Chloroplast Activity and 3′phosphadenosine 5′phosphate Signaling Regulate Programmed Cell Death in Arabidopsis

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3′-phosphoadenosine 5′-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5′-3′ exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.Programmed cell death (PCD) is a universal process in multicellular organisms, contributing to the controlled and active degradation of the cell. In plants, PCD is required for processes as diverse as development, self-incompatibility, and stress response. One well-documented example is the induction of PCD upon pathogen attack, allowing the confinement of the infection, and resistance of the plant. The signaling events leading to the onset of PCD have been extensively studied: pathogen recognition triggers activation of mitogen-activated protein kinase cascades, as well as production of reactive oxygen species (ROS) and salicylic acid (SA), which lead to a hypersensitive response (Coll et al., 2011).From a cellular point of view, several classes of plant PCD have been described and compared with the ones found in animal cells (van Doorn, 2011). PCD is thought to have evolved independently in plants and animals, and genes underlying these mechanisms are therefore poorly conserved between the two kingdoms. However, most cellular features are conserved between plant and animal PCD that are both characterized by cell shrinkage, chromatin condensation, DNA laddering, mitochondria permeabilization, and depolarization (Dickman and Fluhr, 2013). In animal cells, mitochondria play a central role in the regulation of apoptosis (Czabotar et al., 2014; Mariño et al., 2014), and this role is likely shared between the two kingdoms (Lord and Gunawardena, 2012). That said, additional mitochondria-independent PCD pathways have clearly evolved in plants.Genetic approaches have greatly contributed to our understanding of cellular pathways governing PCD in plants. For example, the isolation of lesion mimic mutants (LMMs), in which cell death occurs spontaneously, has allowed the identification of several negative regulators of cell death (for review, see Bruggeman et al., 2015b). Interestingly, lesion formation is light dependent in several of these mutants, which include one of the best characterized LMMs—lesions simulating disease1 (lsd1; Dietrich et al., 1994). The LSD1 protein is required for plant acclimation to excess excitation energy (Mateo et al., 2004): when plants are exposed to excessive amounts of light, the redox status of the plastoquinone pool in the chloroplastic electron transfer chain is thought to influence LSD1-dependent signaling to modulate cell death (Mühlenbock et al., 2008). Additionally, we have previously identified the myoinositol phosphate synthase1 (mips1) mutant as a LMM, in which lesion formation is also light dependent (Meng et al., 2009). This mutant is deficient in the myoinositol (MI) phosphate synthase that catalyzes the first committed step of MI biosynthesis and displays pleiotropic defects such as reduced root growth, abnormal vein development, and spontaneous cell death on leaves, together with severe growth reduction after lesions begin to develop (Meng et al., 2009; Donahue et al., 2010). The light-dependent PCD in the mips1 mutant, as observed for lsd1, suggests that chloroplasts may play a role in the MI-dependent cell death regulation. Accumulating evidence suggests that chloroplasts may play a central role in PCD regulation like mitochondria in animal cells (Wang and Bayles, 2013). First, as described in the case of lsd1, excess light energy received by the chloroplast can function as a trigger for PCD. Furthermore, singlet oxygen (¹O₂), a ROS, can activate the EXECUTER1 (EX1) and EX2 proteins in the chloroplasts to initiate PCD (Lee et al., 2007). Likewise, ROS generated by chloroplasts play a major role for PCD onset during nonhost interaction between tobacco (Nicotiana tabacum) and Xanthomonas campestris (Zurbriggen et al., 2009). Finally, functional chloroplasts have also been shown to be required for PCD in cell suspensions (Gutierrez et al., 2014) and in a number of LMMs (Mateo et al., 2004; Meng et al., 2009; Bruggeman et al., 2015b). Thus, chloroplasts are now recognized as important components of plant defense response against pathogens (Stael et al., 2015) and are proposed to function with mitochondria in the execution of PCD (Van Aken and Van Breusegem, 2015). However, the exact signaling and metabolic contribution of chloroplasts to PCD remain to be elucidated. Furthermore, cross talk between chloroplasts and mitochondria does occur, such as during photorespiration (Sunil et al., 2013), but whether such communication functions sequentially or in parallel in the control of PCD remains to be determined (Van Aken and Van Breusegem, 2015).To further investigate how chloroplasts contribute to the regulation of cell death, we performed both forward and reverse genetics on the mips1 mutant. An extragenic secondary mutation in divinyl protochlorophyllide 8-vinyl reductase involved in chlorophyll biosynthesis leads to chlorophyll deficiency that abolishes the mips1 cell death phenotype, as do changes in CO₂ availability. These findings provide evidence for a link between photosynthetic activity and PCD induction in mips1. Additionally, we investigated the contribution of several retrograde signaling pathways (Chan et al., 2015) to the control of PCD in mips1. This process was independent of GENOMES UNCOUPLED (GUN) and EX signaling pathways, but we found that the SAL1-PAP_XRN retrograde signaling pathway inhibits cell death as well as basal defense reactions in Arabidopsis (Arabidopsis thaliana).     

The Rab GTPase RabG3b Positively Regulates Autophagy and Immunity-Associated Hypersensitive Cell Death in Arabidopsis

A central component of the plant defense response to pathogens is the hypersensitive response (HR), a form of programmed cell death (PCD). Rapid and localized induction of HR PCD ensures that pathogen invasion is prevented. Autophagy has been implicated in the regulation of HR cell death, but the functional relationship between autophagy and HR PCD and the regulation of these processes during the plant immune response remain controversial. Here, we show that a small GTP-binding protein, RabG3b, plays a positive role in autophagy and promotes HR cell death in response to avirulent bacterial pathogens in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing a constitutively active RabG3b (RabG3bCA) displayed accelerated, unrestricted HR PCD within 1 d of infection, in contrast to the autophagy-defective atg5-1 mutant, which gradually developed chlorotic cell death through uninfected sites over several days. Microscopic analyses showed the accumulation of autophagic structures during HR cell death in RabG3bCA cells. Our results suggest that RabG3b contributes to HR cell death via the activation of autophagy, which plays a positive role in plant immunity-triggered HR PCD.In response to the constant attack by microbial pathogens, plants have developed defense mechanisms to protect themselves against harmful diseases caused by various pathogens. Plants primarily rely on two layers of innate immunity to cope with microbial pathogens (Jones and Dangl, 2006). The first layer of plant immunity, which is triggered by pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin, lipopolysaccharides, and fungal chitin, is designated PAMP-triggered immunity (PTI; Boller and He, 2009). Because pathogens have evolved to overcome PTI, plants have developed a second layer of immunity, referred to as effector-triggered immunity (ETI; Dodds and Rathjen, 2010). ETI depends on specific interactions between plant Resistance proteins and pathogen effectors and is often associated with a form of programmed cell death (PCD) termed the hypersensitive response (HR), which inhibits pathogen growth (Coll et al., 2011).Plants use PCD to regulate developmental and defense responses. In addition to pathogen attack, many abiotic stress factors such as heat and ozone exposure elicit PCD in plants (Hayward and Dinesh-Kumar, 2011). PCD also occurs during various developmental processes, including endosperm development, tracheary element (TE) differentiation, female gametophyte differentiation, leaf abscission, and senescence (Kuriyama and Fukuda, 2002; Gunawardena, 2008). Recently, plant PCD has been classified into two types, “autolytic” PCD and “nonautolytic” PCD, on the basis of the presence or absence of rapid cytoplasm clearance after tonoplast rupture, respectively (van Doorn et al., 2011). Autolytic PCD, which mainly occurs during plant development, falls under “autophagic” PCD in animals because it is associated with the accumulation of autophagy-related structures in the cytoplasm. Some forms of HR PCD classified as nonautolytic PCD in plants are accompanied by increased vacuolization, indicating the progress of autophagy, and therefore can be placed under autophagic PCD (Hara-Nishimura et al., 2005; Hatsugai et al., 2009).Autophagy is an intracellular process in which double membrane-bound autophagosomes enclose cytoplasmic components and damaged or toxic materials and target them to the vacuole or lysosome for degradation (Chung, 2011). In plants, autophagy plays important roles in the responses to nutrient starvation, senescence, and abiotic and biotic stresses (Liu et al., 2005; Xiong et al., 2005, 2007; Bassham, 2007; Hofius et al., 2009). Accumulating evidence indicates that autophagy regulates immune responses in both animals and plants. Autophagy is essential for the direct elimination of pathogens in mammalian systems (Levine et al., 2011). Invading bacteria and viruses are targeted to autophagosomes and then delivered to the lysosome for degradation in a process called xenophagy (Levine, 2005). In addition to its function in directly killing pathogens, xenophagic degradation can provide microbial antigens for major histocompatibility complex class II presentation to the innate and adaptive immune systems (Levine, 2005; Schmid and Münz, 2007). Furthermore, the human surface receptor CD46 was shown to directly induce autophagy through physical interaction with the autophagic machinery (Joubert et al., 2009). The role of autophagy in plant basal immunity to virulent pathogens has been determined (Patel and Dinesh-Kumar, 2008; Hofius et al., 2009; Lai et al., 2011; Lenz et al., 2011). Arabidopsis (Arabidopsis thaliana) plants defective in AUTOPHAGY-RELATED (ATG) genes exhibited enhanced susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola, suggesting that the massive breakdown of cytoplasmic materials provides nutrients for the growth of necrotrophic pathogens or that fungal toxin-induced necrotic cell death is enhanced in atg mutants (Lai et al., 2011; Lenz et al., 2011). However, studies on the responses to the biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000) have yielded contradictory results. Whereas earlier studies reported that bacterial numbers significantly increased in ATG6-antisense (AS) and atg mutant plants (Patel and Dinesh-Kumar, 2008; Hofius et al., 2009), a recent study indicated that atg mutants exhibit increased resistance to Pst DC3000 (Lenz et al., 2011). Although these discrepancies remain to be resolved, salicylic acid (SA) levels and SA-dependent gene expression were both elevated in atg mutants, suggesting that autophagy may negatively regulate SA-associated plant immunity (Yoshimoto et al., 2009; Lenz et al., 2011). These findings indicate that the role of autophagy in plant immunity depends on the lifestyle of the invading pathogens (Lenz et al., 2011).Autophagy plays an important role in the regulation of HR PCD in plant innate immunity (Hayward and Dinesh-Kumar, 2011). Tobacco (Nicotiana tabacum) plants silenced for ATG6/Beclin1 and other ATG genes such as phosphatidylinositol 3-kinase (PI3K)/vacuolar protein sorting34 (VPS34), ATG3, and ATG7 underwent unrestricted HR PCD upon pathogen infection (Liu et al., 2005). ATG6-AS and atg5 mutant Arabidopsis plants also displayed unlimited HR PCD upon infection with the avirulent bacterium Pst DC3000 (AvrRpm1; Patel and Dinesh-Kumar, 2008; Yoshimoto et al., 2009). These studies suggest that autophagy is a “prosurvival” or “antideath” mechanism that negatively regulates HR PCD (Liu and Bassham, 2012). By contrast, a “prodeath” role has been suggested for autophagy in HR PCD regulation (Hofius et al., 2009). Pst DC3000 (AvrRps4)-induced and, to a lesser extent, Pst DC3000 (AvrRpm1)-induced HR PCD was suppressed in atg mutants, suggesting that autophagy plays a positive role and that autophagic cell death is involved in RPS4- and RPM1-mediated HR cell death.We previously showed that the small GTP-binding protein RabG3b, isolated from secretome analysis in Arabidopsis (Oh et al., 2005), functions as a component of autophagy and positively regulates TE differentiation via the activation of autophagic cell death (Kwon et al., 2010a, 2010b). Overexpression of a constitutively active RabG3b (RabG3bCA) in plants significantly increased autophagy during PCD associated with TE differentiation, thereby enhancing TE formation and xylem development. Transgenic poplar (Populus alba × Populus tremula var glandulosa) overexpressing Arabidopsis RabG3bCA was further generated, and these exhibited significant stimulation of xylem development together with autophagic activation, suggesting that RabG3b is a positive regulator of autophagy and xylem development in Populus spp. as well as Arabidopsis (Kwon et al., 2011). We also reported that RabG3b is involved in cell death associated with the fungal pathogen A. brassicicola and infection with the fungal toxin fumonisin B1 (FB1) as well as leaf senescence (Kwon et al., 2009). Here, we extend our work to determine the role of RabG3b and autophagy in immunity-associated HR PCD. We found that RabG3bCA transgenic plants accumulated a large number of autophagic structures and displayed accelerated, expanded cell death against a number of PCD inducers, such as FB1 and the bacterial pathogens Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). Our results suggest that RabG3b plays a positive role in immunity-associated HR PCD via the activation of autophagic cell death.     

Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.Plants have evolved a complex array of defenses when attacked by microbial pathogens. The success of plant resistance first relies on the capacity of the plant to recognize its invader. Among early events, a transient production of reactive oxygen species (ROS), known as the oxidative burst, is characteristic of successful pathogen recognition (Torres, 2010). Perception of pathogen attack then initiates a large array of immune responses, including modification of cell walls, as well as the production of antimicrobial proteins and metabolites like pathogenesis-related (PR) proteins and phytoalexins, respectively (Schwessinger and Ronald, 2012). The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are key players in the signaling networks involved in plant resistance (Bari and Jones, 2009; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate array of defense responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). Whereas SA is considered essential for resistance to (hemi)biotrophic pathogens, it is assumed that JA and ET signaling pathways are important for resistance to necrotrophic pathogens in Arabidopsis (Arabidopsis thaliana; Thomma et al., 2001; Glazebrook, 2005). A successful innate immune response often includes the so-called hypersensitive response (HR), a form of rapid programmed cell death (PCD) occurring in a limited area at the site of infection. This suicide of infected cells is thought to limit the spread of biotrophic pathogens, including viruses, bacteria, fungi, and oomycetes (Mur et al., 2008).During the past decade, significant progress has been made in our understanding of the cellular function of plant sphingolipids. Besides being structural components of cell membranes, sphingolipids are bioactive metabolites that regulate important cellular processes such as cell survival and PCD, occurring during either plant development or plant defense (Dunn et al., 2004; Berkey et al., 2012; Markham et al., 2013). The first evidence of the role of sphingolipids in these processes came from the use of the fungal toxins fumonisin B1 (FB1) and AAL, produced by the necrotrophic agent Alternaria alternata f. sp. lycopersici. These toxins are structural sphingosine (d18:1) analogs and function as ceramide synthase inhibitors. They triggered PCD when exogenously applied to plants. Mutant strains in which the production of such toxins is abrogated failed to infect the host plant, implying that toxin accumulation is required for pathogenicity and that the induction of plant PCD could be considered a virulence tool used by necrotrophic pathogens (Berkey et al., 2012). Moreover, several studies revealed that ceramides (Cers) and long-chain bases (LCBs) are also potent inducers of PCD in plants. For example, exogenously applied Cers and LCBs (d18:0, d18:1, or t18:0) induced PCD either in cell suspension cultures (Liang et al., 2003; Lachaud et al., 2010, 2011; Alden et al., 2011) or in whole seedlings (Shi et al., 2007; Takahashi et al., 2009; Saucedo-García et al., 2011). AAL- and FB1-induced PCD seemed to be due to the accumulation of free sphingoid bases (dihydrosphingosine [d18:0] and phytosphingosine [t18:0]; Abbas et al., 1994; Brandwagt et al., 2000; Shi et al., 2007). Spontaneous cell death in lag one homolog1 or l-myoinositol1-phosphate synthase mutant could be due to trihydroxy-LCB and/or Cer accumulation (Donahue et al., 2010; Ternes et al., 2011). Deciphering of Cer participation in the induction of HR and associated PCD also came from studies on accelerated cell death5 (acd5) and enhancing resistance to powdery mildew8 (RPW8)-mediated hypersensitive response (erh1) mutants, which displayed overaccumulation of Cers. These mutants exhibited spontaneous cell death and resistance to biotrophic pathogens, which seemed to be linked with SA and PR protein accumulation (Liang et al., 2003; Wang et al., 2008).Altogether, these data provide evidence of a link between PCD, defense, and sphingolipid metabolism. However, the fatty acid hydroxylase1/2 (atfah1/atfah2) double mutant that accumulates SA and Cers was more tolerant to the obligate biotrophic fungus Golovinomyces cichoracearum but did not display a PCD-like phenotype, suggesting that Cers alone are not involved in the induction of PCD (König et al., 2012). Moreover, Saucedo-García et al. (2011) postulated that dihydroxy-LCBs, but not trihydroxy-LCBs, might be primary mediators for LCB-induced PCD. The sphingoid base hydroxylase sbh1/sbh2 double mutant completely lacking trihydroxy-LCBs showed enhanced expression of PCD marker genes (Chen et al., 2008). On the contrary, increase in t18:0 was specifically sustained in plant interaction with the avirulent Pseudomonas syringae pv tomato (Pst) strain and correlated with a strong PCD induction in leaves (Peer et al., 2010). Thus, the nature of sphingolipids able to induce PCD is still under debate and may evolve depending on plants and their environment. The phosphorylated form of LCBs (LCB-Ps) could abrogate PCD induced by LCBs, Cers, or heat stress in a dose-dependent manner (Shi et al., 2007; Alden et al., 2011). Furthermore, blocking the conversion of LCBs to LCB-Ps by using specific inhibitors induced PCD in cell suspension culture (Alden et al., 2011). Recently, overexpression of rice (Oryza sativa) LCB kinase in transgenic tobacco (Nicotiana tabacum) plants reduced PCD after treatment with FB1 (Zhang et al., 2013). Genetic mutation on LCB-P lyase encoded by the AtDPL1 gene, modifying the LCB-LCB-P ratio, could impact PCD levels after treatment with FB1 (Tsegaye et al., 2007). Altogether, these data point to the existence of a rheostat between LCBs and their phosphorylated forms that controls plant cell fate toward cell death or survival.Data on plant sphingolipid functions are still fragmentary. Only a few reports have described interconnections between sphingolipids, cell death, and plant defense responses, almost exclusively in response to (hemi)biotrophic pathogens. Knowledge about such relations in response to necrotrophic pathogens is still in its infancy (Rivas-San Vicente et al., 2013; Bi et al., 2014). In this report, the link between sphingolipids, cell death, and plant defense has been explored in response to Botrytis cinerea infection and in comparison with Pst infection. For this purpose, Atdpl1 mutant plants, disturbed in LCB/LCB-P accumulation without displaying any phenotype under standard growth conditions (Tsegaye et al., 2007), have been analyzed after pathogen infection. Our results revealed that modification of sphingolipid contents not only impacted plant tolerance to hemibiotrophs but also greatly affected resistance to necrotrophs. Whereas the SA signaling pathway is globally repressed in Atdpl1-1 compared with wild-type plants, the JA signaling pathway is significantly enhanced. Cell death and ROS accumulation are markedly modified in Atdpl1-1 mutant plants. We further demonstrated that phytosphingosine-1-phosphate (t18:0-P) and d18:0 are key players in pathogen-induced cell death and ROS generation. Here, we thus established a link between JA signaling, PCD, and sphingolipid metabolism.     

Non-Recognition-of-BTH4, an Arabidopsis Mediator Subunit Homolog,Is Necessary for Development and Response to Salicylic Acid

Salicylic acid (SA) signaling acts in defense and plant development. The only gene demonstrated to be required for the response to SA is Arabidopsis thaliana NON-EXPRESSER OF PATHOGENESIS-RELATED GENE 1 (NPR1), and npr1 mutants are insensitive to SA. By focusing on the effect of analogs of SA on plant development, we identified mutants in additional genes acting in the SA response. In this work, we describe a gene necessary for the SA Non-Recognition-of-BTH4 (NRB4). Three nrb4 alleles recovered from the screen cause phenotypes similar to the wild type in the tested conditions, except for SA-related phenotypes. Plants with NRB4 null alleles express profound insensitivity to SA, even more than npr1. NRB4 null mutants are also sterile and their growth is compromised. Plants carrying weaker nrb4 alleles are also insensitive to SA, with some quantitative differences in some phenotypes, like systemic acquired resistance or pathogen growth restriction. When weak alleles are used, NPR1 and NRB4 mutations produce an additive phenotype, but we did not find evidence of a genetic interaction in F1 nor biochemical interaction in yeast or in planta. NRB4 is predicted to be a subunit of Mediator, the ortholog of MED15 in Arabidopsis. Mechanistically, NRB4 functions downstream of NPR1 to regulate the SA response.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.     

Novel Plant Immune-Priming Compounds Identified via High-Throughput Chemical Screening Target Salicylic Acid Glucosyltransferases in Arabidopsis

Plant activators are compounds, such as analogs of the defense hormone salicylic acid (SA), that protect plants from pathogens by activating the plant immune system. Although some plant activators have been widely used in agriculture, the molecular mechanisms of immune induction are largely unknown. Using a newly established high-throughput screening procedure that screens for compounds that specifically potentiate pathogen-activated cell death in Arabidopsis thaliana cultured suspension cells, we identified five compounds that prime the immune response. These compounds enhanced disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants. Pretreatments increased the accumulation of endogenous SA, but reduced its metabolite, SA-O-β-d-glucoside. Inducing compounds inhibited two SA glucosyltransferases (SAGTs) in vitro. Double knockout plants that lack both SAGTs consistently exhibited enhanced disease resistance. Our results demonstrate that manipulation of the active free SA pool via SA-inactivating enzymes can be a useful strategy for fortifying plant disease resistance and may identify useful crop protectants.     

Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

Plant resistance to phytopathogenic microorganisms mainly relies on the activation of an innate immune response usually launched after recognition by the plant cells of microbe-associated molecular patterns. The plant hormones, salicylic acid (SA), jasmonic acid, and ethylene have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids (RLs) are glycolipids produced by bacteria and are involved in surface motility and biofilm development. Here we report that RLs trigger an immune response in Arabidopsis (Arabidopsis thaliana) characterized by signaling molecules accumulation and defense gene activation. This immune response participates to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora arabidopsidis, and the necrotrophic fungus Botrytis cinerea. We show that RL-mediated resistance involves different signaling pathways that depend on the type of pathogen. Ethylene is involved in RL-induced resistance to H. arabidopsidis and to P. syringae pv tomato whereas jasmonic acid is essential for the resistance to B. cinerea. SA participates to the restriction of all pathogens. We also show evidence that SA-dependent plant defenses are potentiated by RLs following challenge by B. cinerea or P. syringae pv tomato. These results highlight a central role for SA in RL-mediated resistance. In addition to the activation of plant defense responses, antimicrobial properties of RLs are thought to participate in the protection against the fungus and the oomycete. Our data highlight the intricate mechanisms involved in plant protection triggered by a new type of molecule that can be perceived by plant cells and that can also act directly onto pathogens.In their environment, plants are challenged by potentially pathogenic microorganisms. In response, they express a set of defense mechanisms including preformed structural and chemical barriers, as well as an innate immune response quickly activated after microorganism perception (Boller and Felix, 2009). Plant innate immunity is triggered after recognition by pattern recognition receptors of conserved pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively) or by plant endogenous molecules released by pathogen invasion and called danger-associated molecular patterns (Boller and Felix, 2009; Dodds and Rathjen, 2010). This first step of recognition leads to the activation of MAMP-triggered immunity (MTI). Successful pathogens can secrete effectors that interfere or suppress MTI, resulting in effector-triggered susceptibility. A second level of perception involves the direct or indirect recognition by specific receptors of pathogen effectors leading to effector-triggered immunity (ETI; Boller and Felix, 2009; Dodds and Rathjen, 2010). Whereas MTI and ETI are thought to involve common signaling network, ETI is usually quantitatively stronger than MTI and associated with more sustained and robust immune responses (Katagiri and Tsuda, 2010; Tsuda and Katagiri, 2010).The plant hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) have emerged as key players in the signaling networks involved in MTI and ETI (Robert-Seilaniantz et al., 2007; Tsuda et al., 2009; Katagiri and Tsuda, 2010; Mersmann et al., 2010; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate spectrum of responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). It is assumed that JA and ET signaling pathways are important for resistance to necrotrophic fungi including Botrytis cinerea and Alternaria brassicicola (Thomma et al., 2001; Ferrari et al., 2003; Glazebrook, 2005). Infection of Arabidopsis (Arabidopsis thaliana) with B. cinerea causes the induction of the JA/ET responsive gene PLANT DEFENSIN1.2 (PDF1.2; Penninckx et al., 1996; Zimmerli et al., 2001). Induction of PDF1.2 by B. cinerea is blocked in ethylene-insensitive2 (ein2) and coronatine-insensitive1 (coi1) mutants that are respectively defective in ET and JA signal transduction pathways. Moreover, ein2 and coi1 plants are highly susceptible to B. cinerea infection (Thomma et al., 1998; Thomma et al., 1999). JA/ET-dependent responses do not seem to be usually induced during resistance to biotrophs, but they can be effective if they are stimulated prior to pathogen challenge (Glazebrook, 2005). Plants impaired in SA signaling are highly susceptible to biotrophic and hemibiotrophic pathogens. Following pathogen infection, SA hydroxylase (NahG), enhanced disease susceptibility5 (eds5), or SA induction-deficient2 (sid2) plants are unable to accumulate high SA levels and they display heightened susceptibility to Pseudomonas syringae pv tomato (Pst), Hyaloperonospora arabidopsidis, or Erysiphe orontii (Delaney et al., 1994; Lawton et al., 1995; Wildermuth et al., 2001; Nawrath et al., 2002; Vlot et al., 2009). Mutants that are insensitive to SA, such as nonexpressor of PATHOGENESIS-RELATED (PR) genes1 (npr1), have enhanced susceptibility to these pathogens (Cao et al., 1994; Glazebrook et al., 1996; Shah et al., 1997; Dong, 2004). According to some reports, plant defense against necrotrophs also involves SA. Arabidopsis plants expressing the nahG gene and infected with B. cinerea show larger lesions compared with wild-type plants (Govrin and Levine, 2002). In tobacco (Nicotiana tabacum), acidic isoforms of PR3 and PR5 gene that are specifically induced by SA (Ménard et al., 2004) are up-regulated after challenge by B. cinerea (El Oirdi et al., 2010). Resistance to some necrotrophs like Fusarium graminearum involves both SA and JA signaling pathways (Makandar et al., 2010). It is assumed that SA and JA signaling can be antagonistic (Bostock, 2005; Koornneef and Pieterse, 2008; Pieterse et al., 2009; Thaler et al., 2012). In Arabidopsis, SA inhibits JA-dependent resistance against A. brassicicola or B. cinerea (Spoel et al., 2007; Koornneef et al., 2008). Recent studies demonstrated that ET modulates the NPR1-mediated antagonism between SA and JA (Leon-Reyes et al., 2009; Leon-Reyes et al., 2010a) and suppression by SA of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway (Leon-Reyes et al., 2010b). Synergistic effects of SA- and JA-dependent signaling are also well documented (Schenk et al., 2000; van Wees et al., 2000; Mur et al., 2006) and induction of some defense responses after pathogen challenge requires intact JA, ET, and SA signaling pathways (Campbell et al., 2003).Isolated MAMPs trigger defense responses that also require the activation of SA, JA, and ET signaling pathways (Tsuda et al., 2009; Katagiri and Tsuda, 2010). For instance, treatment with the flagellin peptide flg22 induces many SA-related genes including SID2, EDS5, NPR1, and PR1 (Ferrari et al., 2007; Denoux et al., 2008), causes SA accumulation (Tsuda et al., 2008; Wang et al., 2009), and activates ET signaling (Bethke et al., 2009; Mersmann et al., 2010). Local application of lipopolysaccharides elevates the level of SA (Mishina and Zeier, 2007). The oomycete Pep13 peptide induces defense responses in potato (Solanum tuberosum) that require both SA and JA (Halim et al., 2009). Although signaling networks induced by isolated MAMPs are well documented, the contribution of SA, JA, and ET in MAMP- or PAMP-induced resistance to biotrophs and necrotrophs is poorly understood.Rhamnolipids (RLs) are glycolipids produced by various bacteria species including some Pseudomonas and Burkholderia species. They are essential for bacterial surface motility and biofilm development (Vatsa et al., 2010; Chrzanowski et al., 2012). RLs are potent stimulators of animal immunity (Vatsa et al., 2010). They have recently been shown to elicit plant defense responses and to induce resistance against B. cinerea in grapevine (Vitis vinifera; Varnier et al., 2009). They also participate to biocontrol activity of the plant beneficial bacteria Pseudomonas aeruginosa PNA1 against oomycetes (Perneel et al., 2008). However, the signaling pathways used by RLs to stimulate plant innate immunity are not known. To gain more insights into RL-induced MTI, we investigated RL-triggered defense responses and resistance to the necrotrophic fungus B. cinerea, the biotroph oomycete H. arabidopsidis, and the hemibiotroph bacterium Pst in Arabidopsis. Our results show that RLs trigger an innate immune response in Arabidopsis that protects the plant against these different lifestyle pathogens. We demonstrate that RL-mediated resistance involves separated signaling sectors that depend on the type of pathogen. In plants challenged by RLs, SA has a central role and participates to the restriction of the three pathogens. ET is fully involved in RL-induced resistance to the biotrophic oomycete and to the hemibiotrophic bacterium whereas JA is essential for the resistance to the necrotrophic fungus.