As sessile organisms, plants are under constant attack from diverse microbes including bacteria, fungi, oomycetes, and viruses. To ward off pathogens, plants activate their immune system to mount multiple defense responses, which are similar to animal innate immunity (Jones and Dangl, 2006). Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors results in PAMP-triggered immunity (PTI). To achieve successful colonization, adapted pathogens can deliver effector molecules directly into the plant cells to suppress PTI, resulting in effector-triggered susceptibility (ETS) (Jones and Dangl, 2006). On the other hand, plants have evolved resistance (R) proteins to detect the presence of certain pathogen effector molecules, inducing effector-triggered immunity (ETI). Activation of PTI or ETI leads to generation of mobile signals, which induce a long-lasting broad-spectrum immune response known as systemic acquired resistance (SAR) (Durrant and Dong, 2004).

The phytohormone salicylic acid (SA) plays an essential role in these defense response pathways (Vlot et al., 2009). Exogenous application of SA or its analogs induces expression of defense genes including PR (pathogenesis-related) genes and disease resistance (White, 1979; Dong, 2004), whereas transgenic plants carrying the bacterial NahG gene, which encodes an SA hydroxylase, are hypersusceptible to pathogen infection and fail to develop SAR (Gaffney et al., 1993; Delaney et al., 1994; Lawton et al., 1995). Furthermore, Arabidopsis mutants with impaired SA biosynthesis during pathogen infection, such as sid2 (salicylic acid induction-deficient2) (Nawrath and Métraux, 1999; Wildermuth et al., 2001), eds5 (enhanced disease susceptibility5) (Nawrath and Métraux, 1999; Nawrath et al., 2002), and pad4 (phytoalexin deficient4) (Zhou et al., 1998; Jirage et al., 1999), show compromised defense responses. In contrast, mutants with elevated levels of SA, such as acd (accelerated cell death) (Greenberg et al., 1994; Rate et al., 1999), cpr (constitutive expressor of PR genes) (Bowling et al., 1997; Clarke et al., 1998), and ssi (suppressor of salicylate insensitivity of npr1-5) (Shah et al., 1999, 2001), display constitutive expression of PR genes and SAR.

Previous research has revealed that plants mainly utilize two distinct enzymatic pathways to synthesize SA, the phenylalanine ammonia-lyase (PAL) pathway and the isochorismate (IC) pathway (Vlot et al., 2009; Dempsey et al., 2011). Both pathways require the primary metabolite chorismate, which is derived from the shikimate pathway. Earlier studies using isotope feeding suggested that SA is synthesized from phenylalanine via either benzoate intermediates or coumaric acid catalyzed by a series of enzymes including PAL, benzoic acid 2-hydroxylase, and other unknown enzymes (León et al., 1995; Dempsey et al., 2011). SA can also be synthesized through isochorismate catalyzed by isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL). Two ICS enzymes, ICS1 and ICS2, exist in Arabidopsis, and ICS1 has been shown to play a major role in SA biosynthesis (Garcion et al., 2008). Intriguingly, no plant genes encoding IPL have been identified. In comparison to the PAL pathway, the IC pathway plays a more important role in synthesis of both basal and induced SA in Arabidopsis (Mauch-Mani and Slusarenko, 1996; Garcion et al., 2008). However, neither pathway has been fully defined so far.

Nawrath and Métraux (1999) conducted a forward genetic screen in Arabidopsis for mutants with altered levels of total SA after infection with the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 carrying the avirulence gene avrRpm1. Two mutants, sid1 and sid2, were identified, which did not accumulate SA during the infection (Nawrath and Métraux, 1999). The sid1 and sid2 mutants were shown to be allelic to eds5 and eds16, respectively, which were identified in another genetic screen for enhanced disease susceptibility (Rogers and Ausubel, 1997; Nawrath and Métraux, 1999). EDS5/SID1 encodes a chloroplast MATE (multidrug and toxin extrusion) transporter (Nawrath et al., 2002), and SID2/EDS16 encodes an SA biosynthetic enzyme ICS1 (Wildermuth et al., 2001). In this screen, an HPLC (high performance liquid chromatography)-based method was used to quantify SA levels in pathogen-infected leaf tissues from about 4500 individual M₂ plants. Obviously, the genetic screen did not reach saturation.

The HPLC-based method used by Nawrath and Métraux (1999) is extremely costly and time-consuming, which would not be practical for a large-scale genetic screen. Recently, an SA biosensor, named Acinetobacter sp. ADPWH_lux, was developed (Huang et al., 2005). This bacterial strain was derived from Acinetobacter sp. ADP1 and contains a chromosomal integration of an SA-inducible lux-CDABE operon, which encodes a luciferase (LuxA and LuxB) and the enzymes that produce its substrate (LuxC, LuxD, and LuxE). In the presence of SA, methylsalicylic acid, and acetylsalicylic acid, the operon is activated, resulting in emission of 490-nm light (Huang et al., 2005). Measurement of SA from tobacco mosaic virus-infected tobacco leaves with the biosensor and gas chromatography/mass spectrometry (GC/MS) yielded similar results, demonstrating that this strain is suitable for quantification of SA in plants (Huang et al., 2006). DeFraia et al. developed an improved methodology for Acinetobacter sp. ADPWH_lux-based SA quantification for both free SA and SA O-β-glucoside (SAG) in crude plant extracts (Defraia et al., 2008). Based on this, Marek et al. (2010) established a further simplified protocol for estimation of free SA levels in crude plant extracts in a high-throughput format (Marek et al., 2010). The efficacy and effectiveness of the newly developed SA biosensor-based method were confirmed by HPLC and verified in a small-scale mutant screen.

To better understand SA biology, we conducted a large-scale forward genetic screen aimed at isolating more Arabidopsis mutants with altered SA accumulation upon pathogen infection. We expected that mutants accumulating significantly altered levels of SA during pathogen infection will help study how SA is synthesized in plant cells and uncover important regulators of plant immunity. This screen allowed us to identify nine new mutants with significantly altered levels of pathogen-induced SA in the npr1-3 genetic background. Among them, seven produced SA levels lower than npr1 (sln) and two displayed increased SA accumulation in npr1 (isn). Enhanced disease resistance tests demonstrated that the seven new sln npr1-3 mutants are more susceptible to bacterial pathogen infection, while both isn npr1-3 mutants are more resistant than npr1-3. We further characterized the sln1 single mutant and found that the sln1 mutation compromised the bacterial pathogen P. syringae pv. maculicola (Psm) ES4326-induced defense responses. Moreover, exogenous SA induced both PR gene expression and disease resistance in sln1, indicating that SLN1 functions upstream of SA. Finally, the sln1 mutation was mapped to a region on the north arm of chromosome I, which contains no known genes involved in regulating pathogen-induced SA accumulation, suggesting that SLN1 encodes a new SA pathway component.

In this study, we performed a forward genetic screen for Arabidopsis mutants with altered SA accumulation during pathogen infection using the newly developed SA biosensor method (Marek et al., 2010). Compared with the HPLC and GC/MS approaches, the SA biosensor method is much faster and less expensive (Malamy et al., 1992; Verberne et al., 2002; Marek et al., 2010). Using this method, we screened a large population (35,000) of M₂ plants in less than 1 year. Approximately 350 putative SA accumulation mutants in the npr1-3 genetic background were identified. Among them, 17 are sln npr1-3 mutants, producing significantly lower levels of SA than npr1-3 after Psm ES4326 infection, and two are isn npr1-3 mutants, accumulating higher levels of SA than npr1-3 (Figures 1A,B). Interestingly, upon Psm ES4326 infection, SA levels accumulated in the remaining putative mutants (in the npr1-3 background) were significantly lower than those in npr1-3, but slightly higher than those in the wild-type plants, suggesting the existence of a larger number of regulatory components involved in pathogen-induced SA accumulation. Indeed, genetic studies have uncovered a complicated signaling network that regulates SA accumulation. This consists of upstream SA signaling components (such as EDS1, PAD4, and NDR1), downstream SA signaling components (such as NPR1), transcription factors (such as CBP60g and SARD1), metabolic enzymes (such as EPS1 and PBS3), and various positive and negative feedback loops (Cao et al., 1997; Ryals et al., 1997; Shah et al., 1997; Zhou et al., 1998; Jirage et al., 1999; Shapiro and Zhang, 2001; Wildermuth et al., 2001; Jagadeeswaran et al., 2007; Lee et al., 2007; Nobuta et al., 2007; Zheng et al., 2009; Zhang et al., 2010; Wang et al., 2011).

The SA accumulation phenotype of the sln mutants is similar to that of eds5, sid2, and pad4 mutants (Zhou et al., 1998; Jirage et al., 1999; Nawrath and Métraux, 1999; Wildermuth et al., 2001; Nawrath et al., 2002). EDS5 and SID2 encode a chloroplast MATE transporter and an SA biosynthetic enzyme ICS1, respectively, which are two important components in the SA biosynthesis pathway. PAD4 is a lipase-like protein involved in an SA positive signal-amplification loop required for activation of defense responses (Jirage et al., 1999). Complementation tests indicated that seven out of the 17 sln mutants are new alleles of eds5, two are alleles of sid2, and one is allelic to pad4, and the other seven sln and two isn mutants are new non-allelic mutants (Table 1). Although this is a large-scale genetic screen, the low frequency of alleles for the new sln and isn mutants indicates that our genetic screen has not been saturated.

Several other recessive mutations have also been reported to impair pathogen-induced SA accumulation. In the eps1-1 mutant, pathogen-induced accumulation of SAG was greatly reduced, but free SA levels were comparable to those in the wild type. EPS1 encodes a novel member of the BAHD acyltransferase superfamily, which is predicted to be directly involved in the synthesis of a precursor or regulatory molecule for SA biosynthesis (Zheng et al., 2009). Similarly, the pbs3-1 mutant displayed decreased pathogen-induced accumulation of SAG, but varied in free SA accumulation between studies (Jagadeeswaran et al., 2007; Lee et al., 2007; Nobuta et al., 2007). PBS3 belongs to the acyl adenylate/thioesterforming enzyme superfamily. The exact functions of both EPS1 and PBS3 in SA biosynthesis, however, have not been clearly defined. Consistent with these studies, we found that free SA levels in the eps1-1 and pbs3-1 mutants were comparable to those in the wild type when assayed with the SA biosensor (Figure S1). Thus, the sln mutations are unlikely located in either EPS1 or PBS3, since these mutations greatly influenced Psm ES4326-induced free SA accumulation (Figures 1A,B). Additionally, although the eds1 mutation significantly affects pathogen-induced accumulation of both free SA and SAG (Falk et al., 1999), the sln mutants are unlikely alleles of eds1, because there are two EDS1 genes lying in tandem on chromosome 3 of the Arabidopsis ecotype Col-0 (Feys et al., 2005). Therefore, the SLN genes may encode new signaling components downstream of recognition of pathogen infection, or new enzymes directly involved in the synthesis of a precursor and/or regulatory molecule for SA biosynthesis.

In addition to components upstream of SA biosynthesis, the downstream component, NPR1 (nonexpressor of PR genes1), which has been shown to be an important regulator of defense responses (Cao et al., 1997; Dong, 2004), also regulates SA levels. Mutations in the NPR1 gene enhance SA accumulation during pathogen infection, suggesting that NPR1 is a feedback inhibitor of SA biosynthesis (Figures 1A,B; Clarke et al., 2000; Wildermuth et al., 2001; Zhang et al., 2010). Here we found that eds5 npr1-3, sid2 npr1-3, pad4 npr1-3, and sln npr1-3 double mutants accumulated significantly lower levels of SA than npr1-3 (Figures 1A,B), suggesting that these mutations (eds5, sid2, pad4, and sln) suppress npr1-mediated SA hyperaccumulation. On the other hand, these double mutants were more susceptible to Psm ES4326 than npr1-3 (Figure 2), indicating that EDS5, SID2, PAD4, and the SLN genes may contribute to NPR1-independent defense responses (Glazebrook, 2001). NPR1-independent defense signaling pathways have been shown to be activated in various Arabidopsis mutants, including sni1 (Li et al., 1999), snc1 (Li et al., 2001), ssi (Shah et al., 1999, 2001), and cpr (Bowling et al., 1997; Clarke et al., 1998). The two isn mutations appear to also activate NPR1-independent disease resistance (Figure 2).

We further isolated and characterized the sln1 single mutant. The sln1 plants exhibited significantly reduced levels of Psm ES4326-induced SA and supported more Psm ES4326 growth than the wild-type plants (Figures 4A,B, 5B), suggesting that SLN1 plays an important role in activation of defense responses against this pathogen. Interestingly, the sln1 mutation appears to differentially influence pathogen-induced PR1, PR2, and PR5 expression. Psm ES4326-induced PR1 expression was greatly reduced in sln1 plants, but induction of PR2 and PR5 was nearly unaffected (Figures 5C–E). In this regard, sln1 is also similar to eds5, sid2, and pad4, which cause reduced induction of PR1, but have no effect on the expression of PR2 and PR5 (Rogers and Ausubel, 1997; Zhou et al., 1998; Nawrath and Métraux, 1999). On the other hand, pathogen-induced expression of PR1, PR2, and PR5 is strongly reduced in NahG transgenic plants (Nawrath and Métraux, 1999), which argues against the idea that an SA-independent pathway exists to control PR2 and PR5 expression. It is possible that the low levels of SA accumulated in the SA biosynthesis mutants are sufficient for induction of PR2 and PR5, but not for PR1.

In summary, we identified a group of new SA accumulation mutants, including seven sln mutants and two isn mutants, in a genetic screen using the newly developed SA biosensor-based method. Further characterization of these sln and isn mutants and cloning of the SLN and ISN genes will shed new light on the molecular mechanisms underlying pathogen-induced SA accumulation and SA-mediated defense signaling in plants.

Yezhang Ding and Zhonglin Mou designed research. Yezhang Ding and Danjela Shaholli performed mutant screen. Yezhang Ding characterized mutants and analyzed data. Yezhang Ding and Zhonglin Mou wrote the manuscript.