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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.

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NPR3 and NPR4 bind SACompetition binding assay of NPR4 (a) and NPR3 (b). NC, no competitor. CSA, 5-chlorosalicylic acid. c.p.m., counts per minute. c, Saturation binding assay of NPR4. Kd=46.2±2.35 nM, h=0.830±0.0314. d, Dissociation assay of NPR4. The dissociation was initiated by addition of 1 mM non-radioactive labelled SA (Cold SA) or by infinite dilution. B0 and Bt are total binding before and after dissociation, respectively. e, Homologous competitive binding assay of NPR3. IC50=1811 nM (Log IC50=3.26±0.0901), h=0.554±0.0612. f, Size exclusion chromatography showing that NPR4 tetramer binds SA (black). Upper panel, elution profile. Green, red, purple and orange peaks correspond to 2000, 158, 75 and 44 kDa, respectively. Lower panel, total binding of [3H]-SA in different fractions. Error bars represent SD (n=2 or 3).
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Figure 3: NPR3 and NPR4 bind SACompetition binding assay of NPR4 (a) and NPR3 (b). NC, no competitor. CSA, 5-chlorosalicylic acid. c.p.m., counts per minute. c, Saturation binding assay of NPR4. Kd=46.2±2.35 nM, h=0.830±0.0314. d, Dissociation assay of NPR4. The dissociation was initiated by addition of 1 mM non-radioactive labelled SA (Cold SA) or by infinite dilution. B0 and Bt are total binding before and after dissociation, respectively. e, Homologous competitive binding assay of NPR3. IC50=1811 nM (Log IC50=3.26±0.0901), h=0.554±0.0612. f, Size exclusion chromatography showing that NPR4 tetramer binds SA (black). Upper panel, elution profile. Green, red, purple and orange peaks correspond to 2000, 158, 75 and 44 kDa, respectively. Lower panel, total binding of [3H]-SA in different fractions. Error bars represent SD (n=2 or 3).

Mentions: Both the Y2H and the in vitro pull-down results strongly suggest that SA directly binds to NPR3 and NPR4 to control their interactions with NPR1. To prove that NPR3 and NPR4 are SA receptors, we measured their SA binding activities using [3H]-SA. We found that both GST-tagged NPR3 and NPR4 recombinant proteins bound [3H]-SA (Fig. 3a, 3b, Supplementary Fig. 2a). Next, we assessed if active or inactive SA analogues could compete with [3H]-SA to bind to GST-NPR3 and GST-NPR4. CSA (chlorosalicylic acid; an active SAR inducer)6 and INA reduced the binding of [3H]-SA to GST-NPR3 and GST-NPR4, whereas 4-HBA had little effect (Fig. 3a and 3b). To assess the binding affinity of NPR3 and NPR4, we performed saturation binding experiments. While NPR4 exhibited a classical saturation curve (Fig. 3c), NPR3 binding could not be saturated even with 1000 nM [3H]-SA, indicating that NPR3 has a lower affinity than NPR4. Accordingly, the binding of SA to NPR3 was slower than NPR4 (Supplementary Fig. 6). Next, we analysed the saturation binding data with GraphPad Prism using different models and found that the model One site-Specific binding with Hill Slope is significantly better than the other models, which indicates that there are multiple binding sites or fractions in NPR3 and NPR4. The Kd value for NPR4 was 46.2±2.35 nM with a Hill coefficient (h) of 0.830±0.0314. To check the cooperativity of different binding sites, we carried out dissociation experiments by addition of 1 mM non-radioactive labelled SA (Cold SA) or by infinite dilution. The dissociation curves (Fig. 3d) indicate that NPR4 has multiple SA binding sites, and the lack of overlap between the two curves suggests negative cooperativity between these binding sites (the first binding reduces the affinity for subsequent binding). The Kd value for NPR3 (981 nM, Supplementary Fig. 7) was significantly higher than 100 nM, which made saturation binding an inappropriate way to calculate the Kd. Therefore, we performed homologous competitive binding assay (Fig. 3e). The IC50 value was calculated to be 1811 nM (Log IC50=3.26±0.0901) with a Hill coefficient of 0.554±0.0612. Through these analyses, we demonstrated that NPR3 and NPR4 bind SA specifically and with different affinities.


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 bind SACompetition binding assay of NPR4 (a) and NPR3 (b). NC, no competitor. CSA, 5-chlorosalicylic acid. c.p.m., counts per minute. c, Saturation binding assay of NPR4. Kd=46.2±2.35 nM, h=0.830±0.0314. d, Dissociation assay of NPR4. The dissociation was initiated by addition of 1 mM non-radioactive labelled SA (Cold SA) or by infinite dilution. B0 and Bt are total binding before and after dissociation, respectively. e, Homologous competitive binding assay of NPR3. IC50=1811 nM (Log IC50=3.26±0.0901), h=0.554±0.0612. f, Size exclusion chromatography showing that NPR4 tetramer binds SA (black). Upper panel, elution profile. Green, red, purple and orange peaks correspond to 2000, 158, 75 and 44 kDa, respectively. Lower panel, total binding of [3H]-SA in different fractions. Error bars represent SD (n=2 or 3).
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Figure 3: NPR3 and NPR4 bind SACompetition binding assay of NPR4 (a) and NPR3 (b). NC, no competitor. CSA, 5-chlorosalicylic acid. c.p.m., counts per minute. c, Saturation binding assay of NPR4. Kd=46.2±2.35 nM, h=0.830±0.0314. d, Dissociation assay of NPR4. The dissociation was initiated by addition of 1 mM non-radioactive labelled SA (Cold SA) or by infinite dilution. B0 and Bt are total binding before and after dissociation, respectively. e, Homologous competitive binding assay of NPR3. IC50=1811 nM (Log IC50=3.26±0.0901), h=0.554±0.0612. f, Size exclusion chromatography showing that NPR4 tetramer binds SA (black). Upper panel, elution profile. Green, red, purple and orange peaks correspond to 2000, 158, 75 and 44 kDa, respectively. Lower panel, total binding of [3H]-SA in different fractions. Error bars represent SD (n=2 or 3).
Mentions: Both the Y2H and the in vitro pull-down results strongly suggest that SA directly binds to NPR3 and NPR4 to control their interactions with NPR1. To prove that NPR3 and NPR4 are SA receptors, we measured their SA binding activities using [3H]-SA. We found that both GST-tagged NPR3 and NPR4 recombinant proteins bound [3H]-SA (Fig. 3a, 3b, Supplementary Fig. 2a). Next, we assessed if active or inactive SA analogues could compete with [3H]-SA to bind to GST-NPR3 and GST-NPR4. CSA (chlorosalicylic acid; an active SAR inducer)6 and INA reduced the binding of [3H]-SA to GST-NPR3 and GST-NPR4, whereas 4-HBA had little effect (Fig. 3a and 3b). To assess the binding affinity of NPR3 and NPR4, we performed saturation binding experiments. While NPR4 exhibited a classical saturation curve (Fig. 3c), NPR3 binding could not be saturated even with 1000 nM [3H]-SA, indicating that NPR3 has a lower affinity than NPR4. Accordingly, the binding of SA to NPR3 was slower than NPR4 (Supplementary Fig. 6). Next, we analysed the saturation binding data with GraphPad Prism using different models and found that the model One site-Specific binding with Hill Slope is significantly better than the other models, which indicates that there are multiple binding sites or fractions in NPR3 and NPR4. The Kd value for NPR4 was 46.2±2.35 nM with a Hill coefficient (h) of 0.830±0.0314. To check the cooperativity of different binding sites, we carried out dissociation experiments by addition of 1 mM non-radioactive labelled SA (Cold SA) or by infinite dilution. The dissociation curves (Fig. 3d) indicate that NPR4 has multiple SA binding sites, and the lack of overlap between the two curves suggests negative cooperativity between these binding sites (the first binding reduces the affinity for subsequent binding). The Kd value for NPR3 (981 nM, Supplementary Fig. 7) was significantly higher than 100 nM, which made saturation binding an inappropriate way to calculate the Kd. Therefore, we performed homologous competitive binding assay (Fig. 3e). The IC50 value was calculated to be 1811 nM (Log IC50=3.26±0.0901) with a Hill coefficient of 0.554±0.0612. Through these analyses, we demonstrated that NPR3 and NPR4 bind SA specifically and with different affinities.

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
Related in: MedlinePlus