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. 2013 Mar 6;32(5):701-12.
doi: 10.1038/emboj.2013.15. Epub 2013 Feb 8.

Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7

Valerie Nicaise  1 , Anna Joe, Byeong-ryool Jeong, Christin Korneli, Freddy Boutrot, Isa Westedt, Dorothee Staiger, James R Alfano, Cyril Zipfel
Affiliations

Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7

Valerie Nicaise et al. EMBO J. .

Abstract

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.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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.
Figure 5
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.
Figure 6
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.
Figure 7
Figure 7
HopU1 inhibits FLS2 protein accumulation during infection. (A) Immunoblots with specific antibodies detecting endogenous FLS2 in Col-0 during bacterial infection after syringe inoculation with Pto DC3000 (WT; inoculum: 5 × 107 cfu/ml), Pto DC3000 ΔhopU1 (inoculum: 108 cfu/ml), Pto DC3000 hrcC− (inoculum: 108 cfu/ml). hpi, hours post infection; CBB, Coomassie Brilliant Blue. Values correspond to signal intensity of the FLS2-specific band from the immunoblots relative to the zero time point. (B) Immunoblots with specific antibodies detecting endogenous FLS2 in Col-0 during bacterial infection after syringe inoculation with Pto DC3000 (WT; inoculum: 5 × 107 cfu/ml), Pto DC3000 ΔhopU1 (inoculum: 108 cfu/ml), Pto DC3000 ΔhopU1 [HopU1] (inoculum: 108 cfu/ml), Pto DC3000 ΔhopU1 [HopU1DD] (inoculum: 108 cfu/ml). hpi, hours post infection; CBB, Coomassie Brilliant Blue. Values correspond to signal intensity of the FLS2-specific band from the immunoblots relative to the zero time point. (C) FLS2 transcript level as measured by quantitative RT–PCR in Col-0 plants during bacterial infection after syringe inoculation with Pto DC3000 (WT; inoculum: 5 × 107 cfu/ml), Pto DC3000 ΔhopU1 (inoculum: 108 cfu/ml), Pto DC3000 hrcC− (inoculum: 108 cfu/ml). hpi, hours post infection. (D) Bacterial growth measured during Pseudomonas infection in Col-0 plants, after spray inoculation (inoculum: 2 × 108 cfu/ml) with Pto DC3000 wild-type (WT) or the derivated strains ΔfliC, ΔhopU1 and ΔhopU1ΔfliC. Growth measured by colony forming units (cfu) 4 days after inoculation. Values are mean±s.e. (n=4). Statistical significance was assessed using the ANOVA test (P<0.001; letters indicate statistically significant differences). dai, days after inoculation. The results shown are representative of three independent experiments.
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