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NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants.

Fu ZQ, Yan S, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel SH, Tada Y, Zheng N, Dong X - Nature (2012)

Bottom Line: Accordingly, the Arabidopsis npr3 npr4 double mutant accumulates higher levels of NPR1, and is insensitive to induction of systemic acquired resistance.Moreover, this mutant is defective in pathogen effector-triggered programmed cell death and immunity.Our study reveals the mechanism of SA perception in determining cell death and survival in response to pathogen challenge.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute-Gordon and Betty Moore Foundation, Department of Biology, PO Box 90338, Duke University, Durham, North Carolina 27708, USA.

ABSTRACT
Salicylic acid (SA) is a plant immune signal produced after pathogen challenge to induce systemic acquired resistance. It is the only major plant hormone for which the receptor has not been firmly identified. Systemic acquired resistance in Arabidopsis requires the transcription cofactor nonexpresser of PR genes 1 (NPR1), the degradation of which acts as a molecular switch. Here we show that the NPR1 paralogues NPR3 and NPR4 are SA receptors that bind SA with different affinities. NPR3 and NPR4 function as adaptors of the Cullin 3 ubiquitin E3 ligase to mediate NPR1 degradation in an SA-regulated manner. Accordingly, the Arabidopsis npr3 npr4 double mutant accumulates higher levels of NPR1, and is insensitive to induction of systemic acquired resistance. Moreover, this mutant is defective in pathogen effector-triggered programmed cell death and immunity. Our study reveals the mechanism of SA perception in determining cell death and survival in response to pathogen challenge.

Show MeSH
NPR3 and NPR4 mediate degradation of NPR1a, NPR1 protein levels in wild type (WT), npr3, npr4, and npr3 npr4 (npr34) plants treated with 0.5 mM SA. The NPR1 level was determined based on the ratio of NPR1 band intensity to that of the non-specific band (asterisk). b, GST-NPR1 degradation in extracts from WT or npr3 npr4 double mutant (npr34) without (-) or with MG115 or with recombinant His-MBP-NPR3 and His-MBP-NPR4 proteins (NPR3, 4). c,In vitro pull-down assay of GST-Cullin 3A and NPR3-HA and NPR4-HA. d, Co-immunoprecipitation of NPR1-GFP and Cullin 3 in npr1 and npr1 npr3 npr4 (npr134) plants.
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Figure 1: NPR3 and NPR4 mediate degradation of NPR1a, NPR1 protein levels in wild type (WT), npr3, npr4, and npr3 npr4 (npr34) plants treated with 0.5 mM SA. The NPR1 level was determined based on the ratio of NPR1 band intensity to that of the non-specific band (asterisk). b, GST-NPR1 degradation in extracts from WT or npr3 npr4 double mutant (npr34) without (-) or with MG115 or with recombinant His-MBP-NPR3 and His-MBP-NPR4 proteins (NPR3, 4). c,In vitro pull-down assay of GST-Cullin 3A and NPR3-HA and NPR4-HA. d, Co-immunoprecipitation of NPR1-GFP and Cullin 3 in npr1 and npr1 npr3 npr4 (npr134) plants.

Mentions: To test our hypothesis that NPR3 and NPR4 are CUL3 adaptors for NPR1 degradation, we examined the accumulation of NPR1 protein in wild type (WT), npr3, npr4, and npr3 npr4 plants. NPR1 protein levels were higher in the npr4 and the npr3 npr4 mutants than in WT in the absence of exogenous SA, and increased faster in the npr3, npr4, and npr3 npr4 mutants compared to WT in response to SA treatment (Fig. 1a). The effects of npr3 and npr4 on NPR1 were likely post-transcriptional as NPR1 transcripts were not increased in these mutants (Supplementary Fig. 4). To further prove our hypothesis, we performed in vitro degradation experiments using purified recombinant GST-tagged NPR1 protein. We found that after 15 minutes of incubation, the recombinant NPR1 protein was degraded in the WT plant extract, but not in npr3 npr4 (Fig. 1b). Addition of purified His-MBP-tagged recombinant NPR3 and NPR4 proteins to the extract complemented the mutant phenotype, supporting a role of NPR3 and NPR4 in mediating NPR1 degradation. This degradation is likely through the proteasome, as application of the proteasome inhibitor MG115 stabilized the protein (Fig. 1b). To further demonstrate that NPR3 and NPR4 serve as adaptors for the CUL3 E3 ligase, we first performed pull-down experiment using in vitro translated NPR3-HA and NPR4-HA. We found that CUL3A could pull down NPR3 and NPR4, with NPR4 showing a stronger interaction (Fig. 1c). Then we performed co-immunoprecipitation (co-IP) assay using transgenic plants constitutively expressing NPR1-GFP in npr1 and npr1 npr3 npr4 mutants. We found that the amount of the endogenous CUL3 protein pulled down by NPR1-GFP was significantly reduced in the npr1 npr3 npr4 triple mutant compared to the npr1 single mutant (Fig. 1d), indicating that the NPR1-GFP interaction with CUL3 requires NPR3 and NPR4. These results further support our hypothesis that NPR4 and NPR3 are CUL3 adaptors for the degradation of NPR1 before and after SA induction, respectively (Fig. 1a).


NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants.

Fu ZQ, Yan S, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel SH, Tada Y, Zheng N, Dong X - Nature (2012)

NPR3 and NPR4 mediate degradation of NPR1a, NPR1 protein levels in wild type (WT), npr3, npr4, and npr3 npr4 (npr34) plants treated with 0.5 mM SA. The NPR1 level was determined based on the ratio of NPR1 band intensity to that of the non-specific band (asterisk). b, GST-NPR1 degradation in extracts from WT or npr3 npr4 double mutant (npr34) without (-) or with MG115 or with recombinant His-MBP-NPR3 and His-MBP-NPR4 proteins (NPR3, 4). c,In vitro pull-down assay of GST-Cullin 3A and NPR3-HA and NPR4-HA. d, Co-immunoprecipitation of NPR1-GFP and Cullin 3 in npr1 and npr1 npr3 npr4 (npr134) plants.
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Figure 1: NPR3 and NPR4 mediate degradation of NPR1a, NPR1 protein levels in wild type (WT), npr3, npr4, and npr3 npr4 (npr34) plants treated with 0.5 mM SA. The NPR1 level was determined based on the ratio of NPR1 band intensity to that of the non-specific band (asterisk). b, GST-NPR1 degradation in extracts from WT or npr3 npr4 double mutant (npr34) without (-) or with MG115 or with recombinant His-MBP-NPR3 and His-MBP-NPR4 proteins (NPR3, 4). c,In vitro pull-down assay of GST-Cullin 3A and NPR3-HA and NPR4-HA. d, Co-immunoprecipitation of NPR1-GFP and Cullin 3 in npr1 and npr1 npr3 npr4 (npr134) plants.
Mentions: To test our hypothesis that NPR3 and NPR4 are CUL3 adaptors for NPR1 degradation, we examined the accumulation of NPR1 protein in wild type (WT), npr3, npr4, and npr3 npr4 plants. NPR1 protein levels were higher in the npr4 and the npr3 npr4 mutants than in WT in the absence of exogenous SA, and increased faster in the npr3, npr4, and npr3 npr4 mutants compared to WT in response to SA treatment (Fig. 1a). The effects of npr3 and npr4 on NPR1 were likely post-transcriptional as NPR1 transcripts were not increased in these mutants (Supplementary Fig. 4). To further prove our hypothesis, we performed in vitro degradation experiments using purified recombinant GST-tagged NPR1 protein. We found that after 15 minutes of incubation, the recombinant NPR1 protein was degraded in the WT plant extract, but not in npr3 npr4 (Fig. 1b). Addition of purified His-MBP-tagged recombinant NPR3 and NPR4 proteins to the extract complemented the mutant phenotype, supporting a role of NPR3 and NPR4 in mediating NPR1 degradation. This degradation is likely through the proteasome, as application of the proteasome inhibitor MG115 stabilized the protein (Fig. 1b). To further demonstrate that NPR3 and NPR4 serve as adaptors for the CUL3 E3 ligase, we first performed pull-down experiment using in vitro translated NPR3-HA and NPR4-HA. We found that CUL3A could pull down NPR3 and NPR4, with NPR4 showing a stronger interaction (Fig. 1c). Then we performed co-immunoprecipitation (co-IP) assay using transgenic plants constitutively expressing NPR1-GFP in npr1 and npr1 npr3 npr4 mutants. We found that the amount of the endogenous CUL3 protein pulled down by NPR1-GFP was significantly reduced in the npr1 npr3 npr4 triple mutant compared to the npr1 single mutant (Fig. 1d), indicating that the NPR1-GFP interaction with CUL3 requires NPR3 and NPR4. These results further support our hypothesis that NPR4 and NPR3 are CUL3 adaptors for the degradation of NPR1 before and after SA induction, respectively (Fig. 1a).

Bottom Line: Accordingly, the Arabidopsis npr3 npr4 double mutant accumulates higher levels of NPR1, and is insensitive to induction of systemic acquired resistance.Moreover, this mutant is defective in pathogen effector-triggered programmed cell death and immunity.Our study reveals the mechanism of SA perception in determining cell death and survival in response to pathogen challenge.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute-Gordon and Betty Moore Foundation, Department of Biology, PO Box 90338, Duke University, Durham, North Carolina 27708, USA.

ABSTRACT
Salicylic acid (SA) is a plant immune signal produced after pathogen challenge to induce systemic acquired resistance. It is the only major plant hormone for which the receptor has not been firmly identified. Systemic acquired resistance in Arabidopsis requires the transcription cofactor nonexpresser of PR genes 1 (NPR1), the degradation of which acts as a molecular switch. Here we show that the NPR1 paralogues NPR3 and NPR4 are SA receptors that bind SA with different affinities. NPR3 and NPR4 function as adaptors of the Cullin 3 ubiquitin E3 ligase to mediate NPR1 degradation in an SA-regulated manner. Accordingly, the Arabidopsis npr3 npr4 double mutant accumulates higher levels of NPR1, and is insensitive to induction of systemic acquired resistance. Moreover, this mutant is defective in pathogen effector-triggered programmed cell death and immunity. Our study reveals the mechanism of SA perception in determining cell death and survival in response to pathogen challenge.

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