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

Perception of SA by its receptors is required for activation of NHP biosynthesis during SAR, plays crucial roles in PTI and ETI, and is involved in regulating SA 5-hydroxylation and glycosylation.

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.

INTRODUCTION

Plants have evolved diverse mechanisms to defend themselves against microbial pathogen infections. Plants perceive pathogens by pattern-recognition receptors or resistance (R) proteins at the infection sites, which triggers the activation of local defenses that lead to a secondary immune response in distal tissues termed systemic acquired resistance (SAR; Jones and Dangl, 2006; Fu and Dong, 2013; Zhou and Zhang, 2020). Pattern-recognition receptors recognize conserved molecules present in groups of microorganisms during pattern-triggered immunity (PTI), collectively known as microbe-associated molecular patterns (Zipfel, 2014). For example, the conserved 22-amino acid flg22 peptide, derived from the N terminus of bacterial flagellin, is recognized by the receptor kinase FLAGELLIN-SENSITIVE2 (Gómez-Gómez and Boller, 2000). At the same time, plant R proteins detect fast-evolving effector proteins secreted from pathogens and used for colonization, leading to the activation of effector-triggered immunity (ETI; Cui et al., 2015; Li et al., 2015). Most R genes encode nucleotide binding leucine-rich repeat receptors (NLRs; Li et al., 2015). Typical NLRs can be classified into two subgroups based on their N-terminal domains: Toll/Interleukin1 Receptor-type NLRs (TNLs) and Coiled Coil-type NLRs (CNLs). Differential signaling events occur downstream of TNLs and CNLs. ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) is essential for immunity mediated by TNLs, while NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) is required for disease resistance activated by some CNLs (Aarts et al., 1998).

Salicylic acid (SA) is a phytohormone with critical roles in both local defense and SAR (Vlot et al., 2009; Zhang and Li, 2019). In Arabidopsis (Arabidopsis thaliana), SA is perceived by two classes of receptors: NONEXPRESSER OF PR GENES1 (NPR1) and NPR1-LIKE PROTEIN 3 (NPR3)/NPR4, which activate two parallel signaling pathways to stimulate the expression of defense-related genes and immunity (Fu et al., 2012; Wu et al., 2012; Ding et al., 2018). As NPR family proteins do not contain DNA binding domains, NPR1 and NPR3/NPR4 must interact with the transcription factors TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) TGA2/TGA5/TGA6 for signal transduction (Zhang et al., 1999; Després et al., 2000; Zhou et al., 2000; Zhang et al., 2006). NPR1 functions as a transcriptional activator (Fan and Dong, 2002; Rochon et al., 2006), and binding of SA to NPR1 promotes its activity (Wu et al., 2012; Ding et al., 2018). NPR3/NPR4, by contrast, serve as transcriptional repressors to repress the expression SA-responsive genes in the absence of pathogen infection. Binding of SA to NPR3/NPR4 inhibits their transcriptional repressor activity, leading to derepression of their target genes and activation of defense (Ding et al., 2018). In addition to transcriptional repression, NPR3/NPR4 were also proposed to function in the regulation of NPR1 stability (Fu et al., 2012).

In land plants, SA is synthesized from shikimate through either Phe or isochorismate (Huang et al., 2020). Upon pathogen infection, SA levels in Arabidopsis plants drastically increase due to enhanced SA biosynthesis through the isochorismate pathway (Wildermuth et al., 2001; Rekhter et al., 2019). The expression of SA-DEFICIENT2 (SID2), which encodes the SA biosynthesis enzyme ISOCHORISMATE SYNTHASE1 (ICS1), is rapidly induced (Wildermuth et al., 2001). The induction of SID2/ICS1 by pathogens is largely facilitated by the transcription factors SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 (SARD1) and CAM BINDING PROTEIN60-LIKE g (CBP60g; Wang et al., 2009, 2011; Zhang et al., 2010). Loss of SARD1 or CBP60g results in dramatic reduction of ICS1 induction and SA biosynthesis. In addition to ICS1, two other SA biosynthesis genes, avrPphB SUSCEPTIBLE3 (PBS3) and EDS5, are also transcriptionally regulated by SARD1 and CBP60g (Sun et al., 2015).

2,5-Dihydroxybenzoic acid (2,5-DHBA) is a major SA catabolite, and its formation through SA hydroxylation plays a critical role in maintaining SA homeostasis (Zhang et al., 2017). DOWNY MILDEW RESISTANT6 (DMR6) encodes an SA 5-hydroxylase (S5H) that converts SA to 2,5-DHBA (Zhang et al., 2017). In dmr6 mutant plants, SA cannot be converted to 2,5-DHBA, leading to elevated SA levels and enhanced disease resistance (van Damme et al., 2008; Zhang et al., 2017). During senescence, Arabidopsis plants also accumulate high levels of 2,3-DHBA (Zhang et al., 2017), due to the conversion of SA catalyzed by an SA 3-hydroxylase (S3H) that is paralogous to DMR6/S5H (Zhang et al., 2013). In the s5h s3h double mutant, SA levels further increase compared with the dmr6 single mutant, leading to severe dwarfism and autoimmunity (Zhang et al., 2017). Despite the importance of DMR6 in maintaining SA levels, it is currently unknown how it is transcriptionally regulated.

In addition to SA, N-hydroxypipecolic acid (NHP) and its precursor pipecolic acid (Pip) also play key roles in plant immune signaling and are required for SAR (Navarova et al., 2012; Chen et al., 2018; Hartmann et al., 2018). Pip is biosynthesized from Lys in two sequential reactions catalyzed by the amino acid transferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) and the reductase SARD4 (Ding et al., 2016; Hartmann et al., 2017). FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) further converts Pip into NHP, which most likely serves as a mobile signal for SAR (Chen et al., 2018; Hartmann et al., 2018). Upon pathogen infection, ALD1, SARD4, and FMO1 expression is dramatically induced, leading to increased biosynthesis of Pip and NHP. The induction of ALD1, SARD4, and FMO1, and thus of Pip and NHP biosynthesis, also depends on SARD1 and CBP60g (Sun et al., 2015, 2018, 2020). Furthermore, the transcription factor WRKY33 is similarly involved in the induction of ALD1 and Pip biosynthesis during infection by Pseudomonas syringae pv tomato (Pto) DC3000 avrRpt2 (Wang et al., 2018).

Here, we systematically examined the roles of SA and its receptors in PTI, ETI, and SAR by taking advantage of the npr1-1 npr4-4D double mutant, which blocks both SA perception pathways. We determined that both activation of NPR1 and inhibition of NPR3/NPR4 by SA are required for SAR and contribute to PTI and ETI. In addition, SA perception also plays critical roles in regulating the biosynthesis and catabolism of NHP and SA.

RESULTS

Regulation of SAR and NHP Levels by the SA Receptors NPR1 and NPR4

Arabidopsis NPR1 was previously shown to be required for SAR (Cao et al., 1994). To test whether SAR is affected in the gain-of-function SA-insensitive mutant npr4-4D, we compared SAR responses in wild-type Columbia-0 (Col-0), npr1-1, npr4-4D, and npr1-1 npr4-4D plants using a previously developed SAR assay (Zhang et al., 2010). As shown in Figure 1, the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) ES4326 induced SAR in wild-type plants, as it conferred strong resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2, whereas npr1-1 plants showed severely compromised SAR, consistent with a previous report (Zhang et al., 2010). Notably, SAR was also severely compromised in npr4-4D and completely lost in the npr1 npr4-4D double mutant, suggesting that constitutive repression of SA signaling in npr4-4D dampens SAR, and both branches of SA signaling are required for SAR.

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.

Although SA is known to be required for SAR, how it affects SAR is unclear. Since NHP serves as the likely mobile signal for SAR, we tested whether SA signaling contributes to the biosynthesis of NHP and its precursor Pip. We measured Pip and NHP levels in the wild type and the npr mutants after infection by Psm ES4326. Pip levels were much lower in npr1-1 and npr4-4D plants than in the wild type and were reduced to trace amounts in the npr1-1 npr4-4D double mutant (Figure 1B), suggesting that NPR1 and NPR4 regulate Pip accumulation in response to pathogen infection. In agreement with a previous report, NHP levels significantly increased in npr1-1 compared with the wild type (Hartmann et al., 2018), whereas npr4-4D plants accumulated similar amounts of NHP as the wild type. However, the npr1-1 npr4-4D double mutant accumulated very little NHP after Psm ES4326 infection (Figure 1C), indicating that NPR1 and NPR4 function in parallel to regulate pathogen-induced NHP accumulation. Further analysis showed that O-glycosylated NHP (NHP-OG) levels significantly decreased in npr1-1 and npr4-4D plants relative to the wild type and dropped further in npr1-1 npr4-4D (Figure 1D).

Regulation of the Expression of NHP Biosynthetic Genes by NPR1 and NPR4

As we observed lower NHP levels in npr1-1 npr4-4D plants, we next tested whether NPR1 and NPR4 regulate the expression of the NHP biosynthesis genes ALD1, SARD4, and FMO1 upon infection by examining their transcript levels in npr1-1, npr4-4D, and npr1 npr4-4D plants infiltrated with Psm ES4326. Consistent with Pip levels, the Psm ES4326-mediated induction of ALD1 and SARD4 expression was greatly reduced in npr4-4D and almost completely abolished in npr1-1 npr4-4D plants (Figures 2A and 2B). By contrast, induction of FMO1 by Psm ES4326 was comparable in npr1-1 and wild-type plants but greatly reduced in npr4-4D and was further decreased in npr1-1 npr4-4D (Figure 2C). These findings suggest that NPR1 and NPR4 independently regulate the expression of NHP biosynthesis genes. As the transcript levels of ALD1, SARD4, and FMO1 are not higher in npr1-1 than in the wild type, the increased NHP accumulation in npr1-1 shown in Figure 1C is unlikely to be due to increased NHP biosynthesis.

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).

To examine whether SA induces the expression of NHP biosynthetic genes, we first checked a transcriptome deep sequencing (RNA-seq) data set for SA-induced gene expression in the wild type and npr mutants (Ding et al., 2018). ALD1, SARD4, and FMO1 were among the genes significantly induced by SA in wild-type plants. To validate the RNA-seq data, we measured the expression of these three genes before and after SA treatment by RT-qPCR. As shown in Figures 2D to 2F, SA strongly induced the expression of these three genes, and the induction was significantly lower in npr1-1 and further reduced in the npr1-1 npr4-4D double mutant. Thus, SA and SA perception are required for pathogen-induced NHP biosynthesis.

As NPR proteins interact with TGA transcription factors for signaling, we next examined whether TGAs might contribute to the induction of NHP biosynthetic genes. Sequence analysis identified a single TGACG motif in the promoter regions of ALD1 and FMO1 but none in the SARD4 promoter (Supplemental Table 1). We performed chromatin immunoprecipitation (ChIP)-qPCR analysis using anti-TGA2 antibodies to determine whether TGA transcription factors are targeted to the ALD1 and FMO1 promoters. As shown in Figure 2G, we detected no significant enrichment of ALD1 or FMO1 promoter DNA fragments in the immunoprecipitated chromatin samples. Together, these data suggest that ALD1 and FMO1, as well as SARD4, are most likely not direct targets of TGA2/TGA5/TGA6.

SARD1 and CBP60g are known to directly regulate the expression of ALD1, SARD4, and FMO1 (Wang et al., 2009; Zhang et al., 2010; Sun et al., 2015, 2018), prompting us to check whether their induction by Psm ES4326 is dependent on SA signaling. As shown in Figures 2H and 2I, the npr1-1 npr4-4D double mutant almost completely blocked the induction of both SARD1 and CBP60g, indicating that NPR1 and NPR4 likely modulate the expression of ALD1, SARD4, and FMO1 through SARD1 and CBP60g.

Perception of SA by NPR1 and NPR4 Is Required for NHP-Induced Immunity

Because NHP induces the expression of SA biosynthetic genes (Chen et al., 2018; Hartmann et al., 2018), we examined whether the perception of SA by NPR1 and NPR4 may be required for NHP-induced immunity. We first infiltrated primary leaves with 1 mM NHP and later spray-inoculated the entire plants with a spore suspension of Hpa Noco2. As shown in Figure 3A, we observed very little growth of the pathogen on wild-type plants pretreated with NHP, suggesting that NHP induces strong resistance against Hpa Noco2. By contrast, NHP-pretreated npr1-1, npr4-4D, npr1-1 npr4-4D, and sid2-1 plants remained susceptible to Hpa Noco2, indicating that SA signaling is required for NHP-induced immunity.

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.

To confirm that SA perception is required for NHP-triggered immunity, we crossed the npr1-1 and npr4-4D mutants with the FMO1-3D mutant, where FMO1 is overexpressed and defense responses are constitutively activated, presumably due to increased NHP biosynthesis (Koch et al., 2006). As shown in Figure 3B, FMO1-3D exhibited morphological phenotypes such as stunted growth and curly leaves typically associated with autoimmunity. These phenotypes were partially suppressed in the npr1-1 FMO1-3D and npr4-4D FMO1-3D double mutants and completely suppressed in the npr1-1 npr4-4D FMO1-3D triple mutant. In addition, the npr1-1 and npr4-4D mutations also blocked the enhanced resistance against Hpa Noco2 normally seen in the FMO1-3D background (Figure 3C). Similarly, the dwarfism and enhanced immunity of FMO1-3D plants were also suppressed by the sid2-1 mutation (Figures 3D and 3E). Together, these data further underscore that SA signaling is required for NHP-activated immunity.

Since the NHP and SA molecules have similar structures, with six-membered rings carrying carboxyl and hydroxyl substituents in a 1,2-constellation, we tested whether NHP might bind to NPR proteins like SA. We performed [³H]SA-NPR binding assays in the presence of excessive amounts of unlabeled substrates. As shown in Figures 3F and 3G, the addition of unlabeled SA outcompeted the binding of [³H]SA to NPR1 and NPR4 proteins, while the addition of unlabeled NHP had no effect, suggesting that NHP probably does not bind directly to NPR1/NPR4, at least not through the same binding sites as SA.

Perception of SA by Both NPR1 and NPR4 Is Required for PTI

Since npr mutants were shown to support enhanced growth of the nonpathogenic bacterium Pto DC3000 hrcC (Ding et al., 2018), we analyzed the contributions of the two branches of SA signaling to PTI, using the sid2-1 mutant as a control. As shown in Figure 4A, flg22-induced protection against Pto DC3000 was compromised in npr1-1 and npr4-4D to a similar extent and was further reduced in npr1-1 npr4-4D, suggesting that perception of SA by NPR1 and NPR4 contributes to flg22-induced immunity. Surprisingly, flg22-induced protection against Pto DC3000 was more drastically reduced in npr1-1 npr4-4D than in sid2-1, suggesting that PTI is more severely compromised in npr1-1 npr4-4D than in sid2-1. Such phenotypic differences between SA biosynthesis and SA perception mutants indicate a major function of the residual SA in sid2-1 for PTI.

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.

In wild-type plants, the expression of SARD1, PATHOGENESIS-RELATED1 (PR1), and PR2 is strongly induced by treatment with Pto DC3000 hrcC. Here, we documented a significant reduction of this induction in npr1-1 and npr4-4D and an almost complete loss in npr1-1 npr4-4D (Figures 4B to 4D), suggesting that the perception of SA by NPR1 and NPR4 is required for the expression of defense genes during PTI. Although sid2-1 blocked Pto DC3000 hrcC-induced PR1 expression, we still detected some induction of SARD1 and PR2 in sid2-1, suggesting that the residual SA contributes to defense gene expression during PTI.

Perception of SA by NPR1 and NPR4 Is Required for ETI

Next, we examined the contribution of the two SA signaling pathways in ETI by comparing the growth of the avirulent bacterial strains Pto DC3000 AvrRpt2 and Pto DC3000 AvrRps4 in wild-type, npr1-1, npr4-4D, npr1-1 npr4-4D, and sid2-1 plants. These two bacterial strains trigger immunity mediated by the CNL RPS2 and the TNL RPS4, respectively. As shown in Figure 4E, the growth of both pathogens significantly increased in npr1-1 and npr4-4D relative to the wild type and increased even further in npr1-1 npr4-4D, suggesting that perception of SA by NPR1 and NPR4 is important for ETI. The npr1-1 npr4-4D double mutant supported a 34-fold higher growth of Pto DC3000 AvrRpt2 and a 12-fold higher growth of Pto DC3000 AvrRps4 than the sid2-1 mutant, indicating that defense conferred by the residual SA in sid2-1 is also critical for ETI.

Treatment with Pto DC3000 AvrRpt2 or Pto DC3000 AvrRps4 in the wild type dramatically induced the expression of the defense-related genes SARD1, PR1, and PR2 (Figures 4F to 4H). Induction of all three genes was drastically reduced in npr1-1 npr4-4D. By contrast, the induction of SARD1 and PR2 remained largely unaffected in sid2-1 and only the expression of PR1 was SID2-dependent, suggesting that the residual SA in sid2-1 contributes to defense gene expression during ETI.

PTI and ETI Are More Severely Compromised in npr1-1 npr4-4D Than in fmo1-1 sid2-1 Mutants

Since mutations in npr1-1 and npr4-4D not only directly affect SA perception but also indirectly influence NHP production during pathogen infection (Figure 1C), we tested whether the severe PTI and ETI defects in npr1-1 npr4-4D are due to synergistic effects between SA signaling defects and reduced NHP levels. To this end, we compared npr1-1 npr4-4D with the fmo1-1 sid2-1 double mutant, in which NHP- and pathogen-induced SA biosynthesis are blocked. As shown in Figure 5, growth of Pto DC3000 hrcC did not change in fmo1-1, sid2-1, and fmo1-1 sid2-1 but reached considerably higher levels in npr1-1 npr4-4D. flg22-induced protection against Pto DC3000 was reduced in both npr1-1 npr4-4D and fmo1-1 sid2-1 but was much more pronounced in npr1-1 npr4-4D (Figure 5B). Consistent with a previous report in which mutations in fmo1-1 and sid2-1 had additive effects on ETI (Bartsch et al., 2006), growth of Pto DC3000 AvrRpt2 and Pto DC3000 AvrRps4 was significantly higher in the fmo1-1 sid2-1 double mutant than in the single mutants (Figures 5C and 5D). However, growth of the two avirulent bacterial strains was even higher in npr1-1 npr4-4D than in fmo1-1 sid2-1. These results further underscore the important roles played by residual SA in sid2-1 during PTI and ETI.

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.

NPR1 and NPR4 Regulate SA Hydroxylation by Controlling DMR6 Expression

Arabidopsis npr1 mutant plants have been shown to accumulate higher levels of SA than the wild type (Delaney et al., 1995); however, it was unclear whether this increase is only due to enhanced biosynthesis or also due to reduced catabolism. To test whether NPR1 and NPR4 are involved in the regulation of SA catabolism, we quantified 2,5-DHBA levels in wild-type, npr1-1, npr4-4D, and npr1-1 npr4-4D plants. As shown in Figure 6, basal 2,5-DHBA levels were significantly lower in npr1-1 and npr4-4D and greatly reduced in npr1-1 npr4-4D plants compared with the wild type. Similarly, 2,5-DHBA levels after pathogen infection also decreased in npr1-1 and npr4-4D single mutants and reached much lower levels in the npr1-1 npr4-4D double mutant. These results suggest that both NPR1 and NPR4 are involved in regulating the production of 2,5-DHBA, which is a major SA catabolite.

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).

Next, we examined whether the expression of DMR6, which encodes a 5-hydroxylase converting free SA into 2,5-DHBA (Zhang et al., 2017), is regulated by NPR1 and NPR4. Analysis of a previously generated RNA-seq data set (Ding et al., 2018) showed that the expression of DMR6 was induced by SA in wild-type plants, which we confirmed by RT-qPCR analysis (Figure 6B). The induction of DMR6 by SA was completely blocked in npr1-1 npr4-4D (Figure 6B). We also measured the expression levels of DMR6 in wild-type and npr mutant plants treated with Pto DC3000 AvrRpt2. In the mock treatment, the expression of DMR6 was significantly lower in npr1-1 and npr4-4D than in the wild type and was further reduced in npr1-1 npr4-4D (Figure 6C). Following infection by Pto DC3000 AvrRpt2, DMR6 expression was strongly induced in the wild type, and this induction was reduced in npr1-1 as well as npr4-4D and completely blocked in the npr1-1 npr4-4D double mutant. Together, these data suggest that NPR1 and NPR4 regulate SA catabolism by modulating DMR6 expression.

Since both NPR1 and NPR4 work with the redundant TGA transcription factors TGA2, TGA5, and TGA6 to regulate SA-induced gene expression (Zhang et al., 1999, 2003, 2006; Després et al., 2000), we examined the expression of DMR6 in the tga2 tga5 tga6 triple mutant with or without SA treatment. Consistent with the requirement of TGA2/TGA5/TGA6 for the transcriptional repression of SA-responsive genes by NPR3/NPR4 (Ding et al., 2018), the basal expression level of DMR6 was much higher in the tga2 tga5 tga6 triple mutant than in the wild type (Figure 6D). However, treatment with SA did not further induce DMR6 in tga2 tga5 tga6 plants, suggesting that TGAs may be involved in the induction of DMR6 by SA.

In the promoter region of DMR6, we identified two TGACG motifs that are 9 bp apart (Supplemental Table 1). To determine whether DMR6 is a direct target of TGAs, we performed ChIP-qPCR experiments on wild-type and tga2 tga5 tga6 plants using anti-TGA2 antibodies. qPCR analysis of the immunoprecipitated DNA revealed that DMR6 promoter fragments were significantly enriched by anti-TGA2 antibodies in wild-type plants but not in tga2 tga5 tga6 plants (Figure 6E), suggesting that the transcription factors TGA2/TGA5/TGA6 directly bind to the DMR6 promoter region and thus, together with the SA receptors, regulate the expression of DMR6.

NPR1 and NPR4 Regulate the Production of SAG and SA

As SA can also be glycosylated into SA 2-O-β-D-glucoside (SAG), we examined whether NPR1 and NPR4 regulate the production of SA as well as SAG. We measured and compared the levels of free SA and SAG in the wild type, npr1-1, npr4-4D, and npr1 npr4-4D. As shown in Figure 6F, under mock treatment, SAG levels were significantly higher in npr1-1, npr4-4D, and npr1 npr4-4D than in the wild type, suggesting that decreased SA 5-hydroxylation in npr1-1 and npr4-4D mutant plants may be compensated by increased SA glycosylation. The levels of free SA after infection by Pto DC3000 AvrRpt2 were comparable in the wild type, npr4-4D, and npr1 npr4-4D but were considerably higher in npr1-1, consistent with a previous report (Delaney et al., 1995). SAG levels after Pto DC3000 AvrRpt2 infection were similar in the wild type and npr1-1 but were significantly lower in npr4-4D and were further reduced in the npr1-1 npr4-4D double mutant. These results suggest that NPR1 and NPR4 regulate SA accumulation as well as the conversion of SA to SAG.

There are at least three UDP-glucosyltransferases (UGTs) involved in the conversion of SA to SAG in Arabidopsis. Of those, UGT74F1 and UGT76B1 have high glucosyltransferase activity, whereas UGT74F2 shows very low glucosyltransferase activity (Noutoshi et al., 2012). Therefore, we focused our analysis on UGT74F1 and UGT76B1. As shown in Supplemental Figure 1, the expression of UGT74F1 was not induced upon infection by Pto DC3000 AvrRpt2, consistent with previous findings that UGT74F1 is expressed at low but constitutive levels and is barely induced by biotic stresses (Noutoshi et al., 2012). In comparison, the expression of UGT76B1 was dramatically induced by Pto DC3000 AvrRpt2 in wild-type plants, but the induction was greatly reduced in npr1-1 and npr4-4D and was almost completely blocked in npr1-1 npr4-4D (Figure 6G). While SA also induced UGT76B1 expression in the wild type, this induction was greatly reduced in npr1-1 and npr4-4D and was completely blocked in npr1-1 npr4-4D (Figure 6H), suggesting that NPR1 and NPR4 are involved in regulating the expression of UGT76B1.

We further analyzed the expression levels of UGT76B1 l in the tga2 tga5 tga6 triple mutant before and after SA treatment. As shown in Figure 6I, the expression level of UGT76B1 in tga2 tga5 tga6 was much higher than in the wild type but was not further induced by SA treatment. There are two TGACG motifs in the UGT76B1 promoter region (Supplemental Table 1), and ChIP-qPCR analysis showed that DNA from this region was enriched by anti-TGA2 antibodies in samples from the wild type but not tga2 tga5 tga6 (Figure 6J). These results suggest that TGA2, TGA5, and TGA6 directly regulate the expression of UGT76B1 together with the SA receptors.

We also tested whether NPR1 and NPR4 regulate the expression of SA biosynthetic genes. As shown in Figures 6K and 6L, the expression of ICS1, EDS5, and PBS3 was dramatically induced by Pto DC3000 AvrRpt2 in the wild type, but this induction was significantly reduced in the npr1-1 npr4-4D double mutant. SA treatment also induced the expression of ICS1, EDS5, and PBS3 in the wild type but not in npr1-1 npr4-4D (Supplemental Figures 2A to 2C), suggesting that SA positively regulates its own biosynthesis through its receptors NPR1 and NPR4. Notably, the expression levels of ICS1 and PBS3 were significantly higher in npr1-1 than in the wild type after infection by Pto DC3000 AvrRpt2, indicating a negative role of NPR1 besides its positive feedback function on SA biosynthesis.

Even though TGACG motifs are present in the promoter regions of ICS1, EDS5, and PBS3 (Supplemental Table 1), DNA from the TGACG motif-containing regions was not significantly enriched by anti-TGA2 antibodies in ChIP-qPCR experiments (Supplemental Figures 2D to 2F), suggesting that NPR1 and NPR4 regulate the expression of SA biosynthetic genes either indirectly or by interacting with other transcription factors that bind to their promoter regions. The induction of ICS1, EDS5, and PBS3 was previously shown to be directly regulated by the transcription factors SARD1 and CBP60g. As shown in Figure 4F and Supplemental Figure 3, the expression of SARD1 and CBP60g was significantly reduced in the npr1-1 npr4-4D double mutant upon infection by Pto DC3000 AvrRpt2, suggesting that NPR1 and NPR4 likely regulate pathogen-induced SA biosynthesis by modulating the expression of SARD1 and CBP60g.

DISCUSSION

Since the early characterization of transgenic plants expressing the salicylate hydroxylase gene NahG from Pseudomonas putida, in which SA is converted to catechol (Gaffney et al., 1993; Delaney et al., 1994), it has long been established that SA is required for SAR. However, how SA contributes to SAR activation is still not fully understood. Here, analysis of the SA perception-deficient npr1-1 npr4-4D double mutant revealed that SA perception is essential for the induction of NHP biosynthetic genes and the production of NHP during pathogen infection. This is most likely through the SA-mediated upregulation of SARD1 and CBP60g, which encode two transcription factors that directly control the expression of the NHP biosynthesis genes ALD1, SARD4, and FMO1 (Sun et al., 2015, 2018). Since NHP functions as a mobile signal for SAR (Chen et al., 2018; Hartmann et al., 2018), one of the contributions of SA to SAR is to induce the production of the mobile signal in local tissue (Figure 7). As SAR induced by NHP treatment and resistance against Hpa Noco2 in FMO1-3D are blocked by mutations in npr1-1 and npr4-4D, the perception of SA by NPR1 and NPR4 is also required for NHP-induced defense responses, in addition to promoting NHP biosynthesis (Figure 7). NHP was previously shown to induce the expression of SARD1 and CBP60g as well as SA biosynthetic genes (Chen et al., 2018; Hartmann et al., 2018). Most likely, NHP activates SA-mediated immunity by promoting SA biosynthesis.

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.

In npr1-1 mutant plants infected with Psm ES4326, the expression of FMO1 is unaffected but the NHP level is considerably higher than in the wild type (Figure 1C). The increased NHP accumulation in npr1-1 may be due to reduced conversion of NHP to NHP-OG, as NHP-OG abundance in npr1-1 is significantly lower than in the wild type (Figure 1D). In npr4-4D mutant plants infected with Psm ES4326, although the expression of FMO1 is dramatically reduced, NHP levels are similar to those in wild-type plants, which may be explained by reduced conversion of NHP to NHP-OG in npr4-4D (Figure 1C). UGT76B1 was recently reported to function as an NHP glucosyltransferase (Bauer et al., 2020; Mohnike et al., 2020; Sattely et al., 2020). Since SA induces the expression of UGT76B1 (Figure 6H), the reduced conversion of NHP to NHP-OG in the npr1-1 and npr4-4D mutants is most likely due to reduced expression of UGT76B1.

As flg22-induced resistance against Pto DC3000 is compromised in the npr1-1 and npr4-4D mutants and further reduced in the npr1-1 npr4-4D double mutant, both NPR1-dependent and NPR4-dependent SA signaling are important, and both contribute additively to PTI. This hypothesis is supported by the reduced induction of defense gene expression (Figures 4B to 4D) and the increased growth of Pto DC3000 hrcC in npr1-1, npr4-4D, and npr1-1 npr4-4D (Ding et al., 2018). Interestingly, the reduction of flg22-induced resistance against Pto DC3000 in npr1-1 npr4-4D is even more dramatic than in the SA-deficient mutant sid2-1. The npr1-1 npr4-4D double mutant also supports considerably higher growth of Pto DC3000 hrcC than sid2-1, and the induction of SARD1 and PR2 by Pto DC3000 hrcC is significantly lower in npr1-1 npr4-4D than in sid2-1. These data suggest that perception of the residual SA in sid2-1 by the SA receptors contributes to a SID2-independent PTI response.

It was shown previously that npr1-1 npr4-4D exhibits enhanced cell death upon Pto DC3000 AvrRpt2 infection, suggesting that SA signaling negatively regulates cell death during ETI (Radojičić et al., 2018). An analysis of the growth of Pto DC3000 AvrRpt2 and Pto DC3000 AvrRps4 showed that they are modestly increased in the npr1-1 and npr4-4D single mutants but are much higher in the npr1-1 npr4-4D double mutant (Figure 4E), suggesting that both NPR1-dependent and NPR4-dependent SA signaling pathways contribute to resistance against avirulent pathogens. Notably, growth of the two bacterial strains is over 10-fold higher in npr1-1 npr4-4D than in sid2-1, suggesting that the perception of a basal level of SA in sid2-1 by NPR1 and NPR4 is also critical for ETI. This observation is further supported by the much lower expression of defense-related genes in npr1-1 npr4-4D than in sid2-1 during infection by the avirulent bacteria (Figures 4F to 4H).

Since SID2 is required for pathogen-induced SA biosynthesis (Wildermuth et al., 2001), immune responses in sid2 mutant plants have been considered as evidence of SA-independent defense in previous studies. However, our findings showed that blocking SA perception has a much more severe effect on both ETI and PTI than loss of SID2 function, suggesting that residual SA content in sid2-1 plays critical roles in plant immunity. As SA perception promotes NHP production, and since the fmo1-1 sid2-1 double mutant is more susceptible to Pto DC3000 AvrRpt2 and Pto DC3000 AvrRps4 than the sid2-1 single mutant, the severely compromised ETI in npr1-1 npr4-4D may be partially due to reduced NHP accumulation. However, both ETI and PTI are more severely compromised in npr1-1 npr4-4D than in fmo1-1 sid2-1, suggesting that residual SA in sid2-1 also contributes to plant defenses independently of NHP. Considering that defense-related genes controlled by NPR1 and NPR3/NPR4 are not up-regulated in the absence of pathogen, even though the in vitro binding constants of NPR1 and NPR3/NPR4 (ranging from ∼20 to 200 nM) are considerably lower than the estimated basal levels of free SA (∼1.4 µM; Ding et al., 2018), the activities of the SA receptors are most likely subject to posttranscriptional regulation in planta. It is possible that certain protein modifications induced at early stages of pathogen attack enable them to respond to basal levels of SA.

In addition to their roles in PTI, ETI, and SAR, we determined that NPR1 and NPR4 are also involved in regulating the conversion of SA to 2,5-DHBA and SAG, two major SA derivatives contributing to SA homeostasis. DMR6 and UGT76B1, which encode an S5H and an SA glycosyltransferase, respectively (Noutoshi et al., 2012; Zhang et al., 2017), are target genes of the NPR1/NPR4-interacting transcription factors TGA2/TGA5/TGA6. Their expression is strongly induced by SA, suggesting that they are directly regulated by the SA receptors. Consistent with the expression levels of DMR6, 2,5-DHBA levels in npr1-1 and npr4-4D are significantly lower than in the wild type and are further reduced in the npr1-1 npr4-4D double mutant. In npr1-1 npr4-4D, induction of UGT76B1 and accumulation of SAG following infection by Pto DC3000 AvrRpt2 are also dramatically reduced. These findings indicate that NPR1 and NPR4 play key roles in the negative feedback regulation of SA accumulation by controlling the production of 2,5-DHBA and SAG (Figure 7).

In the npr1-1 npr4-4D double mutant, the induction of SA biosynthetic genes and the accumulation of SA upon induction by Pto DC3000 AvrRpt2 are significantly lower than in the wild type. The lower induction of SA biosynthetic genes in npr1-1 npr4-4D is most likely due to reduced expression of the genes encoding the transcription factors SARD1 and CBP60g (Figure 4F; Supplemental Figure 3), which directly regulate the expression of ICS1, EDS5, and PBS3, whose transcripts are also induced by SA. These findings suggest a positive feedback amplification loop in which the perception of SA stimulates its own biosynthesis (Figure 7). The involvement of SA receptors in both positive regulation of SA biosynthesis and negative regulation of SA accumulation through SA hydroxylation and glycosylation is likely important for spatial and temporal regulation of SA levels, which is an interesting area to explore in the future.

It was previously shown that SA levels in npr1 mutant plants are significantly higher than in the wild type, indicating that NPR1 plays a negative role in regulating SA levels (Delaney et al., 1995). However, the mechanism by which NPR1 affects SA levels was unclear. The reduced accumulation of 2,5-DHBA in npr1-1 suggests that increased SA accumulation in npr1 mutant plants is partly due to the reduced hydroxylation of SA by DMR6. SAG levels in npr1-1 after Pto DC3000 AvrRpt2 infection are comparable to the free SA levels, in contrast to wild-type plants, which accumulate much higher SAG than SA, suggesting that the mutant has also reduced conversion of SA to SAG, contributing to the increased free SA accumulation. Furthermore, increased SA biosynthesis most likely also contributes to higher SA levels in npr1 mutants, as the expression levels of ICS1 and PBS3 following infection by Pto DC3000 AvrRpt2 are significantly higher in npr1-1 than in the wild type. How loss of function of NPR1 leads to increased ICS1 and PBS3 expression remains to be determined in the future.

In summary, the SA receptors NPR1 and NPR3/NPR4 play diverse roles in plant immunity by regulating SA-responsive defense-related genes (Figure 7). The perception of SA promotes both PTI and ETI and is required for activating NHP biosynthesis in local tissue and NHP-induced defense responses during SAR. In addition, the SA receptors are involved in positive feedback amplification of SA biosynthesis as well as SA 5-hydroxylation and glycosylation, enabling fine-tuned regulation of SA levels during plant immunity.

METHODS

Plant Materials and Growth Conditions

All plants used in this study are in the Arabidopsis (Arabidopsis thaliana) accession Col-0 background. The npr1-1, npr4-4D, npr1-1 npr4-4D, tga2 tga5 tga6, fmo1-3D, fmo1-1, sid2-1, fmo1-1 sid2-1, ndr1-1, and eds1-2 mutants were described previously (Cao et al., 1994; Century et al., 1995; Wildermuth et al., 2001; Zhang et al., 2003; Bartsch et al., 2006; Koch et al., 2006; Ding et al., 2018). The double and triple mutants npr1-1 FMO1-3D, npr4-4D FMO1-3D, and npr1-1 npr4-4D FMO1-3D were isolated from the F2 progeny of a cross between npr1-1 npr4-4D and FMO1-3D, while sid2-1 FMO1-3D was isolated from the F2 progeny of a cross between sid2-1 and FMO1-3D. Primers used for genotyping are listed in Supplemental Table 2. For SA-induced gene expression assays, plants were grown on half-strength Murashige and Skoog medium plates with 0.6% (w/v) agar at 23°C under cycles of 16 h of fluorescent light/8 h of dark (80 µE; Sylvania Octron 4100K, FO32/741/ECO bulbs). For pathogen-induced gene expression, measurement of metabolites, and pathogen growth assays, plants were grown on soil at 23°C under cycles of 12 h of fluorescent light/12 h of dark (100 µE; Philips Master TL5 HO 54W/840 bulbs).

Gene Expression Analysis

For RNA preparation, we collected ∼50 mg of tissue from 4-week-old plants (four leaves from four different plants per replicate, for three independent biological replicates from different sets of plants) grown on soil or from 2-week-old seedlings (five to six whole seedlings per sample; three samples per genotype per treatment) grown on half-strength Murashige and Skoog medium plates. To analyze pathogen-induced gene expression, we collected the leaves of 4-week-old plants 24 h after infiltration with Pseudomonas syringae pv maculicola ES4326 (OD₆₀₀ = 0.001), 12 h after infiltration with Pseudomonas syringae pv tomato DC3000 hrcC (OD₆₀₀ = 0.05) or 10 mM MgCl₂ (mock), or 16 h after infiltration with Pto DC3000 AvrRpt2 (OD₆₀₀ = 0.005), Pto DC3000 AvrRps4 (OD₆₀₀ = 0.005), or 10 mM MgCl₂. Total RNA was extracted from all samples using the EZ-10 Spin Column Plant RNA Mini-Preps Kit (BIO BASIC CANADA). RT was performed using the EasyScript Reverse Transcriptase (ABM), and qPCR was performed using the Takara SYBR Premix Ex (Clontech) following the manufacturers’ instructions. We normalized relative expression values to ACTIN1. Primer sequences used for qPCR are listed in Supplemental Table 2.

Measurement of Pip and NHP

To induce the production of Pip and NHP, we collected the leaves of 4-week-old plants 24 h after infiltration with Psm ES4326 (OD₆₀₀ = 0.001) or 10 mM MgCl₂ (mock). Each treatment consisted of three independent samples from each genotype, each constituting ∼75 mg of treated leaf tissue from six individual plants; a total of 18 plants per genotype/treatment were sampled. We extracted Pip using the EZ:faast free amino acid analysis kit, with norvalene as internal standard (Phenomenex). Pip detection and quantification were performed on a gas chromatography-mass spectrometry system (Agilent, 5973N) as previously described, with minor modifications (Návarová et al., 2012). A ZB-AAA capillary column (Zebron, Phenomenex; 10 m × 0.25 mm i.d.) and the following oven program were used: on-column injection at 50°C, oven temperature maintained at 50°C for 2 min, raised by 30°C/min to 320°C, and held for 2 min at 320°C. A constant helium flow of 1.4 mL/min was maintained during the entire program.

We extracted NHP using a previously described procedure (Hartmann et al., 2018) with some modifications. Briefly, ∼75 mg of leaf tissue was ground and extracted once with 0.6 mL of 90% (v/v) methanol, with 0.5 µg of 2-hydroxy-cyclohexanecarboxylic acid (2-HCC; Sigma-Aldrich) added to each sample as internal standard. After gently shaking at 4°C for 2 h, the samples were centrifuged at 12,000g for 5 min and the supernatants were transferred to new tubes. We extracted the remaining pellets again with 0.6 mL of methanol and centrifuged as above, and the new supernatant was combined with the first. We took out an aliquot of 300 μL per sample, evaporated the solvent under a stream of nitrogen, and subjected the resulting residue to chemical derivatization by adding 20 μL of pyridine and 20 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich). The reaction mixtures were incubated at 70°C for 30 min, cooled and kept at room temperature for 30 min, and finally diluted with 60 μL of hexane and transferred into gas chromatography vials. We injected 0.2 μL of each sample for analysis on a gas chromatography-mass spectrometry system (Agilent, 5973N) equipped with an HP-1 capillary column (Agilent; 30 m × 0.32 mm i.d.) using the following oven program: on-column injection at 70°C, oven held for 2 min at 70°C, raised by 10°C/min to 320°C, and held for 5 min at 320°C.

NHP, 2-HCC, and NHP-OG were analyzed based on their characteristic fragment ions m/z 172, 273, and 652, respectively. To determine the amount of individual metabolites, we integrated their peak areas in selected ion chromatograms using MSD ChemStation software (Agilent) and compared them with the peak area of the corresponding internal standard (IS): Pip (m/z 170)/IS norvalene (m/z 158); NHP (m/z 172)/IS 2-HCC (m/z 273), using correction factors for each pair of compounds experimentally determined with authentic standards. The presence of NHP-OG was determined based on its mass spectral characteristics (m/z 172 and 652). Because an authentic standard for NHP-OG was not available, relative NHP-OG abundances were determined by quantifying peak areas of its molecular ion m/z 652 relative to those of 2-HCC fragment m/z 273 in respective selected ion chromatograms.

2,5-DHBA and SA Quantification

For 2,5-DHBA and SA measurements, we collected two leaves each from six 4-week-old plants 16 h after infiltration with Pto DC3000 AvrRpt2 (OD₆₀₀ = 0.005) or 10 mM MgCl₂ (mock). For each treatment, four independent samples were collected, each sample constituting ∼100 mg of leaf tissue from six individual plants, for a total of 24 plants per genotype/treatment. 2,5-DHBA, free SA, and total SA were extracted using previously described protocols (Zhang et al., 2017). Samples were analyzed using an HPLC device (1200 series, Agilent) equipped with a C18 column (5 µm, 4.6 ×150 mm; Eclipse XDB, Agilent) and a fluorescence detector (G1321A, Agilent). 2,5-DHBA, free SA, and total SA were quantified using ISs and as previously described with modifications (Zhang et al., 2017). The mobile phase contained 0.2 M KOAc, 0.5 mM EDTA, pH 5.0, and methanol. For SA analysis, we included 6% (v/v) methanol and selected 295 and 405 nm as excitation and emission wavelengths, respectively. For 2,5-DHBA measurement, methanol was maintained at 3% (v/v), with excitation and emission wavelengths of 320 and 449 nm, respectively. The flow rate was fixed at 1 mL/min, and 5 μL of sample was injected in both cases. We determined target compound concentrations by calculating the peak areas in plant samples relative to those of corresponding synthetic standards.

ChIP-qPCR Analysis

ChIP assays were performed following a previously described protocol (Sun et al., 2015). Specifically, we cross-linked 2-week-old seedlings from Col-0 and tga2 tga5 tga6 and collected the tissue for ChIP. Anti-TGA2 antibodies specifically recognizing proteins of clade II members of the TGA family, TGA2, TGA5, and TGA6 (Ding et al., 2018), and protein A-agarose beads (GE Healthcare) or protein A-agarose beads alone (no-antibody control) were used to pull down the chromatin complexes containing TGA2/5/6 proteins. Two independent batches of samples were used, and each sample was divided into four aliquots, two for no-antibody controls and two with anti-TGA2 antibodies for the ChIP samples. We performed qPCR with the immunoprecipitated DNA as template using primers specific to promoters of selected genes. Sequences of primers for ChIP-qPCR are listed in Supplemental Table 2.

Bacterial Pathogen Infection Assays

For bacterial pathogen infection assays, two leaves each from two 4-week-old plants were infiltrated with bacterial suspension in 10 mM MgCl₂: Pto DC3000 hrcC (OD₆₀₀ = 0.002), Pto DC3000 AvrRpt2 (OD₆₀₀ = 0.0005), and Pto DC3000 AvrRps4 (OD₆₀₀ = 0.0005). We collected infected leaves 1 h (on day 0) and 3 d after inoculation. Two leaf discs from two infected leaves of one plant were collected as one sample, and six samples were analyzed for each genotype for day 3, while three to four samples were used for day 0. Samples were ground, serially diluted, and plated on Luria-Bertani agar plates to determine colony-forming units. For flg22-triggered protection assays, two leaves each from 4-week-old plants were infiltrated with water or 1 μM flg22. After 24 h, we infiltrated the pretreated leaves with Pto DC3000 at a cell density of OD₆₀₀ = 0.001 in 10 mM MgCl₂. After inoculation with Pto DC3000 for 3 d, we determined bacterial titers in infected leaves as above.

Oomycete Pathogen Infection Assays

We performed the SAR assay with the oomycete pathogen Hyaloperonospora arabidopsidis Noco2 as previously reported (Zhang et al., 2010). Briefly, two primary leaves from 3-week-old plants were infiltrated with Psm ES4326 (OD₆₀₀ = 0.001) or 10 mM MgCl₂. After 2 d, we sprayed whole plants with Hpa Noco2 spore suspension at a titer of 50,000/mL in water, using 15 plants per genotype per treatment. Inoculated plants were covered with a clear plastic dome and incubated at 18°C in a 12-h/12-h light/dark cycle in a growth chamber for 1 week. Disease symptoms were scored by counting the number of conidiophores on the distal leaves; we assigned disease rating scores as described in the legend of Figure 1A.

For NHP-induced immunity against Hpa Noco2, we infiltrated two primary leaves from 3-week-old plants with 1 mM NHP or water (mock). After 24 h, plants were sprayed with Hpa Noco2 spore suspension (50,000/mL in water), using 15 plants for each treatment. Disease symptoms were scored 7 d after inoculation as described above.

For Hpa Noco2 infection on seedlings, 2-week-old soil-grown seedlings were sprayed with Hpa Noco2 spore suspension (50,000/mL in water). Inoculated plants were covered with a clean dome and grown at 18°C under a 12-h/12-h light/dark cycle in a growth chamber. Hpa Noco2 sporulation was counted 7 d later.

[³H]SA Binding Assays

We purified recombinant His₆-MBP-NPR1 and His₆-MBP-NPR4 proteins for [³H]SA binding assay by size-exclusion chromatography, as previously described (Ding et al., 2018). Size-exclusion columns were prepared by adding 0.13 g of Sephadex G-25 (GE Healthcare) to a Qiagen shredder column and preequilibrated with PBS buffer containing 0.1% (v/v) Tween 20 overnight at 4°C; excess buffer was removed by spinning at 735g for 2 min before use.

The binding reactions were performed by incubating 0.4 mg/mL (w/v) His₆-MBP-NPR1 or His₆-MBP-NPR4 protein with 200 nM [³H]SA (American Radiolabeled Chemicals; specific activity 30 Ci/mmol) in 50 μL of PBS buffer on ice for 1 h. Unlabeled SA or NHP was added to the reaction mixture at 2 mM (10,000-fold excess over [³H]SA). After incubation, the reaction mixtures were loaded onto the columns and centrifuged immediately as above. The radioactivity in the flow-through fraction (bound [³H]SA) was measured using a scintillation counter (LS6500, Beckman Coulter).

Accession Numbers

Sequence data for most genes studied in this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: ACTIN1 (At2g37620), ALD1 (At2g13810), CBP60g (At5g26920), DMR6 (At5g24530), EDS5 (At4g39030), FMO1 (At1g19250), NPR1 (At1g64280), NPR4 (At4g19660), PBS3 (At5g13320), PR1 (At2g14610), PR2 (At3g57260), SARD1 (At1g73105), SID2/ICS1 (At1g74710), UGT76B1 (At3g11340), UGT74F1 (At2g43840), and UGT74F2 (At2g43820).

Supplemental Data

• Supplemental Figure 1. Induction of UGT74F1 in wild-type, npr1-1, npr4-4D and npr1-1 npr4-4D plants by Pto DC3000 AvrRpt2. (Supports Figure 6).

• Supplemental Figure 2. Induction of ICS1, EDS5 and PBS3 expression by SA and ChIP-PCR analysis of binding of TGA2 to their promoter regions.  (Supports Figure 6)

• Supplemental Figure 3. Induction of CBP60g in Col-0 (wild type), npr1-1, npr4-4D and npr1-1 npr4-4D plants by Pto DC3000 AvrRpt2.(Supports Figure 6).

• Supplemental Table 1. TGACG motifs in promoter regions of genes involved in NHP/SA biosynthesis and metabolism. 

• Supplemental Table 2. Primers used in this study.

DIVE Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• PR1 Gramene: AT2G14610

• PR1 Araport: AT2G14610

• UGT74F1 Gramene: AT2G43840

• UGT74F1 Araport: AT2G43840

• NPR1 Gramene: At1g64280

• NPR1 Araport: At1g64280

• DMR6 Gramene: AT5G24530

• DMR6 Araport: AT5G24530

• UGT74F2 Gramene: AT2G43820

• UGT74F2 Araport: AT2G43820

• CBP60g Gramene: AT5G26920

• CBP60g Araport: AT5G26920

• ETI Gramene: Effector-triggered immunity

• ETI Araport: Effector-triggered immunity

• PTI Gramene: Pattern triggered immunity

• PTI Araport: Pattern triggered immunity

• NPR4 Gramene: AT4G19660

• NPR4 Araport: AT4G19660

• TGA2 Gramene: AT5G06950

• TGA2 Araport: AT5G06950

• SARD1 Gramene: AT1G73805

• SARD1 Araport: AT1G73805

• UGT76B1 Gramene: AT3G11340

• UGT76B1 Araport: AT3G11340

• FMO1 Gramene: AT1G19250

• FMO1 Araport: AT1G19250