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

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.

Figure 1. NPR3 and NPR4 mediate degradation of NPR1

a, 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.

Figure 2. SA directly regulates interactions between NPR proteins

a, Interaction between NPR1 and NPR3 in yeast two-hybrid (Y2H) assay. b, Interaction between NPR1 and NPR4 in Y2H. c, Interaction between NPR3 and NPR4 in Y2H. In a, b, and c, diploid yeast cells were spotted on plates (SD-Trp-Leu-His + 3 mM 3-aminotriazole) without (Control) or with 100 μM SA, INA (2,6-dichloroisonicotinic acid), or 4-HBA (4-hydroxybenzoic acid). AD, activation domain; BD, DNA-binding domain. 1, NPR1; 3, NPR3; 4, NPR4. d, In vitro pull-down assays between His-MBP-NPR1 and GST-NPR3 and GST-NPR4 in the presence or absence of 100 μM SA.

Figure 3. NPR3 and NPR4 bind SA

Competition 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. K_d=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 B_t 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).

Figure 4. SA receptors control NPR1 stability to regulate SAR and ETI

a, SAR test in WT, npr3, npr4, npr3 npr4 (npr34), npr1 npr3 npr4 (npr134), and npr1. b-d, ETI test in different mutants using Psm ES4326/avrRpt2. b, The hypersensitive response phenotype 2 and 3 days post inoculation (dpi). c, Ion leakage measurement. Error bars represent SD (n=4). d, Growth of Psm ES4326/avrRpt2. In a and d, Error bars represent 95% confidence intervals (n=6-8). cfu, colony forming units. *, P<0.05; ***, P<0.001. e-g, Close-up images of an infection site by Psm ES4326/avrRpt2. Yellow colour in g and h indicates dead cells and green colour indicates npr1C82A-GFP. Arrows point to intact cells inside the inoculated area. h, Image of the whole infection site showing high npr1C82A-GFP accumulation surrounding the PCD zone. The rectangle shows the area from which the close-up images in e-g were taken.