Volume 26, Issue 18, Pages 2399-2411 (September 2016) Immunity to Rice Blast Disease by Suppression of Effector-Triggered Necrosis  Ruyi Wang, Yuese Ning, Xuetao Shi, Feng He, Chongyang Zhang, Jiangbo Fan, Nan Jiang, Yu Zhang, Ting Zhang, Yajun Hu, Maria Bellizzi, Guo-Liang Wang  Current Biology  Volume 26, Issue 18, Pages 2399-2411 (September 2016) DOI: 10.1016/j.cub.2016.06.072 Copyright © 2016 Elsevier Ltd Terms and Conditions

Current Biology 2016 26, 2399-2411DOI: (10.1016/j.cub.2016.06.072) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 AvrPiz-t Interacts with APIP5 In Vitro and In Vivo (A) Interaction between AvrPiz-t and APIP5 in yeast. Yeast strains containing AvrPiz-t and APIP5 were grown on an SD-Leu-Trp-His selective medium containing 35 mM 3-amino-1,2,4-triazole (3AT) and were stained with X-gal solution. (B) GST pull-down assay between AvrPiz-t and APIP5. GST or GST-APIP5 was incubated with an equal amount of bacterial lysates containing MBP-AvrPiz-t for 2 hr at 25°C before Glutathione Sepharose 4B beads were added and the preparation was incubated further. Pulled-down proteins were examined by western blots using anti-GST antibody or anti-MBP antibody. (C) In vivo coIP assay for AvrPiz-t and APIP5. Protein isolated from N. benthamiana tissues co-infiltrated with the indicated plasmid combinations was extracted and immunoprecipitated with anti-GFP antibody. Immunoblot analysis was performed using anti-RFP and anti-GFP antibodies. (D) LCI assays show that APIP5 interacts with AvrPiz-t in N. benthamiana leaves. The leaves of N. benthamiana were infiltrated with Agrobacterium strains containing the following construct pairs: NLuc+CLuc, AvrPiz-t-NLuc+CLuc, NLuc+CLuc-APIP5, or AvrPiz-t-NLuc+CLuc-APIP5. (E) The APIP5 N terminus is sufficient for interaction with AvrPiz-t. APIP5 was divided into three regions: N, M, and C. Five constructs were constructed: full-length APIP5 (FL; aa 1–273), APIP5N-terminus (N; aa 1–92), APIP5M (M; aa 93–148), APIP5C-terminus (C; aa 149–273), and APIP5NM (NM; aa 1–148). See also Figure S1. Current Biology 2016 26, 2399-2411DOI: (10.1016/j.cub.2016.06.072) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Subcellular Localization of APIP5 and Its Co-localization with AvrPiz-t in Rice Protoplasts (A) Subcellular localization of APIP5 in rice protoplasts. APIP5-CFP (CFP was fused to the C terminus of APIP5) and mCherry empty plasmids were transfected into rice protoplasts. The images were captured with a confocal microscope 16 hr after transfection. (B) Detection of APIP5 in the cell fractions by western blot analysis. Total (total protein), cytoplasmic (soluble cytoplasmic fraction), and nuclear (nuclear fraction) were extracted from 4-week-old Nipponbare rice plants. HSP protein was used as a cytoplasmic marker. Histone H3 protein was used as a nuclear protein marker. (C) Subcellular localization of AvrPiz-t in rice protoplasts. AvrPiz-t-mCherry (mCherry was fused to C terminus of AvrPiz-t) was transfected into rice protoplasts. Autofluorescence indicates chloroplasts (blue). (D) Co-localization of APIP5-CFP and AvrPiz-t-mCherry in rice protoplasts. Autofluorescence indicates chloroplasts (blue). Scale bars in (A), (C), and (D) represent 10 μm. See also Figures S2 and S3. Current Biology 2016 26, 2399-2411DOI: (10.1016/j.cub.2016.06.072) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Suppression of APIP5 Causes Cell Death Phenotypes and Changes in Defense Gene Expression (A–C) Phenotypes of APIP5 RNAi transgenic plants at the tillering stage. (A) Leaves were collected from 6-week-old APIP5 RNAi transgenic plants (line #71 and #76) and wild-type (WT) plants grown in a growth chamber. (B) Cell death phenotypes of APIP5 RNAi transgenic plants. The first leaf from the top (1); The second leaf from the top (2); The third leaf from the top (3). (C) Reactive oxygen species (ROS) accumulate in the developing lesions on leaves from APIP5 RNAi plants. H2O2 accumulation was detected by DAB staining and observed in the leaves of 6-week-old APIP5 RNAi plants. (D) Transcript level of APIP5 in the APIP5 RNAi transgenic plants. The expression level of APIP5 in the RNAi plants was determined by qRT-PCR. The expression level of the ubiquitin gene was used as an internal control. (Student’s t test, ∗p < 0.05). (E) The protein level of APIP5 in the APIP5 RNAi plants was determined by immunoblotting. Total protein was extracted from rice seedlings and subjected to SDS-PAGE, followed by immunoblot analysis using anti-ACTIN and anti-APIP5 antibodies. The relative band intensity was quantified by ImageJ software. (F) Expression levels of cell death- and defense-related genes in the APIP5 RNAi transgenic and WT plants by qRT-PCR. An asterisk indicates a significant difference between WT and APIP5 RNAi transgenic plants according to Student’s t test (∗∗p < 0.01). See also Figure S4. Current Biology 2016 26, 2399-2411DOI: (10.1016/j.cub.2016.06.072) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Ectopic Expression of AvrPiz-t Enhances the Cell Death Phenotype of APIP5 RNAi Plants (A and B) Cell death phenotypes and DAB staining of APIP5 RNAi∗AvrPiz-t leaves in the F2 generation. The second leaf from the top from 6-week-old plants (WT, APIP5 RNAi, APIP5 RNAi∗AvrPiz-t, and AvrPiz-t) grown in a growth chamber was sampled and photographed. (C and D) Transcript and protein levels of APIP5 in the 6-week-old WT, APIP5 RNAi, APIP5 RNAi∗AvrPiz-t, and AvrPiz-t plants. The relative band intensities in (D) were quantified by ImageJ software. (E) The transcript levels of OsMT2b and OsHLM1 as determined by qRT-PCR. (F) The schematic diagram shows the constructs used in the transient expression assays. (G) The APIP5 transcriptional activation activity assay in Arabidopsis protoplast. HOS15, a transcription suppressor, and ARF5M, an activator, were used as controls. An asterisk indicates a significance between APIP5N and empty vector (Student’s t test, ∗∗p < 0.01). (H) Attenuation of APIP5 transactivation activity by AvrPiz-t. An asterisk indicates a significant difference between APIP5N and APIP5N-AvrPiz-t combination (Student’s t test, ∗∗p < 0.01). See also Figure S5. Current Biology 2016 26, 2399-2411DOI: (10.1016/j.cub.2016.06.072) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Piz-t Partially Suppresses APIP5 Silencing-Induced Cell Death (A) Cell death phenotypes of APIP5 RNAi∗Piz-t-HA leaves in the F2 generation. The second leaf from the top from 6-week-old plants grown in a growth chamber (WT, APIP5 RNAi, APIP5 RNAi∗Piz-t-HA, and Piz-t-HA) was used for photograph. (B) ROS accumulation in the developing lesions on the second leaf from the top of 6-week-old plants grown in a growth chamber (WT, APIP5 RNAi, APIP5 RNAi∗Piz-t-HA, and Piz-t-HA). H2O2 accumulation was detected by DAB staining. (C and D) Protein and transcript levels of APIP5 in 6-week-old plants (WT, APIP5 RNAi, APIP5 RNAi∗Piz-t-HA, and Piz-t-HA). The relative band intensities were quantified with ImageJ software. (E) Transcript level of Piz-t as determined by qRT-PCR. (F) Transcript levels of OsMT2b and OsHLM1 as determined by qRT-PCR. Current Biology 2016 26, 2399-2411DOI: (10.1016/j.cub.2016.06.072) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 APIP5 Interacts with Piz-t In Vitro and In Vivo (A) GST pull-down assay between APIP5 and Piz-t-HA. GST or GST-APIP5 was incubated with an equal amount of total protein containing Piz-t-HA extracted from Piz-t-HA transgenic rice plants for 2 hr at 25°C before Glutathione Sepharose 4B beads were added and the preparation was further incubated. Pulled-down proteins were examined by western blots using anti-GST and anti-HA antibodies, respectively. (B) GST pull-down assay between APIP5 and Piz-t-NT. GST or GST-APIP5 was incubated with an equal amount of bacterial lysates containing MBP-Piz-t-NT for 2 hr at 25°C before Glutathione Sepharose 4B beads were added and the preparation was further incubated. Pulled-down proteins were examined by immunoblots using the anti-GST and anti-MBP antibodies, respectively. (C) Interaction between APIP5 and Piz-t-NT in N. benthamiana as detected by LCI assay. The leaves of N. benthamiana were infiltrated with Agrobacterium strains containing the indicated construct pairs: AvrPiz-t-NLuc+CLuc-APIP5 (positive control), AvrPiz-t-NLuc+CLuc-AvrPiz-t, Piz-t-NT-NLuc+CLuc-AvrPiz-t, or Piz-t-NT-NLuc+CLuc-APIP5. (D) Interaction between Piz-t-NT and APIP5 truncated fragments in N. benthamiana leaves as detected by LCI assay. The leaves of N. benthamiana were infiltrated with Agrobacterium strains containing the following construct pairs: Piz-t-NT-NLuc+CLuc-APIP5, Piz-t-NT-NLuc+CLuc-APIP5N, Piz-t-NT-NLuc+CLuc-APIP5M, or Piz-t-NT-NLuc+CLuc-APIP5C. See also Figure S6. Current Biology 2016 26, 2399-2411DOI: (10.1016/j.cub.2016.06.072) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 APIP5 and Piz-t Protein Accumulation in the Resistant and Susceptible Reactions and a Working Model for the Interactions among AvrPiz-t, APIP5, and Piz-t (A and B) APIP5 and Piz-t protein levels in Piz-t-HA (A) and NPB (B; non-Piz-t) plants inoculated with M. oryzae isolate RB22 carrying AvrPiz-t. The leaf tissue was harvested at 0, 24, 48, 72, and 96 hr after inoculation. APIP5 and Piz-t protein levels were determined by immunoblotting using anti-APIP5 and anti-HA antibodies, respectively. HSP protein was used as the internal control. The relative band intensities were quantified with ImageJ software. (C–E) Working model. (C) In the absence of M. oryzae infection, the transcription factor APIP5 molecules are dimerized, and the dimerized APIP5 proteins regulate gene expression to prevent cell death. (D) During the biotrophic stage of M. oryzae infection in rice cells that lack the Piz-t gene, AvrPiz-t secreted from M. oryzae binds to APIP5 and suppresses its transcriptional activity and protein accumulation, which causes effector-triggered necrosis (ETN). (E) During the biotrophic stage of M. oryzae infection in rice cells that contain the Piz-t gene, Piz-t binds to APIP5 in order to stabilize APIP5 activity and protein level. At the same time, APIP5 is required for the accumulation of Piz-t, which activates the effector-triggered immunity (ETI) responses. See also Figure S7. Current Biology 2016 26, 2399-2411DOI: (10.1016/j.cub.2016.06.072) Copyright © 2016 Elsevier Ltd Terms and Conditions