Volume 20, Issue 6, Pages 758-769 (December 2016) An E3 Ubiquitin Ligase-BAG Protein Module Controls Plant Innate Immunity and Broad- Spectrum Disease Resistance  Quanyuan You, Keran Zhai, Donglei Yang, Weibing Yang, Jingni Wu, Junzhong Liu, Wenbo Pan, Jianjun Wang, Xudong Zhu, Yikun Jian, Jiyun Liu, Yingying Zhang, Yiwen Deng, Qun Li, Yonggen Lou, Qi Xie, Zuhua He  Cell Host & Microbe  Volume 20, Issue 6, Pages 758-769 (December 2016) DOI: 10.1016/j.chom.2016.10.023 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2016 20, 758-769DOI: (10.1016/j.chom.2016.10.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Phenotypes of Wild-Type and ebr1 Mutant Plants (A) The ebr1 mutant enhanced resistance to multiple virulent strains of bacterial blight (Xoo) compared with wild-type (WT) control. Lesions were photographed at 14 days postinfection (dpi). (B) Bacterial blight lesion lengths of wild-type and ebr1 plants inoculated with PXO99A, PXO86, PXO71, PXO112, and PXO341. Asterisks indicate significant difference compared to the wild-type by Student’s t test (∗∗p < 0.01). (C) ebr1 enhanced resistance to rice fungal blast (virulent isolate TH12). Photos were taken at 7 dpi. (D) ebr1 displayed lesion mimic phenotype under field conditions. (E) Detection of cell death by trypan blue staining in ebr1 leaves. (F) DAB staining indicated H2O2 accumulation in ebr1 leaves. (G) The expression of the pathogenesis-related gene PBZ1 was strongly elevated in ebr1, indicating constitutive defense activation. The rice ACTIN1 was used as a control to normalize expression levels. Values are means ± SD (B, n > 50; G, n=3). Scale bars, 1 cm (C–F). Experiments were carried out three times with similar results (A–C and G). See also Figures S1 and S2. Cell Host & Microbe 2016 20, 758-769DOI: (10.1016/j.chom.2016.10.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Cloning and Functional Identification of EBR1 (A) EBR1 was initially mapped to the centromeric region of chromosome 5 using a small population (n = 288), and was further narrowed down to a 2,800-kb region with a large population (n = 9,000). (B) EBR1 was finally located through a microarray approach. Sequence comparison revealed a substitution of T to A in the 5′ splice site of intron 3 of EBR1 in ebr1. (C) Altered splicing of the EBR1 transcript in ebr1. The wild-type fragment is 374 bp in length, whereas RT-PCR generated three fragments with 374, 384, and 460 bp in ebr1; the latter two are derived from incorrectly spliced transcripts. Rice Ubi-1 was used as a loading control. (D) Protein levels of EBR1 in leaves of wild-type, ebr1, and pEBR1::EBR1/ebr1 plants detected with an anti-EBR1 antibody. Actin was detected as a loading control. Note that the EBR1 protein levels were much lower in ebr1 compared with the wild-type. (E–I) Genetic complementation of ebr1. Wild-type, ebr1, and complemented plants (pEBR1::EBR1/ebr1) were inoculated with Xoo (E) and M. oryzae (H). Lesion length (F), bacterial growth (G), and lesion mimics (I) were restored in the complementation plants. Values are means ± SD (F, n > 50; G, n = 3). Asterisks indicate significance difference compared with the wild-type by Student’s t test (∗∗p < 0.01). Scale bar, 1 cm (H and I). Experiments were conducted three times with similar results (E–H). See also Figures S1 and S2 and Tables S1 and S4. Cell Host & Microbe 2016 20, 758-769DOI: (10.1016/j.chom.2016.10.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 EBR1 Negatively Regulates Programmed Cell Death and Defense (A) Transcript levels of EBR1 revealed by qRT-PCR in leaves of the wild-type and EBR1-RNAi lines. The rice ACTIN1 was used as a control to normalize expression levels. (B) Protein levels of EBR1 in leaves detected by immunoblotting with an anti-EBR1 antibody. The rice actin was detected as a loading control. (C) Xoo inoculation of wild-type and EBR1-RNAi plants with strain PXO99A. Note that EBR1-RNAi plants developed a strong lesion mimic phenotype, similar to the ebr1 mutant. (D and E) Disease resistance to Xoo in the EBR1-RNAi lines, measured by length (D) and bacterial growth (E). (F) Increased blast resistance in the EBR1-RNAi lines. Photos were taken at 7 dpi (isolate TH12). (G) Cell death in the EBR1-RNAi plants detected by trypan blue staining. (H) ROS accumulation in the EBR1-RNAi plants detected by DAB staining. Note that line 5-1 had less EBR1 accumulation and likely displayed stronger phenotypes than line 2-6. Values are means ± SD. n = 3 (A and E), n > 50 (D). Asterisks represent significance difference determined by Student’s t test (∗∗p < 0.01). Scale bars, 1 cm (F–H). Experiment was repeated three times with similar results (A and C–F). See also Figures S1, S2, and S3. Cell Host & Microbe 2016 20, 758-769DOI: (10.1016/j.chom.2016.10.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 EBR1 Is an E3 Ligase and Interacts with OsBAG4 to Trigger Degradation of OsBAG4 (A) EBR1 exhibits E3 ubiquitin ligase activity. GST-EBR1 and its mutant (EBR1H22Y) fusion proteins were assayed for E3 activity in the presence of human E1, human E2 (UBCH5b), and Flag tag ubiquitin (Ub). Ubiquitinated proteins were detected by western blot using an anti-ubiquitin antibody. (B) Y2H assay indicates that OsBAG4 interacts with EBR1. (C) GST-OsBAG4 could pull down His-EBR1 in vitro. Fusion proteins were detected by immunoblotting using anti-His and anti-GST antibodies, respectively. (D) A luciferase biomolecular complementation assay shows the OsBAG4- EBR1 interaction in N. benthamiana leaf cells. Fluorescence signal intensity is indicated. (E) CoIP assay of OsBAG4 with EBR1 in rice. The OsBAG4-Flag and EBR1-Myc fusion proteins were constitutively expressed in transgenic rice plants, and coIP was conducted with the anti-Flag (for OsBAG4-Flag), anti-Myc (for EBR1-Myc), and anti-EBR1 (for both EBR1-Myc and endogenous EBR1) antibodies. The asterisk indicates EBR1-Myc coimmunoprecipitated by OsBAG4-Flag, and the band around 100 kDa indicates endogenous EBR1. (F) Cell-free degradation assay showed the proteasome-dependent degradation of OsBAG4. MG132 (50 μM) inhibited the proteasome and thus delayed OsBAG4 degradation in the wild-type extract. (G) Cell-free degradation assay showed the delayed degradation of OsBAG4 in ebr1 mutant extract. (H) Immunodetection of the endogenous OsBAG4 levels with an anti-OsBAG4 antibody. (I) In vitro ubiquitination of OsBAG4 by EBR1. The polyubiquitination of OsBAG4-Flag by EBR1 was detected by immunoblotting with the anti-Flag antibody. The mutated EBR1 protein (EBR1H22Y) was used as a negative control. Actin served as a loading control (F–H). The numbers under lanes indicate relative protein abundance (F and G). See also Figures S4 and S5 and Tables S2 and S4. Cell Host & Microbe 2016 20, 758-769DOI: (10.1016/j.chom.2016.10.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Ectopic Expression of OsBAG4 Causes PCD and Enhances Disease Resistance (A) Immunoblotting to detect the OsBAG4-Flag fusion protein in two representative OsBAG4 overexpression lines (OsBAG4-OX 3-1 and 7-2) using the anti-OsBAG4 antibody. Actin was detected as a loading control. (B–D) PCD was induced by OsBAG4 overaccumulation in the OsBAG4-OX plants, with ebr1-like lesion mimics (B), cell death (C), and H2O2 accumulation (D). (E) Blast symptoms were greatly reduced in the OsBAG4 overexpression lines compared with the wild-type. Photos were taken at 7 dpi (isolate TH12). (F–H) OsBAG4-OX plants enhanced disease resistance to Xoo. Two-month-old plants were inoculated with Xoo (virulent strain PXO99A) inoculation. Lesions were photographed at 14 dpi (F). Lesion length was significantly reduced in the OsBAG4-OX plants (G), with significant decrease in bacterial growth (H) compared with the wild-type. Values are means ± SD. n > 50 (G), n = 3 (H). Asterisks indicate significance difference in comparison with the wild-type by Student’s t test (∗∗p < 0.01). Scale bars, 1 cm (B–E). Experiment was repeated three times with similar results (E–H). See also Figures S1 and S6. Cell Host & Microbe 2016 20, 758-769DOI: (10.1016/j.chom.2016.10.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 OsBAG4 Accumulation Underlies the Autoimmunity Caused by the ebr1 Mutation (A) Two representative OsBAG4-RNAi/ebr1 lines showed decreased OsBAG4 protein levels as detected by western blot. Actin was detected as the loading control. (B) OsBAG4 RNAi largely suppressed the lesion mimic phenotype in the ebr1 background. (C) Bacterial blight symptoms in the OsBAG4-RNAi/ebr1 plants compared with ebr1 at 14 dpi. (D) Lesion lengths of Xoo. Values are means ± SD (n > 50). (E) Bacterial growth in the plants at 14 dpi. Values are means ± SD (n = 3). (F) Decreased blast resistance in the OsBAG4-RNAi/ebr1 lines compared with ebr1. Photos were taken at 7 dpi (isolate TH12). (G) Transcript levels of OsBAG4 revealed by qRT-PCR in OsBAG4-RNAi protoplasts, with empty vector as the control. (H) Transcript levels of OsJAR1, OsAOS2, CEBiP, Cht1, OsPAL1, OsPAL2, PR3, PR4, PR5, and PBZ1 were reduced in OsBAG4-RNAi protoplasts compared with the empty vector control. Asterisks indicate significant difference in comparison with the control (Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Scale bars, 1 cm (B and F). Values are means ± SD (G,H, n = 3). Experiment was repeated three times with similar results (C–H). See also Figures S1, S3, and S6. Cell Host & Microbe 2016 20, 758-769DOI: (10.1016/j.chom.2016.10.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 The EBR1-OsBAG4 Module Fine-Tunes Homeostasis of Immunity Likely through Regulating PTI (A and B) Transient expression of Flag-OsBAG4 (circle 2) also induced PCD in N. benthamiana leaves (A) and H2O2 accumulation (B), which was suppressed by co-expression of HA-EBR1 (circle 3), but not the HA-EBR1H22Y mutant protein (circle 4). (C) EBR1 promoted OsBAG4 degradation in the tobacco system as revealed by immunodetection. OsBAG4 fusion protein levels were detected using the anti-OsBAG4 antibody, and EBR1 fusion protein levels were detected using an anti-HA antibody. pK7WGF2 is the control construct for GFP expression. (D and E) OsBAG4-OX and ebr1 plants simultaneously accumulated higher levels of SA (D) and JA (E) in comparison with the wild-type control. (F) Lesions of wild-type, ebr1, pEBR1::EBR1/ebr1, EBR1-OX, and OsBAG4-OX plants inoculated with avirulent strain PXO339 at 14 dpi. Scale bar, 1 cm. (G) Lesion lengths of wild-type, ebr1, pEBR1::EBR1/ebr1, EBR1-OX, and OsBAG4-OX plants inoculated with PXO339. Note that EBR1 overexpression did not decrease resistance to PXO339. Asterisks indicate significant difference in comparison with wild-type (Student’s t test; ∗p < 0.05, ∗∗p < 0.01) (D, E, and G). Values are means ± SD (D, E, n = 3; G, n>50). Experiments were carried out three times with similar results (F and G). See also Figure S7 and Table S3. Cell Host & Microbe 2016 20, 758-769DOI: (10.1016/j.chom.2016.10.023) Copyright © 2016 Elsevier Inc. Terms and Conditions