RESUMEN
Beneficial interactions between plant roots and Trichoderma species lead to both local and systemic enhancements of the plant immune system through a mechanism known as priming of defenses. Previously, we have reported a number of genes and proteins that are differentially regulated in distant tissues of maize plants following inoculation with Trichoderma atroviride. To further investigate the mechanisms involved in the systemic activation of plant responses, here we have further evaluated the regulatory aspects of a selected group of genes when priming is triggered in maize plants. Time-course experiments from the beginning of the interaction between T. atroviride and maize roots followed by leaf infection with Colletotrichum graminicola allowed us to identify a gene set regulated by priming in the leaf tissue. In the same experiment, phytohormone measurements revealed a decrease in jasmonic acid concentration while salicylic acid increased at 2 d and 6 d post-inoculation. In addition, chromatin structure and modification assays showed that chromatin was more open in the primed state compared with unprimed control conditions, and this allowed for quicker gene activation in response to pathogen attack. Overall, the results allowed us to gain insights on the interplay between the phytohormones and epigenetic regulatory events in the systemic and long-lasting regulation of maize plant defenses following Trichoderma inoculation.
Asunto(s)
Trichoderma , Zea mays , Zea mays/genética , Zea mays/metabolismo , Trichoderma/genética , Trichoderma/metabolismo , Ácido Salicílico/metabolismo , Hojas de la Planta/metabolismo , Enfermedades de las Plantas/genética , Raíces de Plantas/metabolismoRESUMEN
MAIN CONCLUSION: Trichoderma activates plant proteins to counteract Fusarium infection. Comparison between proteomic and transcriptomic data suggests differential response regulation. Proteins from the phenylpropanoid pathway are activated to quickly respond to pathogen attack. Trichoderma species can stimulate local and distant immune responses in colonized plant tissues to prevent future pathogenic attacks. Priming of plant defenses is characterized by changes in transcriptional, metabolic, and epigenetic states after stimulus perception. We have previously investigated transcriptional reprogramming in silk tissues from maize plants inoculated with Trichoderma atroviride and challenged with Fusarium verticillioides (Agostini et al., Mol Plant-Microbe In 32:95-106, 2019). To better understand the molecular changes induced by T. atroviride in maize, a proteomic approach was conducted in this instance. Several proteins belonging to different metabolic categories were detected as priming-involved proteins. However, we detected a very low correlation with those priming-modulated transcripts suggesting the importance of regulatory events a posteriori of the transcriptional process to accomplish the final goal of blocking pathogen entry. Specifically, we focused on the phenylpropanoid pathway, since we detected several proteins that are upregulated in the priming state and might explain cell wall reinforcement as well as the increase in flavonoid and lignin content in maize silks after activation of induced systemic resistance.
Asunto(s)
Trichoderma , Zea mays , Fusarium , Hypocreales , Enfermedades de las Plantas , Proteoma , Proteómica , SedaRESUMEN
Detoxification of methylglyoxal, a toxic by-product of central sugar metabolism, is a major issue for all forms of life. The glyoxalase pathway evolved to effectively convert methylglyoxal into d-lactate via a glutathione hemithioacetal intermediate. Recently, we have shown that the monomeric glyoxalase I from maize exhibits a symmetric fold with two cavities, potentially harboring two active sites, in analogy with homodimeric enzyme surrogates. Here we confirm that only one of the two cavities exhibits glyoxalase I activity and show that it adopts a tunnel-shaped structure upon substrate binding. Such conformational change gives rise to independent binding sites for glutathione and methylglyoxal in the same active site, with important implications for the molecular reaction mechanism, which has been a matter of debate for several decades. DATABASE: Structural data are available in The Protein Data Bank database under the accession numbers 6BNN, 6BNX, and 6BNZ.
Asunto(s)
Lactoilglutatión Liasa/química , Sustancias Macromoleculares/química , Conformación Proteica , Zea mays/enzimología , Secuencia de Aminoácidos/genética , Dominio Catalítico/genética , Lactoilglutatión Liasa/genética , Lactoilglutatión Liasa/ultraestructura , Sustancias Macromoleculares/ultraestructura , Pliegue de Proteína , Piruvaldehído/química , Especificidad por Sustrato , Azúcares/metabolismoRESUMEN
The glyoxalase system is ubiquitous among all forms of life owing to its central role in relieving the cell from the accumulation of methylglyoxal, a toxic metabolic byproduct. In higher plants, this system is upregulated under diverse metabolic stress conditions, such as in the defence response to infection by pathogenic microorganisms. Despite their proven fundamental role in metabolic stresses, plant glyoxalases have been poorly studied. In this work, glyoxalase I from Zea mays has been characterized both biochemically and structurally, thus reporting the first atomic model of a glyoxalase I available from plants. The results indicate that this enzyme comprises a single polypeptide with two structurally similar domains, giving rise to two lateral concavities, one of which harbours a functional nickel(II)-binding active site. The putative function of the remaining cryptic active site remains to be determined.
Asunto(s)
Lactoilglutatión Liasa/química , Zea mays/química , Zea mays/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Lactoilglutatión Liasa/genética , Lactoilglutatión Liasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Níquel/metabolismo , Conformación Proteica , Alineación de Secuencia , Zea mays/genética , Zea mays/metabolismoRESUMEN
Fusarium verticillioides causes ear rot and grain mycotoxins in maize (Zea mays L.), which are harmful to human and animal health. Breeding and growing less susceptible plant genotypes is one alternative to reduce these detrimental effects. A better understanding of the resistance mechanisms would facilitate the implementation of strategic molecular agriculture to breeding of resistant germplasm. Our aim was to identify genes and metabolites that may be related to the Fusarium reaction in a resistant (L4637) and a susceptible (L4674) inbred. Gene expression data were obtained from microarray hybridizations in inoculated and non-inoculated kernels from both inbreds. Fungal inoculation did not produce considerable changes in gene expression and metabolites in L4637. Defense-related genes changed in L4674 kernels, responding specifically to the pathogen infection. These results indicate that L4637 resistance may be mainly due to constitutive defense mechanisms preventing fungal infection. These mechanisms seem to be poorly expressed in L4674; and despite the inoculation activate a defense response; this is not enough to prevent the disease progress in this susceptible line. Through this study, a global view of differential genes expressed and metabolites accumulated during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize.
Asunto(s)
Resistencia a la Enfermedad/genética , Fusarium/patogenicidad , Enfermedades de las Plantas/genética , Transcriptoma/fisiología , Zea mays/genética , Zea mays/metabolismo , Cruzamiento , Ergosterol/metabolismo , Fumonisinas/metabolismo , Fusarium/metabolismo , Regulación de la Expresión Génica de las Plantas , Semillas/metabolismo , Zea mays/microbiologíaRESUMEN
The Escherichia coli genes pta and eutD encode proteins containing the phosphate-acetyltransferase domain. EutD is composed only by this domain and belongs to the ethanolamine operon. This enzyme has not been characterized yet, and its relationship to the multimodular E. coli phosphotransacetylase (Pta) remains unclear. In the present work, a detailed characterization of EutD from E. coli (EcEutD) was performed. The enzyme is a more efficient phosphotransacetylase than E. coli Pta (EcPta) in catalyzing its reaction in either direction and assembles as a dimer, being differentially modulated by EcPta effectors. When comparing EutD and Pta, both from E. coli, certain divergent regions of the primary structure responsible for their unique properties can be found. The growth on acetate of the E. coli pta acs double-mutant strain, was complemented by either introducing EcEutD or by inducing the eut operon with ethanolamine. In this case, the expression of a phosphotransacetylase different from Pta was confirmed by activity assays. Overall, the results indicate that EcEutD and Pta, although able to catalyse the same reaction, display differential efficiency and regulation, and also differ in the induction of their expression. However, under certain growth conditions, they can fulfil equal roles in E. coli metabolism.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Etanolamina/metabolismo , Fosfato Acetiltransferasa/metabolismo , Acetatos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Redes y Vías Metabólicas , Modelos Biológicos , Operón , Fosfato Acetiltransferasa/genética , Multimerización de ProteínaRESUMEN
The metallo-ß-lactamase (MßL) superfamily is a functionally diverse group of metalloproteins sharing a distinctive αß/αß fold and a characteristic metal binding motif. A large number of open reading frames identified in genomic sequencing efforts have been annotated as members of this superfamily through sequence comparisons. However, structural and functional studies performed on purified proteins are normally needed to unequivocally include a newly discovered protein in the MßL superfamily. Here we report the spectroscopic characterization of recombinant YcbL, a gene product annotated as a member of the MßL superfamily whose function in vivo remains unknown. By taking advantage of the structural features characterizing the MßL superfamily metal binding motif, we performed spectroscopic studies on Zn(II)- and Co(II)-substituted YcbL to structurally interrogate the metal binding site. The dinuclear center in Co(II)-YcbL was shown to display characteristic electronic absorption features in the visible region, which were also observed in an engineered MßL aimed at mimicking this metal site. Thus, the spectroscopic features reported herein can be employed as a signature to readily identify and characterize the presence of these ubiquitous metal binding sites.
Asunto(s)
Cobalto/química , Metaloproteínas/química , Metaloproteínas/metabolismo , Zinc/química , beta-Lactamasas/química , beta-Lactamasas/metabolismo , Sitios de Unión , Cobalto/metabolismo , Espectroscopía de Resonancia Magnética , Metaloproteínas/genética , Modelos Moleculares , Conformación Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometría , Estereoisomerismo , Espectroscopía de Absorción de Rayos X , Zinc/metabolismo , beta-Lactamasas/genéticaRESUMEN
Glyoxalase II (GLX2, EC 3.1.2.6., hydroxyacylglutathione hydrolase) is a metalloenzyme involved in crucial detoxification pathways. Different studies have failed in identifying the native metal ion of this enzyme, which is expressed with iron, zinc and/or manganese. Here we report that GloB, the GLX2 from Salmonella typhimurium, is differentially inhibited by glutathione (a reaction product) depending on the bound metal ion, and we provide a structural model for this inhibition mode. This metal-dependent inhibition was shown to occur in metal-enriched forms of the enzyme, complementing the spectroscopic data. Based on the high levels of free glutathione in the cell, we suggest that the expression of the different metal forms of GLX2 during Salmonella infection could be exploited as a mechanism to regulate the enzyme activity.
Asunto(s)
Metales Pesados/farmacología , Tioléster Hidrolasas/antagonistas & inhibidores , Glutatión/farmacología , Hierro , Cinética , Manganeso , Metaloproteínas/antagonistas & inhibidores , Infecciones por Salmonella , Salmonella typhimurium , ZincRESUMEN
Glyoxalase II is a hydrolytic enzyme part of the glyoxalase system, responsible for detoxifying several cytotoxic compounds employing glutathione. Glyoxalase II belongs to the superfamily of metallo-beta-lactamases, with a conserved motif able to bind up to two metal ions in their active sites, generally zinc. Instead, several eukaryotic glyoxalases II have been characterized with different ratios of iron, zinc, and manganese ions. We have expressed a gene coding for a putative member of this enzyme superfamily from Salmonella typhimurium that we demonstrate, on the basis of its activity, to be a glyoxalase II, named GloB. Recombinant GloB expressed in Escherichia coli was purified with variable amounts of iron, zinc, and manganese. All forms display similar activities, as can be shown from protein expression in minimal medium supplemented with specific metal ions. The crystal structure of GloB solved at 1.4 A shows a protein fold and active site similar to those of its eukaryotic homologues. NMR and EPR experiments also reveal a conserved electronic structure at the metal site. GloB is therefore able to accommodate these different metal ions and to carry out the hydrolytic reaction with similar efficiencies in all cases. The metal promiscuity of this enzyme (in contrast to other members of the same superfamily) can be accounted for by the presence of a conserved Asp residue acting as a second-shell ligand that is expected to increase the hardness of the metal binding site, therefore favoring iron uptake in glyoxalases II.
Asunto(s)
Metales/metabolismo , Salmonella typhimurium/enzimología , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismo , Sitios de Unión , Escherichia coli/genética , Cinética , Espectroscopía de Resonancia Magnética , Metales/química , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Especificidad por Sustrato , Tioléster Hidrolasas/genética , Zinc/química , Zinc/metabolismoRESUMEN
Malic enzymes catalyze the oxidative decarboxylation of l-malate to yield pyruvate, CO(2), and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are involved in a wide range of metabolic pathways. The C(4)-specific NADP-ME has evolved from C(3)-type malic enzymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and regulatory properties. In the present study, the mature C(4)-specific NADP-ME of maize was expressed in Escherichia coli. The recombinant enzyme has essentially the same physicochemical properties and K(m) for the substrates as those of the naturally occurring NADP-ME previously characterized. However, the k(cat) was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inactive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and purified to homogeneity. The participation of these residues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimensional model of maize C(4) NADP-ME based on crystallographic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.