were sampled at 3 dpi. Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated three times with similar results. (F) TCV-stop-inoculated Col-0, Di-17, HRT drb4, and HRT crt1 leaves with absence of visual HR. Plants were photographed at 4 dpi. This experiment was repeated three times with similar results. (G) RNA (upper three panels; PR-1, CP, and rRNA) and protein (lower two panels; α-CP and Ponceau-S) gel blot analyses showing expression of PR-1 and CP transcripts and CP protein levels in TCV-stop-inoculated plants. TCV-inoculated Col-0 plants were included as a positive control in protein gel blot. Total RNA and protein were extracted from inoculated leaves at 3 dpi. This experiment was repeated two times with similar results. Ethidium bromide staining of rRNA and Ponceau-S staining of protein were used as the loading controls. (H) Levels of TCV-CP-specific small RNA in Col-0, Di-17, HRT drb4, and HRT crt1 plants inoculated with TCV, TCV-stop, or R8A. Leaves were sampled at 3 dpi. This experiment was repeated four times with similar results. See also Figures S2 and S3. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure 4 DRB4 Interacts with and Is Required for the Stability of HRT (A and B) Western blots showing relative levels of HRT-FLAG in Di-17 (designated HRT) and HRT drb4 (A) or HRT crt1 (B) plants. Ponceau-S staining of the western blots was used as the loading control. This experiment was repeated four times with similar results. (C) Western blot showing WT-like levels of HRT-FLAG in drb4 plants expressing HRT-FLAG and DRB4-MYC. The F2 plants were derived from a cross between drb4::HRT-FLAG and drb4::DRB4-MYC transgenic plants. Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated twice with similar results. (D) Confocal micrographs showing BiFC for indicated proteins. Agroinfiltration was used to express protein in transgenic Nicotiana benthamiana plants expressing the nuclear marker CFP-H2B (scale bar, 10 μM). The micrographs shown are YFP and CFP overlay images. Individual YFP and CFP micrographs are shown in Figure S3E. Arrow and arrowhead indicates nucleus and endosomes, respectively. All interactions were confirmed in three separate experiments. (E) Coimmunoprecipitation (IP) of DRB4-MYC with HRT-FLAG. Total protein extracted from the transgenic Arabidopsis plants expressing HRT-FLAG or DRB4-MYC or both was immunoprecipitated using anti-FLAG affinity beads, and the immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. The experiment was repeated three times with similar results. (F) CoIP of DRB4-MYC with HRT-FLAG. N. benthamiana plants were agroinfiltrated, and immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. Right panel shows IP assay of DCL4-MYC with HRT-FLAG. HRT and DRB4 were expressed using their respective native promoters, and DCL4 was expressed under 35S promoter. This experiment was repeated twice with similar results. (G) Confocal micrographs showing localization of DRB4-GFP, DRB4-NLS-GFP, DRB4-nls-GFP (mutant NLS), DRB4-NES-GFP, and DRB4-nes-GFP (mutant NES) in N. benthamiana (scale bar, 10 μM). The experiment was repeated four times with similar results. (H) CoIP of HRT-FLAG with DRB4-NLS or DRB4-NES/nes proteins. N. benthamiana plants were agroinfiltrated and immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. HRT and DRB4 were expressed under the 35S promoter. This experiment was repeated twice with similar results. See also Figures S3 and S4. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure 5 TCV Inoculation Increases the Cytosolic Pool of DRB4 (A) Western blot showing DRB4-GFP levels in mock- and WT-TCV-/R8A-inoculated and water- or SA- (500 μM) treated transgenic Di-17 plants expressing DRB4-GFP under its native promoter. Pathogen- and SA-treated leaves were sampled at 3 dpi and 2 days posttreatment, respectively. This experiment was repeated three times with similar results. (B) Confocal micrographs showing localization of DRB4-GFP in untreated, mock-, or TCV-/CPB-/R8A-inoculated Arabidopsis plants. The leaves were analyzed at 3 dpi. The experiment was repeated three times with similar results. Scale bars, 10 μM. Bottom-right panel is an enlarged view of micrograph (shown by circle, R8A panel). (C) Confocal micrographs showing localization of DRB4-GFP in TCV-inoculated Arabidopsis plants at 1–3 dpi (upper panel). The bottom panel shows corresponding trypan-blue-stained leaves. The experiment was repeated two times with similar results. Scale bars, 10 μM. Arrows indicate nucleus (upper panel) and dead cells (bottom panel). (D) Confocal micrographs showing localization of DRB4-GFP, DRB4-NLS-GFP, DRB4-NES-GFP, CP-RFP, and R8A-RFP in N. benthamiana (Scale bar, 10 μM). Arrows and arrowheads indicate nucleus and inclusion structures, respectively. (E) Confocal micrographs showing localization of the indicated proteins when coexpressed in N. benthamiana (scale bars, 10 μM). Experiments shown in (D) and (E) were carried out together. Arrows and arrowheads indicate nucleus and inclusion structures, respectively. This experiment was repeated three times with similar results. See also Figure S5. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure 6 TCV Coat Protein Prevents HRT-DRB4 Complex Formation (A) CoIP of TCV-CP with DRB4-MYC. The transgenic Arabidopsis plants expressing DRB4-MYC were inoculated with buffer (indicate by −) or TCV (indicated by +), and the total protein extracted at 3 dpi was immunoprecipitated using anti-MYC affinity beads and analyzed with α-MYC and α-CP. TCV-inoculated Col-0 plants were used as an additional control. This experiment was repeated three times with similar results. (B) IP of DRB4-MYC with HRT-FLAG in mock- and TCV-inoculated plants. Total protein extracted from the transgenic Arabidopsis plants expressing DRB4-MYC and HRT-FLAG was immunoprecipitated using anti-FLAG affinity beads and analyzed with α-MYC and α-FLAG. Double the amount of total protein (2 mg/ml) extracted from TCV-inoculated plants was used for IP. The experiment was repeated three times with similar results. (C–F) CoIP of DRB4-MYC with HRT-FLAG (C), DRB4-MYC with DCL4-FLAG (D), CRT1-MYC with HRT-FLAG (E), and EDS1-MYC with HRT-FLAG (F), in the presence or absence of CP. N. benthamiana plants were agroinfiltrated, and total extracts and immunoprecipitated proteins were analyzed with α-MYC, α-FLAG, and α-CP. The experiments shown in (C)–(F) were repeated three times with similar results. (G) Visual phenotype of N. benthamiana leaves expressing HRT+DRB4 or HRT+DRB4+avrB. Arrows indicate cell death lesions. Agroinfiltration was used to express HRT and DRB4. For avrB, the N. benthamiana plants were inoculated with Pseudomonas syringae expressing avrB. The leaves were photographed at 3 days posttreatment. (H) CoIP of DRB4-MYC with HRT-FLAG in the presence or absence of CP or avrB. Total and immunoprecipitated proteins were analyzed with α-MYC, α-FLAG, α-CP, and α-avrB. Arrow indicates band corresponding to avrB. The experiment was repeated two times with similar results. (I) CoIP of DRB4-MYC with HRT-FLAG in the presence or absence of CP or R8A. N. benthamiana plants were agroinfiltrated and immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. This experiment was repeated two times with similar results. See also Figures S6 and S7. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure 7 DRB4 Is Required for RPS2- and RPM1-Mediated Resistance to Bacterial Pathogens (A and B) Growth of avirulent (avrRpt2, A; avrRpm1, B) P. syringae (Pst) strains on indicated genotypes. Error bars indicate SD. Asterisks indicate data statistically significant from that of control (Col-0) (p < 0.05, n = 4). (C) Morphological phenotype of Col-0 and drb4 plants inoculated with 105 or 106 CFU/ml avrRpt2 Pst. The leaves were photographed at 3 dpi. (D) Electrolyte leakage in Col-0, drb4, and rps2 plants infiltrated with MgCl2 or avrRpt2 Pst. Error bars represent SD (n = 6). (E) RNA gel blot analysis showing expression of PR-1 in Col-0 and drb4 plants after inoculation with MgCl2 (mock) or avrRpt2 Pst. Total RNA was extracted at indicated hours postinoculation. Ethidium bromide staining of rRNA was used as the loading control. The experiment was repeated twice with similar results. (F) CoIP of DRB4-MYC with RPS2-FLAG (left panel) or RPM1-FLAG (right panel). N. benthamiana plants were agroinfiltrated and immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. This experiment was repeated twice with similar results. (G) Confocal micrographs showing BiFC for DRB4-RPM1 and DRB4-RPS2. Agroinfiltration was used to express protein in transgenic Nicotiana benthamiana plants expressing the nuclear marker CFP-H2B (scale bar, 10 μM). The micrographs shown are YFP and CFP overlay images. These interactions were confirmed in three separate experiments. (H–J) Western blots showing relative levels of RPM1-MYC (H), RPS2-HA (I), or RAR1 (J) in Col-0 and drb4 plants. The rar1-21 mutant was used as a negative control for Figure 7J. Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated four (H and I) or two (J) times with similar results. The arrow in Figure 7J indicates band corresponding to RAR1. See also Figure S8. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure S1 The rdr6 and dcl4 Mutations Compromise HRT-Mediated Resistance to TCV, Related to Figure 2 (A) RNA gel-blot analysis showing CP transcript levels in uninoculated tissues of mock- or TCV-inoculated Di-17 plants and TCV-inoculated F2 progeny derived from various crosses (see Table S1). The uninoculated tissues (inflorescence and/or bolt tissues) were sampled at 18 dpi. Ethidium bromide staining of rRNA was used as the loading control. The experiment was repeated with at least six other F2s from each cross and showed similar results. (B) HRT-FLAG levels in four-week-old Di-17, HRT rdr6 and HRT dcl4 plants. Wild-type Col-0 plants were used as a negative control. Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated twice with similar results. (C) HRT-FLAG levels in total (T), soluble (S) and membrane (M) fractions extracted from indicated genotypes. Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated twice with similar results. (D) HRT-FLAG levels in TCV inoculated plants sampled at indicated hours post-inoculation (hpi). Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated twice with similar results. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure S2 The drb4 Mutation Compromises Resistance to TCV, Related to Figure 3 (A) Quantitative RT-PCR analysis showing relative levels of PR-1 transcripts in mock- and TCV-inoculated Di-17 plants. The samples were collected at 1 and 3 dpi and this experiment was repeated twice using two independent cDNA preparations as templates. (B) Typical morphological phenotypes of TCV inoculated Col-0 leaves. (C) Western blot showing relative levels of CP in TCV or R8A inoculated Di-17 and HRT drb4 plants sampled at 1 and 3 dpi. Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated twice with similar results. (D) A time-course analysis showing relative CP levels in the inoculated leaves of Col-0, drb4 and dcl4 sampled at 1, 2 and 3 dpi. Enzyme-linked immunosorbent assay was used to quantify the CP levels. This experiment was repeated three times with similar results. The error bars indicate SD. (E) Western blot showing relative CP levels in TCV inoculated Col-0 and drb4 plants at 1 and 3 dpi. Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated two times with similar results. (F) Western blot showing relative CP levels in the distal bolt tissues (influorescence) of TCV or R8A inoculated Col-0 and drb4 plants sampled at 6 dpi. Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated three times with similar results. (G) Levels of TCV-CP specific small RNA in indicated genotypes inoculated with buffer (mock), TCV, CPB or R8A viruses. Leaves were sampled at 3 dpi. This experiment was repeated two times with similar results. (H) Typical morphological phenotypes of TCV inoculated Di-17, HRT drb4 and Col-0 plants. Plants were photographed at 18 dpi. (I) SA and SAG levels in buffer (Mock)- or TCV-inoculated indicated genotypes at 3 dpi. Asterisks indicate statistical significance of difference in data from the corresponding mock-inoculated plants (p < 0.05, n = 3). The error bars indicate SD. This experiment was repeated twice with similar results. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure S3 HRT Interacts with DRB4 and DRB4-NES but Not DRB4-NLS, Related to Figure 4 (A) Quantitative RT-PCR analysis showing relative levels of HRT transcript in Di-17 and HRT drb4 plants. This experiment was repeated twice using two independent cDNA preparations as templates. (B) HR formation in TCV-inoculated Di-17, and HRT cry2 plants at 3, 6 and 10 dpi. The HR phenotype was evaluated in ∼20 plants that were analyzed in two separate experiments. (C) Trypan blue stained leaves showing microscopic cell death phenotype at 10 dpi with TCV. Scale bars, 270 microns. At least five independent leaves were analyzed with similar results. (D) Western blot showing relative CP levels in TCV inoculated genotypes. Leaves were sampled at 3 dpi. Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated three times with similar results. (E) Confocal micrographs showing BiFC for indicated proteins. Agroinfiltration was used to express protein in transgenic N. benthamiana plants expressing the nuclear marker CFP-H2B (Scale bar, 10 μM). All interactions were confirmed using both combinations of reciprocal N-EYFP/C-EYFP fusion proteins in two separate experiments (three replicates per experiment). (F) Co-IP showing no detectable interaction between DRB4 and GST proteins. N. benthamiana plants were agroinfiltrated and total extracts and immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. The DRB4 and GST proteins were expressed under the 35S promoter. This experiment was repeated twice with similar results. (G) Confocal micrographs showing BiFC between HRT and DRB4-NES or DRB4-NLS. Agroinfiltration was used to express proteins in transgenic N. benthamiana plants (Scale bar, 10 μM). The micrographs shown are YFP images. All interactions were confirmed using both combinations of reciprocal N-EYFP/C-EYFP fusion proteins in two separate experiments (three replicates per experiment). (H) Western blot showing DRB4-NES/NLS levels in N. benthamiana plants shown in (G). Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated two times with similar results. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure S4 DRB4 Interacts with CC, NBS, and LRR Domains of HRT, Related to Figure 4 (A) Confocal micrographs showing BiFC for DRB4 with CC, NBS and LRR domains of HRT and of HRT domains among themselves or GST. Agroinfiltration was used to express proteins in transgenic N. benthamiana plants expressing the nuclear marker CFP-H2B (Scale bar, 10 μM). The micrographs shown are YFP and CFP overlay images. All interactions were confirmed using both combinations of reciprocal N-EYFP/C-EYFP fusion proteins in two separate experiments (three replicates per experiment). (B) Co-IP of DRB4-MYC with CC-FLAG, NBS-FLAG and LRR-FLAG domains of HRT. N. benthamiana plants were agroinfiltrated and total extracts and immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. This experiment was repeated twice with similar results. (C) Hypothetical model showing possible arrangement of HRT and DRB4 proteins. Yellow, orange and blue indicate coiled coil (CC), nucleotide binding site (NBS), and leucine rich repeat (LRR) domains of HRT, respectively. (D) IP of HRT-MYC with HRT-FLAG in N. benthamiana (left panels) and Arabidopsis (right panels). The N. benthamiana plants were agroinfiltrated and total extracts and immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. The Arabidopsis plants coexpressing HRT-FLAG and HRT-MYC transgenes were generated by crossing the individual transgenic plants. This experiment was repeated twice with similar results. (E) IP of DRB4-MYC with DRB4-FLAG. N. benthamiana plants were agroinfiltrated and total extracts and immunoprecipitated proteins were analyzed with α-MYC and α-FLAG. This experiment was repeated three times with similar results. (F and G) Yeast-two hybrid assay showing growth on selection medium (F) and β-glactosidase assay (G). Yeast colonies co-expressing bait (pGADT7) and prey (pGBKT7) plasmids were streaked on plates without (-) leucine and tryptophane or without leucine, tryptophane, and histidine. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure S5 DRB4 Is Induced in TCV Inoculated Plants, Related to Figure 5 (A) Quantitative RT-PCR analysis showing relative levels of DRB4 and PR-1 transcripts in mock- and TCV-inoculated Di-17 plants. The samples were collected at 2 dpi and this experiment was repeated twice using two independent cDNA preparations as templates. (B) RNA gel blot analysis showing expression of DRB4 transcript in mock- and TCV-inoculated Di-17 plants. Same RNA preparation was used in (A) and (B). Ethidium bromide staining of rRNA was used as the loading control. (C) Confocal micrographs showing localization of DRB4-GFP in CPC inoculated Arabidopsis plants. The leaves were analyzed at 3 dpi. (D) Visual phenotypes in mock- and TCV-inoculated pDRB4-DRB4-GFP leaves sampled at indicated dpi. Arrow indicates HR phenotype that starts to appear as small specks at 2 dpi and is prominent at 3 dpi. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure S6 The DRB-nes/nls-GFP Proteins Localize Similar to DRB4-GFP, Related to Figure 5 (A) Typical visual phenotypes in R8A inoculated Di-17 leaf at 3 dpi. At least 35 plants were analyzed in four separate experiments carried together with genotypes shown in Figure 1A. (B) Trypan blue stained leaves from Di-17 plants showing microscopic cell death. Leaves were inoculated with wt-TCV, or R8A strains and sampled at 3 dpi. Arrows indicate isolated cell death zones corresponding to visible HR lesions. This experiment was repeated three times with similar results. (C) RNA gel blot analysis showing expression of PR-1 in TCV or R8A inoculated Di-17 and Col-0 plants. Total RNA was extracted from inoculated leaves at 3 dpi. Ethidium bromide staining of rRNA was used as the loading control. The experiment was repeated three times with similar results. (D) Partial sequence of CP indicating mono- (asterisks) and bi-partite (underlined) nuclear localization sequence. Amino acid mutated in R8A is indicated in blue. (E) Confocal micrographs showing localization of DRB4-nes-GFP and DRB4-nls-GFP (upper panel) and of the indicated proteins when coexpressed in N. benthamiana (lower panel). Arrows and arrowheads indicate nucleus and inclusion structures, respectively (Scale bars, 10 μM). This experiment was carried out together with analysis shown in Figures 5D and 5E and repeated three times (three replicates per experiment) with similar results. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure S7 DRB4 Promotes HRT-Mediated Cell Death, Related to Figure 6 (A) Visual phenotype of Nicotiana benthamiana leaves expressing indicated proteins. Agroinfiltration was used to express HRT, CP, EDS1 (E90-At3g48090), and DRB4 proteins and the leaf was photographed at 4 days post treatment. (B) Electrolyte leakage in N. benthamiana leaves infiltrated with buffer (150 μM acetosyringone, 10 mM MES, 10 mM MgCl2, pH 5.6), or Agrobacterium cultures (suspended in the same buffer) expressing HRT and CP, HRT, CP and EDS1 (E90-At3g48090) or HRT, CP and DRB4. Error bars represent SD (n = 6). Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions

Figure S8 DRB4 Is Required for RPS2- and RPM1-Mediated Resistance to Bacterial Pathogens, Related to Figure 7 (A) Growth of virulent P. syringae (Pst) on Col-0 and drb4. Error bars indicate SD. Asterisks indicate data statistically significant from that of Col-0 (p = 0.04, n = 4). (B) Morphological and microscopic phenotypes of Col-0 and drb4 plants inoculated with 105 or 106 CFU/ml avrRpm1 Pst. The leaves were photographed at 3 dpi. (C) Electrolyte leakage in Col-0 plants infiltrated with MgCl2 or avrRpm1 Pst. Error bars represent SD (n = 6). (D) RNA gel blot analysis showing expression of PR-1 in Col-0 and drb4 plants after inoculation with MgCl2 (mock), avrRpt2 or avrRpm1 Pst. Total RNA was extracted from inoculated leaves at 48 hr post inoculation. Ethidium bromide staining of rRNA was used as the loading control. The experiment was repeated twice with similar results. (E) Quantitative RT-PCR analysis showing relative levels of RPM1 and RPS2 transcripts in Col-0 and drb4 plants. This experiment was repeated twice using two independent cDNA preparations as templates. NS indicates data not statistically significant compared to Col-0. (F and G) SAR response in Col-0 and drb4 plants. Primary leaves were inoculated with MgCl2 or P. syringae expressing avrRpt2 (F) or avrRpm1 (G). The distal leaves were inoculated with the virulent P. syringae and growth of the virulent bacteria was monitored at 3 dpi. Cell Reports 2013 4, 1168-1184DOI: (10.1016/j.celrep.2013.08.018) Copyright © 2013 The Authors Terms and Conditions