Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7
2013; Springer Nature; Volume: 32; Issue: 5 Linguagem: Inglês
10.1038/emboj.2013.15
ISSN1460-2075
AutoresValérie Nicaise, Anna Joe, Byeong‐ryool Jeong, Christin Korneli, Freddy Boutrot, Isa Westedt, Dorothee Staiger, James R. Alfano, Cyril Zipfel,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoArticle8 February 2013free access Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7 Valerie Nicaise Valerie Nicaise The Sainsbury Laboratory, Norwich Research Park, Norwich, UK Search for more papers by this author Anna Joe Anna Joe School of Biological Sciences, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA Search for more papers by this author Byeong-ryool Jeong Byeong-ryool Jeong Department of Plant Pathology, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USAPresent address: Biology Department, Creighton University, 2500 California Plaza, Omaha, NE 68178, USA. Search for more papers by this author Christin Korneli Christin Korneli Molecular Cell Physiology and Cebitec, University of Bielefeld, Bielefeld, Germany Search for more papers by this author Freddy Boutrot Freddy Boutrot The Sainsbury Laboratory, Norwich Research Park, Norwich, UK Search for more papers by this author Isa Westedt Isa Westedt The Sainsbury Laboratory, Norwich Research Park, Norwich, UKPresent address: Research Center for Infectious Diseases, University of Wuerzburg, 97080 Wuerzburg, Germany. Search for more papers by this author Dorothee Staiger Dorothee Staiger Molecular Cell Physiology and Cebitec, University of Bielefeld, Bielefeld, Germany Search for more papers by this author James R Alfano Corresponding Author James R Alfano Department of Plant Pathology, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA Search for more papers by this author Cyril Zipfel Corresponding Author Cyril Zipfel The Sainsbury Laboratory, Norwich Research Park, Norwich, UK Search for more papers by this author Valerie Nicaise Valerie Nicaise The Sainsbury Laboratory, Norwich Research Park, Norwich, UK Search for more papers by this author Anna Joe Anna Joe School of Biological Sciences, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA Search for more papers by this author Byeong-ryool Jeong Byeong-ryool Jeong Department of Plant Pathology, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USAPresent address: Biology Department, Creighton University, 2500 California Plaza, Omaha, NE 68178, USA. Search for more papers by this author Christin Korneli Christin Korneli Molecular Cell Physiology and Cebitec, University of Bielefeld, Bielefeld, Germany Search for more papers by this author Freddy Boutrot Freddy Boutrot The Sainsbury Laboratory, Norwich Research Park, Norwich, UK Search for more papers by this author Isa Westedt Isa Westedt The Sainsbury Laboratory, Norwich Research Park, Norwich, UKPresent address: Research Center for Infectious Diseases, University of Wuerzburg, 97080 Wuerzburg, Germany. Search for more papers by this author Dorothee Staiger Dorothee Staiger Molecular Cell Physiology and Cebitec, University of Bielefeld, Bielefeld, Germany Search for more papers by this author James R Alfano Corresponding Author James R Alfano Department of Plant Pathology, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA Search for more papers by this author Cyril Zipfel Corresponding Author Cyril Zipfel The Sainsbury Laboratory, Norwich Research Park, Norwich, UK Search for more papers by this author Author Information Valerie Nicaise1,‡, Anna Joe2,‡, Byeong-ryool Jeong3, Christin Korneli4, Freddy Boutrot1, Isa Westedt1, Dorothee Staiger4, James R Alfano 3 and Cyril Zipfel 1 1The Sainsbury Laboratory, Norwich Research Park, Norwich, UK 2School of Biological Sciences, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA 3Department of Plant Pathology, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA 4Molecular Cell Physiology and Cebitec, University of Bielefeld, Bielefeld, Germany ‡These authors contributed equally to this work. *Corresponding authors. Department of Plant Pathology, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE 68588-0660, USA. Tel.:+1 402 472 0395; Fax:+1 402 472 3139; E-mail: [email protected] Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK. Tel.:+44 1603 452056; Fax:+44 1603 450011; E-mail: [email protected] The EMBO Journal (2013)32:701-712https://doi.org/10.1038/emboj.2013.15 PDFDownload PDF of article text and main figures. 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 Pathogens target important components of host immunity to cause disease. The Pseudomonas syringae type III-secreted effector HopU1 is a mono-ADP-ribosyltransferase required for full virulence on Arabidopsis thaliana. HopU1 targets several RNA-binding proteins including GRP7, whose role in immunity is still unclear. Here, we show that GRP7 associates with translational components, as well as with the pattern recognition receptors FLS2 and EFR. Moreover, GRP7 binds specifically FLS2 and EFR transcripts in vivo through its RNA recognition motif. HopU1 does not affect the protein–protein associations between GRP7, FLS2 and translational components. Instead, HopU1 blocks the interaction between GRP7 and FLS2 and EFR transcripts in vivo. This inhibition correlates with reduced FLS2 protein levels upon Pseudomonas infection in a HopU1-dependent manner. Our results reveal a novel virulence strategy used by a microbial effector to interfere with host immunity. Introduction An important aspect of innate immunity is the perception of pathogen-associated molecular patterns (PAMPs) by specific pattern recognition receptors (PRRs) leading to PAMP-triggered immunity (PTI; Dodds and Rathjen, 2010; Segonzac and Zipfel, 2011). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases (LRR-RKs) FLS2 and EFR recognize bacterial flagellin (or its derived peptide flg22) and EF-Tu (or its derived peptide elf18), respectively (Gomez-Gomez and Boller, 2000; Zipfel et al, 2006), while perception of fungal chitin depends on the LysM-RK CERK1 (Miya et al, 2007; Wan et al, 2008). In addition to its role in chitin perception, CERK1 is required for peptidoglycan perception (Willmann et al, 2011). Perception of flg22, elf18 or chitin induces a series of early immune responses, including a rapid burst of reactive oxygen species (ROS), phosphorylation events, gene expression, as well as late responses such as callose deposition at the plant cell wall and resistance to pathogens (Segonzac and Zipfel, 2011). Plant PRRs are key to immunity, as their inhibition or loss of function leads to enhanced susceptibility to adapted and non-adapted pathogens (Segonzac and Zipfel, 2011; Willmann et al, 2011). Pathogens must block or avoid PTI to cause disease. A potent strategy to inhibit PTI is via the action of secreted effectors delivered into the host cells leading to effector-triggered susceptibility (ETS; Dodds and Rathjen, 2010). The genome of the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) encodes >30 type III-secreted effectors (T3SEs). Recently, several T3SEs from different P. syringae strains were shown to be virulence factors. Corresponding host targets have been identified only for a few of them, but they revealed that T3SEs interfere with key components of PTI (Block and Alfano, 2011). For instance, AvrPto is a kinase inhibitor that blocks PTI signalling by interfering with the activation of PRRs and/or the complex formation of PRRs with their associated proteins (Xing et al, 2007; Shan et al, 2008; Xiang et al, 2008, 2011). AvrPtoB is a multifunctional protein carrying E3 ubiquitin ligase activity that leads to the degradation of several PRRs and the inhibition of the PRR-associated LRR-RK BAK1 (Gohre et al, 2008; Shan et al, 2008; Gimenez-Ibanez et al, 2009; Cheng et al, 2011; Zeng et al, 2012). Other Pto DC3000 T3SEs target signalling components downstream of PRR activation (Block and Alfano, 2011; Feng et al, 2012). The Pto DC3000 T3SE HopU1 is a mono-ADP-ribosyltransferase (mono-ADP-RT) required for full virulence in Arabidopsis (Fu et al, 2007). Ectopic expression of HopU1 in A. thaliana suppresses callose deposition induced by flg22 in a manner dependent on its mono-ADP-RT activity (Fu et al, 2007). HopU1 targets at least five different A. thaliana RNA-binding proteins (RBPs), including Glycine-Rich Protein 7 (GRP7) and GRP8 (Fu et al, 2007). HopU1 mono-ADP-ribosylates an arginine at position 49 (R49) located in the conserved ribonucleoprotein consensus sequence 1 (RNP-1) motif of the RNA recognition motif (RRM) of GRP7, and this modification affects GRP7's ability to bind RNA in vitro (Jeong et al, 2011). Although HopU1 targets several RBPs, grp7 null mutant plants produce less ROS and callose in response to flg22, elf18 and chitin (Fu et al, 2007; Jeong et al, 2011), indicating that GRP7 regulates both early and late PAMP responses. In addition, grp7 plants are more susceptible to Pto DC3000 (Fu et al, 2007; Jeong et al, 2011). A mutation in R49 blocks the ability of GRP7 to complement these phenotypes (Jeong et al, 2011). These results demonstrate the importance of GRP7 in plant innate immunity and the potency of mono-ADP-ribosylation to block GRP7 function. However, as in the case for many targets of pathogenic effectors, the exact role of GRP7 in innate immunity and therefore the molecular mechanism underlying PTI suppression by HopU1 are still unclear. Here, we illustrate a function for GRP7 in PTI that is inhibited by HopU1. We show that GRP7 associates with translational components in vivo, and more surprisingly that GRP7 associates with the PRRs FLS2 and EFR. However, HopU1 does not affect the associations between GRP7, FLS2 and translational components. Instead, we reveal that GRP7 binds FLS2 and EFR mRNA in vivo, and that HopU1 blocks this interaction. The FLS2 and EFR transcripts provide the first examples of in vivo targets for GRP7 with a clear biological function. This inhibition correlates with reduced FLS2 protein levels in planta upon infection with Pto DC3000 in a HopU1-dependent manner. Our results reveal a novel virulence strategy used by a microbial effector to interfere with host immunity. Results Modulation of GRP7 level and activity affects early and late immune responses Previous results conclusively showed that loss of GRP7 impairs PTI and resistance to Pto DC3000 infection (Fu et al, 2007; Jeong et al, 2011). To investigate the consequences of ectopic GRP7 expression, we monitored PTI and pathogen response in transgenic A. thaliana plants expressing untagged GRP7 under the control of the constitutive promoter 35S (GRP7ox lines) (Streitner et al, 2008). An immunoblot analysis using a specific anti-GRP7 antibody confirmed higher GRP7 levels in transgenic homozygous GRP7ox plants in comparison to the wild-type (WT) Col-2 ecotype (Supplementary Figure S1). A. thaliana Col-2 (WT) and GRP7ox plants were treated with flg22, elf18 or chitin, which resulted in substantially higher ROS production in GRP7ox plants compared to WT (Figure 1A). Similarly, callose deposition was increased in GRP7ox plants compared to WT plants after all three treatments (Figure 1B). Figure 1.GRP7 overexpression enhances significantly PTI responses and resistance to Pseudomonas infection. (A) Oxidative burst triggered by 1 μM flg22, 1 μM elf18, 100 μg/ml chitin or in absence of PAMP treatment in Col-2 and transgenic A. thaliana plants overexpressing GRP7 (GRP7ox). ROS production is presented as total photon count during 25 min of treatment and measured in relative light units (RLUs). Values are mean±s.e. (n=6). Statistical significance was assessed using the ANOVA test (P<0.001). (B) Callose deposition induced by 1 μM flg22, 1 μM elf18, 100 μg/ml chitin or in absence of PAMP treatment, directly infiltrated in Col-2 and transgenic A. thaliana plants overexpressing GRP7 (GRP7ox). Values are mean±s.e. (n=24). Statistical significance was assessed using the ANOVA test (P<0.001). ND, non-detectable. (C) Growth of Pseudomonas syringae pv. tomato (Pto) DC3000 on Col-2 and GRP7ox plants as measured by colony forming units (cfu). Bacterial growth was measured 4 days after spray inoculation with the wild-type strain (WT) or the hrcC− strain. Values are mean±s.e. (n=4). dai, days after inoculation. Statistical significance was assessed using the ANOVA test (P<0.001; letters indicate statistically significant differences). (D) Disease symptoms on Col-2 and GRP7ox plants, 4 days after spray infection with Pto DC3000 WT. All results shown are representative of at least three independent experiments. Download figure Download PowerPoint The A. thaliana GRP7ox plants were used in pathogenicity assays with Pto DC3000 or the Pto DC3000 hrcC− mutant that does not secrete any T3SEs and is therefore severely hypo-virulent. Plants were spray inoculated and bacteria were enumerated at 0 and 4 days after inoculation. Interestingly, GRP7ox plants were more resistant to infection by Pto DC3000 than WT plants (Figures 1C and D). The Pto DC3000 hrcC− mutant exhibited unaltered growth on GRP7ox plants. This may be due to the strongly reduced virulence of the Pto DC3000 hrcC− mutant to which the endogenous GRP7 seems to be sufficient to confer high resistance. The increased resistance to Pto DC3000 infection observed in plants overexpressing GRP7 clearly demonstrates its important role in innate immunity. GRP7 is required for full immunity to Pto DC3000 WT and hrcC−, and HopU1 targets GRP7 (Fu et al, 2007; Jeong et al, 2011). To assess the extent to which HopU1 inhibits PTI responses, we analysed early and late responses triggered by flg22 in transgenic A. thaliana lines constitutively expressing HopU1 C-terminally tagged with haemagglutinin (HA) under the control of the 35S promoter (Fu et al, 2007). In HopU1 plants, the ROS burst induced by flg22 and elf18 treatment was reduced compared to WT plants (Supplementary Figure S2A). Next, we confirmed that HopU1 leaves exhibit less callose deposition upon flg22 treatment (Supplementary Figure S2B; Fu et al, 2007). The reduced flg22 responsiveness of the HopU1 plants correlated with reduced FLS2 protein levels observed in three out of four independent biological experiments (Supplementary Figure S2C). These results were further validated using A. thaliana transgenic lines expressing HopU1-HA under the control of an estradiol-inducible promoter (ind_HopU1) (Supplementary Figures S2D and E). Together, this demonstrates that HopU1 affects both early and late flg22-induced responses. To test whether in planta HopU1 expression affects A. thaliana disease resistance, we assayed bacterial growth after spray inoculation with the Pto DC3000 strains WT, hrcC− or ΔhopU1 that is hypo-virulent (Fu et al, 2007). HopU1 plants were more susceptible to all the strains tested (Supplementary Figure S2F), albeit to a lesser extent than fls2 null mutant plants consistent with the reduced flg22 sensitivity of HopU1 plants (Supplementary Figures S2A–E). Consistent with the notion that GRP7 is a main target of HopU1 in reducing plant immunity, GRP7ox plants were as resistant to Pto DC3000 as WT plants to Pto DC3000 ΔhopU1 (Supplementary Figure S2G). These results, together with previous results (Fu et al, 2007; Jeong et al, 2011), indicate that the abundance and/or activity of GRP7 are both required and limited for triggering optimal early and late PTI responses. GRP7 associates with the immune receptors FLS2 and EFR at the plasma membrane The importance of GRP7 for early PTI responses suggests that GRP7 may affect directly PRRs and/or associated proteins, or indirectly the expression and/or biogenesis of such proteins. Notably, we identified GRP7 in an unbiased yeast two-hybrid screen for EFR-interacting proteins (Supplementary Figure S3A). Importantly, we could confirm this interaction in co-immunoprecipitation experiments after transient co-expression of EFR and GRP7 as C-terminally tagged fusion proteins with HA and enhanced green fluorescent protein (GFP) tags (EFR-3 × HA and GRP7-eGFP, respectively) in Nicotiana benthamiana (Figure 2A). Similarly, GRP7 and FLS2 also interacted when transiently co-expressed as fusion proteins (FLS2-3 × myc and GRP7-eGFP) in N. benthamiana (Figure 2B). However, GRP7-eGFP did not interact under similar conditions with the LRR-RK BAK1 (BAK1-HA) (Figure 2C), which is an important positive regulator of PTI responses downstream of FLS2 and EFR (Chinchilla et al, 2007; Heese et al, 2007; Roux et al, 2011). Figure 2.GRP7 associates with FLS2 at the plasma membrane. (A–C) Co-immunoprecipitation assay performed after transient co-expression of GRP7-eGFP or eGFP with EFR-3 × HA (A), FLS2-3 × myc (B) or BAK1-HA (C) in N. benthamiana plants. Total proteins (input) were subjected to immunoprecipitation with GFP Trap beads followed by immunoblot analysis. (D) Co-immunoprecipitation of GRP7 and FLS2 in A. thaliana. Co-immunoprecipitation assay performed on Col-0 and GRP7-GFP plants untreated (−) or treated (+) with 1 μM flg22 for 15 min. Total proteins (input) were subjected to immunoprecipitation with GFP Trap beads followed by immunoblot analysis. (E) Bimolecular fluorescence complementation assays between GRP7 and FLS2. YFPn, GRP7-YFPn, YFPc and FLS2-YFPc, as well as the reverse combinations YFPc, GRP7-YFPc, YFPn and FLS2-YFPn, were transiently co-expressed in N. benthamiana leaves. Plasmolysis experiment was performed in the presence of 5% NaCl for 5 min. Arrows indicate Hechtian strands. The chlorophyll autofluorescence appears in red. Scale bar corresponds to 20 μm. Photographs were taken 2 days after infiltration and are representative of the total observations (n=60). All results shown are representative of three independent experiments. Download figure Download PowerPoint We confirmed the GRP7–FLS2 association by co-immunoprecipitation in an A. thaliana transgenic line expressing GRP7 C-terminally tagged with a GFP epitope (GRP7-GFP) under the control of the 35S promoter (Kim et al, 2008) and using an anti-FLS2 antibody recognizing the native FLS2 protein (Figure 2D). The GRP7–FLS2 interaction occurred independently of elicitation and was unaltered by flg22 treatment (Figure 2D). The presence of EFR and FLS2 proteins that may correspond to their glycosylated forms (migrating at ∼150 kDa and ∼175 kDa, respectively Nekrasov et al, 2009; Haweker et al, 2010) in the GRP7 immunoprecipitates (Figures 2A, B and D) suggests that the association between GRP7 and PRRs occurs at the plasma membrane once the mature and functional PRRs have migrated through the secretory pathway. GRP7-GFP shows a nucleo-cytoplasmic subcellular localization in A. thaliana, tobacco (Nicotiana tabacum) and N. benthamiana cells upon stable or transient expression (Supplementary Figures S4A and B; Ziemienowicz et al, 2003; Fu et al, 2007; Kim et al, 2008; Lummer et al, 2011). Bimolecular fluorescence complementation (BiFC) experiments using split-yellow fluorescent protein (YFP) following transient expression in N. benthamiana suggest that GRP7 and FLS2 closely associate (Figure 2E). This interaction occurs most likely at the plasma membrane, as indicated by the presence of the reconstituted YFP signal in typical cell wall–plasma membrane connections (called Hechtian strands) after cell plasmolysis (arrows in Figure 2E). An interaction at the cell periphery between GRP7 and EFR could also be observed (Supplementary Figure S3B). GRP7 associates with translational components In exploratory experiments to identify GRP7 interactors in planta by immunoprecipitation using an A. thaliana transgenic line expressing GRP7 C-terminally tagged with HA under the control of its native promoter (GRP7-HA) (Jeong et al, 2011), we identified by mass-spectrometry analysis of the GRP7-HA immunoprecipitates several components of the 43S complex involved in protein translation (Supplementary Table S1). Before the initiation of active translation, the 43S complex recruits both mRNAs and ribosomes, and is composed of several initiation factors in addition to the cap-binding protein eIF4E and the ribosomal 40S subunit (Pestova et al, 2001). Co-immunoprecipitation experiments using the A. thaliana GRP7-GFP transgenic line (Kim et al, 2008) and specific antibodies further revealed the presence of eIF4E and the ribosomal subunit S14 in complex with GRP7 (Figure 3). We used here the GRP7-GFP line for consistency with previous targeted co-immunoprecipitation experiments (Figure 2). Interestingly, slower migrating bands of eIF4E were enriched in GRP7-GFP immunoprecipitates in comparison to the main form detected in the input (see asterisks in Figure 3). Strikingly, treatment with flg22 induced the dissociation of elF4E and S14 from the GRP7 complex (Figure 3), indicating a potential dynamic link between GRP7, ligand-activated PRRs and components of the translational machinery. Figure 3.GRP7 associates with translational components in Arabidopsis. Co-immunoprecipitation of GRP7 and translational components in A. thaliana. Co-immunoprecipitation assay performed on Col-0 and GRP7-GFP plants untreated (−) or treated (+) with 1 μM flg22 for 15 min. Total proteins (input) were subjected to immunoprecipitation with GFP Trap beads followed by immunoblot analysis with anti-GFP antibodies to detect GRP7-GFP or specific antibodies recognizing the translation initiation factor elF4E and the ribosomal protein S14. Asterisks mark eIF4E slower migrating bands. The results shown are representative of three independent experiments. Download figure Download PowerPoint HopU1 does not affect interactions between GRP7, PRRs and translational components Next, we tested if HopU1 could directly affect FLS2 or the GRP7–FLS2 interaction. HopU1 did not interact with FLS2 in vivo as determined by co-immunoprecipitation and split-YFP experiments in N. benthamiana (Supplementary Figures S5A and B). Consistently, HopU1 did not mono-ADP-ribosylate FLS2 in vitro (Supplementary Figure S5C). Although HopU1 directly interacts with GRP7 in vivo (Supplementary Figure S6), HopU1 did not affect the interaction between GRP7 and FLS2 in an A. thaliana transgenic line expressing both GRP7-GFP and HopU1-HA (Figure 4). In addition, HopU1 did not interfere, at least qualitatively, with the association or ligand-induced dissociation between GRP7-GFP and either elF4E or S14 (Supplementary Figure S7). Therefore, the effect of HopU1 on PTI responses is most likely not mediated by direct inhibition of PRRs or protein–protein interactions with GRP7. Figure 4.HopU1 does not affect the protein–protein interactions between GRP7, FLS2 and translational components. Co-immunoprecipitation of GRP7-associated proteins in the presence of HopU1 in A. thaliana. Co-immunoprecipitation assay performed on Col-0 and HopU1 plants expressing or not GRP7-GFP. Total proteins (input) were subjected to immunoprecipitation with GFP Trap beads followed by immunoblot analysis with anti-GFP antibodies to detect GRP7 or specific antibodies recognizing FLS2, the translation initiation factor elF4E, or the ribosomal protein S14. Asterisks mark slower migrating band forms. The results shown are representative of three independent experiments. Download figure Download PowerPoint GRP7 associates with FLS2 and EFR transcripts in planta Next, we investigated the role of GRP7 in PTI in relation to its capacity to bind RNA by testing if GRP7 could bind PRR transcripts. Quantitative RNA-immunoprecipitation assays using the A. thaliana GRP7-HA (Jeong et al, 2011) transgenic line revealed that GRP7 binds FLS2 mRNAs in vivo independently of flg22 treatment (Figure 5A). As positive controls, we confirmed that GRP7 binds its own transcripts as well as transcripts of its closest paralogue GRP8 (Figure 5A), as previously reported in vitro (Staiger et al, 2003; Schoning et al, 2008). GRP7 binds the 3′-UTR of its own transcript and of the GRP8 mRNA (Schoning et al, 2007). Similarly, we identified the 3′-UTR as a binding region of GRP7 in the FLS2 mRNA (Figures 5B and C). In addition to the FLS2 mRNA, GRP7 could also bind the EFR transcript in vivo (Supplementary Figure S8A), consistent with the importance of GRP7 for responses triggered by both flg22 and elf18 (Jeong et al, 2011; Figure 1). However, transcripts of the regulatory LRR-RK BAK1 were not enriched in GRP7 immunoprecipitates (Figure 5A; Supplementary Figure S8A), revealing a certain degree of specificity. Interestingly, GRP8, which is also targeted by HopU1 (Fu et al, 2007), is also able to bind FLS2 and EFR mRNAs (Supplementary Figure S9). These results demonstrate that GRP7, as well as GRP8, bind transcripts of the important PRRs FLS2 and EFR. Figure 5.GRP7 binds FLS2 transcript. (A) RNA immunoprecipitation in grp7-1 and grp7-1/GRP7-HA A. thaliana lines treated for 30 min with water or 1 μM flg22. Total proteins were subjected to immunoprecipitation with anti-HA antibodies followed by quantitative RT–PCR analysis of FLS2, BAK1, GRP7 and GRP8 transcripts with specific primers. Values are mean±s.e. (n=4). The results shown are representative of three independent experiments. (B, C) GRP7 binds the 3′UTR of FLS2 transcripts in vitro. Electrophoretic shift assays performed on the 3′UTR of FLS2 RNAs, in presence of increasing concentrations of GRP7-GST (B). Competition assay was performed with increasing quantity of unlabelled FLS2 3′UTR transcripts to GRP7-GST and 32P-labelled FLS2 3'UTR transcripts (C). The results shown are representative of three independent experiments. Download figure Download PowerPoint HopU1 disrupts the association between GRP7 and PRR transcripts Next, we investigated if HopU1 could affect the ability of GRP7 to bind its target mRNAs, including FLS2 and EFR transcripts. Using an A. thaliana transgenic line expressing both GRP7-GFP and HopU1-HA in quantitative RNA-immunoprecipitation assays, we found that the amount of FLS2 and EFR transcripts bound to GRP7-GFP was strongly reduced in the presence of HopU1 (Figure 6A; Supplementary Figure S8A). A similar effect was observed on the interaction between GRP7 and its own mRNA (Figure 6A; Supplementary Figure S8A). Furthermore, a GRP7(R49K) variant, which carries a mutation in a conserved arginine residue within the RRM RNA-binding domain that is mono-ADP-ribosylated by HopU1 (Jeong et al, 2011), is strongly impaired in its ability to bind FLS2, EFR, GRP7 and GRP8 transcripts (Figure 6B; Supplementary Figure S8B). Furthermore, ADP ribosylation of GRP7 by HopU1 (but not by the catalytically inactive HopU1DD variant; Fu et al, 2007; Jeong et al, 2011) completely blocks the binding of GRP7 to the 3′-UTR of FLS2 mRNA in vitro (Figure 6C). Together, our results suggest that the mono-ADP ribosylation of GRP7 by HopU1 disrupts in planta the ability of GRP7 to bind mRNAs of the PRRs FLS2 and EFR. Figure 6.HopU1 disrupts GRP7–FLS2 transcripts interactions. (A) RNA immunoprecipitation in HopU1, GRP7-GFP and GRP7-GFP/HopU1 A. thaliana lines. Total proteins were subjected to immunoprecipitation with GFP Trap beads followed by quantitative RT–PCR analysis of BAK1, FLS2 and GRP7 transcripts with specific primers. Values are mean±s.e. (n=4). (B) RNA immunoprecipitation in grp7, grp7/GRP7-HA and grp7/GRP7(R49K)-HA A. thaliana lines. Total proteins were subjected to immunoprecipitation with anti-HA matrix beads followed by quantitative RT–PCR analysis of BAK1, FLS2, GRP7 and GRP8 transcripts with specific primers. Values are mean±s.e. (n=4). (C) Electrophoretic shift assays in the presence of HopU1 and its inactive version HopU1DD. Standard ADP-ribosylation reaction was performed with 4 μM GRP7-GST in the presence of 1 μM HopU1 or HopU1DD. The corresponding GRP7-GST was then added to the 3′-UTR of FLS2 transcript binding assay. The results shown are representative of three independent experiments. Download figure Download PowerPoint HopU1 inhibits the pathogen-induced FLS2 protein accumulation during Pseudomonas infection Because HopU1 inhibits GRP7–FLS2 mRNA binding (Figure 6), we asked whether HopU1's action could ultimately affect FLS2 protein levels after translocation into A. thaliana cells during Pto DC3000 infection, which would correspond to the most biologically relevant observation. We observed that the amount of FLS2 protein increases (3.5- to 3.8-fold) over 24 h in leaves infected with Pto DC3000 hrcC− (unable to secrete any T3SEs and therefore unable to dampen PTI) (Figure 7A), consistent with the previous observation that the expression of FLS2, EFR and other potential PRR-encoding genes is PAMP inducible (Zipfel et al, 2004, 2006). Notably, this PAMP-induced accumulation is attenuated by T3SEs (Figure 7A; compare hrcC− with WT). However, this T3SE-mediated suppression was much less marked after inoculation with Pto DC3000 ΔhopU1 (Figure 7A; compare ΔhopU1 with WT). Importantly, expression of HopU1 in trans on a plasmid in Pto DC3000 ΔhopU1 restored the inhibition of FLS2 accumulation during infection, while trans-complementation with the catalytically inactive HopU1DD variant (Fu et al, 2007; Jeong et al, 2011) did not (Figure 7B). In addition, HopU1 does not affect BAK1 levels during infection (Supplementary Figure S10); consistent with our previous finding that GRP7 does not bind BAK1 mRNA (Figures 5 and 6; Supplementary Figure S8). Figure 7.HopU1 inhibits
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