Volume 5, Issue 5, Pages 1125-1137 (September 2012) PAPP2C Interacts with the Atypical Disease Resistance Protein RPW8.2 and Negatively Regulates Salicylic Acid-Dependent Defense Responses in Arabidopsis  Wen-Ming Wang, Xian-Feng Ma, Yi Zhang, Ming-Cheng Luo, Guo-Liang Wang, Maria Bellizzi, Xing-Yao Xiong, Shun-Yuan Xiao  Molecular Plant  Volume 5, Issue 5, Pages 1125-1137 (September 2012) DOI: 10.1093/mp/sss008 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 PAPP2C Interacts with RPW8.2 in Yeast and In Planta. (A) Yeast-two-hybrid (Y2H) assay. The interaction of PAPP2C with full-length RPW8.2 or RPW8.2 without the N-terminal 22 amino acid (▵22RPW8.2) was revealed by the activation of both the LEU and the LacZ reporter genes, which was indicated by the growth of blue-colored yeast cells on synthetic drop-out medium lacking leucine and containing X-Gal. Yeast cells expressing 14–3–3 kappa in fusion with the AD domain (42AD–GF14κ) was used as negative control (Yang et al., 2009). (B) Co-immunoprecipitation (Co-IP) assay. Anti-HA antibody can pull down RPW8.2–YFP from total protein of Col-gl leaves expressing 35S:HA–PAPP2C and NP:RPW8.2–YFP (Lane 1) but not YFP alone from Col-gl leaves expressing 35S:HA–PAPP2C plus 35S:YFP (Lane 2). RPW8.2–YFP was detected as a faint band (*) but YFP as a strong band (#) in the total protein extracts as input (I). RPW8.2–YFP (*) but not YFP was detected in the blot with the pull-downs (P). The presence of the anti-HA light chain (arrowhead) served as a loading control. Note a higher-molecular-weight band (arrow) was detected for HA–PAPP2C in the pull-down containing RPW8.2–YFP (also see Supplemental Figure 3). (C) BiFC assay. Leaves of 6-week-old Col-gl plants transgenic for both NP:RPW8.2–YFPC and 35S:YFPN–PAPP2C or NP:RPW8.2–YFPC and 35S:YFPN (as control) were examined by LSCM. The in planta PAPP2C–RPW8.2 interaction was reported by the reconstitution of YFP in fluorescent punctate spots in a representative epidermal cell. Size bar = 20 μm. Molecular Plant 2012 5, 1125-1137DOI: (10.1093/mp/sss008) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 PAPP2C Is Induced by SA and Pathogens. (A) Expression of PAPP2C was detected in roots (R), stems (S), leaves (L), and flowers (F) by Northern blot analysis. (B) Expression of PAPP2C was induced to higher levels by SA, but not by JA, as measured by qRT–PCR. Six-week-old Col-0 plants were sprayed with 1 mM SA or 50 μM JA. The mRNA levels of PAPP2C were calculated relative to that of Col-0 sprayed with water at 24 h. ACT2 was used as an internal control. Data were means ± S.E. calculated based on results from three duplicated experiments. (C) Expression of PAPP2C was not or only slightly induced to higher levels by infiltration of 10 mM MgCl2 solution. (D) Expression of PAPP2C was induced to higher levels by pathogens. Seven-week-old plants of Col-0 were inoculated via leaf-infiltration of OD600 = 0.0002 bacterial suspension of the virulent P. syringae pv. maculicola ES4326 wild-type strain (Psm Wt) or the congenic avirulent strain carrying avrRpm1 in 10 mM MgCl2 solution. The same-aged Col-0 plants were inoculated with G. cichoracearum UCSC1 (Gc), and 3-week-old Col-0 plants were inoculated with H. arabidopsidis Noco2 (Ha). Total RNA from inoculated leaves collected at the indicated time points was subject to Northern blot analysis for PAPP2C and PR-1. Mock inoculated (c) or un-inoculated plants were used as control. Molecular Plant 2012 5, 1125-1137DOI: (10.1093/mp/sss008) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Down-Regulation of PAPP2C Expression Results in Cell Death and SA- Dependent Powdery Mildew Resistance. (A) Plant phenotypes of representative Col-gl lines transgenic for a PAPP2C–RNAi construct (line I-1) or an amiRNA construct specifically targeting PAPP2C (line M-1) in comparison with Col-gl wild-type. (B) Trypan blue-stained leaf sections of Col-gl and Col-gl transgenic for PAPP2C–RNAi (I-1) showing spontaneous cell death in clusters (arrows). Size bar = 100 μm. (C, D) Enhanced mildew resistance and fungus-induced cell death of PAPP2C–RNAi plants shown by plants (C) or trypan blue-stained leaf sections (D) from a representative RNAi line I-1 in comparison with Col-gl wild-type. Size bar = 100 μm. (E) PAPP2C negatively regulates SA-signaling. The PAPP2C–RNAi transgene in I-1 was introduced into the indicated genotypes by crossing. One representative leaf of each indicated genotype infected by powdery mildew was shown to reflect the infection phenotypes at 8 dpi. (F, G) Silencing Os04g0452000, a homolog of PAPP2C in rice, resulted in stunted growth and severe leaf cell death. One representative of six RNAi lines with similar silencing effect (see Figure 4B for details) is shown. Molecular Plant 2012 5, 1125-1137DOI: (10.1093/mp/sss008) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Measurement of mRNA Levels of Relevant Arabidopsis and Rice Lines. (A) Down-regulation of PAPP2C expression by RNAi results in constitutive expression of PR1. Reduced expression of PAPP2C correlated with induction of PR-1 in Col-gl lines transgenic for PAPP2C–RNAi (I-1 to I-4) or PAPP2C–amiRNA (M-1 and M-2). Relative levels of PAPP2C and PR-1 of the indicated genotypes were measured by qRT–PCR using ACT2 as an internal control (Xiao et al., 2003). Data were means ± S.E. calculated based on results from three duplicated experiments. (B) Down-regulation of Os04g0452000, a homolog of PAPP2C in rice by RNAi. Levels of Os04g0452000 in six rice RNAi lines along with the wild-type rice cultivar (Oryza sativa L. spp. japonica, var. nippobare) were measured by RT–PCR (28 PCR cycles), using ubiquitin as an internal control (25 PCR cycles). Molecular Plant 2012 5, 1125-1137DOI: (10.1093/mp/sss008) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Down-Regulation of PAPP2C in Plants Expressing RPW8 Results in Massive Spontaneous HR-Like Cell Death. (A) Generation of S6 lines transgenic for 35S:HA–PAPP2C. A Western blot analysis using anti-HA antibody (α-HA) revealed the expression levels of HA–PAPP2C in individual T1 lines. Note HA–PAPP2C was barely detectable in line 1 and T2 derived from this line (S6/HA–PAPP2C–CS1) were subject to further analyses. (B, C) Lack of HA–PAPP2C expression correlated with more severe cell death. Twelve T2 individuals of line S6/HA–PAPP2C-CS1 were subjected to Western blot analysis using anti-HA antibody (B) and phenotypic examination at 8 weeks old (C). Notes #1 and #6, which were phenotypically similar to S6, showed expression of HA–PAPP2C, whilst other lanes show background signal. (D) Phenotype and cell death comparison between S6 and S6/PAPP2C–CS1. More extensive cell death in S6/PAPP2C–CS1 was observed in comparison with that of S6. Six-week-old plants were examined by trypan blue staining. A representative leaf section from each genotype is shown. Size bar = 200 μm. (E) Phenotype and cell death comparison between S5 and S5/PAPP2C–I1. Size bar = 200 μm. Molecular Plant 2012 5, 1125-1137DOI: (10.1093/mp/sss008) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Down-Regulation of PAPP2C Leads to Up-Regulation of RPW8.2 (A) and HA–PAPP2C Has PP2C Activity (B, C). (A) Relative mRNA levels of RPW8.2 of the indicated genotypes were measured by qRT–PCR using ACT2 as an internal control (Xiao et al., 2003). Data were means ± S.E. calculated based on results from three duplicated experiments. (B) Expression of HA–PAPP2C in tobacco. N. benthamiana leaves were infiltrated with Agrobacterial cells (strain GV3101) harboring the 35S:HA–PAPP2C construct, or a mixture of Agrobacterial cells containing either the 35S:HA–PAPP2C or the NP:RPW8.2 construct (XE3.8). Total protein was extracted from infiltrated tissues at 3 d post infiltration and subject to Western blot analysis using an anti-HA antibody. (C) Protein phosphatase activity assay. HA–PAPP2C was immunoprecipitated by anti-HA monoclonal antibody from N. benthamiana leaves agro-infiltrated as in (B). About 50 ng of HA–PAPP2C pull-down from each sample was incubated with 32P-labeled casein at 30°C for the indicated times. Equal volume of the pull-down product from leaves infiltrated with Agrobacterial cells carrying an empty vector was used as a negative control. Phosphatase activity was measured by the amount of free 32Pi released. Molecular Plant 2012 5, 1125-1137DOI: (10.1093/mp/sss008) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions