Salicylic acid-mediated plant defense: Recent developments, missing links, and future outlook

Abstract

BACKGROUND: Plant pathogens are responsible for many of history’s greatest famines. Understanding how plants defend themselves against pathogens is crucial to preventing future famines. Salicylic acid (SA)-mediated plant defense is a key defense pathway, which plants use to defend against biotrophic and hemi-biotrophic pathogens. As a master regulator of SA-mediated plant defense, NPR1 interacts with TGA and WRKY transcription factor families, individual members of which positively or negatively regulate plant defense.

OBJECTIVE:In this review we describe the recent developments and predict future directions of research on the involvement of circadian rhythm-, autophagy-, and viral RNA silencing-related genes in SA-mediated plant defense on SA, on plant defense, the induction effects of PR proteins, and the mechanisms by which NPR1 regulates defense-related genes.

METHODS:We performed an extensive search of current and past literature using the PubMed, Google Scholar, and Google search engines. Our search terms included: “SA-mediated plant defense,” and “NPR1 [AND] salicylic acid.” Other search terms, wildcards, and Boolean operators were paired with “NPR1” or “plant defense” as needed to research more detailed information related to specific topics covered within this review. We also used Google to search for, “economic impact citrus greening,” “aspirin,” “Irish potato famine,” and “rice blast,” among other terms, to gather background information on the history and impact of plant diseases, and the historical use of aspirin.

RESULTS:Of 148 sources found, 132 were directly related to plant defense. The remaining sources are related to the historical and economic impact of plant diseases and the historical use and mechanism of action of aspirin or salicylate. All reviewed sources have been documented in the references section.

CONCLUSION:The topic of salicylic acid-mediated plant defense is broad, and new research is expanding our understanding of this topic quickly. In this review, we give a basic overview of the historical economic impact of plant diseases, and how an understanding of SA-mediated plant defense can prevent future famines. We provide a basic overview of plant defense, then discuss how SA acts as a defense signaling molecule.We discuss how SA regulates NPR1, which goes on to activate expression of SA-related genes includingPRgenes. Later, we discuss current research topics, including the role of NPR1 and SA in autophagy, circadian rhythmicity, viral gene silencing, SA biosynthesis, and SAR. We also discuss the potential roles of PR proteins, other SA binding proteins, WRKY and TGA family transcription factors, Elongator, and ER transport proteins in plant defense. Finally, we discuss the potential future routes that research into this topic could take, in order to further our understanding of role SA plays in plant defense.

Keywords

salicylic acid / plant defense / SA-mediated defense

Introduction

Diseases caused by invading pathogens are a common problem for all species. Plant diseases caused by plant pathogens, such as fungi, bacteria, oomycetes, viruses and nematodes, are responsible for a substantial loss of crop yield each year. One well-known example is the potato late blight disease, caused by the oomycete pathogenPhytophthora infestans (Ó Gráda, 2007). Late blight is responsible for the infamous Great Irish Potato Famine, which lasted from 1845 to 1852, and resulted in the death of over one million people, and the emigration of another million (Ross, 2002). Another example is rice blast, caused by the fungus Magnaporthe grisea (Talbot, 2003). Rice blast is estimated to destroy enough rice to feed 60 million people annually, and is now known to be present in 85 countries (Scardaci et al., 2016). Further, the citrus greening disease, also known as Huanglongbing, is caused by a psyllid-transmitted bacterial pathogen known asCandidatusLiberibacter spp. (Lin and Lin, 1956). This devastating disease recently appeared in the citrus fields of California, Mississippi, Louisiana, Georgia, Alabama, Florida, South Carolina, and Texas (Kunta et al., 2012). California alone boasts a citrus industry of $3.38 B, as of 2015, which is threatened if the pathogen continues to spread (U.S.D.A. and N.A.S., 2015). As of 2015, Florida produced only 96.8 million boxes of oranges, an eight percent reduction from the previous year, a 60% reduction from the 242 million boxes produced a decade ago (U.S.D.A. and N.A.S., 2015). The Florida citrus industry is a $10B industry that has been losing about $1B each year since 2005 due to citrus greening disease (Spreen, 2012).

To defend against these microscopic invaders, organisms have evolved different defense mechanisms to protect themselves. In plants, these mechanisms generally rely upon the successful detection of pathogens, followed by physiologic changes that result in increased defense against the invader. These changes include the formation and release of reactive oxygen species and other antibiotic substances, the deposit of molecules to strengthen the cell wall, and programmed cell death, known as the hypersensitive response (HR) (Glazebrook, 2005).

Plant defense mechanisms are generally classified into two tiers: MAMP-triggered immunity (MTI) and ETI (Effector-triggered immunity). MTI, also known as microbe-associated molecular pattern (MAMP)-triggered immunity is associated with the detection of highly conserved, signature, pathogen associated molecules known as microbe-associated molecular patterns (MAMPs), including flagellin, chitin, and elongation factor T_u, among others (Boller and Felix, 2009). ETI, is a more severe defense mechanism, and supplements MTI. ETI is associated with HR in resistant hosts, and it is triggered by direct or indirect recognition of pathogenic effector proteins by plant resistance (R) proteins (Tsuda and Katagiri, 2010), for example: the type III effector AvrR2 functions as a cysteine protease that cleaves the negative regulator of plant defense, RIN4. Recognition of degradation product of RIN4 by the resistance protein RPS2 activates ETI and triggers HR (Axtell et al., 2001; Mackey et al., 2003) (See Fig. 1). Activation of ETI will not only produce rapid cell death in the infection zone, but also induce the production of the plant defense hormone salicylic acid (SA), and activate systemic acquired resistance (SAR) in the entire plant (Fu and Dong, 2013; Wu et al., 2014).

SA is well known as a precursor of aspirin, the active ingredient of which is acetylsalicylic acid. Aspirin is among the oldest, cheapest, and most widely used medicines in human history; it is broadly used as fever-reducer, pain-reliever, and anti-inflammatory medicine (Myers, 2007). Studies have shown that long-term use of aspirin may reduce the risk of stroke, cardiovascular disease, and heart attack. In addition, non-acetylated salicylate shows effectiveness in treating type II diabetes (Goldfine et al., 2013). In humans, aspirin irreversibly inhibits cyclooxygenase 1 (COX-1) (DeWitt et al., 1990), and modifies the enzymatic activity of COX-2, both of which catalyze the production of prostaglandin H2 from arachidonic acid, involved in inflammation, and thromboxane A₂, involved in blood clotting (Preston et al., 1981; Smith et al., 1996). SA and its derivatives also inhibit IkB kinase (Yin et al., 1998), NF-kB (Kopp and Ghosh, 1994), and activate AMP-activated protein kinase (Hawley et al., 2012).

As one of the major plant hormones, SA plays a regulatory role in many physiologic processes, such as seed germination, storage, and fruit maturity (Raskin, 1992). In addition, SA plays roles in regulating flowering development, sex differentiation, stomatal movement, and photoperiod. SA is both required and sufficient to induce a defense response against pathogens (Raskin, 1992). Transgenic plants overexpressing the NahG transgene from Pseudomonas putida, encoding SA-degrading hydroxylase, have been proven to be more susceptible to a variety of pathogens (Delaney et al., 1994).

During pathogen infection, SA is synthesized in the chloroplast, primarily through the isochorismate pathway inArabidopsis. Isochorismate synthases one and two (ICS1/2) are localized in the plastid, and ICS1 is responsible for the majority of SA accumulation in response to the presence of hemi- and biotrophic pathogens (Strawn et al., 2007; Fragnière et al., 2011). Arabidopsis ics1 mutant plants are significantly reduced in SA level, and as a consequence, these mutants are more susceptible to pathogen infection.

In addition to inducing a local defense response, SA promotes systemic acquired resistance (SAR) after an invading pathogen is recognized (An and Mou, 2011). SAR protects the plant against further pathogen colonization by causing a systemic defense reaction including the production of pathogensis related (PR) proteins, phytoalexins, and the strengthening of cell walls. SA is also responsible for regulating these later responses to pathogenic invasion (Lu et al., 2016), and application of SA is sufficient to induce plant defense including SAR (Anand et al., 2008).

It was suggested that methyl salicylate (MeSA) is the mobile signal that can induce SAR in Nicotiniana benthamiana. MeSA moves through phloem from infected tissue to healthy tissue, acting as a long distance defense signal. MeSA is a gaseous product of SA, produced in infected tissue, which is converted back to SA in target tissues (Shulaev et al., 1997; Chen et al., 2003); however, MeSA does not seem to be a mobile defense signal in Arabidopsis (Attaran et al., 2009). Research indicates that Arabidopsis plants lacking salicylic acid methyltransferase fail to accumulate MeSA, but still possess the ability to systemically accumulate SA and activate SAR (Attaran et al., 2009). Arabidopsis mutants that cannot respond to or synthesize SA cannot effectively induce SAR, and their basal defense is also compromised (Durrant and Dong, 2004). In addition to inducing defense, secreted SA acts as an intercellular antimicrobial agent againstPseudomonas spp. and fungal pathogens in Arabidopsis (Cameron and Zaton, 2004; Carviel et al., 2009; Carviel et al., 2014).

SA is important for defense against biotrophs and hemi-biotrophs, but is generally not effective against necrotrophic pathogens, because these pathogens are not impeded by cell death nor HR. The jasmonic acid and ethylene signaling pathways activate defenses against necrotrophic pathogens (Glazebrook, 2005).

Plants and plant pathogens are locked in an evolutionary arms race to develop more advanced proteins to enhance or subvert plant defense, respectively. EDS1 is a positive regulator of basal resistance to biotrophic pathogens (Wiermer et al., 2005), required by many Arabidopsis TIR-NB-LRR class R proteins to activate ETI (Feys et al., 2001). EDS1 is required for accumulation of SA in response to a pathogen (Parker et al.,1996), and it has been reported that the reduced levels of SA in eds1 and pad4 mutants results in increased susceptibility to pathogen infection (Falk et al., 1999; Jirage et al.,1999; Nawrath et al., 2002). In addition to EDS1, PAD4 also serves as a regulator of basal plant immunity. EDS1 forms heterocomplexes with PAD4 in the nucleus and cytoplasm, which are required for HR and pathogen resistance (Feys et al., 2001) (See Fig. 1). In addition to PAD4, EDS1 also interacts with SAG101 (SENESCENCE ASSOCIATED PROTEIN 101), which can form a ternary complex with EDS1 and PAD4, and plays a pivotal role in pathogen resistance (Feys et al., 2005; Zhu et al., 2011; Wagner et al., 2013). It is also demonstrated that EDS1 interacts with SRFR1 (SUPPRESSOR OF RPS4-RLD1), which is a tetratricopeptide repeat containing a negative regulator of ETI (Kim et al., 2009; Kwon et al., 2009) (See Fig. 1). EDS1 also interacts with TIR-NB-LRR proteins RPS4 and RPS6, and the interactions between them are disrupted in the presence of AvrRps4 and HopA1 respectively (Bhattacharjee et al., 2011; Heidrich et al., 2011) (See Fig. 1).

PBS3 is a member of the GH3 family of acyl-adenylate/thioester-forming enzymes, which when mutated, causes SA to fail to accumulate, no induction ofPR1 defense gene, and increased pathogen susceptibility (Nobuta et al., 2007). EDS1, PBS3, and PAD4 proteins are critical to SA-mediated plant defense, and likely targets for pathogen effectors.

Recent developments in SA-mediated plant defense

SA biosynthesis

In addition to the primary ICS1/2 SA biosynethesis pathway, another less often used, redundant pathway exists for SA synthesis: the phenylalanine ammonia-lyase (PAL) pathway. This pathway converts phenylalanine into trans-cinnamic acid, then to benzic acid, and finally to salicylic acid (Mauch-Mani and Slusarenko, 1996). Basal levels of SA vary widely between plant species, with up to 100-fold differences recorded between species (Raskin et al., 1990). In addition, most of the SA produced by plants is glycosylated or methylated or both. Glycosylation at the hydroxyl group yields SA 2-O-β-D-glucoside (SAG), which is speculated to serve as an inactive storage form of SA that can be released from the cell’s vacuole as needed (Dean and Mills, 2004; Dean et al., 2005).

In Arabidopsis, the isochorismate pathway is the main biosynthetic pathway for pathogen induced SA; however, current research suggests that the PAL pathway may play a more important role in SA biosynthesis in other plants. Shine, et al., examined the PAL pathway in soybean. They discovered that, unlikeArabidopsis, knocking out proteins in either the PAL or ICS pathways caused a similar reduction in defense response to invading pathogens. This research indicates that both pathways are equally important to plant defense (Shine et al., 2016). Huang et al. (2010) generated a pal1/2/3/4 quadruple mutant Arabidopsis, and found that this mutant could accumulate only substantially reduced levels of SA, and was much more susceptible toPseudomonas syringae than its wild type counterparts, further suggesting an overlapping role of PAL genes in defense. More research is needed to determine if the PAL pathway plays a role in pathogen-induced SA biosynthesis inArabidopsis and in other plants.

Redox and post-transcriptional regulation of NPR1

The SA signaling pathway is regulated by NPR1, which co-activates 95% of SA-related defense genes (Pajerowska-Mukhtar et al., 2013). After pathogen infection, SA accumulates in the cytosol of infected cells, altering the redox state of those cells (Mou et al., 2003). This altered redox state causes NPR1 to dissociate from an oligomeric state to a monomeric state, and move to the nucleus (Tada et al., 2008). In this state, NPR1 interacts with TGA-bZIP transcription factors in the nucleus. This leads to the expression of several SA-dependent genes includingPR1/2/5 (Zhang et al., 1999; Zhou et al., 2000) (Fan and Dong, 2002). Depending on the level of SA present, NPR1 is poly-ubiquitinated by CUL3, with NPR3 and NPR4 acting as adaptors (Fu et al., 2012), and degraded by the 26S proteasome to prevent the activation of immune responses in naïve cells without infection or SA (Spoel et al., 2006). NPR1’s degradation in the nucleus is dependent on its interaction with NPR3 and NPR4, both of which only bind NPR1 when a high level or a low level of SA is present, respectively (Fu et al., 2012). SA-mediated plant defense is, therefore, biphasic. NPR1 may only accumulate in the nucleus at high level when a moderate level of SA is present (Fu et al., 2012) (See Fig. 1). Additionally, the phytohormone abscisic acid (ABA) and SA antagonistically affect NPR1 levels during pathogen infection. While SA can prevent NPR1’s proteasome-mediated degradation via CUL3, ABA promotes this degradation (Ding et al., 2016). Indeed, a deficiency of ABA has been found to cause constitutive PR gene expression (Mosher et al., 2010), suggesting that ABA can prevent defense gene activation in the absence of a pathogen infection.

As a transcriptional co-activator, the nuclear localization of NPR1 is required for NPR1’s function in plant defense (Tada et al., 2008). Redox changes induced by SA can induce transcriptional reprogramming of jasmonic acid (JA) induced genes by affecting NPR1 and the TGA family of transcription factors (Ndamukong et al., 2007). These redox changes can determine the proteins’ sub-cellular localization or DNA binding ability to modify gene expression. Also, SA can cause proteins associated with JA signaling, like ORA59, to be degraded (Van der Does et al., 2013), and can induce negative regulators of JA signaling, specifically WRKY70 and WRKY33 (Li et al., 2004; Zheng et al., 2006). This cross-talk between SA and JA is important to plant defense, because SA-mediated defense is generally more effective against hemi- and biotrophic pathogens, while JA-mediated defense allowing is effective against herbivory and necrotrophic pathogens, the plant to mount a quick, targeted defense against any type of pathogen (Pieterse et al., 2012).

Several proteins assist in the homeostasis of NPR1. Recent research suggests that NPR1 is S-nitrosylated at cysteine-156 by S-nitrosoglutathione (GSNO), which facilitates its oligomerization. In addition, NPR1’s monomerization is facilitated by several thioredoxins (TRXs). These reactions regulate NPR1 by sequestering it in the cytosol, or conversely, allowing the free monomer to enter the nucleus (See Fig. 1) (Tada et al., 2008).

Sumoylation is another method by which the plant cell can post-transcriptionally regulate the function of NPR1. Saleh, et al., discovered that NPR1 is sumoylated after application of SA, dependent on NPR1’s phosphorylation. NPR1’s sumoylation causes it to switch from interacting with WRKY transcription factors, to interacting with the TGA family of transcription factors that activate transcription of defense-related genes. Sumoylation also causes NPR1 to become degraded, which is essential for its function in fully regulating SA-mediated plant defense (Spoel et al., 2009; Saleh et al., 2015).

Expression of NPR1 is regulated by a number of WRKY DNA binding proteins. The 5′-UTR of NPR1 contains two W-box sequences, which are recognized by WRKY proteins that are induced by SA and pathogen infection. These W-boxes are necessary for induction ofNPR1 and activation of SA-mediated plant defense (Yu et al., 2001). Specifically, Chai et al. found that WRKY6 directly binds to the NPR1 promoter, and that the level of NPR1 mRNA is reduced in wrky6 mutants (Chai et al., 2014). Further, they demonstrated that MPK6 works upstream WRKY6, and that SA-induced activation of MPK6 leads to higher expression of WRKY6, ultimately increasing the level ofNPR1 mRNA (Chai et al., 2014).

TGA defense signaling pathway

TGA transcription factors are found in all eukaryotes, and belong to the group of basic region/leucine zipper (bZIP) transcription factors. These transcription factors have a basic region that binds DNA, and a leucine zipper dimerization motif. TGA transcription factors can bind specifically to the canonical sequence TGACGTCA (Jakoby et al., 2002). TGA Transcription factors mediate SAR and PR gene expression (Fan and Dong, 2002). NPR1 interacts with TGA transcription factors in the nucleus, stimulating their ability to bind to SA-responsive genetic elements. Functional NPR1 is required for TGA transcription factors to bind to these elements (Després et al., 2000).

There are ten TGA transcription factors, which have been confirmed in Arabidopsis (Jakoby et al., 2002). Of those ten, TGA1-TGA7 have been found to interact with NPR1. Those seven TGA transcription factors could be further divided into three classes based on their sequence homology: TGA1 and TGA4; TGA2, TGA5, and TGA6; and TGA3 and TGA7 (Xiang et al., 1997). TGA2/3/5/6/7 have consistent interaction with NPR1 in yeast, and can be transiently expressed in planta; however, in SA-induced leaves, TGA1 can bind NPR1, while no interaction was found in yeast. Later, it was confirmed that reduction of two Cys residues in TGA1 determined this SA-dependent interaction (Després et al., 2003). Contrary to previous negative results, Tada, et al., found that NPR1 and TGA4 indeed interact using a GAL4-based yeast system (Tada et al., 2008). Després et al. confirmed that TGA4 weakly interacts with NPR1 using co-immunoprecipation and western blotting techniques in vitro (Després et al., 2000). The latest studies show that PR gene expression did not change significantly when TGA4 and TGA7 were individually disrupted. There is conflicting evidence regarding TGA2. Pontier et al. reported that TGA2 can enhancePR-1 expression and SAR ( Pontier et al., 2001 ). TGA5 has been reported to enhance defense against the oomycete parasite, Peronospora parasitica, independent of interaction with NPR1 and SA (Kim and Delaney, 2002).

PR genes and their antimicrobial effects

Upon pathogen infection, a group of plant defense genes, called Pathogenesis-Related (PR) genes, which encode low molecular proteins with antimicrobial activities, are significantly induced (Carr et al., 1989). The PR1 family in Arabidopsis is composed of 22 genes, only one of which is upregulated in response to pathogen infection (van Loon et al., 2006). Interestingly, in rice, which possesses 12 PR1 genes, all 12 were induced in response to infection with rice blast fungal pathogen, Magnaporthe grisea (Mitsuhara et al., 2008). In tobacco, PR-1a encodes an acidic secreted protein that was found to bind to ergosterols in fungal membranes, causing cellular leakage. The PR-1a ortholog, P14c, in tomato encodes a basic protein, which is stored in the vacuole. These proteins share similar indiscriminate sterol binding capability; however, P14c presumably is released from the vacuole after the cell is lysed in order to flood the pathogen invader and damage its plasma membrane before its sterol biosynthesis mechanisms can adjust (Gamir et al., 2016). The PR-2 family of proteins have β-1,3-glucanase activity in Xanthi-nc (NN) tobacco (Kauffmann et al., 1987), and PR-3 proteins are putative chitinases (Legrand et al., 1987).

Among PRs, PR-1, PR-2, and PR-5 have SA-dependent expression in Arabidopsis, and are required for defense against the biotrophic fungus Peronospora parasitica (Curto et al., 2006). van Verk et al. (2011), identified a novel tobacco WRKY transcription factor (NtWRKY12) that can bind the promoter ofPR-1a, which is induced by SA. Several classes of PR proteins, including PR-3, -4, -8, and -11, are believed to be different types of chitinases, conferring resistance to fungal pathogens (Kasprzewska, 2003). Transgenic tomato plants overexpressing either a class I tobacco β-1,3-glucanase or a class I chitinase show no increased resistance to pathogen infection; however, transgenic tomato seed endosperm tissue overexpressing both the β-1,3-glucanase and chitinase shows significant resistance to pathogen infections, indicating a synergistic effect (Wu et al., 2001).

ER transport genes required for defense and SAR

Recent evidence indicates that endoplasmic reticulum (ER) genes, involved in the protein secretion pathway, are also involved in SA-mediated defense, and are required for systemic acquired resistance (Wang et al., 2005). These secretion-related genes include those encoding the Sec61 translocon complex, a protein complex that allows proteins to cross the ER membrane, and a signal recognition particle receptor, which can recognize proteins with the ER transport signal, then direct them to the translocon complex. It has been reported that the production and secretion of defense proteins depend on the functions of the ER, whose secretory functions can be altered in the presence of SA (Trombetta and Parodi, 2003; Vitale and Denecke, 1999). Many genes encoding ER-resident chaperones and co-chaperones are also induced by NPR1, such asBiP2, GRP94, DAD1, CNXs, CRTs, and PDIs. The chaperone proteins encoded by these genes are crucial for determining protein co-translational folding and modification in various organelles (Fu and Kreibich, 2000; Wang et al., 2005).

NAD(P) and its role in SA-related defense gene expression

In mammalian tissues, NAD(P) is released into the extracellular compartment during stress, where it activates transmembrane signaling, likely through purinoreceptors (Billington et al., 2006). Zhang and Mou hypothesized that the same may be true of plant tissues during pathogen attack. After infiltrating leaf tissues with NADP⁺ , they found that expression of PR1, PR2, and PR5 could be induced in a dose-dependent manner. Similarly, they also infiltrated NADP⁺, NAD⁺, and ATP, but observed no induction of PR gene expression when using ATP, and a similar induction when using NADP⁺ and NAD⁺. This indicates that the induction does not depend on the oxidation state of NADP⁺, and that the induction depends on receptors for pyridine nucleotides. Additionally, treatment with these pyridine nucleotides causes SA to accumulate in treated tissues and enhanced resistance toPsm ES4326; however, there is a difference in induction capabilities between NAD⁺ and NADP⁺. It seems that NADP⁺ can only partially induce resistance in sid2 and npr1 mutants, while NAD⁺ can fully restore resistance against bacterial pathogens in these mutants (Zhang and Mou, 2009). These data demonstrate the importance of the presence and perception of extracellular molecules in SA-mediated plant defense signaling.

After screening for mutants that were insensitive to extracellular NAD, An, et al., identified ien2, which has a mutation in the gene encoding ELONGATA3 (ELO3), an Elongator complex subunit. Elongator subunit mutations result in diverse phenotypes including: resistance to oxidative stress, abnormal root development, increased pathogen susceptibility, severe auxin phenotypes, and sensitivity to abscisic acid. ELO3 has been shown to be responsible for the rapid induction of transcriptional reprogramming in response toPst DC3000, acting as an epigenetic regulator of defense genes (An et al., 2016).

WRKY transcription factors involved in plant defense signaling

WRKY proteins are defined as a class of DNA binding proteins, which possess a conserved N-terminal amino acid sequence, WRKYGQK, and a novel C-terminal zinc finger motif. WRKY proteins recognize TTGAC(C/T) W-box elements, which are often located in the promoters of plant defense-related genes, such as thePR genes (Dong et al., 2003). Most of the analyzed WRKY genes are capable of responding to pathogen defense and are involved in defense signaling (Eulgem and Somssich, 2007). Several WRKY protein-regulated genes were found to be induced after application of SA in Arabidopsis (Dong et al., 2003). SA-induced WRKY proteins also regulate the expression of NPR1 and other regulatory proteins (Yu et al., 2001). The Arabidopsis WRKY family contains 74 genes, while the rice WRKY family has more than 90 members (Eulgem et al., 2000). Phylogenetic analysis demonstrated that the functions of WRKY proteins are conserved in monocots and dicots (Mangelsen et al., 2008). Many reports demonstrate that WRKY transcription factors are involved in disease resistance. For example, overexpression ofWRKY18 causes increased resistance against two bacterial pathogens in plants (Chen C. and Chen Z., 2002). Plants overexpressing WRKY70 exhibited strengthened SA-mediated resistance with impaired resistance mediated by jasmonic acid (Li et al., 2004). WRKY53 is involved in leaf senescence (Miao et al., 2004). Among 64 genes, WRKY18/38/53/54/58/59/66/70 were identified as direct transcriptional targets of NPR1. WRKY18 showed positive regulation for full induction of SAR, while WRKY58 negatively regulated SAR (Wang et al., 2006).

Additional SA receptors and SA binding proteins

In N. benthamiana, three SA binding proteins (SABPs) have been isolated. SABP1 was found to be a catalase enzyme, possibly involved in the HR response in local tissue. SABP2 was shown to be an SA-stimulated lipase (Delaney, 2005). SABP3 has been identified as a chloroplast carbonic anhydrase, which exhibits both enzymatic and SA binding activities (Slaymaker et al., 2002). Current research estimates that there are 30 SABP’s, most of which play a role in plant immunity (Tian et al., 2012).

NPR3 and NPR4 both bind SA, and serve to modulate SA-mediated plant defense in the presence of jasmonic acid (JA). During ETI, both SA and JA are induced to high levels. JA, which is normally associated with defense against necrotrophic pathogens, is induced during ETI in order to protect against simultaneous attack by biotrophic and necrotrophic pathogens in areas where programmed cell death has occurred. In fact, this phenomenon explains why plant tissue adjacent to areas of programmed cell death are not susceptible to necrotrophic pathogen infection (Spoel et al., 2007). Liu, et al., recently discovered that NPR3 and NPR4 mediate concurrent induction of SA and JA by causing degradation of the JAZ proteins, which repress transcription of JA-responsive genes. This represents a non-canonical activation of the JA signaling pathway, through SA receptors NPR3 and NPR4, rather than through activation of JA receptor COI1 (CORONATINE INSENSITIVE 1) (Liu et al., 2016).

Choi et al. recently discovered that High Mobility Group Box 3 (HMGB3) binds SA. HMGB3 is a Damage-Associated Molecular Pattern (DAMP) that plays a role in regulating MT2, including inducing defense related genes, increasing callose deposition, and enhancing pathogen resistance. HMGB3 is released into the apoplast following infection by a necrotrophic pathogen such asB. cinerea. After its release, HMGB3 interacts with an unknown LRR-RLK PRR associated with BAK1 and BKK1, similarly to MAMPs. In the apoplast, HMGB3 binds SA, and its DAMP activity is reduced. This indicates that the jasmonic acid and ethylene pathways, which are associated with defense against necrotrophic pathogens, and the SA-mediated defense pathway are intertwined. HMGB3’s effect against hemi- and biotrophic pathogens is negligible, and the presence of SA seems to be responsible for this (Choi et al., 2016).

Connection between MTI and SA-mediated plant immunity

Plant biologists studying plant defense have sought to discover the link between ETI and MTI, but it seems that Kong, et al., have made one step closer to discovering the link. Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 are kinases that are known to interact with the PRR FLS2. These researchers identified apcrk1 pcrk2 double mutant that was deficient in SA-mediated plant defense. The double mutant can only accumulate about half the amount of SA compared with WT after pathogen inoculation. They discovered that PCRK1/2 are rapidly phosphorylated after the plant is exposed to the synthetic flg22 peptide, and act downstream on two transcription factors, SARD1 and CBP60g, both of which induce expression ofICS1, thereby influencing SA mediated plant defense by altering SA biosynthesis (Kong et al., 2016).

Research suggests that application of SA can prime plant defense upon detection of flg22, the synthetic flagellin epitope. Yi and Kwoon measured the flg22-triggered oxidative burst in Col-0,sid2, and eds5 Arabidopsis. They found that the oxidative burst was suppressed in the sid2 and eds5 mutants. To confirm their results, they measured the induction ofWRKY29 and FRK1 using RT-qPCR, and found that the induction of these defense-related genes was reduced by 50% in thesid2 mutant. After pre-treatment with SA, they discovered that in addition to amplifying the flg22-triggered oxidative burst in Col-0Arabidopsis, SA increased the level of FLS2 mRNA (Yi and Kwon, 2014).

Circadian clock-related genes and SA-mediated plant defense

The circadian clock is a highly conserved system of proteins that coordinate the physiology and behavior of living things with daily environmental change, regulating plant growth and development (Edgar et al., 2012). The core components of the circadian clock, a central self-sustaining oscillator with a period of about 24 h, consist of various negative feedback loops, which are related to each other inArabidopsis (McClung, 2008; Harmer, 2009). Those loops are responsible for responding to external stimuli individually. Among clock components, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and its homolog LATE ELONGATED HYPOCOTYL (LHY) are grouped into transcription factor families, which are responsible for multiple feedback loops and orchestrate clock activity (Alabadí et al., 2002; Mizoguchi et al., 2002; Lu et al., 2009; Wang et al., 2011b). Recently, crosstalk between the circadian clock and plant innate immunity has been revealed. CCA1 and LHY co-regulate basal and resistance gene-mediated defense againstPseudomonas syringae and Hyaloperonospora arabidopsidis (Wei et al., 2011). In overexpresssion mutants of CCA1 and LHY, Arabidopsis showed severely increased susceptibility to P. syringae. GRP7, a downstream target of CCA1 and LHY, is regulated by the circadian clock and demonstrated to affect plant defense and stomatal activity (Zhang et al., 2013).

NPR1 has also been found to be involved in regulating the circadian clock genes. By regulating the expression of TIMING OF CAB2 EXPRESSION 1 (TOC1), LHY, and CCA1, but not changing their phase, NPR1 can reinforce the circadian clock. It is thought that the reinforcement of the circadian clock enables the plant to increase photosynthesis and gate the immune response to the morning, while preserving the ability to grow for night (Zhou et al., 2015).

PHOSPHATE TRANSPORTER 4;1 (PHT4;1) is another protein regulated by the circadian clock. The dominant pht4;1-1 mutant is more susceptible to several virulent Pseudomonas syringae strains. This susceptibility can be suppressed by treating the mutant with SA, indicating that PHT4;1 is upstream of SA signaling.PHT4;1 possesses two CCA1 binding sites in its promotor, and its RNA transcript is degraded in the dark, which indicates that PHT4;1 is under control of the circadian clock, and that light is required for its expression (Wang et al., 2011a).

SA-primed viral gene silencing

SA induces genes involved in gene silencing to suppress viral pathogens. Researchers studying the plum pox virus (PPV) discovered thatNicotiana tabacum plants expressing NahG, salicylate hydroxylase, developed systemic PPV infections (Alamillo et al., 2006). This pathogen normally cannot spread systemically in this host, although it can cause local cell death at its point of entry. They also showed that accumulation of PPV in infected tissues was higher in plants that were deficient in SA. In addition, theNahG plants showed reduced viral derived small RNAs. Taken together, this research indicates that a SA-mediated defense mechanism and a RNA-silencing mechanism work together to limit the spread of this virus (Alamillo et al., 2006). In tomato, several RNA silencing genes are induced after ToMV and CEVd viral infection: Arabidopsis orthologs ToDCL1, ToDCL2, ToDCL4, and ToRDR1. In their study, Campos, et al. found ToDCL1, ToDCL2, ToRDR1, and ToRDR2 are significantly induced after SA treatment, resulting in a delay in accumulation of pathogenic RNA in inoculated plants. This indicates that SA has the ability to prime defense by pre-inducing RNA silencing genes (Campos et al., 2014).

Cmv2b is a viral protein produced by the Cucumber mosaic virus that is required for systemic viral infection in cucumber plants, and is found to be involved in suppressing RNA silencing in tobacco. Ji et al. discovered that by deletingCmv2b, the systemic spread of the virus could be halted in older N. glutinosa plants, with very mild symptoms, and reduced viral accumulation appearing in younger plants. When they pre-treated their tobacco plants with SA, they observed a reduction of CMV RNAs in systemic leaves by up to 7-fold (Ji and Ding, 2001). ALTERNATIVE OXIDASE (Aox) is a gene involved in SA-mediated virus resistance that is rapidly induced by SA. Its induction is suppressed in systemic leaves after infection with WT CMV, but not CMV lacking the Cmv2b protein (Ji and Ding, 2001). This research seems to indicate that viral proteins target SA-mediated viral resistance by blocking induction of SA-induced defense genes.

Another recent study using Tomato Ringspot Virus (ToRSV) revealed that SA treatment can reduce the size of viral lesions found on infected tobacco leaves, and that this reduction is abolished inNahG expressing plants. They also found that, because SA can induce RDR1, SA treatment can restrict the systemic spread of the virus. This data indicates that SA is critical to the induction of antiviral plant defenses, which include RNA silencing, HR, and a moderate form of SAR (Jovel et al., 2011).

Regulation of autophagy by SA and NPR1

Autophagy is a degradation process, which delivers cytoplasmic ingredients to the vacuole or lysosome. The molecular mechanism for autophagy has been well studied and 18 autophagy-related (ATG) genes for autophagosome formation are identified in yeast (Saccharomyces cerevisiae). Arabidopsis possesses 30 autophagy-related genes, which correspond to 14 yeast ATG genes. Four out of 30 AtATG genes have not been characterized (Hanaoka et al., 2002). The remaining genes are found to be highly conserved in plant and animal kingdoms. In addition, autophagy activity is lost inArabidopsis ATG-deficient mutants, indicating that ATG genes encode proteins for plant autophagy. Inatg mutants, accelerated programmed cell death in senescence and immunity were found to be SA signaling dependent. When treatment of SA is performed, senescence and cell death occur in SA-deficientatg mutants; however, atg npr1 plants show normal growth. This research suggests that the early senescence phenotype is caused by an over-accumulation of ubiquitinated defense protein aggregates, causing stress to the ER, and disruption of cellular homeostasis. This disruption leads to the accumulation of SA, causing the over-accumulation of defense-related transcripts, which is NPR1-dependent. This phenomenon confirms that the cell death inatg mutants is dependent on NPR1 (Munch et al., 2014).

Missing links in current research

Although scientists are learning more about how NPR1 regulates salicylic acid-mediated plant defense, much is still unknown. The point of cross-talk between MTI and ETI remains elusive (Tully et al., 2014). Current research suggests that NPR1 plays a role in MTI. Plants treated with low levels of SA exhibited an enhanced level of MAPK3 and MAPK6 activation when exposed to flg22, indicating that NPR1 is involved in the MTI pathway, and is required for an enhanced response to flg22 (Yi et al., 2015), but the feedback regulator of FLS2, which senses bacterial flagellin and the synthetic epitope flg22, is unknown. The precise role of effectors targeting proteins involved in SA biosynthesis or SA receptors remains largely unknown. Current research suggests that NPR1 and SA are involved in regulating programmed cell death during immune responses and senescence, but the mechanism of this regulation is unknown (Yoshimoto et al., 2009) .

Interestingly, the necrotrophic pathogen, Botrytis cinerea, fails to induce SAR (Govrin and Levine, 2002). This pathogen is non-specific, infecting over 200 species of plants, including many crops (Prins et al., 2000). Additional research is needed to understand how B. cinerea can infect the plant without inducing SAR, and why prior inoculation with P. syringea to induce SAR does not suppress lesion formation nor growth of B. cinerea (Govrin and Levine, 2002).

The role of SA in roots against soil-borne pathogens also remains elusive, but current research suggests that SA produced by non-pathogenic rhizobacteria aids in disease resistance. In a study ofPseudomonas aeruginosa 7NSK2, it was found that the SA produced by this bacterium was necessary to induce resistance to the previously mentioned necrotrophic bacterium Botrytis cinerea in bean (De Meyer, 1997).

Future research into NPR1’s role in SA-mediated plant defense may progress further down many avenues. One prospective avenue is the investigation of heterotrimeric G-proteins and their role in regulating plant defense. Heterotrimeric G-proteins have been found to interact with many different proteins in many different biological processes, including regulating cell wall modification and saline stress in plants (Klopffleisch et al., 2011; Colaneri et al., 2014). G-proteins have also been implicated in transducing transmembrane signals from receptor-like kinases (Ishikawa, 2009; Liu et al.,2013). G-protein mutants show impaired resistance to various pathogenic microbes, and reduced sensitivity to various exogenous MAMPs (Maeda et al., 2009; Zeng and He, 2010; Lee et al., 2013; Liu et al., 2013; Lorek et al., 2013; Torres et al., 2013). Further research is required to discover if NPR1 also plays a role in G-protein MAMP signaling or if these G-proteins are involved in SA-mediated plant defense.

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