RESUMO
The nonexpressor of pathogenesis-related (NPR) gene family is well known to play a crucial role in transactivation of TGA transcription factors for salicylic acid (SA)-responsive genes, including pathogenesis-related protein 1 (PR1), during plants' immune response after pathogen attack in the model dicot Arabidopsis thaliana. However, little is known about NPR gene functions in monocots. We therefore explored the functions of NPRs in SA signaling in the model monocot Brachypodium distachyon. BdNPR1 and BdNPR2/3 share structural similarities with A. thaliana AtNPR1/2 and AtNPR3/4 subfamilies, respectively. The transcript level of BdNPR2 but not BdNPR1/3 appeared to be positively regulated in leaves in response to methyl salicylate. Reporter assays in protoplasts showed that BdNPR2 positively regulated BdTGA1-mediated activation of PR1. This transactivation occurred in an SA-dependent manner through SA binding at Arg468 of BdNPR2. In contrast, BdNPR1 functioned as a suppressor of BdNPR2/BdTGA1-mediated transcription of PR1. Collectively, our findings reveal that the TGA-promoted transcription of SA-inducible PR1 is orchestrated by the activator BdNPR2 and the repressor BdNPR1, which function competitively in B. distachyon.
Assuntos
Arabidopsis , Brachypodium , Arabidopsis/genética , Arabidopsis/metabolismo , Brachypodium/genética , Brachypodium/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácido Salicílico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genéticaRESUMO
JAV1-associated ubiquitin ligase 1 (JUL1) is a RING-type E3 ubiquitin ligase that catalyzes ubiquitination of JAV1, a jasmonate signaling repressor, in Arabidopsis thaliana in response to herbivore attack. Here we present a new insight into the nature of JUL1 as a multi-targeting enzyme for not only JAV1 but also transcription factors (TFs) screened using in vitro and in vivo protein interaction assays. Reporter assays using protoplasts showed that the JUL1-interacting TFs (JiTFs), including ERF15, bZIP53 and ORA59, were involved in transcriptional activation of jasmonate-responsive PDF1.2 and abscisic acid-responsive GEA6. Likewise, assays using mutant plants suggested that the 3 JiTFs were indeed responsible for transcriptional regulation of PDF1.2 and/or GEA6, and ERF15 and ORA59 were substantially responsible for the anti-herbivore trait. In vitro protein ubiqutination assays showed that JUL1 catalyzed ubiqutination of JAV1 but not any of the TFs. This was in accord with the finding that JUL1 abolished JAV1's interference with ERF15 function, according to the reporter assay. Moreover, of great interest is our finding that ERF15 but not bZIP53 or ORA59 serves as a scaffold for the JAV1/JUL1 system, indicating that there is narrow selectivity of the transcriptional reprogramming by the JAV1/JUL1 system.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ubiquitina-Proteína Ligases , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Quitina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fosforilação/genética , Fosforilação/fisiologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Lysin motif (LysM) receptor-like kinase CERK1 is a co-receptor essential for plant immune responses against carbohydrate microbe-associated molecular patterns (MAMPs). Concerning the immediate downstream signaling components of CERK1, receptor-like cytoplasmic kinases such as PBL27 and other RLCK VII members have been reported to regulate immune responses positively. In this study, we report that a novel CERK1-interacting E3 ubiquitin ligase, PUB4, is also involved in the regulation of MAMP-triggered immune responses. Knockout of PUB4 resulted in the alteration of chitin-induced defense responses, indicating that PUB4 positively regulates reactive oxygen species generation and callose deposition but negatively regulates MAPK activation and defense gene expression. On the other hand, detailed analyses of a double knockout mutant of pub4 and sid2, a mutant of salicylic acid (SA) synthesis pathway, showed that the contradictory phenotype of the pub4 mutant was actually caused by abnormal accumulation of SA in this mutant and that PUB4 is a positive regulator of immune responses. The present and recent findings on the role of PUB4 indicate that PUB4 is a unique E3 ubiquitin ligase involved in the regulation of both plant immunity and growth/development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Doenças das Plantas , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina/metabolismoRESUMO
The two-spotted spider mite (Tetranychus urticae) is a plant-sucking arthropod herbivore that feeds on a wide array of cultivated plants. In contrast to the well-characterized classical chewing herbivore salivary elicitors that promote plant defense responses, little is known about sucking herbivores' elicitors. To characterize the sucking herbivore elicitors, we explored putative salivary gland proteins of spider mites by using an Agrobacterium-mediated transient expression system or protein infiltration in damaged bean leaves. Two candidate elicitors (designated as tetranin1 (Tet1) and tetranin2 (Tet2)) triggered early leaf responses (cytosolic calcium influx and membrane depolarization) and increased the transcript abundances of defense genes in the leaves, eventually resulting in reduced survivability of T. urticae on the host leaves as well as induction of indirect plant defenses by attracting predatory mites. Tet1 and/or Tet2 also induced jasmonate, salicylate and abscisic acid biosynthesis. Notably, Tet2-induced signaling cascades were also activated via the generation of reactive oxygen species. The signaling cascades of these two structurally dissimilar elicitors are mostly overlapping but partially distinct and thus they would coordinate the direct and indirect defense responses in host plants under spider mite attack in both shared and distinct manners.
Assuntos
Phaseolus/parasitologia , Doenças das Plantas/parasitologia , Solanum melongena/parasitologia , Tetranychidae/fisiologia , Agrobacterium tumefaciens , Animais , Cálcio , Bases de Dados Genéticas , Feminino , Regulação da Expressão Gênica , Phaseolus/imunologia , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Folhas de Planta/parasitologia , Espécies Reativas de Oxigênio , Solanum melongena/imunologiaRESUMO
Plant cell surface receptor-like kinases (RLKs) mediate the signals from microbe-associated molecular patterns (MAMPs) that induce immune responses. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, is a common MAMP perceived by animals and plants; however, the plant receptors/co-receptors are unknown except for LORE, a bulb-type lectin S-domain RLK (B-lectin SD1-RLK) in Arabidopsis. OsCERK1 is a multifunctional RLK in rice that contains lysin motifs (LysMs) and is essential for the perception of chitin, a fungal MAMP, and peptidoglycan, a bacterial MAMP. Here, we analyzed the relevance of OsCERK1 to LPS perception in rice. Using OsCERK1-knockout mutants (oscerk1), we evaluated hydrogen peroxide (H2 O2 ) production and gene expression after LPS treatment. We also examined the LPS response in knockout mutants for the B-lectin SD1-RLK genes in rice and for all LysM-protein genes in Arabidopsis. Compared with wild-type rice cells, LPS responses in oscerk1 cells were mostly diminished. By contrast, rice lines mutated in either of three B-lectin SD1-RLK genes and Arabidopsis lines mutated in the LysM-protein genes responded normally to LPS. From these results, we conclude that OsCERK1 is an LPS receptor/co-receptor and that the LPS perception systems of rice and Arabidopsis are significantly different.
Assuntos
Lipopolissacarídeos/farmacologia , Oryza/imunologia , Imunidade Vegetal/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação/genética , Oryza/efeitos dos fármacos , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile122 in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc)8 induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc)8-induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcNß1,4GlcNAc)4, which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitin-elicitor receptor kinase-1.
Assuntos
Quitina/química , Oryza/imunologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Epitopos/imunologia , Ligantes , Lisina/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Oryza/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de Aminoácidos , NicotianaRESUMO
Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Quitina/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Treonina/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Mutação , Fosforilação/efeitos dos fármacos , Imunidade Vegetal/efeitos dos fármacos , Plantas Geneticamente Modificadas , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacosRESUMO
Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called sclerotinia culture filtrate elicitor1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to receptor-like protein30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases brassinosteroid insensitive1-associated receptor kinase1 (BAK1) and Suppressor of BIR1-1/evershed (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.
Assuntos
Proteínas de Arabidopsis/imunologia , Arabidopsis/imunologia , Ascomicetos/patogenicidade , Doenças das Plantas/genética , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/imunologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Resistência à Doença/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/microbiologia , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Recognition of microbe-associated molecular patterns (MAMPs) initiates pattern-triggered immunity in host plants. Pattern recognition receptors (PRRs) and receptor-like cytoplasmic kinases (RLCKs) are the major components required for sensing and transduction of these molecular patterns. However, the regulation of RLCKs by PRRs and their specificity remain obscure. In this study we show that PBL27, an Arabidopsis ortholog of OsRLCK185, is an immediate downstream component of the chitin receptor CERK1 and contributes to the regulation of chitin-induced immunity in Arabidopsis. Knockout of PBL27 resulted in the suppression of several chitin-induced defense responses, including the activation of MPK3/6 and callose deposition as well as in disease resistance against fungal and bacterial infections. On the other hand, the contribution of PBL27 to flg22 signaling appears to be very limited, suggesting that PBL27 selectively regulates defense signaling downstream of specific PRR complexes. In vitro phosphorylation experiments showed that CERK1 preferentially phosphorylated PBL27 in comparison to BIK1, whereas phosphorylation of PBL27 by BAK1 was very low compared with that of BIK1. Thus, the substrate specificity of the signaling receptor-like kinases, CERK1 and BAK1, may determine the preference of downstream RLCKs.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/imunologia , Transdução de Sinais , Alternaria/fisiologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Quitina/metabolismo , Técnicas de Inativação de Genes , Glucanos/metabolismo , Modelos Biológicos , Fosforilação , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Reconhecimento de Padrão , Especificidade por Substrato , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/fisiologiaRESUMO
OsCERK1 is a rice receptor-like kinase that mediates the signal of a fungal cell wall component, chitin, by coordinating with a lysin motif (LysM)-containing protein CEBiP. To further elucidate the function of OsCERK1 in the defense response, we disrupted OsCERK1 using an Agrobacterium-mediated gene targeting system based on homologous recombination. In OsCERK1-disrupted lines, the generation of hydrogen peroxide and the alteration of gene expression in response to a chitin oligomer were completely abolished. The OsCERK1-disrupted lines also showed lowered responsiveness to a bacterial cell wall component, peptidoglycan. Yeast two-hybrid analysis indicated that OsCERK1 interacts with the LysM-containing proteins LYP4 and LYP6, which are known to participate in the peptidoglycan response in rice. Observation of the infection behavior of rice blast fungus (Magnaporthe oryzae) revealed that disruption of OsCERK1 led to increased hyphal growth in leaf sheath cells. Green fluorescent protein-tagged OsCERK1 was localized around the primary infection hyphae. These results demonstrate that OsCERK1 is indispensable for chitin perception and participates in innate immunity in rice, and also mediates the peptidoglycan response. It is also suggested that OsCERK1 mediates the signaling pathways of both fungal and bacterial molecular patterns by interacting with different LysM-containing receptor-like proteins.
Assuntos
Quitina/metabolismo , Magnaporthe/fisiologia , Oryza/enzimologia , Peptidoglicano/metabolismo , Doenças das Plantas/imunologia , Proteínas de Plantas/imunologia , Motivos de Aminoácidos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Marcação de Genes , Genes Reporter , Peróxido de Hidrogênio/metabolismo , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Oryza/genética , Oryza/imunologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
Plants defend against folivores by responding to folivore-derived elicitors following activation of signaling cascade networks. In Arabidopsis, HAK1, a receptor-like kinase, responds to polysaccharide elicitors (Frα) that are present in oral secretions of Spodoptera litura larvae to upregulate defense genes (e.g., PDF1.2) mediated through downstream cytoplasmic kinase PBL27. Here, we explored whether other protein kinases, including CPKs and CRKs, function with PBL27 in the intracellular signaling network for anti-herbivore responses. We showed that CRK2 and CRK3 were found to interact with PBL27, but CPKs did not. Although transcripts of PDF1.2 were upregulated in leaves of wild-type Arabidopsis plants in response to mechanical damage with Frα, this failed in CRK2- and PBL27-deficient mutant plants, indicating that the CRK2/PBL27 system is predominantly responsible for the Frα-responsive transcription of PDF1.2 in S. litura-damaged plants. In addition to CRK2-phosphorylated ERF13, as shown previously, ethylene signaling in connection to CRK2-phosphorylated PBL27 was predicted to be responsible for transcriptional regulation of a gene for ethylene response factor 13 (ERF13). Taken together, these findings show that CRK2 regulates not only ERF13 phosphorylation but also PBL27-dependent de novo synthesis of ERF13, thus determining active defense traits against S. litura larvae via transcriptional regulation of PDF1.2.
RESUMO
Chitin is a major molecular pattern for various fungi, and its fragments, chitin oligosaccharides, are known to induce various defense responses in plant cells. A plasma membrane glycoprotein, CEBiP (chitin elicitor binding protein) and a receptor kinase, CERK1 (chitin elicitor receptor kinase) (also known as LysM-RLK1), were identified as critical components for chitin signaling in rice and Arabidopsis, respectively. However, it is not known whether each plant species requires both of these two types of molecules for chitin signaling, nor the relationships between these molecules in membrane signaling. We report here that rice cells require a LysM receptor-like kinase, OsCERK1, in addition to CEBiP, for chitin signaling. Knockdown of OsCERK1 resulted in marked suppression of the defense responses induced by chitin oligosaccharides, indicating that OsCERK1 is essential for chitin signaling in rice. The results of a yeast two-hybrid assay indicated that both CEBiP and OsCERK1 have the potential to form hetero- or homo-oligomers. Immunoprecipitation using a membrane preparation from rice cells treated with chitin oligosaccharides suggested the ligand-induced formation of a receptor complex containing both CEBiP and OsCERK1. Blue native PAGE and chemical cross-linking experiments also suggested that a major portion of CEBiP exists as homo-oligomers even in the absence of chitin oligosaccharides.
Assuntos
Quitina/metabolismo , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Oryza/genética , Multimerização Proteica , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
Chitinase treatment of a commercial laminarioligosaccharide preparation from a mushroom resulted in a loss of previously reported elicitor activity in rice cells, indicating that the activity was attributable not to the laminarioligosaccharide but rather to the contaminating chitin fragments. This suggests that the elicitor activity of laminarioligosacchraides from such sources containing chitinaceous substances should be carefully interpreted.
Assuntos
Quitina/química , Contaminação de Medicamentos , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Oryza/citologia , Oryza/efeitos dos fármacosRESUMO
The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.
Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Receptores de Superfície Celular/isolamento & purificação , Marcadores de Afinidade , Biotinilação , Reagentes de Ligações Cruzadas , Daucus carota/química , Oryza/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Chitin is a major component of fungal cell walls and serves as a microbe-associated molecular pattern (MAMP) for the detection of various potential pathogens in innate immune systems of both plants and animals. We recently showed that chitin elicitor-binding protein (CEBiP), plasma membrane glycoprotein with LysM motifs, functions as a cell surface receptor for chitin elicitor in rice. The predicted structure of CEBiP does not contain any intracellular domains, suggesting that an additional component(s) is required for signaling through the plasma membrane into the cytoplasm. Here, we identified a receptor-like kinase, designated CERK1, which is essential for chitin elicitor signaling in Arabidopsis. The KO mutants for CERK1 completely lost the ability to respond to the chitin elicitor, including MAPK activation, reactive oxygen species generation, and gene expression. Disease resistance of the KO mutant against an incompatible fungus, Alternaria brassicicola, was partly impaired. Complementation with the WT CERK1 gene showed cerk1 mutations were responsible for the mutant phenotypes. CERK1 is a plasma membrane protein containing three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain with autophosphorylation/myelin basic protein kinase activity, suggesting that CERK1 plays a critical role in fungal MAMP perception in plants.
Assuntos
Alternaria , Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Quitina/metabolismo , Doenças das Plantas/genética , Proteínas Serina-Treonina Quinases/fisiologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Membrana Celular/enzimologia , Expressão Gênica , Teste de Complementação Genética , Genoma de Planta , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mutação , Doenças das Plantas/microbiologia , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genéticaRESUMO
Cell cultures established from various plant species have been used for a range of physiological and biochemical studies. Homogeneity of cell types and size of clusters in the cell culture often gave a clearer and simpler results compared to those obtained with the whole plant. On the other hand, possible variability of physiological conditions and responsiveness to external stimuli between the cell lines could be problematic for comparative studies. Aiming at combining the usefulness of plant cell culture with the rich information and genetic resources of Arabidopsis, we systemically examined the methods/conditions to establish cell lines for comparative studies, which could be applicable to a variety of genetic resources. Arabidopsis cell lines thus established from the meristem of mature seeds showed reproducible and comparable MAMP responses such as ROS generation and defense-related gene expression. MAMP responses of the cultured cells showed the specificity depending on the presence/absence of the corresponding MAMP receptor. Pharmacological study with a protein kinase inhibitor, K252a, also showed the usefulness of the cell culture for such studies. These results indicated the usefulness of the method to establish Arabidopsis cell lines, which are useful for comparative studies between genetic resources.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quitina/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Plants respond to herbivory by perceiving herbivore danger signal(s) (HDS(s)), including "elicitors", that are present in herbivores' oral secretions (OS) and act to induce defense responses. However, little is known about HDS-specific molecules and intracellular signaling. Here we explored soybean receptor-like kinases (RLKs) as candidates that might mediate HDS-associated RLKs' (HAKs') actions in leaves in response to OS extracted from larvae of a generalist herbivore, Spodoptera litura. Fractionation of OS yielded Frα, which consisted of polysaccharides. The GmHAKs composed of their respective homomultimers scarcely interacted with Frα. Moreover, Arabidopsis HAK1 homomultimers interacted with cytoplasmic signaling molecule PBL27, resulting in herbivory resistance, in an ethylene-dependent manner. Altogether, our findings suggest that HAKs are herbivore-specific RLKs mediating HDS-transmitting, intracellular signaling through interaction with PBL27 and the subsequent ethylene signaling for plant defense responses in host plants.
Assuntos
Arabidopsis/genética , Glycine max/genética , Defesa das Plantas contra Herbivoria/genética , Proteínas de Plantas/genética , Polissacarídeos/fisiologia , Spodoptera/fisiologia , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cadeia Alimentar , Herbivoria , Larva/crescimento & desenvolvimento , Larva/fisiologia , Proteínas de Plantas/metabolismo , Transdução de Sinais , Glycine max/metabolismo , Spodoptera/crescimento & desenvolvimentoRESUMO
Reactive oxygen species generation is one of the most popular index of plant immune responses. Leaf disk assay has been commonly used for MAMP/elicitor-induced ROS analysis by many groups. However, the reproducibility of the leaf disk assay relies on the skills of the people engaged in the experiments and the experiment itself seems not suitable for some plant species, which had a tough leaf structure and lower penetration efficiency of MAMPs/elicitors. In this study, we prepared a handmade leaf cutter to cut out the leaf fragments with uniform size and slits. The use of such fragments obtained by the new leaf cutter as well as the increase of the number of leaf fragments for each experiment improved the reliability and reproducibility of the leaf disk assay. This cutter was also successfully applied to rice leaf disk assay, indicating the applicability to other plant spices.