Salmonella enterica Flagellin Is Recognized via FLS2 and Activates PAMP-Triggered Immunity in Arabidopsis thaliana

Infections with Salmonella enterica belong to the most prominent causes of food poisoning and infected fruits and vegetables represent important vectors for salmonellosis. Recent evidence indicates that plants recognize S. enterica and raise defense responses. Nonetheless, the molecular mechanisms controlling the interaction of S. enterica with plants are still largely unclear. Here, we show that flagellin from S. enterica represents a prominent pathogenassociated molecular pattern （PAMP） in Arabidopsis thaliana, which induces PAMP-triggered immunity （PTI） via the recognition of the fig22 domain by the receptor kinase FLS2. The Arabidopsis fls2 mutant shows reduced though not abolished PTI activation, indicating that plants rely also on recognition of other S. enterica PAMPs. Interestingly, the S. enterica type III secretion system （T3SS） mutant prgH- induced stronger defense gene expression than wild-type bacteria in Arabidopsis, suggesting that T3SS effectors are involved in defense suppression. Furthermore, we observe that S. enterica strains show variation in the fig22 epitope, which results in proteins with reduced PTI-inducing activity. Altogether, these results show that S. enterica activates PTI in Arabidopsis and suggest that, in order to accomplish plant colonization, S. enterica evolved strategies to avoid or suppress PTI.     

FLS2-BAK1 Extracellular Domain Interaction Sites Required for Defense Signaling Activation

Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.     

ESCRT-I Mediates FLS2 Endosomal Sorting and Plant Immunity

The plant immune receptor FLAGELLIN SENSING 2 (FLS2) is present at the plasma membrane and is internalized following activation of its ligand flagellin (flg22). We show that ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT)-I subunits play roles in FLS2 endocytosis in Arabidopsis. VPS37-1 co-localizes with FLS2 at endosomes and immunoprecipitates with the receptor upon flg22 elicitation. Vps37-1 mutants are reduced in flg22-induced FLS2 endosomes but not in endosomes labeled by Rab5 GTPases suggesting a defect in FLS2 trafficking rather than formation of endosomes. FLS2 localizes to the lumen of multivesicular bodies, but this is altered in vps37-1 mutants indicating compromised endosomal sorting of FLS2 by ESCRT-I loss-of-function. VPS37-1 and VPS28-2 are critical for immunity against bacterial infection through a role in stomatal closure. Our findings identify that VPS37-1, and likewise VPS28-2, regulate late FLS2 endosomal sorting and reveals that ESCRT-I is critical for flg22-activated stomatal defenses involved in plant immunity.     

1O2-Mediated and EXECUTER-Dependent Retrograde Plastid-to-Nucleus Signaling in Norflurazon-Treated Seedlings of Arabidopsis thaliana

Chloroplast development depends on the synthesis and import of a large number of nuclear-encoded pro- teins. The synthesis of some of these proteins is affected by the functional state of the plastid via a process known as retrograde signaling. Retrograde plastid-to-nucleus signaling has been often characterized in seedlings of Arabidopsis thaliana exposed to norflurazon （NF）, an inhibitor of carotenoid biosynthesis. Results of this work suggested that, throughout seedling development, a factor is released from the plastid to the cytoplasm that indicates a perturbation of plastid homeostasis and represses nuclear genes required for normal chloroplast development. The identity of this factor is still under debate. Reactive oxygen species （ROS） were among the candidates discussed as possible retrograde signals in NF-treated plants. In the present work, this proposed role of ROS has been analyzed. In seedlings grown from the very beginning in the presence of NF, ROS-dependent signaling was not detectable, whereas, in seedlings first exposed to NF after light-dependent chloroplast formation had been completed, enhanced ROS production occurred and, among oth- ers, 1O2-mediated and EXECUTER-dependent retrograde signaling was induced. Hence, depending on the developmental stage at which plants are exposed to NF, different retrograde signaling pathways may be activated, some of which are also active in non-treated plants under light stress.     

Ethylene Signaling Regulates Accumulation of the FLS2 Receptor and Is Required for the Oxidative Burst Contributing to Plant Immunity

Reactive oxygen species (ROS) are potent signal molecules rapidly generated in response to stress. Detection of pathogen-associated molecular patterns induces a transient apoplastic ROS through the function of the NADPH respiratory burst oxidase homologs D (RbohD). However, little is known about the regulation of pathogen-associated molecular pattern-elicited ROS or its role in plant immunity. We investigated ROS production triggered by bacterial flagellin (flg22) in Arabidopsis (Arabidopsis thaliana). The oxidative burst was diminished in ethylene-insensitive mutants. Flagellin Sensitive2 (FLS2) accumulation was reduced in etr1 and ein2, indicating a requirement of ethylene signaling for FLS2 expression. Multiplication of virulent bacteria was enhanced in Arabidopsis lines displaying altered ROS production at early but not late stages of infection, suggesting an impairment of preinvasive immunity. Stomatal closure, a mechanism used to reduce bacterial entry into plant tissues, was abolished in etr1, ein2, and rbohD mutants. These results point to the importance of flg22-triggered ROS at an early stage of the plant immune response.A rapid and transient increase in reactive oxygen species (ROS), termed an “oxidative burst,” is often associated with responses to abiotic and biotic stresses and could trigger changes in stomatal aperture or programmed cell death in defense against pathogens (Kwak et al., 2003; Torres and Dangl, 2005). ROS production can occur extracellularly through activities of plasma membrane-resident NADPH oxidases (Kangasjärvi et al., 2005; Torres and Dangl, 2005). In plants, Rboh proteins, which are homologs of mammalian NADPH oxidase 2, were shown to be the predominant mediators of apoplastic ROS production (Torres et al., 1998; Galletti et al., 2008). Respiratory burst oxidase homologs D and F (RbohD and RbohF) were identified by mutation to be the responsible oxidases in Arabidopsis (Arabidopsis thaliana) defense responses (Torres et al., 2002). While most ROS generated in response to avirulent Pseudomonas syringae bacteria and Hyaloperonospora oomycete pathogens depend on RbohD function, the induced cell death response by these pathogens appears to be mostly regulated by RbohF. Cell death provoked upon infection with the necrotizing fungus Alternaria, however, is under the control of RbohD (Pogány et al., 2009). The contribution of NADPH oxidases to plant immunity was also described in barley (Hordeum vulgare) and tobacco (Nicotiana benthamiana), where resistance to powdery mildew fungi and the oomycete Phytophthora infestans, respectively, was dependent on Rboh functions (Yoshioka et al., 2003; Trujillo et al., 2006).An early layer of active plant defense is mediated by pattern recognition receptors, which sense microbes according to conserved constituents, so-called pathogen-associated molecular patterns (PAMPs). These initiate a plethora of defense responses referred to as PAMP-triggered immunity (Boller and Felix, 2009). The Arabidopsis receptor kinase Flagellin Sensitive2 (FLS2) recognizes and physically interacts with flg22, the elicitor-active epitope of bacterial flagellin (Felix et al., 1999; Gomez-Gomez and Boller, 2000; Chinchilla et al., 2006). FLS2 is plasma membrane localized and expressed throughout the plant (Robatzek et al., 2006). FLS2 requires the receptor kinase BRI1-Associated Kinase1 (BAK1), which forms a heteromeric complex upon flg22 binding (Chinchilla et al., 2007). Subsequently, a rapid and transient flg22-stimulated oxidative burst occurs that is dependent on RbohD (Zhang et al., 2007). In addition, flg22 triggers early responses, such as ethylene biosynthesis, activation of mitogen-activated protein (MAP) kinase cascades, and changes in gene expression (Felix et al., 1999; Asai et al., 2002; Zipfel et al., 2004). Late flg22 responses include the accumulation of salicylic acid (SA), callose deposition, and an arrest of seedling growth (Gomez-Gomez et al., 1999; Mischina and Zeier, 2007). This collectively contributes to plant immunity (Zipfel et al., 2004; Melotto et al., 2006).Little is known about the regulatory components of FLS2-activated early flg22 responses and their relevance in plant resistance to pathogens. Here, we investigated flg22-triggered ROS production in Arabidopsis seedlings and have identified ethylene signaling as a critical component of the oxidative burst in response to flg22, partly through promoting the accumulation of FLS2. We further provide evidence that the flg22-triggered oxidative burst is required for resistance to bacterial infection at the point of pathogen entry through stomata.     

Both the Extracellular Leucine-Rich Repeat Domain and the Kinase Activity of FLS2 Are Required for Flagellin Binding and Signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

Honokiol Inhibits Non-Small Cell Lung Cancer Cell Migration by Targeting PGE2-Mediated Activation of β-Catenin Signaling

Lung cancer remains a leading cause of death due to its metastasis to distant organs. We have examined the effect of honokiol, a bioactive constituent from the Magnolia plant, on human non-small cell lung cancer (NSCLC) cell migration and the molecular mechanisms underlying this effect. Using an in vitro cell migration assay, we found that treatment of A549, H1299, H460 and H226 NSCLC cells with honokiol resulted in inhibition of migration of these cells in a dose-dependent manner, which was associated with a reduction in the levels of cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂). Celecoxib, a COX-2 inhibitor, also inhibited cell migration. Honokiol inhibited PGE₂-enhanced migration of NSCLC cells, inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A549 and H1299 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited migration of NSCLC cells. PGE₂ has been shown to activate β-catenin signaling, which contributes to cancer cell migration. Therefore, we checked the effect of honokiol on β-catenin signaling. It was observed that treatment of NSCLC cells with honokiol degraded cytosolic β-catenin, reduced nuclear accumulation of β-catenin and down-regulated matrix metalloproteinase (MMP)-2 and MMP-9, which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. Honokiol enhanced: (i) the levels of casein kinase-1α, glycogen synthase kinase-3β, and (ii) phosphorylation of β-catenin on critical residues Ser⁴⁵, Ser^33/37 and Thr⁴¹. These events play important roles in degradation or inactivation of β-catenin. Treatment of celecoxib also reduced nuclear accumulation of β-catenin in NSCLC cells. FH535, an inhibitor of Wnt/β-catenin pathway, inhibited PGE₂-enhanced cell migration of A549 and H1299 cells. These results indicate that honokiol inhibits non-small cell lung cancer cells migration by targeting PGE₂-mediated activation of β-catenin signaling.     

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling

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Chimeric receptors of the Arabidopsis thaliana pattern recognition receptors EFR and FLS2

FLS2 and EFR are pattern recognition receptors in Arabidopsis thaliana perceiving the bacterial proteins flagellin and Elongation factor Tu (EF-Tu). Both receptors belong to the >200 membered protein family of Leucine-Rich Repeat Receptor Kinases (LRR-RKs) in Arabidopsis. FLS2 and EFR are engaged in the activation of a common intracellular signal output and they belong to the same subfamily of LRR-RKs, sharing structural features like the intracellular kinase domain and the ectodomain organized in LRRs. On the amino acid sequence level, however, they are only <50% identical even in their kinase domains. In our recently published paper¹ we demonstrated that it is possible to create chimeric receptors of EFR and FLS2 that are fully functional in ligand binding and receptor activation. Chimeric receptors consisting of the complete EFR ectodomain and the FLS2 kinase domain proved to be sensitive to elf18, the minimal peptide required for EF-Tu recognition, similar to the native EFR. In chimeric receptors where parts of the FLS2 ectodomain were swapped into the EFR LRR-domain, the receptor function was strongly affected even in cases with only small fragments exchanged. In this addendum we want to address problems and limits but also possibilities and chances of studying receptor functions using a chimeric approach.Key words: pattern recognition receptors, chimeric receptors, MAMP, flagellin perception, FLS2, EFRIn the Arabidopsis genome exist >600 genes that are predicted to encode for receptor-like kinases (RLKs).^2,3 More than 200 of them have ectodomains with LRRs. Physiological functions have been attributed only to a rather small percentage of them. Examples for known receptor-ligand pairs in A. thaliana include the well studied BRI1/Brassionlide,^4,5 AtPEPR1/Pep25,⁶ HAESA/IDA⁷ or CLV1/CLV3.⁸ While these LRR-RKs detect endogenous ligands, other members of this family function as immunoreceptors that detect ligands indicative of ‘non-self,’ such as pathogen associated molecular patterns (PAMPs). Examples of such LRR-RKs include FLS2 (Flagellin Sensing 2) and EFR (EF-Tu Receptor) from Arabidopsis and XA21 from rice.^9–11 The corresponding ligands have been identified as the flg22-epitope of bacterial flagellin for FLS2, the N-terminus of bacterial EF-Tu represented by the elf18 peptide for EFR, and the sulfated Avr21 peptide from Xanthomonas for XA21, respectively. LRR-ectodomains with related function in pathogen recognition occur also in so-called receptor-like proteins that lack the cytoplasmic kinase domains. Well studied examples include several Cf-receptor proteins which confer resistance against the fungus Cladosporium fulvum (Cf) in a gene-for-gene dependent manner. Thereby, different Cf-proteins function as recognition systems with specificity for factors determined by corresponding AvrCf products of the fungal pathogen.^12,13Receptor activation of the well studied receptor BRI1 by its ligand brassinolide involves interaction with a further receptor kinase, BAK1 (BRI1-associated receptor kinase 1).^5,14 Most interestingly, BAK1, or one of the four BAK1-related receptor kinases of the SERK protein family, also acts as a co-receptor for the ligand-dependent activation of FLS2, AtPEPR1 and EFR.^15–17 It seems that the co-receptor BAK1 plays an important role in activation of receptor kinases, serving different intracellular signaling pathways and output programs.¹⁸Up to now, little is known about the molecular details of ligand binding by the ectodomain in the apoplast and how this process leads to activation of the output signaling by the kinase moiety in the cytoplasm. The interaction with the co-receptor BAK1 suggests an activation process involving a ligand-induced intramolecular conformational change of the LRR-RK that then allows heterodimerization with the co-receptor BAK1. An initial task in elucidation of this activation process consists in defining the exact sites in the ectodomains of the receptors that interact with their corresponding ligands. So far, the clearest results for mapping ligand binding sites on LRR-receptor proteins were obtained with directed point mutations within the LRR domains as performed with the tomato receptor-like protein Cf-9,^19,20 and the Arabidopsis FLS2. There, a series of directed point mutations helped to map the LRRs 9–15 as a subdomain essential for interaction with the ligand flg22.²¹ Another interesting and promising approach consists in swaps of receptor sub-domains or exchanges of LRRs. In a remarkable, pioneering experiment this approach was used to produce chimeric receptors with the ectodomain of the brassinosteroid receptor BRI1 from Arabidopsis and the kinase domain of the immunoreceptor XA21 from rice.²² This chimera was reported to recognize the “developmental signal” brassinolide but to trigger characteristic cellular defense responses. In a recent publication²³ a domain swap between the ectodomain of the Wall Associated Kinase 1 (WAK1) and EFR was used to gain evidence for a function of the WAK1 ectodomain as a pectin receptor. Chimeric forms of the Cf receptor-like protein were used to identify subdomains carrying the specificity for the corresponding effectors from the C. fulvum pathogens.²⁴ However, as a limitation of this analysis, for none of these tomato resistance proteins a direct interaction with the corresponding effector proteins of the pathogen could be demonstrated so far.²⁵In our work, recently published in the Journal of Biochemistry,¹ we used the Arabidopsis thaliana receptors FLS2 and EFR to generate receptor chimeras. The main goal was to study the elf18 binding site in the EFR LRR-domain. In initial attempts we used EFR-constructs lacking some of the LRRs to narrow down the interaction site on the ectodomain. However, all of these truncated ectodomain versions lacking the transmembrane domain or more turned out to be unable in binding elf18 and triggering responses. In a second approach, we used the replacement of receptor parts with fragments from the structurally related receptor AtFLS2. These chimeras were tested for proper expression, localization, functionality in several plant defence related assays and affinity for the ligand elf18 in binding assays. The chimera with the complete EFR ectodomain swapped to the Kinase of FLS2 was fully functional as EF-Tu receptor. Since both receptors are known to trigger the same set of defense responses this might be not unexpected. Nevertheless, it is noteworthy that the two receptors show ∼45% sequence identity in their kinase domain, a degree of identity also shared with the kinase domains of receptors involved in other output programs, like BRI1. The 21 LRRs of EFR are sufficient for specifying full affinity for the elf18 as a ligand (

Chimeric FLS2 receptors reveal the basis for differential flagellin perception in Arabidopsis and tomato

The flagellin receptor of Arabidopsis thaliana, At-FLAGELLIN SENSING2 (FLS2), has become a model for mechanistic and functional studies on plant immune receptors. Here, we started out with a comparison of At-FLS2 and the orthologous tomato (Solanum lycopersicum) receptor Sl-FLS2. Both receptors specifically responded to picomolar concentrations of the genuine flg22 ligand but proved insensitive to >10(6)-fold higher concentrations of CLV3 peptides that have recently been reported as a second type of ligand for At-FLS2. At-FLS2 and Sl-FLS2 exhibit species-specific differences in the recognition of shortened or sequence-modified flg22 ligands. To map the sites responsible for these species-specific traits on the FLS2 receptors, we performed domain swaps, substituting subsets of the 28 leucine-rich repeats (LRRs) in At-FLS2 with the corresponding LRRs from Sl-FLS2. We found that the LRRs 7 to 10 of Sl-FLS2 determine the high affinity of Sl-FLS2 for the core part RINSAKDD of flg22. In addition, we discovered importance of the LRRs 19 to 24 for the responsiveness to C-terminally modified flagellin peptides. These results indicate that ligand perception in FLS2 is a complex molecular process that involves LRRs from both the outermost and innermost LRRs of the FLS2 ectodomain.     

Roles of Peroxinectin in PGE2-Mediated Cellular Immunity in Spodoptera exigua

Background

Prostaglandins (PGs) mediate insect immune responses to infections and invasions. Although the presence of PGs has been confirmed in several insect species, their biosynthesis in insects remains a conundrum because orthologs of the mammalian cyclooxygenases (COXs) have not been found in the known insect genomes. PG-mediated immune reactions have been documented in the beet armyworm, Spodoptera exigua. The purpose of this research is to identify the source of PGs in S. exigua.

Principal Findings

Peroxidases (POXs) are a sister group of COX genes. Ten putative POXs (SePOX-A ∼ SePOX-J) were expressed in S. exigua. Expressions of SePOX-F and -H were induced by bacterial challenge and expressed in the hemocytes and the fat body. RNAi of each POX was performed by hemocoelic injection of their specific double-stranded RNAs. dsPOX-F or, separately, dsPOX-H, but not the other eight dsRNA constructs, specifically suppressed hemocyte-spreading behavior and nodule formation; these two reactions were also inhibited by aspirin, a COX inhibitor. PGE₂, but not arachidonic acid, treatment rescued the immunosuppression. Sequence analysis indicated that both POX genes were clustered with peroxinectin (Pxt) and their cognate proteins shared some conserved domains corresponding to the Pxt of Drosophila melanogaster.

Conclusions

SePOX-F and -H are Pxt-like genes associated with PG biosynthesis in S. exigua.