PARylation of the forkhead‐associated domain protein DAWDLE regulates plant immunity
2016; Springer Nature; Volume: 17; Issue: 12 Linguagem: Inglês
10.15252/embr.201642486
ISSN1469-3178
AutoresBaomin Feng, Shisong Ma, Sixue Chen, Ning Zhu, Shuxin Zhang, Bin Yu, Yu Yu, Brandon H. Le, Xuemei Chen, Savithramma P. Dinesh‐Kumar, Libo Shan, Ping He,
Tópico(s)Plant Pathogenic Bacteria Studies
ResumoArticle18 October 2016free access Transparent process PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity Baomin Feng Baomin Feng Department of Biochemistry and Biophysics, Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA Search for more papers by this author Shisong Ma Shisong Ma Department of Plant Biology, University of California, Davis, CA, USA Search for more papers by this author Sixue Chen Sixue Chen Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, USA Search for more papers by this author Ning Zhu Ning Zhu Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, USA Search for more papers by this author Shuxin Zhang Shuxin Zhang School of Biological Sciences & Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA Search for more papers by this author Bin Yu Bin Yu School of Biological Sciences & Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA Search for more papers by this author Yu Yu Yu Yu Department of Botany and Plant Sciences, Institute of Integrative Genome Biology and Howard Hughes Medical Institute, University of California, Riverside, CA, USA Search for more papers by this author Brandon Le Brandon Le Department of Botany and Plant Sciences, Institute of Integrative Genome Biology and Howard Hughes Medical Institute, University of California, Riverside, CA, USA Search for more papers by this author Xuemei Chen Xuemei Chen Department of Botany and Plant Sciences, Institute of Integrative Genome Biology and Howard Hughes Medical Institute, University of California, Riverside, CA, USA Search for more papers by this author Savithramma P Dinesh-Kumar Savithramma P Dinesh-Kumar Department of Plant Biology, University of California, Davis, CA, USA Search for more papers by this author Libo Shan Libo Shan Department of Plant Pathology and Microbiology and Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA Search for more papers by this author Ping He Corresponding Author Ping He [email protected] orcid.org/0000-0002-5926-8349 Department of Biochemistry and Biophysics, Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA Search for more papers by this author Baomin Feng Baomin Feng Department of Biochemistry and Biophysics, Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA Search for more papers by this author Shisong Ma Shisong Ma Department of Plant Biology, University of California, Davis, CA, USA Search for more papers by this author Sixue Chen Sixue Chen Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, USA Search for more papers by this author Ning Zhu Ning Zhu Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, USA Search for more papers by this author Shuxin Zhang Shuxin Zhang School of Biological Sciences & Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA Search for more papers by this author Bin Yu Bin Yu School of Biological Sciences & Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA Search for more papers by this author Yu Yu Yu Yu Department of Botany and Plant Sciences, Institute of Integrative Genome Biology and Howard Hughes Medical Institute, University of California, Riverside, CA, USA Search for more papers by this author Brandon Le Brandon Le Department of Botany and Plant Sciences, Institute of Integrative Genome Biology and Howard Hughes Medical Institute, University of California, Riverside, CA, USA Search for more papers by this author Xuemei Chen Xuemei Chen Department of Botany and Plant Sciences, Institute of Integrative Genome Biology and Howard Hughes Medical Institute, University of California, Riverside, CA, USA Search for more papers by this author Savithramma P Dinesh-Kumar Savithramma P Dinesh-Kumar Department of Plant Biology, University of California, Davis, CA, USA Search for more papers by this author Libo Shan Libo Shan Department of Plant Pathology and Microbiology and Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA Search for more papers by this author Ping He Corresponding Author Ping He [email protected] orcid.org/0000-0002-5926-8349 Department of Biochemistry and Biophysics, Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA Search for more papers by this author Author Information Baomin Feng1, Shisong Ma2, Sixue Chen3, Ning Zhu3, Shuxin Zhang4, Bin Yu4, Yu Yu5, Brandon Le5, Xuemei Chen5, Savithramma P Dinesh-Kumar2, Libo Shan6 and Ping He *,1 1Department of Biochemistry and Biophysics, Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA 2Department of Plant Biology, University of California, Davis, CA, USA 3Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, USA 4School of Biological Sciences & Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA 5Department of Botany and Plant Sciences, Institute of Integrative Genome Biology and Howard Hughes Medical Institute, University of California, Riverside, CA, USA 6Department of Plant Pathology and Microbiology and Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA *Corresponding author. Tel: +1 979 458 1368; E-mail: [email protected] EMBO Reports (2016)17:1799-1813https://doi.org/10.15252/embr.201642486 See also: FLH Menke (December 2016) AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Protein poly(ADP-ribosyl)ation (PARylation) primarily catalyzed by poly(ADP-ribose) polymerases (PARPs) plays a crucial role in controlling various cellular responses. However, PARylation targets and their functions remain largely elusive. Here, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation assays to globally identify PARylation targets in plants. Consistent with the essential role of PARylation in plant immunity, the forkhead-associated (FHA) domain protein DAWDLE (DDL), one of PARP2 targets, positively regulates plant defense to both adapted and non-adapted pathogens. Arabidopsis PARP2 interacts with and PARylates DDL, which was enhanced upon treatment of bacterial flagellin. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of DDL by PARP2. Genetic complementation assays indicate that DDL PARylation is required for its function in plant immunity. In contrast, DDL PARylation appears to be dispensable for its previously reported function in plant development partially mediated by the regulation of microRNA biogenesis. Our study uncovers many previously unknown PARylation targets and points to the distinct functions of DDL in plant immunity and development mediated by protein PARylation and small RNA biogenesis, respectively. Synopsis Protein poly(ADP-ribosyl)ation plays crucial roles in various cellular processes, but few PARylation targets have been identified so far in plants. This study reports on a proteome-wide screen for PARP targets in Arabidopsis and identifies PARylation of DAWDLE as essential for its function in plant immunity. The forkhead-associated (FHA) domain protein DAWDLE (DDL) positively regulates plant immunity. Biochemical and genetic analyses indicate that PARylation of DDL is required for its function in pathogen defense. PARylation of DDL is not essential for its function in plant development. Introduction In addition to the preformed physical barriers, the innate immune system is essential for sessile plants to ward off potential infections 1. Perception of microbe-associated molecular patterns (MAMPs) by cell surface-resident pattern recognition receptors (PRRs) activates the first line of plant immunity, termed as pattern-triggered immunity (PTI) 23. Bacterial flagellin, lipopolysaccharide (LPS), peptidoglycan (PGN), elongation factor Tu (EF-Tu), and fungal chitin are among well-characterized MAMPs that elicit robust defense responses in plants. Recognition of flg22, a 22 amino acid peptide derived from bacterial flagellin, by the cognate PRR flagellin-sensing 2 (FLS2) initiates immune signaling via heterodimerization with brassinosteroid (BR)-insensitive 1 (BRI1)-associated kinase 1 (BAK1) in Arabidopsis 45. Members of Botrytis-induced kinase 1 (BIK1) family receptor-like cytoplasmic kinases (RLCKs) are rapidly phosphorylated by the FLS2/BAK1 complex upon flg22 perception to transduce intracellular signaling 67. BIK1 directly phosphorylates NADPH oxidase respiratory burst oxidase homolog D (RBOHD), thereby contributing to the production of reactive oxygen species (ROS) 89. In addition, MAMP perception triggers rapid activation of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), calcium influx, stomatal closure, callose deposition, and production of antimicrobial compounds and defense hormones 210. The robust defense responses are intertwined with MAMP-induced massive transcriptional reprogramming which are orchestrated by the coordinated action of gene-specific transcription factors and the general transcriptional machinery 111213. Protein poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification process that plays a crucial role in a broad array of cellular responses such as DNA damage, cell death, and inflammation 1415. PARylation is primarily catalyzed by poly(ADP-ribose) polymerases (PARPs), which transfer ADP-ribose moieties from donor nicotinamide adenine dinucleotide (NAD+) molecules to target proteins often at glutamate (Glu), aspartate (Asp), or in some cases lysine (Lys) residues, resulting in the formation of linear or branched poly(ADP-ribose) (PAR) polymers on target proteins 1415. PARylation is a reversible reaction and the covalently attached PAR on the target proteins can be hydrolyzed to free PAR or mono-(ADP-ribose) by poly(ADP-ribose) glycohydrolases (PARGs). PAR activities and PARPs have been found in a wide range of organisms from archaebacteria to mammals and plants 16. Similar with their animal counterparts, plant PARPs and PARGs are important regulators in plant growth, genotoxic and abiotic stress responses, circadian clock, and immunity to pathogen infections 1617. In contrast to animals, which often contain more than a dozen of PARP family members in the genome, the Arabidopsis genome encodes three PARPs, PARP1, PARP2, and PARP3. Although AtPARP1 bears the highest sequence homology to human hsPARP-1, which accounts for more than 90% of PARP activities in humans, PARP2 appears to be the most active and important PARP enzyme in Arabidopsis 1819. Protein PARylation regulates multiple PTI responses, such as immune gene activation and callose deposition. Importantly, MAMP treatment enhances PARP2 auto-PARylation activity, suggesting that PARylation is an integral part of plant innate immunity 18. However, protein PARylation targets remain largely unknown in plants. To identify PARylation substrates, we deployed a high-density Arabidopsis protein microarray coupled with in vitro PARylation assays and identified 54 proteins as the putative substrates of PARP2. Several of these candidates were confirmed with in vivo PARylation assays. DAWDLE (DDL), a forkhead-associated (FHA) domain protein, was found to play an important role in plant defense responses. DDL was previously reported to control plant growth and development partially via regulating small RNA biogenesis 2021. We showed that the ddl mutants exhibited enhanced susceptibility to the infections by several bacterial pathogens accompanied with the compromised MAMP-induced callose deposition and defense gene activation at late time points. PARP2 directly interacts with DDL in a PARylation-dependent manner. Mass spectrometry (MS), mutagenesis, and complementation analyses indicate that protein PARylation of DDL is essential for its function in plant immunity, but dispensable for its function in plant development. Thus, we identified DDL as a physiological target of protein PARylation in plant immunity, which is uncoupled from its function in plant development. Results Identification of PARylated proteins using Arabidopsis protein microarray We have previously established in vitro and in vivo PARylation assays of Arabidopsis PARPs and PARGs, and demonstrated that PARP2 possesses a robust polymerase activity 18. To identify PARylated Arabidopsis proteins, we employed a high-density Arabidopsis protein microarray containing 10,048 proteins and performed in vitro PARylation assays using recombinant proteins of PARP2 fused with maltose-binding protein (MBP). The protein microarray chips were incubated with MBP-PARP2 proteins in the presence of NAD+, the donor of ADP-ribose groups. The chips incubated without NAD+ served as a negative control. After extensive washing, the protein chips were incubated with α-PAR antibody, which detects the PAR polymer of PARylated proteins, and then Cy3 fluorophore-conjugated secondary antibody. The fluorescence signals were scanned and imaged by GenePix4100A scanner (Fig 1A). As the same proteins were duplicated as two individual spots on the protein chips, the proteins with both spots consistently exhibiting significantly higher signals than those on the negative control chips were identified as PARylated proteins by PARP2 (Fig 1B, Appendix Fig S1 and Dataset EV1). We have detected 54 candidates that are likely PARylated by PARP2 using this approach (Appendix Table S1). Notably, based on the prediction of protein subcellular localization by TAIR, 56% of candidates are predicated as nuclear-localized proteins (Fig 1C) compared to 34% of total proteins on the chip to be nuclear-localized (Dataset EV1), consistent with the nuclear localization of PARP2 18. Gene Ontology (GO) analysis indicates that proteins associated with DNA/RNA metabolism, response to stresses, response to biotic/abiotic stimuli, and transcription are enriched among candidates compared to the total proteins on the chip (Fig 1D and Dataset EV1). This is consistent with the notion that protein PARylation is often involved in stress responses, DNA damage repair, chromatin modification, and gene transcriptional regulation 22. Figure 1. Identification of PARylation targets using Arabidopsis protein microarray Scheme of identification of Arabidopsis proteins PARylated by PARP2 using protein microarray. Same proteins were spotted as duplicates on the chips. Protein chips were first incubated with MBP-PARP2 proteins in the PARylation reaction buffer with NAD+ (left) or without NAD+ (right). The PARylated proteins indicated as the orange dotted chain were detected with α-PAR antibody hybridization followed by Cy3 fluorophore-conjugated secondary antibody. The fluorescence signals were scanned and imaged by GenePix4100A scanner. Scanned images from a pair of representative protein chips after in vitro PARylation reaction. Subcellular localization of identified candidates based on TAIR prediction. The percentage of each subgroup among the total candidates was presented. GO analysis of identified candidates (dark gray bars) compared to the total proteins on the chip (light gray bars). The proteins were categorized based on the GO annotation by TAIR. The x-axis shows the percentage of genes in a specific functional category; the y-axis represents different categories. Confirmation of PARylated proteins with an in vivo PARylation assay. FLAG-tagged candidate proteins were expressed in protoplasts and immunoprecipitated by α-FLAG antibody after feeding protoplasts with 32P-NAD+. The immunoprecipitated proteins were separated in 10% SDS–PAGE and detected by autoradiography (top panel). The input of FLAG-tagged proteins is shown in an α-FLAG immunoblot (middle panel), and the protein loading control is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). The experiments were repeated three times with similar results. Download figure Download PowerPoint To validate whether some of the candidates are PARylated in vivo, we transiently expressed the candidate genes with a FLAG-epitope tag in Arabidopsis protoplasts fed with radiolabeled 32P-NAD+ as the ADP-ribose donor. The candidate proteins were immunoprecipitated with α-FLAG antibody and subjected to SDS–PAGE autoradiograph. The PARylated proteins were detected as a ladder-like smear due to the incorporation of radiolabeled ADP-ribose with different length of PAR polymer formation. As shown in Fig 1E, DAWDLE (DDL, AT3G20550), PLANT TUDOR-LIKE PROTEIN (AT1G06340), HYALURONAN/mRNA-BINDING PROTEIN (AT5G47210), and METHYL-CPG-BINDING DOMAIN 11 (MBD11, AT3G15790) were PARylated in vivo compared with the transfection of a control plasmid (Fig 1E) or a FLAG-tagged nuclear protein, AT5G03660 (Fig 1E, left lane). The compromised disease resistance to bacterial pathogens in the ddl mutants Protein PARylation has been implicated to play an important role in plant defense 181923. To determine whether any identified PARylation target candidates are involved in plant defense responses, we characterized homozygous T-DNA insertional mutants of several candidates in response to the infection by a virulent bacterial pathogen, Pseudomonas syringae pv. tomato (Pst) DC3000. Among five homozygous T-DNA insertional mutants examined (ddl, mbd11, ubc13b, at1 g10520, and at4 g22150) (Appendix Fig S2A), the ddl-6 (SALK_045025) mutant exhibited high susceptibility to Pst DC3000 infection compared to Col-0 WT and other mutant plants (Appendix Fig S2B). We failed to identify the homozygous mutants for AT5G47210 and AT1G06340 that were PARylated in vivo (Fig 1E). The transcript of MBD11 and UBC13B was not altered in the corresponding T-DNA insertion plants (Appendix Fig S2A). Quantitative RT–PCR (qRT–PCR) analysis indicated that DDL transcripts were substantially reduced in ddl-6 (Fig 2A and B). The bacterial multiplication of Pst DC3000 was more than tenfold higher in the ddl-6 mutant than that in WT plants 3 days post-inoculation (dpi) (Fig 2C and Appendix Fig S2B). The disease symptom development with yellowing and necrosis leaves was more pronounced in the ddl-6 mutant than that in WT plants (Fig 2C). We also isolated another allele of ddl T-DNA insertional mutants, ddl-7 (SAIL_1281_F08), which had about fourfold reduction in DDL transcripts compared to WT Col-0 plants (Fig 2A and B). The ddl-7 mutant also exhibited enhanced susceptibility to Pst DC3000 infections compared to WT plants although to a less extend than ddl-6 (Fig 2C). The previously reported ddl1-1 and ddl1-2 mutants in the Ws background were dwarf with severely stunted growth under the 12-h light/12-h dark growth condition 20 (Appendix Fig S3A). However, the severity of growth defects of ddl-6 and ddl-7 mutants in the Col-0 background was less pronounced than that of ddl-1 and ddl-2 in the Ws background (Appendix Fig S3A). Clearly, ddl-6 and ddl-7 mutants were much bigger than ddl-1 and ddl-2 at 4 weeks after germination when we performed bacterial infection assays (Appendix Fig S3A). Similar with ddl-1 and ddl-2, ddl-6 displayed delayed flowering and reduced apical dominance compared with Col-0 WT plants (Appendix Fig S3B). The ddl-1 mutant was also more susceptible to Pst DC3000 infections than Ws plants (Fig 2D). In addition, ddl-6 was more susceptible to another virulent bacterial pathogen P. syringae pv. maculicola (Psm) ES4326 (Fig 2E), non-pathogenic bacterium Pst DC3000 hrcC, a type III secretion mutant of Pst DC3000 (Figs 2F and EV1A), and non-adaptive pathogen P. syringae pv. phaseolicola (Psp) (Fig 2G). Expression of DDL under the control of the native promoter in ddl-6 restored the disease susceptibility to Pst DC3000 infection to the WT level in two independent transgenic lines W7 and W18 (Fig 2H). The data indicate that DDL plays an important role in plant defense to bacterial infections. Figure 2. DDL is involved in plant defense Scheme of DDL protein domains and gene structures with molecular lesions of five mutants. N-terminal NLS (nuclear localization signal) and C-terminal FHA domains are labeled. The dark gray bars indicate exons, and lines in between indicate introns. The light gray bars indicate 5′ and 3′ UTRs. The ddl-1, ddl-2, and ddl-3 mutants are in the Ws background, whereas ddl-6 (SALK_045025) and ddl-7 (SAIL_1281_F08) are in the Col-0 background. ddl-1, ddl-2, ddl-6, and ddl-7 are T-DNA insertional lines, whereas ddl-3 is a TILLING line with a point mutation. The DDL transcripts and growth phenotype of ddl-6 and ddl-7. DDL transcripts were detected in fully expanded leaves of 4-week-old plants by qRT–PCR (left panel). The expression of DDL in WT Col-0 was set as 1. Plants grown under 12-h light/12-h dark condition for 4 weeks are shown (right panel). Scale bar = 1 cm. ddl-6 and ddl-7 are more susceptible to Pst DC3000 infections. Leaves of 4-week-old plants were inoculated with Pst DC3000 at OD600 = 5 × 10−4. Bacterial numbers were counted at 0 and 3 dpi. Leaf pictures were taken at 3 dpi. Scale bar = 1 cm. The ddl-1 mutant in the Ws background is more susceptible to Pst DC3000 infection. ddl-6 is more susceptible to Psm infection. Leaves of 4-week-old plants were inoculated with Psm at OD600 = 5 × 10−4. Increased bacterial growth of Pst DC3000 hrcC in ddl-6. Leaves of 4-week-old plants were inoculated with Pst DC3000 hrcC at OD600 = 5 × 10−4. Increased bacterial growth of Psp in ddl-6. Leaves of 4-week-old plants were inoculated with Psp at OD600 = 5 × 10−4. Complementation of DDL in disease resistance. Two independent T3 homozygous lines carrying pDDL::DDL-FLAG (W7 and W18) in the ddl-6 background were assayed for the susceptibility to Pst DC3000. Data information: The data are shown as mean ± SD (n = 3) with one-way ANOVA analysis and Tukey test (P < 0.05). Different letters, a, b, or c above the bars, indicate significant differences. One representative result from three independent repeats is shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Defense responses in ddl-6 Increased bacterial growth of Pst DC3000 hrcC in ddl-6. Leaves of 4-week-old plants were inoculated with Pst DC3000 hrcC at OD600 = 5 × 10−4. The data are shown as mean ± SD (n = 3) from three independent repeats with one-way ANOVA analysis and Tukey test (P < 0.05). Different letters, a or b, indicate significant differences. flg22-induced PTI marker gene expression in Col-0 and ddl-6. Ten-day-old seedlings were treated with 100 nM flg22 for 60 min for qRT–PCR analysis. Data are shown as mean ± SD from three independent repeats. Pst DC3000-mediated induction of some late responsive genes is compromised in the ddl-6 and parp1,2 mutants. Fully expanded leaves of 4-week-old plants were inoculated with Pst DC3000 (OD600 = 0.01) and collected at 0, 8, and 24 hpi for qRT–PCR. Data are shown as mean ± SD from three independent repeats. BIK1 phosphorylation upon flg22 treatment in Col-0 and ddl-6. BIK1-HA was transiently expressed in Col-0 or ddl-6 protoplasts, and its phosphorylation upon flg22 treatment was detected as the band shift by an immunoblot with the α-HA antibody. Download figure Download PowerPoint To investigate the potential mechanisms underlying the compromised disease resistance in ddl mutants, we systematically examined various PTI responses in the ddl-6 mutant. Callose deposition is a relatively late response upon MAMP perception. The ddl-6 mutants showed reduced callose deposits at 24 h post-inoculation (hpi) with flg22 or Pst DC3000 hrcC treatment compared to WT plants (Fig 3A). Consistent with an important role of protein PARylation in MAMP-induced callose deposition, the parp1,2 double mutant also showed reduced callose deposits upon flg22 or Pst DC3000 hrcC treatment (Fig 3A). The parp1,2 mutant showed the reduced induction of some early MAMP responsive genes 18. It appears that the flg22-mediated induction of FRK1, WRKY30, AT1G07160, and AT2G17740 by 1 h did not change significantly in the ddl-6 mutant (Fig EV1B). We also investigated the induction of some MAMP responsive genes at late time points, such as 3, 8, or 24 h after treatment 24. Interestingly, the induction of PPDK (AT4G15530), VSR7 (AT4G20110), AT3G08870, and PEN3 (AT1G59870) at 24 hpi after Pst DC3000 hrcC treatment was reduced in the ddl-6 mutant compared to that in WT plants. The induction of these genes by Pst DC3000 hrcC treatment was also reduced in the parp1,2 mutant (Fig 3B). Similarly, Pst DC3000-mediated induction of these genes was reduced in both ddl-6 and parp1,2 mutants (Fig EV1C). The PR1 and PR5 induction by Pst DC3000 was also diminished dramatically in the ddl-6 mutant at 24 hpi (Fig 3C), suggesting that DDL is required for regulating some late responsive or secondary defense genes. The ddl-6 mutant did not affect flg22-induced BIK1 phosphorylation (Fig EV1D), MAPK activation (Fig 3D), or ROS burst (Fig 3E), which is consistent with the dispensable role of protein PARylation in these early responses 18. The data suggested that the compromised plant immunity in the ddl mutant might be partly due to altered PTI responses, especially those occurring at the late time points. Figure 3. Differential PTI responses in ddl-6 flg22-induced callose deposition is reduced in ddl-6 and parp1,2. Leaves of 4-week-old plants were inoculated with 0.5 μM flg22 for 20 h or hrcC at OD600 = 0.2 for 24 h and stained with aniline blue. Callose deposits were visualized under UV light and quantified by ImageJ. The data are shown as mean ± SE (n = 12). Scale bar = 100 μm. The hrcC-mediated induction of some late responsive genes is compromised in the ddl-6 and parp1,2 mutants. Fully expanded leaves of 4-week-old plants were hand-inoculated with hrcC (OD600 = 0.5) and collected at 0, 3, and 24 hpi for qRT–PCR. The data are shown as mean ± SE from three independent repeats. Pst DC3000-induced PR1 and PR5 transcripts are blocked in ddl-6 and parp1,2. Fully expanded leaves of 4-week-old plants were inoculated with Pst DC3000 (OD600 = 0.01) and collected at 0, 8, and 24 hpi for qRT–PCR. The data are shown as mean ± SE from three independent repeats. flg22-induced MAPK activation in Col-0 and ddl-6. Ten-day-old seedlings were treated with 100 nM flg22 and collected at the indicted time points. MAPK activation was analyzed by an immunoblot (IB) with α-pERK antibody (top panel), and the protein loading is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). flg22-induced ROS in Col-0 and ddl-6. Leaf disks from 4-week-old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min. The data are show as mean ± SE (n = 24). Data information: Experiments were repeated three times with similar results. Download figure Download PowerPoint Flg22 treatment potentiates DDL–PARP2 interaction Since DDL is PARylated by PARP2, we further investigated whether DDL directly interacted with PARP2 and whether this interaction is modulated upon MAMP elicitation. We expressed FLAG-epitope tagged DDL and HA-epitope tagged PAPR2 in Arabidopsis protoplasts. DDL-FLAG could immunoprecipitate PARP2-HA (Fig 4A). Apparently, the association was enhanced upon flg22 treatment (Fig 4A). The flg22-induced DDL-HA and PARP2-FLAG association was also observed when they were transiently expressed in Nicotiana benthamiana (Fig 4B). Consistently, bimolecular fluorescence complementation (BiFC) assay also indicated the association of DDL and PARP2 in nuclei with co-expression of PARP2 fused to amino-terminal half of YFP (yellow fluorescence protein) (nYFP-PARP2) and DDL fused to the carboxyl-terminal half of YFP (cYFP-DDL) in Col-0 protoplasts (Fig 4C). JAM3, another nuclear protein 25, did not interact with PARP2 or DDL with BiFC assay (Fig EV2A). To test whether DDL directly interacts with PARP2, we performed an in vitro pull-down assay with PARP2 fused to glutathione-S-transferase (GST) immobilized on Sepharose beads as a bait against DDL fused to 6×HIS-SUMO at N-terminus and an HA tag at C-terminus. As shown in Fig 4D, HIS-SUMO-DDL-HA could be pulled down by GST-PARP2, but not GST itself. Interestingly, when GST-PARP2 and HIS-SUMO-DDL-HA were first subjected to a PARylation reaction before the pull-down assay (Fig 4D, right panels), the interaction between DDL and PARP2 was markedly enhanced (Fig 4D, left panels), suggesting that PARylation may enhance the association of DDL and PARP2. The data are in line with the flg22-induced DDL–PARP2 association (Fig 4A and B). We have shown that flg22 treatment enhanced PARylation activity of PARP2 18. The enhanced PARP2 activity may promote PARP2 and DDL interaction. Consistent with the direct interaction between DDL and PARP2, PARP2 PARylated DDL in vitro (Fig 4E). DDL-FLAG immunoprecipitated from protoplasts could be PARylated by GST-PARP2 in the presence of biotin-NAD+ as detected by a Western blot using the streptavidin-HRP antibody (Fig 4E). The PARylation of DDL, as well as PARP2, could be suppressed by the PARP inhibitor, 3-aminobenzamide (3-AB), or removed by GST-PARG1
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