Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity

Abstract

The plant defense hormone salicylic acid (SA) is perceived by two classes of receptors, NPR1 and NPR3/NPR4. They function in two parallel pathways to regulate SA-induced defense gene expression. To better understand the roles of the SA receptors in plant defense, we systematically analyzed their contributions to different aspects of Arabidopsis (Arabidopsis thaliana) plant immunity using the SA-insensitive npr1-1 npr4-4D double mutant. We found that perception of SA by NPR1 and NPR4 is required for activation of N-hydroxypipecolic acid biosynthesis, which is essential for inducing systemic acquired resistance. In addition, both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are severely compromised in the npr1-1 npr4-4D double mutant. Interestingly, the PTI and ETI attenuation in npr1-1 npr4-4D is more dramatic compared with the SA-induction deficient2-1 (sid2-1) mutant, suggesting that the perception of residual levels of SA in sid2-1 also contributes to immunity. Furthermore, NPR1 and NPR4 are involved in positive feedback amplification of SA biosynthesis and regulation of SA homeostasis through modifications including 5-hydroxylation and glycosylation. Thus, the SA receptors NPR1 and NPR4 play broad roles in plant immunity.

Figure 1.

Regulation of SAR and NHP Levels by NPR1 and NPR4. (A) Growth of Hpa Noco2 on the distal leaves of wild-type Col-0, npr1-1, npr4-4D, and npr1-1 npr4-4D plants in a SAR assay. Two primary leaves of 3-week-old plants were infiltrated with Psm ES4326 (OD₆₀₀ = 0.001) or 10 mM MgCl₂ (mock) 2 d before the plants were sprayed with Hpa Noco2 spore suspension (50,000/mL in water). We included 15 plants for each treatment. Disease symptoms were scored 7 d later by counting the number of conidiophores on the distal leaves. Disease ratings are as follows: 0, no conidiophores on plants; 1, one leaf is infected with no more than five conidiophores; 2, one leaf is infected with more than five conidiophores; 3, two leaves are infected but with no more than five conidiophores on each infected leaf; 4, two leaves are infected with more than five conidiophores on each infected leaf; 5, more than two leaves are infected with more than five conidiophores. The experiment was repeated three times with independently grown plants, yielding similar results. (B) and (C) Amounts of Pip (B) and NHP (C) in leaf tissue for the indicated genotypes 24 h after infiltration with Psm ES4326 (OD₆₀₀ = 0.001) or 10 mM MgCl₂ (Mock). (D) Amounts of NHP-OG in leaf samples 24 h after treatment with Psm ES4326 (OD₆₀₀ = 0.001) or 10 mM MgCl₂ (Mock). For (B) to (D), error bars represent sd of three independent biological replicates from 4-week-old plants. Different letters indicate samples with statistical differences (P < 0.05, Student’s t test; n = 3). FW, fresh weight; nd, not detectable. These experiments were repeated twice with independently grown plants, yielding similar results.

Figure 2.

Regulation of ALD1, SARD4, and FMO1 Transcript Levels by NPR1 and NPR4. (A) to (C) Induction of ALD1 (A), SARD4 (B), and FMO1 (C) expression in the leaves of 4-week-old wild-type Col-0, npr1-1, npr4-4D, and npr1-1 npr4-4D plants 24 h after infiltration with Psm ES4326 (OD₆₀₀ = 0.001) or 10 mM MgCl₂ (Mock). (D) to (F) Induction of ALD1 (D), SARD4 (E), and FMO1 (F) expression in 2-week-old seedlings for wild-type Col-0, npr1-1, npr4-4D, and npr1-1 npr4-4D before (0 h) and after (1 h) treatment with 50 μM SA. (G) Binding of TGA2 to the ALD1 and FMO1 promoter regions, as determined by ChIP experiments. ChIP was performed using anti-TGA2 antibodies and protein A-agarose beads or protein A-agarose beads alone (no-antibody control). For each genotype, we calculated the fold change of ChIP signal for anti-TGA2 antibodies relative to the no-antibody control. Data represent measurements of four samples from two independent experiments. No statistical differences were detected between ChIP signals from the wild type and tga2 tga5 tga6 for each promoter tested (Student’s t test; n = 4). (H) and (I) Induction of SARD1 (H) and CBP60g (I) expression in leaf tissue of 4-week-old wild-type Col-0, npr1-1, npr4-4D, and npr1-1 npr4-4D plants 24 h after infiltration with Psm ES4326 (OD₆₀₀ = 0.001) or 10 mM MgCl₂ (Mock). Values were normalized to ACTIN1 expression. Error bars represent sd of three independent biological replicates. Different letters indicate samples with statistical differences: P < 0.01 ([A] to [C]) and P < 0.05 ([D] to [F], [H], and [I]), Student’s t test (n = 3).

Figure 3.

Roles of NPR1 and NPR4 in NHP-Induced Immunity. (A) NHP-induced immunity against Hpa Noco2 in wild-type Col-0, npr1-1, npr4-4D, npr1-1 npr4-4D, sid2-1, and fmo1-1 plants. Two primary leaves from 3-week-old plants were infiltrated with 1 mM NHP or water 1 d before plants were sprayed with Hpa Noco2 spore suspension (50,000/mL in water). A total of 15 plants were scored for each treatment. Disease symptoms were scored 7 d later using the disease rating scores (0 to 5) described in Figure 1A. (B) Morphology of 3-week-old wild-type Col-0, FMO1-3D, npr1-1 FMO1-3D, npr4-4D FMO1-3D, and npr1-1 npr4-4D FMO1-3D plants. Bar = 1 cm. (C) Growth of Hpa Noco2 in 2-week-old seedlings of the indicated genotypes. (D) Morphology of 3-week-old wild-type Col-0, FMO1-3D, and sid2-1 FMO1-3D plants. Bar = 1 cm. (E) Growth of Hpa Noco2 on 2-week-old wild-type Col-0, FMO1-3D, sid2-1 FMO1-3D, and sid2-1 seedlings. Error bars in (C) and (E) represent sd of four independent biological replicates. Different letters indicate samples with statistical differences (P < 0.01, Student’s t test; n = 4). FW, fresh weight. For (A), (C), and (E), the experiments were repeated twice using independently grown plants, with similar results. (F) and (G) Effects of excessive amounts of unlabeled SA or NHP on binding of recombinant NPR1 (F) and NPR4 (G) proteins to [³H]SA in size-exclusion chromatography. A total of 0.4 mg/mL purified His6-MBP-NPR1 or His6-MBP-NPR4 protein was incubated with 200 nM [³H]SA in 50 μL of PBS buffer with or without a 10,000-fold excess amount of unlabeled SA or NHP. A sample with no protein added (No protein) was used as a negative control. Error bars represent sd of three independent reactions. Different letters indicate samples with statistical differences (P < 0.01, Student’s t test; n = 3). Experiments were repeated twice using different batches of recombinant proteins, with similar results.

Figure 4.

Regulation of PTI and ETI by NPR1 and NPR4. (A) Growth of Pto DC3000 on the leaves of 4-week-old wild-type Col-0, npr1-1, npr4-4D, npr1-1 npr4-4D, and sid2-1 plants after treatment with water or 1 µM flg22. After 24 h, the treated leaves were infiltrated with Pto DC3000 (OD₆₀₀ = 0.001). Samples were taken 3 d after Pto DC3000 inoculation. Error bars represent sd from six biological replicates. The reduction of bacterial titer after flg22 treatment in each genotype was regarded as flg22-induced protection. The flg22-induced protection among different genotypes was compared using a two-way ANOVA test, and different letters indicate genotypes with statistical differences (P < 0.05, Student’s t test; n = 6). The experiment was repeated twice with independently grown plants, with similar results. CFU, colony-forming units. (B) to (D) Induction of SARD1 (B), PR1 (C), and PR2 (D) expression in the indicated genotypes 12 h after infiltration with Pto DC3000 hrcC or 10 mM MgCl₂ (Mock). (E) Growth of Pto DC3000 AvrRpt2 and Pto DC3000 AvrRps4 in the indicated genotypes. Error bars represent sd from six biological replicates. Different letters indicate samples with statistical differences (P < 0.05, Student’s t test; n = 6). The experiment was repeated three times with independently grown plants, with similar results. (F) to (H) Induction of SARD1 (F), PR1 (G), and PR2 (H) expression in the indicated genotypes 16 h after infiltration with 10 mM MgCl₂ (Mock), Pto DC3000 AvrRpt2, and Pto DC3000 AvrRps4. In (B) to (D) and (F) to (H), values were normalized to ACTIN1. Error bars represent sd from three independent biological replicates. Different letters indicate samples with statistical differences (P < 0.05, Student’s t test; n = 3). Plants used in all assays were 4 weeks old.

Figure 5.

Analysis of Immune Defects in npr1-1 npr4-4D and fmo1-1 sid2-1 Double Mutants. (A) Growth of Pto DC3000 hrcC in wild-type Col-0, npr1-1 npr4-4D, fmo1-1 sid2-1, fmo1-1, and sid2-1 plants. (B) Growth of Pto DC3000 in the indicated genotypes after treatment with water or flg22. The experiment was performed as described in Figure 4A. The flg22-induced protection (the reduction of bacterial titer after flg22 treatment in each genotype) among different genotypes was compared using a two-way ANOVA test. (C) and (D) Growth of Pto DC3000 AvrRpt2 (C) and Pto DC3000 AvrRps4 (D) for the indicated genotypes. Error bars represent sd from six biological replicates. Different letters indicate samples with statistical differences (P < 0.05, Student’s t test; n = 6). Experiments were repeated twice with independently grown plants, with similar results. Plants used in all assays were 4 weeks old. CFU, colony-forming units.

Figure 6.

Regulation of SA Biosynthesis, Hydroxylation of SA, and Conversion of SA to SAG by NPR1 and NPR4. (A) Levels of 2,5-DHBA in 4-week-old wild-type Col-0, npr1-1, npr4-4D, and npr1-1 npr4-4D plants treated with 10 mM MgCl₂ (Mock) or Pto DC3000 AvrRpt2. (B) SA-induced expression of DMR6 in 2-week-old seedlings of the indicated genotypes. (C) Induction of DMR6 expression in the leaves of 4-week-old plants of the indicated genotypes 16 h after infiltration with 10 mM MgCl₂ (Mock) or Pto DC3000 AvrRpt2. (D) SA-induced expression of DMR6 in 2-week-old wild-type Col-0 and tga2 tga5 tga6 seedlings. (E) Binding of TGA2 to the DMR6 promoter region, as determined by ChIP-qPCR. (F) Levels of free SA and SAG in 4-week-old plants of the indicated genotypes treated with 10 mM MgCl₂ (Mock) or Pto DC3000 AvrRpt2. (G) Induction of UGT76B1 expression in the leaves of 4-week-old plants of the indicated genotypes 16 h after infiltration with 10 mM MgCl₂ (Mock) or Pto DC3000 AvrRpt2. (H) SA-induced expression of UGT76B1 in 2-week-old seedlings of the indicated genotypes. (I) SA-induced expression of UGT76B1 in 2-week-old wild-type Col-0 and tga2 tga5 tga6 seedlings. (J) Binding of TGA2 to the UGT76B1 promoter region, as determined by ChIP-qPCR. (K) to (M) Induction of ICS1 (K), EDS5 (L), and PBS3 (M) expression in the leaves of 4-week-old plants of the indicated genotypes 16 h after infiltration with 10 mM MgCl₂ (Mock) or Pto DC3000 AvrRpt2. For (A) and (F), error bars represent sd from four independent biological replicates. Different letters indicate samples with statistical differences (P < 0.05, Student’s t test; n = 4). FW, fresh weight. These experiments were repeated three times with similar results. For (B), (D), (H), and (I), 2-week-old seedlings were sprayed with 50 μM SA. RNA samples were collected before (−SA) and 1 h after (+SA) treatment. For (B) to (D), (G) to (I), and (K) to (M), values were normalized to ACTIN1. Error bars represent sd from three independent biological replicates. Different letters indicate samples with statistical differences (P < 0.05, Student’s t test; n = 3). For (E) and (J), ChIP was performed using anti-TGA2 antibodies and protein A-agarose beads or protein A-agarose beads with no antibody added (no-antibody control). For each genotype, fold change of the ChIP signal for anti-TGA2 antibodies was calculated relative to the no-antibody control. The results represent measurements of four samples from two independent experiments. Different letters indicate samples with statistical differences (P < 0.01, Student’s t test; n = 4).

Figure 7.

A Working Model Summarizing the Broad Roles of SA Receptors in Plant Immunity. SA is perceived by two classes of receptors: NPR1 and NPR3/NPR4. Binding of SA abolishes the transcriptional repression activity of NPR3/NPR4 and enhances the transcriptional activation activity of NPR1, leading to the upregulation of SA-responsive defense regulators. The induction of SA biosynthetic genes (ICS1, EDS5, and PBS3) promotes SA production, whereas the induction of UGT76B1 and DMR6 stimulates the conversion of SA to 2,5-DHBA and SAG, respectively. In local tissues, the expression of SA-responsive defense regulators promotes both PTI and ETI and stimulates the production of the SAR mobile signal NHP by activating the expression of NHP biosynthetic genes (ALD1, SARD4, and FMO1). In distal tissues, NHP promotes SA biosynthesis and SA-induced resistance.