RESUMEN
The green leaf volatiles (GLVs) Z-3-hexen-1-ol (Z3-HOL) and Z-3-hexenyl acetate (Z3-HAC) are airborne infochemicals released from damaged plant tissues that induce defenses and developmental responses in receiver plants, but little is known about their mechanism of action. We found that Z3-HOL and Z3-HAC induce similar but distinctive physiological and signaling responses in tomato seedlings and cell cultures. In seedlings, Z3-HAC showed a stronger root growth inhibition effect than Z3-HOL. In cell cultures, the two GLVs induced distinct changes in MAP kinase (MAPK) activity and proton fluxes as well as rapid and massive changes in the phosphorylation status of proteins within 5 min. Many of these phosphoproteins are involved in reprogramming the proteome from cellular homoeostasis to stress and include pattern recognition receptors, a receptor-like cytoplasmic kinase, MAPK cascade components, calcium signaling proteins and transcriptional regulators. These are well-known components of damage-associated molecular pattern (DAMP) signaling pathways. These rapid changes in the phosphoproteome may underly the activation of defense and developmental responses to GLVs. Our data provide further evidence that GLVs function like DAMPs and indicate that GLVs coopt DAMP signaling pathways.
Asunto(s)
Células Vegetales , Compuestos Orgánicos Volátiles , Células Vegetales/metabolismo , Plantones/metabolismo , Plantas/metabolismo , Transducción de Señal , Hojas de la Planta/metabolismo , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
The Arabidopsis MAP Kinases (MAPKs) MPK6 and MPK3 and orthologs in other plants function as major stress signaling hubs. MAPKs are activated by phosphorylation and are negatively regulated by MAPK-inactivating phosphatases (MIPPs), which alter the intensity and duration of MAPK signaling via dephosphorylation. Unlike in other plant species, jasmonic acid (JA) accumulation in Arabidopsis is apparently not MPK6- and MPK3-dependent, so their role in JA-mediated defenses against herbivorous insects is unclear. Here we explore whether changes in MPK6/3 phosphorylation kinetics in Arabidopsis MIPP mutants lead to changes in hormone synthesis and resistance against herbivores. The MIPPs MKP1, DsPTP1, PP2C5, and AP2C1 have been implicated in responses to infection, drought, and osmotic stress, which all impinge on JA-mediated defenses. In loss-of-function mutants, we found that the four MIPPs alter wound-induced MPK6/3 phosphorylation kinetics and affect the accumulation of the defense hormones JA, abscisic acid, and salicylic acid, as compared to wild type plants (Col-0). Moreover, MPK6/3 misregulation in MIPP or MAPK mutant plants resulted in slight changes in the resistance to Trichoplusia ni and Spodoptera exigua larvae as compared to Col-0. Our data indicate that MPK6/3 and the four MIPPs moderately contribute to wound signaling and defense against herbivorous insects in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Herbivoria , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteínas Tirosina FosfatasasRESUMEN
Plants perceive insect herbivores via a sophisticated surveillance system that detects a range of alarm signals, including herbivore-associated molecular patterns (HAMPs). Fatty acid-amino acid conjugates (FACs) are HAMPs present in oral secretions (OS) of lepidopteran larvae that induce defense responses in many plant species. In contrast to eggplant (Solanum melongena), tomato (S. lycopersicum) does not respond to FACs present in OS from Manduca sexta (Lepidoptera). Since both plants are found in the same genus, we tested whether loss of sensitivity to FACs in tomato may be a domestication effect. Using highly sensitive MAP kinase (MAPK) phosphorylation assays, we demonstrate that four wild tomato species and the closely related potato (S. tuberosum) do not respond to the FACs N-linolenoyl-L-glutamine and N-linolenoyl-L-glutamic acid, excluding a domestication effect. Among other genera within the Solanaceae, we found that bell pepper (Capsicum annuum) is responsive to FACs, while there is a differential responsiveness to FACs among tobacco (Nicotiana) species, ranging from strong responsiveness in N. benthamiana to no responsiveness in N. knightiana. The Petunia lineage is one of the oldest lineages within the Solanaceae and P. hybrida was responsive to FACs. Collectively, we demonstrate that plant responsiveness to FACs does not follow simple phylogenetic relationships in the family Solanaceae. Instead, sensitivity to FACs is a dynamic ancestral trait present in monocots and eudicots that was repeatedly lost during the evolution of Solanaceae species. Although tomato is insensitive to FACs, we found that other unidentified factors in M. sexta OS induce defenses in tomato.
Asunto(s)
Aminoácidos/metabolismo , Antibiosis , Ácidos Grasos/metabolismo , Herbivoria , Manduca/fisiología , Solanaceae/fisiología , Animales , Larva , Especificidad de la EspecieRESUMEN
Plant responses to the environment and developmental processes are mediated by a complex signaling network. The Arabidopsis thaliana mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 and their orthologs in other plants are shared signal transducers that respond to many developmental and environmental signals and thus represent highly connected hubs in the cellular signaling network. In animals, specific MAPK signaling complexes are assembled which enable input-specific protein-protein interactions and thus specific signaling outcomes. In plants, not much is known about such signaling complexes. Here, we report that MPK3, MPK6, and MPK10 orthologs in tomato, tobacco, and Arabidopsis as well as tomato MAPK kinase 4 (MKK4) associate with high molecular weight (~250-550 kDa) multiprotein complexes. Elicitation by the defense-associated peptides flg22 and systemin resulted in phosphorylation and activation of the monomeric MAPKs, whereas the complex-associated MAPKs remained unphosphorylated and inactive. In contrast, treatment of tomato cells with a phosphatase inhibitor resulted in association of phosphorylated MPK1/2 with the complex. These results demonstrate that plant MAPKs and MAPKKs dynamically assemble into stable multiprotein complexes and this may depend on their phosphorylation status. Identification of the constituents of these multiprotein complexes promises a deeper understanding of signaling dynamics.
Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas de Plantas/genética , Arabidopsis/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Peso Molecular , Complejos Multiproteicos , Proteínas de Plantas/metabolismoRESUMEN
AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Pared Celular/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/citología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Pared Celular/genética , Ciclopentanos/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Inflorescencia/genética , Inflorescencia/crecimiento & desarrollo , Meristema/genética , Meristema/metabolismo , Mutación , Oxilipinas/metabolismo , Pectinas/genética , Pectinas/metabolismo , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/patogenicidad , Ácido Salicílico/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/genéticaRESUMEN
Methanol is a byproduct of cell wall modification, released through the action of pectin methylesterases (PMEs), which demethylesterify cell wall pectins. Plant PMEs play not only a role in developmental processes but also in responses to herbivory and infection by fungal or bacterial pathogens. Molecular mechanisms that explain how methanol affects plant defenses are poorly understood. Here we show that exogenously supplied methanol alone has weak effects on defense signaling in three dicot species, however, it profoundly alters signaling responses to danger- and microbe-associated molecular patterns (DAMPs, MAMPs) such as the alarm hormone systemin, the bacterial flagellum-derived flg22 peptide, and the fungal cell wall-derived oligosaccharide chitosan. In the presence of methanol the kinetics and amplitudes of DAMP/MAMP-induced MAP kinase (MAPK) activity and oxidative burst are altered in tobacco and tomato suspension-cultured cells, in Arabidopsis seedlings and tomato leaf tissue. As a possible consequence of altered DAMP/MAMP signaling, methanol suppressed the expression of the defense genes PR-1 and PI-1 in tomato. In cell cultures of the grass tall fescue (Festuca arundinacea, Poaceae, Monocots), methanol alone activates MAPKs and increases chitosan-induced MAPK activity, and in the darnel grass Lolium temulentum (Poaceae), it alters wound-induced MAPK signaling. We propose that methanol can be recognized by plants as a sign of the damaged self. In dicots, methanol functions as a DAMP-like alarm signal with little elicitor activity on its own, whereas it appears to function as an elicitor-active DAMP in monocot grasses. Ethanol had been implicated in plant stress responses, although the source of ethanol in plants is not well established. We found that it has a similar effect as methanol on responses to MAMPs and DAMPs.
RESUMEN
Protein kinase-driven phosphorylation constitutes the core of cellular signaling. Kinase components of signal transduction pathways are often targeted for inactivation by pathogens. The study of kinases and immune signal transduction in the model crop tomato (Solanum lycopersicum) would benefit from the availability of community-wide resources for large scale and systems-level experimentation. Here, we defined the tomato kinome and performed a comprehensive comparative analysis of the tomato kinome and 15 other plant species. We constructed a tomato kinase library (TOKN 1.0) of over 300 full-length open reading frames (ORF) cloned into a recombination-based vector. We developed a high-throughput pipeline to isolate and transform tomato protoplasts. A subset of the TOKN 1.0 library kinases were expressed in planta, were purified, and were used to generate a functional tomato protein microarray. All resources created were utilized to test known and novel associations between tomato kinases and Pseudomonas syringae DC3000 effectors in a large-scale format. Bsk7 was identified as a component of the plant immune response and a candidate effector target. These resources will enable comprehensive investigations of signaling pathways and host-pathogen interactions in tomato and other Solanaceae spp.
Asunto(s)
Enfermedades de las Plantas/inmunología , Proteínas Quinasas/metabolismo , Pseudomonas syringae/metabolismo , Transducción de Señal , Solanaceae/fisiología , Solanum lycopersicum/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biología Computacional , Biblioteca de Genes , Prueba de Complementación Genética , Interacciones Huésped-Patógeno , Luciferasas , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Solanum lycopersicum/inmunología , Sistemas de Lectura Abierta , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análisis por Matrices de Proteínas , Proteínas Quinasas/genética , Protoplastos , Pseudomonas syringae/genética , Solanaceae/enzimología , Solanaceae/genética , Solanaceae/inmunologíaRESUMEN
The COP9 signalosome (CSN) is a multiprotein complex that regulates the activity of CULLIN-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate substrate proteins and thus target them for proteasomal degradation. This post-translational modification of proteins is arguably as important as reversible protein phosphorylation. The number of putative CRLs that recognize specific substrate proteins is vast, and known CRL substrates are involved in many cellular plant processes such as hormone signaling, the cell cycle, and regulation of growth, development, and defenses. By controlling the activity of CRLs, the CSN may integrate and fine-tune all of these processes. Recent research has unraveled in great mechanistic detail how the two multiprotein complexes CSN and CRL interact. As a consequence of CSN pleiotropy, complete loss of CSN function results in seedling lethality. However, recent work on plants that exhibit a partial loss of CSN function, has uncovered a role of the CSN during later life stages in processes such as development and defenses against pathogens and herbivorous insects. Not all aspects of development and defense are affected equally by CSN silencing, probably due to the differential participation and importance of CSN-regulated CRLs in these processes. This review will provide an overview of the highly complex regulation of CRL activity by CSN, and the many roles of the CSN in plant development and defense.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Fosforilación , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/fisiología , Transducción de Señal/fisiología , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Forage and turf grasses are continually cut and grazed by livestock, however very little is known concerning the perception or molecular responses to wounding. Mechanical wounding rapidly activated a 46 kDa and a 44 kDa mitogen-activated protein kinase (MAPK) in six different grass species. In the model grass species Lolium temulentum, the 46 kDa MAPK was rapidly activated within 5 min of wounding both locally and systemically in an adjacent unwounded tiller. This indicates that wounding generates a rapidly propagated long-distance signal that activates a MAPK in the distal portions of the plant. This 46 kDa MAPK activity was not enhanced by the addition of the pathogen-associated signal salicylic acid (SA) to the wound site nor induced when exposed to methyl jasmonate (MJ), which is a potent inducer of the wound response in dicotyledonous plants. However, pretreatment with MJ increased the wound-induced activity of the 44 kDa MAPK over the activity in control plants.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Poaceae/metabolismo , Acetatos/metabolismo , Catalasa/metabolismo , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas Quinasas Activadas por Mitógenos/genética , Oxilipinas/metabolismo , Hojas de la Planta/fisiología , Proteínas de Plantas/metabolismo , Poaceae/enzimología , Poaceae/genética , Ácido Salicílico/metabolismoRESUMEN
The COP9 signalosome (CSN) is a multi-protein complex that regulates the activities of cullin-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate proteins in order to target them for proteasomal degradation. The CSN is required for proper plant development. Here we show that the CSN also has a profound effect on plant defense responses. Silencing of genes for CSN subunits in tomato plants resulted in a mild morphological phenotype and reduced expression of wound-responsive genes in response to mechanical wounding, attack by Manduca sexta larvae, and Prosystemin over-expression. In contrast, expression of pathogenesis-related genes was increased in a stimulus-independent manner in these plants. The reduced wound response in CSN-silenced plants corresponded with reduced synthesis of jasmonic acid (JA), but levels of salicylic acid (SA) were unaltered. As a consequence, these plants exhibited reduced resistance against herbivorous M. sexta larvae and the necrotrophic fungal pathogen Botrytis cinerea. In contrast, susceptibility to tobacco mosaic virus (TMV) was not altered in CSN-silenced plants. These data demonstrate that the CSN orchestrates not only plant development but also JA-dependent plant defense responses.
Asunto(s)
Ciclopentanos/metabolismo , Complejos Multiproteicos/fisiología , Oxilipinas/metabolismo , Péptido Hidrolasas/fisiología , Inmunidad de la Planta/genética , Proteínas de Plantas/fisiología , Solanum lycopersicum/fisiología , Animales , Botrytis/inmunología , Botrytis/patogenicidad , Complejo del Señalosoma COP9 , Ciclopentanos/análisis , Regulación de la Expresión Génica de las Plantas/inmunología , Silenciador del Gen , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Solanum lycopersicum/parasitología , Manduca/inmunología , Manduca/patogenicidad , Complejos Multiproteicos/genética , Oxilipinas/análisis , Péptido Hidrolasas/genética , Fenotipo , Enfermedades de las Plantas , Proteínas de Plantas/genética , Ácido Salicílico/análisis , Ácido Salicílico/metabolismo , Virus del Mosaico del Tabaco/inmunología , Virus del Mosaico del Tabaco/patogenicidad , Heridas y LesionesRESUMEN
Transgenic tomato (Solanum lycopersicum) plants that overexpress the Prosystemin gene (35S::PS) and plants with a mutation in the JA biosynthetic pathway (def1) are known to exhibit a constitutive or reduced wound response, respectively. Here it is demonstrated that several independent 35S::PS lines emit high levels of specific volatiles in addition to increased accumulation of proteinase inhibitors (PIs). Furthermore, the temporal dynamics of systemically induced volatile compounds including green-leaf volatiles, terpenes, and shikimic acid-derivatives from 35S::PS and def1 plants in response to herbivore wounding and treatment with jasmonic acid (JA) are described. Application of JA induced defense protein accumulation and volatile emissions in wild type plants, but did not further increase systemic volatile emissions from 35S::PS plants. Wounding by Manduca sexta larvae induced synthesis of defense proteins and emission of volatiles in wild type plants, but not in def1 plants. Application of jasmonic acid restored the local and systemic accumulation of defense proteins in def1, as well as enhanced herbivore-induced volatile emissions. These results provide strong support for the role of prosystemin- and JA-signaling in the regulation of volatile emissions in tomato plants.
Asunto(s)
Ciclopentanos/metabolismo , Manduca/fisiología , Oxilipinas/metabolismo , Péptidos/fisiología , Solanum lycopersicum/fisiología , Compuestos Orgánicos Volátiles/metabolismo , Animales , Secuencia de Bases , Ciclopentanos/análisis , Regulación de la Expresión Génica de las Plantas , Larva/genética , Larva/fisiología , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Manduca/embriología , Estructura Molecular , Oxilipinas/análisis , Péptidos/análisisRESUMEN
Systemin is a wound signaling peptide from tomato that is important for plant defenses against herbivory. The systemin receptor was initially identified as the tomato homolog of the brassinosteroid receptor BRI1, but genetic evidence argued against this finding. However, we found that BRI1 may function as an inappropriate systemin binding protein that does not activate the systemin signaling pathway. Here we provide evidence that systemin perception is localized in a tissue-type specific manner. Mesophyll protoplasts were not sensitive to systemin, while they responded to other elicitors. We hypothesize that the elusive systemin receptor is a protein with high similarity to BRI1 which is specifically localized in vascular tissue like the systemin precursor prosystemin. Binding of systemin to BRI1 may be an artifact of transgenic BRI1-overexpressing plants, but does not take place in wild type tomato cells.
Asunto(s)
Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Superficie Celular/metabolismo , Solanum lycopersicum/metabolismo , Inmunidad Innata , Enfermedades de las Plantas , Unión Proteica , Protoplastos/metabolismo , Transducción de SeñalRESUMEN
The tomato wound signal systemin is perceived by a specific high-affinity, saturable, and reversible cell surface receptor. This receptor was identified as the receptor-like kinase SR160, which turned out to be identical to the brassinosteroid receptor BRI1. Recently, it has been shown that the tomato bri1 null mutant cu3 is as sensitive to systemin as wild type plants. Here we explored these contradictory findings by studying the responses of tobacco plants (Nicotiana tabacum) to systemin. A fluorescently-labeled systemin analog bound specifically to plasma membranes of tobacco suspension-cultured cells that expressed the tomato BRI1-FLAG transgene, but not to wild type tobacco cells. On the other hand, signaling responses to systemin, such as activation of mitogen-activated protein kinases and medium alkalinization, were neither increased in BRI1-FLAG-overexpressing tobacco cells nor decreased in BRI1-silenced cells as compared to levels in untransformed control cells. Furthermore, in transgenic tobacco plants BRI1-FLAG became phosphorylated on threonine residues in response to brassinolide application, but not in response to systemin. When BRI1 transcript levels were reduced by virus-induced gene silencing in tomato plants, the silenced plants displayed a phenotype characteristic of bri1 mutants. However, their response to overexpression of the Prosystemin transgene was the same as in control plants. Taken together, our data suggest that BRI1 can function as a systemin binding protein, but that binding of the ligand does not transduce the signal into the cell. This unusual behavior and the nature of the elusive systemin receptor will be discussed.
Asunto(s)
Membrana Celular/metabolismo , Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Brasinoesteroides , Células Cultivadas , Colestanoles/metabolismo , Colestanoles/farmacología , Colorantes Fluorescentes/química , Silenciador del Gen , Immunoblotting , Inmunoprecipitación , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Fosforilación/efectos de los fármacos , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Quinasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esteroides Heterocíclicos/metabolismo , Esteroides Heterocíclicos/farmacología , Treonina/metabolismo , Nicotiana/citología , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
In this study, we introduce the Micro-Electrode Ion Flux Estimation technique as a sensitive and accurate technique to study systemin-induced changes in ion fluxes from isolated nearly intact plant tissues. Our results demonstrate the effectiveness and value of the Micro-Electrode Ion Flux Estimation technique to monitor and characterize those elicitor-induced ion flux changes from intact tissues. We used the method to monitor the systemin-induced changes in ion fluxes from leaf tissue of various plant species, including wild-type and cu3 mutant tomato (Solanum pimpinellifolium) plants, and confirm previous observations, but now in intact leaf tissue. Upon exposure of leaf tissue of plant species from the subtribe solaneae to systemin, the H(+) influx and K(+) efflux were transiently strongly increased. Plant species of other clades did not show a response upon systemin exposure. Although it has been reported that the gene containing the cu3 null mutation is identical to the SR160/tBRI1 gene, which encodes the systemin/brassinosteroid receptor and is essential in systemin and brassinosteroid perception, we observed no differences in the response of H(+) and K(+) fluxes from both wild-type and mutant leaf tissue to systemin. Also, the effects of various pharmacological effectors on systemin-induced flux changes were similar. Moreover, a SR160/tBRI1 transgene-containing tobacco (Nicotiana tabacum) line was insensitive to systemin, whereas both this line and its wild-type predecessor were responsive to the elicitor flg22. Our results support the conclusion that the Cu3 receptor of tomato is not the systemin receptor, and, hence, another receptor is the principal systemin receptor.
Asunto(s)
Péptidos/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Solanum/efectos de los fármacos , Solanum/genética , Carbazoles/farmacología , Conductividad Eléctrica , Electrodos , Regulación de la Expresión Génica de las Plantas/fisiología , Alcaloides Indólicos/farmacología , Iones/metabolismo , Lantano/farmacología , Mutación , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Potasio/metabolismo , Protones , Solanum/metabolismo , Suramina/farmacología , Factores de Tiempo , Nicotiana/genética , Nicotiana/crecimiento & desarrolloRESUMEN
Systemin is a wound-signaling peptide that mediates defenses of tomato plants against herbivorous insects. Perception of systemin by the membrane-bound receptor SR160 results in activation of MAPKs, synthesis of jasmonic acid (JA), and expression of defense genes. To test the function of MAPKs in the response to systemin, we used virus-induced gene silencing (VIGS) in plants that overexpress the systemin precursor prosystemin (35S::prosys plants). These transgenic plants accumulate high levels of defense proteins and exhibit increased resistance to herbivorous insects. Cosilencing of the MAPKs MPK1 and MPK2 reduced MPK1/2 kinase activity, JA biosynthesis, and expression of JA-dependent defense genes. Application of methyl-JA restored the full defense response. These data show that MPK1 and MPK2 are essential components of the systemin signaling pathway and most likely function upstream of JA biosynthesis. MPK1 and MPK2 are 95% identical at the amino acid level. Specific VIGS of only MPK1 or MPK2 resulted in the same reduction of defense gene expression as cosilencing of MPK1 and MPK2, indicating that gene dosage effects may be important for MPK signaling. In addition, VIGS of the closely related MPK3 also reduced systemin-induced defense responses. The function of MPK1/2 and orthologs in pathogen-induced defenses is well established. Here we show that cosilencing of MPK1 and MPK2 compromised prosystemin-mediated resistance to Manduca sexta (Lepidoptera) herbivory, demonstrating that MPK1 and MPK2 are also required for successful defenses against herbivorous insects.
Asunto(s)
Manduca/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Péptidos/metabolismo , Solanum lycopersicum/enzimología , Solanum lycopersicum/inmunología , Animales , Ciclopentanos/metabolismo , Activación Enzimática , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Inmunidad Innata , Larva , Solanum lycopersicum/genética , Solanum lycopersicum/parasitología , OxilipinasRESUMEN
Tomato (Solanum lycopersicum) plants with the Cf-4 resistance gene recognize strains of the pathogenic fungus Cladosporium fulvum that secrete the avirulence protein Avr4. Transgenic tomato seedlings coexpressing Cf-4 and Avr4 mount a hypersensitive response (HR) at 20 degrees C, which is suppressed at 33 degrees C. Within 120 min after a shift from 33 degrees C to 20 degrees C, tomato mitogen-activated protein (MAP) kinase (LeMPK) activity increases in Cf-4/Avr4 seedlings. Searching tomato genome databases revealed at least 16 LeMPK sequences, including the sequence of LeMPK1, LeMPK2, and LeMPK3 that cluster with biotic stress-related MAP kinase orthologs from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). LeMPK1, LeMPK2, and LeMPK3 are simultaneously activated in Cf-4/Avr4 seedlings, and, to reveal whether they are functionally redundant or not, recombinant LeMPKs were incubated on PepChip Kinomics slides carrying peptides with potential phosphorylation sites. Phosphorylated peptides and motifs present in them discriminated between the phosphorylation specificities of LeMPK1, LeMPK2, and LeMPK3. LeMPK1, LeMPK2, or LeMPK3 activity was specifically suppressed in Cf-4-tomato by virus-induced gene silencing and leaflets were either injected with Avr4 or challenged with C. fulvum-secreting Avr4. The results of these experiments suggested that the LeMPKs have different but also overlapping roles with regard to HR and full resistance in tomato.
Asunto(s)
Cladosporium/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Solanum lycopersicum/enzimología , Solanum lycopersicum/microbiología , Familia de Multigenes , Fosforilación , Enfermedades de las Plantas , Proteínas de Plantas/metabolismo , Transducción de Señal/fisiología , Especificidad por SustratoRESUMEN
Leaf wounding and the wound signaling peptide systemin induce expression of wound response genes while the fungal toxin fusicoccin (FC) induces expression of pathogenesis-related genes. Consistent with their functional differences, FC and systemin regulate the extracellular pH in opposite ways, with systemin inducing an alkalinization and FC an acidification response. Here we show that systemin, wounding and FC activate the same mitogen-activated protein kinases (MAPKs; MPKs) MPK1 and 2 in tomato (Lycopersicon esculentum) leaves and L. peruvianum suspension-cultured cells. Wounding and FC activated an additional MAPK, MPK3. Pronounced differences were observed with regard to MAPK activation kinetics. FC induced prolonged, and systemin transient activity of the MAPKs. This shows that functionally different elicitors engage the same signaling components, yet induce signal-specific activation dynamics. A comparative analysis of pH effects and MAPK activity in response to specific treatments revealed that the kinetics of pH changes and MAPK activation did not correlate. Simultaneous application of FC and systemin did not lead to immediate pH changes but resulted in rapid increases in MAPK activity. Furthermore, changes in extracellular pH could be induced without concomitant MAPK activation by exchanging conditioned medium with fresh medium. This shows that changes in the extracellular pH are neither required nor sufficient for MAPK activation, suggesting that signaling pathways involving MAPKs and extracellular pH changes operate in parallel and are not part of the same linear pathway.
Asunto(s)
Glicósidos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Péptidos/farmacología , Solanum lycopersicum/metabolismo , Activación Enzimática , Líquido Extracelular/química , Concentración de Iones de Hidrógeno , Micotoxinas/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Proteínas de Plantas/farmacologíaRESUMEN
Plant respiratory burst oxidase homologs (Rboh) are homologs of the human neutrophil pathogen-related gp91(phox). Antisense technology was employed to ascertain the biological function of Lycopersicon esculentum (tomato) Rboh. Lines with diminished Rboh activity showed a reduced level of reactive oxygen species (ROS) in the leaf, implying a role for Rboh in establishing the cellular redox milieu. Surprisingly, the antisense plants acquired a highly branched phenotype, switched from indeterminate to determinate growth habit, and had fasciated reproductive organs. Wound-induced systemic expression of proteinase inhibitor II was compromised in the antisense lines, indicating that ROS intermediates supplied by Rboh are required for this wound response. Extending these observations by transcriptome analysis revealed ectopic leaf expression of homeotic MADS box genes that are normally expressed only in reproductive organs. In addition, both Rboh-dependent and -independent wound-induced gene induction was detected as well as transcript changes related to redox maintenance. The results provide novel insights into how the steady state cellular level of ROS is controlled and portrays the role of Rboh as a signal transducer of stress and developmental responses.
Asunto(s)
NADPH Oxidasas/metabolismo , Solanum lycopersicum/metabolismo , Secuencia de Bases , ADN sin Sentido/genética , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , NADPH Oxidasas/genética , Fenotipo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
We tested whether signaling pathways induced by systemin, oligosaccharide elicitors (OEs), and ultraviolet (UV)-B radiation share common components in Lycopersicon peruvianum suspension-cultured cells. These stress signals all induce mitogen-activated protein kinase (MAPK) activity. In desensitization assays, we found that pretreatment with systemin and OEs transiently reduced the MAPK response to a subsequent treatment with the same or a different elicitor. In contrast, MAPK activity in response to UV-B increased after pretreatment with systemin and OEs. These experiments demonstrate the presence of signaling components that are shared by systemin, OEs, and UV-B. Based on desensitization assays, it is not clear if the same or different MAPKs are activated by different stress signals. To identify specific stress-responsive MAPKs, we cloned three MAPKs from a tomato (Lycopersicon esculentum) leaf cDNA library, generated member-specific antibodies, and performed immunocomplex kinase assays with extracts from elicited L. peruvianum cells. Two highly homologous MAPKs, LeMPK1 and LeMPK2, were activated in response to systemin, four different OEs, and UV-B radiation. An additional MAPK, LeMPK3, was only activated by UV-B radiation. The common activation of LeMPK1 and LeMPK2 by many stress signals is consistent with the desensitization assays and may account for substantial overlaps among stress responses. On the other hand, MAPK activation kinetics in response to elicitors and UV-B differed substantially, and UV-B activated a different set of LeMPKs than the elicitors. These differences may account for UV-B-specific responses.