A Nitrilase-Like Protein Interacts with GCC Box DNA-Binding Proteins Involved in Ethylene and Defense Responses

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.     

LIK1, A CERK1-Interacting Kinase,Regulates Plant Immune Responses in Arabidopsis

Chitin, an integral component of the fungal cell wall, is one of the best-studied microbe-associated molecular patterns. Previous work identified a LysM receptor-like kinase (LysM-RLK1/CERK1) as the primary chitin receptor in Arabidopsis. In order to identify proteins that interact with CERK1, we conducted a yeast two-hybrid screen using the intracellular kinase domain of CERK1 as the bait. This screen identified 54 putative CERK1-interactors. Screening mutants defective in 43 of these interacting proteins identified only two, a calmodulin like protein (At3g10190) and a leucine-rich repeat receptor like kinase (At3g14840), which differed in their response to pathogen challenge. In the present work, we focused on characterizing the LRR-RLK gene where mutations altered responses to chitin elicitation. This LRR-RLK was named LysM RLK1-interacting kinase 1 (LIK1). The interaction between CERK1 and LIK1 was confirmed by co-immunoprecipitation using protoplasts and transgenic plants. In vitro experiments showed that LIK1 was directly phosphorylated by CERK1. In vivo phosphorylation assays showed that Col-0 wild-type plants have more phosphorylated LIK1 than cerk1 mutant plants, suggesting that LIK1 may be directly phosphorylated by CERK1. Lik1 mutant plants showed an enhanced response to both chitin and flagellin elicitors. In comparison to the wild-type plants, lik1 mutant plants were more resistant to the hemibiotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic pathogen Sclerotinia sclerotiorum. Consistent with the enhanced susceptibility to necrotrophs, lik1 mutants showed reduced expression of genes involved in jasmonic acid and ethylene signaling pathways. These data suggest that LIK1 directly interacts with CERK1 and regulates MAMP-triggered innate immunity.     

LIM and SH3 Protein -1 Modulates CXCR2-Mediated Cell Migration

Background

The chemokine receptor CXCR2 plays a pivotal role in migration of neutrophils, macrophages and endothelial cells, modulating several biological responses such as angiogenesis, wound healing and acute inflammation. CXCR2 is also involved in pathogenesis of chronic inflammation, sepsis and atherosclerosis. The ability of CXCR2 to associate with a variety of proteins dynamically is responsible for its effects on directed cell migration or chemotaxis. The dynamic network of such CXCR2 binding proteins is termed as “CXCR2 chemosynapse”. Proteomic analysis of proteins that co-immunoprecipitated with CXCR2 in neutrophil-like dHL-60 cells revealed a novel protein, LIM and SH3 protein 1 (LASP-1), binds CXCR2 under both basal and ligand activated conditions. LASP-1 is an actin binding cytoskeletal protein, involved in the cell migration.

Methodology/Principal Findings

We demonstrate that CXCR2 and LASP-1 co-immunoprecipitate and co-localize at the leading edge of migrating cells. The LIM domain of LASP-1 directly binds to the carboxy-terminal domain (CTD) of CXCR2. Moreover, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4. Using a site-directed and deletion mutagenesis approach, Iso323-Leu324 of the conserved LKIL motif on CXCR2-CTD was identified as the binding site for LASP-1. Interruption of the interaction between CXCR2-CTD and LIM domain of LASP-1 by dominant negative and knock down approaches inhibited CXCR2-mediated chemotaxis. Analysis for the mechanism for inhibition of CXCR2-mediated chemotaxis indicated that LASP-1/CXCR2 interaction is essential for cell motility and focal adhesion turnover involving activation of Src, paxillin, PAK1, p130CAS and ERK1/2.

Conclusions/Significance

We demonstrate here for the first time that LASP-1 is a key component of the “CXCR2 chemosynapse” and LASP-1 interaction with CXCR2 is critical for CXCR2-mediated chemotaxis. Furthermore, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4, suggesting that LASP-1 is a general mediator of CXC chemokine mediated chemotaxis. Thus, LASP-1 may serve as a new link coordinating the flow of information between chemokine receptors and nascent focal adhesions, especially at the leading edge. Thus the association between the chemokine receptors and LASP-1 suggests to the presence of a CXC chemokine receptor-LASP-1 pro-migratory module in cells governing the cell migration.     

Ethylene Receptors Function as Components of High-Molecular-Mass Protein Complexes in Arabidopsis

Background

The gaseous plant hormone ethylene is perceived in Arabidopsis thaliana by a five-member receptor family composed of ETR1, ERS1, ETR2, ERS2, and EIN4.

Methodology/Principal Findings

Gel-filtration analysis of ethylene receptors solubilized from Arabidopsis membranes demonstrates that the receptors exist as components of high-molecular-mass protein complexes. The ERS1 protein complex exhibits an ethylene-induced change in size consistent with ligand-mediated nucleation of protein-protein interactions. Deletion analysis supports the participation of multiple domains from ETR1 in formation of the protein complex, and also demonstrates that targeting to and retention of ETR1 at the endoplasmic reticulum only requires the first 147 amino acids of the receptor. A role for disulfide bonds in stabilizing the ETR1 protein complex was demonstrated by use of reducing agents and mutation of Cys4 and Cys6 of ETR1. Expression and analysis of ETR1 in a transgenic yeast system demonstrates the importance of Cys4 and Cys6 of ETR1 in stabilizing the receptor for ethylene binding.

Conclusions/Significance

These data support the participation of ethylene receptors in obligate as well as ligand-dependent non-obligate protein interactions. These data also suggest that different protein complexes may allow for tailoring of the ethylene signal to specific cellular environments and responses.     

An Abscisic Acid-Independent Oxylipin Pathway Controls Stomatal Closure and Immune Defense in Arabidopsis

Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.     

Trans-spliced Heat Shock Protein 90 Modulates Encystation in Giardia lamblia

Background

Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear.

Principle Finding

In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5′ UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential “Arg” in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation.

Significance

Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.     

Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana

Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30–45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.Brassicaceae plants such as oilseed rape (Brassica napus), turnip (Brassica rapa), and white mustard (Sinapis alba) as well as the model plant Arabidopsis thaliana contain a group of secondary metabolites known as glucosinolates (GSLs)² (1, 2). These are β-thioglucoside N-hydroxysulfates with a sulfur-linked β-d-glucopyranose moiety and a variable side chain that is derived from one of eight amino acids or their methylene group-elongated derivatives. Aliphatic GSLs are derived from alanine, leucine, isoleucine, valine, or predominantly methionine. Tyrosine or phenylalanine give aromatic GSLs, and tryptophan-derived GSLs are called indolic GSLs (for review, see Ref. 3). Although more than 120 different GSLs have been identified in total so far, individual plant species usually contain only a few GSLs (2). Quantitative and qualitative differences of GSL profiles are also observed within a species, such as, for example, for different A. thaliana ecotypes (4–6). In addition, GSL composition varies among organs and during the life cycle of plants (7, 8) and is affected by external factors (9).Intact GSLs are mostly considered to be biologically inactive. Most GSL degradation products have toxic effects on insect, fungal, and bacterial pests, serve as attractants for specialist insects, or may have beneficial health effects for humans (10–15). The enzymatic degradation of GSLs (Fig. 1A), which occurs massively upon tissue damage, is catalyzed by plant thioglucosidases called myrosinases (EC 3.2.1.147; glycoside hydrolase family 1). Depending on several factors (e.g. GSL structure, proteins, cofactors, pH) myrosinase-catalyzed hydrolysis of GSLs can lead to a variety of products (Fig. 1B; for review, see Refs. 16 and 17). Of these, isothiocyanates are the most common as their formation only requires myrosinase activity. Thiocyanates on the other hand are only produced from a very limited number of GSLs, and their formation necessitates the presence of a thiocyanate-forming factor in addition to myrosinase (18). A thiocyanate-forming protein (TFP) has recently been identified in Lepidium sativum (19). Alkenyl GSLs, a subgroup of aliphatic GSLs containing a terminal unsaturation in their side chain, can lead to the production of epithionitriles through the cooperative action of myrosinase and a protein called epithiospecifier protein (ESP (20)) in a ferrous ion-dependent way (21–23). Both TFP and ESP contain a series of Kelch repeats (19). Kelch repeats are involved in protein-protein interactions, and Kelch repeat-containing proteins are involved in a number of diverse biological processes (24). In addition to isothiocyanates, nitriles are the major group of GSL hydrolysis products. Although ESP and TFP activities can generate nitriles (19, 21, 25, 26), indications for an ESP-independent nitrile-specifier activity exist. The GSL hydrolysis profile of A. thaliana roots, an organ that does not show ESP expression or activity (27), reveals predominantly the presence of nitriles (28). In addition, leaf tissue of A. thaliana ecotypes supposedly devoid of ESP activity produces a certain amount of nitriles upon autolysis (21). Under acidic buffer conditions, a non-enzymatic production of nitriles from GSLs is observed (Ref. 29 and references therein). Increasing Fe²⁺ concentrations have also been shown to favor nitrile formation over isothiocyanate formation from a number of GSLs in the presence of myrosinase and absence of ESP (21, 22). Therefore, a non-enzymatic origin of this nitrile production cannot be excluded, although the presence of a nitrile-specifier protein is a tempting alternative. Although ESP is able to generate nitriles, it has also been shown that the conversion rates of GSLs to nitriles are lower than those of GSLs to epithionitriles for ESP (21, 22).Open in a separate windowFIGURE 1.Simplified scheme of enzymatic GSL hydrolysis (A) and structures and names of GSLs and their hydrolysis products that are mentioned in the article. (B). A, myrosinase acts on GSLs to form an unstable aglycone intermediate that can rearrange spontaneously to form an isothiocyanate. Hydrolysis can be diverted from this default route under certain conditions (e.g. the presence of NSPs, ferrous ions, or at pH < 5) to give the corresponding nitrile. ESP is responsible for the formation of epithionitriles from alkenyl GSLs in a ferrous ion-dependent mechanism. B, the general structure of GSLs, indicating the variable side chain as R, is given as well as the three major classes of hydrolysis products (i.e. isothiocyanates, nitriles, and epithionitriles). The listed GSLs are the ones mentioned in this article and are arranged according to the class of GSLs they belong to and with an increase in chain length or complexity. The names of the respective hydrolysis products are given for a better understanding of the present article, and not all were encountered during our studies.A nitrile-specifier protein (NSP) that is able to redirect the hydrolysis of GSLs toward nitriles has been cloned from the larvae of the butterfly Pieris rapae (30). This protein does not, however, exhibit sequence similarity to plant ESP, and a corresponding plant nitrile-specifier protein has not yet been identified. We report here the identification of a group of six A. thaliana genes with some sequence similarity to A. thaliana ESP, providing evidence for a new family of nitrile-specifier proteins and a more detailed characterization of one member that possesses nitrile-specifier activity in vitro, when applied exogenously to plant tissue and after ectopic expression in the two A. thaliana ecotypes Col-0 and C24. Despite its sequence homology to A. thaliana epithiospecifier protein (AtESP), it does not possess epithiospecifier activity under similar conditions. Therefore, we propose to designate this protein as A. thaliana nitrile-specifier protein 2 (AtNSP2). Although the biological roles of AtNSP2 and related proteins are not yet known, their specificities and distinctive expression patterns indicate the presence of a fine-tuned mechanism for GSL degradation controlling the outcome of an array of biologically active molecules.     

Tyro3 Modulates Mertk-Associated Retinal Degeneration

Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.     

Activation of Salicylic Acid–Induced Protein Kinase, a Mitogen-Activated Protein Kinase, Induces Multiple Defense Responses in Tobacco

The activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses in plants challenged by avirulent pathogens or cells treated with pathogen-derived elicitors. Expression of a constitutively active MAPK kinase, NtMEK2^DD, in tobacco induces the expression of defense genes and hypersensitive response–like cell death, which are preceded by the activation of two endogenous MAPKs, salicylic acid–induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK). However, the roles that SIPK and WIPK each play in the process are unknown. Here we report that SIPK alone is sufficient to activate these defense responses. In tobacco leaves transiently transformed with SIPK under the control of a steroid-inducible promoter, the induction of SIPK expression after the application of dexamethasone, a steroid, leads to an increase of SIPK activity. The increase of SIPK activity is dependent on the phosphorylation of newly synthesized SIPK by its endogenous upstream kinase. In contrast, the expression of WIPK under the same conditions fails to increase its activity, even though the protein accumulates to a similar level. Studies using chimeras of SIPK and WIPK demonstrated that the C terminus of SIPK contains the molecular determinant for its activation, which is rather surprising because the N termini of SIPK and WIPK are more divergent. SIPK has been implicated previously in the regulation of both plant defense gene activation and hypersensitive response–like cell death based on evidence from pharmacological studies using kinase inhibitors. This gain-of-function study provided more direct evidence for its role in the signaling of multiple defense responses in tobacco.     

Innate,translation‐dependent silencing of an invasive transposon in Arabidopsis

Co‐evolution between hosts’ and parasites’ genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral‐like silencing response. To investigate this response’s activation, we studied the single‐copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full‐length genomic flGAG‐POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG‐POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5’OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA‐DEPENDENT‐RNA‐POLYMERASE‐6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto‐unrecognized “translation‐dependent silencing” (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.