RESUMO
Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the QS regulation in the bacteria-yeast interactions.
Assuntos
Percepção de Quorum , Saccharomycetales , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteômica , Saccharomycetales/genética , Saccharomycetales/metabolismoRESUMO
Plasmids are widely distributed in rhizobia, a group of bacteria able to establish symbiotic relationships with the roots of legume plants. Two types of conjugative transfer (CT) regulation of these elements have been described in more detail. The most prevalent is through Quorum-Sensing (QS), mediated by the interaction of the TraR regulator protein and its cognate acyl-homoserine lactone (AHL) synthesized by TraI. In this study, we analyzed rhizobial plasmids classified according to their TraR regulators into four different groups. Each group has a particular genomic architecture. In one of the groups (I-C), represented by pLPU83a from Rhizobium favelukesii LPU83, CT induction requires TraR. With manual annotation, a traI was located in the plasmid distant to the traR gene. These features make pLPU83a an interesting plasmid for studying novel mechanisms of CT regulation. We mutagenized the traI gene, and found that it does not participate in CT regulation. Furthermore, we studied whether pLPU83a is subject to QS regulation by determining CT at different growth stages (cell densities). Our results showed no positive correlation between increase in culture densities and CT induction, on the contrary a slight decrease in CT was found at higher culture densities, unlike other TraR-depending plasmids. Our results show that transfer of pLPU83a is not regulated in a QS-dependent manner, and suggest that molecules not yet identified may activate its CT. Also, accumulation of a putative inhibitor cannot be disregarded.
Assuntos
Agrobacterium tumefaciens/genética , Conjugação Genética , Plasmídeos/química , Percepção de Quorum/genética , Rhizobium/genética , Acil-Butirolactonas/metabolismo , Agrobacterium tumefaciens/metabolismo , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fabaceae/microbiologia , Anotação de Sequência Molecular , Filogenia , Raízes de Plantas/microbiologia , Plasmídeos/classificação , Plasmídeos/metabolismo , Rhizobium/metabolismo , Simbiose/genéticaRESUMO
AIMS: The aim of this research was to analyse the quorum-sensing (QS) and quorum-quenching (QQ) mechanisms based on N-acyl-l-homoserine lactones (AHLs) in Azospirillum brasilense Az39, a strain with remarkable capacity to benefit a wide range of crops under agronomic conditions. METHODS AND RESULTS: We performed an in silico and in vitro analysis of the quorum mechanisms in A. brasilense Az39. The results obtained in vitro using the reporter strains Chromobacterium violaceum and Agrobacterium tumefaciens and liquid chromatography coupled with mass-mass spectrometry analysis showed that although Az39 does not produce AHL molecules, it is capable of degrading them by at least two hypothetical enzymes identified by bioinformatics approach, associated with the bacterial cell. In Az39 cultures supplemented with 500 nmol l-1 of the C3 unsubstituted AHLs (C4, C6, C8, C10, C12, C14), AHL levels were lower than in noninoculated LB media controls. Similar results were observed upon the addition of AHLs with hydroxy (OH-) and keto (oxo-) substitutions in C3. These results not only demonstrate the ability of Az39 to degrade AHLs. They also show the wide spectrum of molecules that can be degraded by this bacterium. CONCLUSIONS: Although A. brasilense Az39 is a silent bacterium unable to produce AHL signals, it is able to interrupt the communications between other bacteria and/or plants by a QQ activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report confirming by unequivocal methodology the ability of A. brasilense, one of the most agriculturally used benefic bacteria around the world, to degrade AHLs by a QQ mechanism.
Assuntos
Acil-Butirolactonas/metabolismo , Azospirillum brasilense/fisiologia , Percepção de Quorum/fisiologia , Agrobacterium tumefaciens/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida , Chromobacterium/metabolismo , Espectrometria de Massas , Percepção de Quorum/genéticaRESUMO
The constant demand for new antibiotic drugs has driven efforts by the scientific community to prospect for peptides with a broad spectrum of action. In this context, antimicrobial peptides (AMPs) have acquired great scientific importance in recent years due to their ability to possess antimicrobial and immunomodulatory activity. In the last two decades, plants have attracted the interest of the scientific community and industry as regards their potential as biofactories of heterologous proteins. One of the most promising approaches is the use of viral vectors to maximize the transient expression of drugs in the leaves of the plant Nicotiana benthamiana. Recently, the MagnifectionTM expression system was launched. This sophisticated commercial platform allows the assembly of the viral particle in leaf cells and the systemic spread of heterologous protein biosynthesis in green tissues caused by Agrobacterium tumefaciens "gene delivery method". The system also presents increased gene expression levels mediated by potent viral expression machinery. These characteristics allow the mass recovery of heterologous proteins in the leaves of N. benthamiana in 8 to 10 days. This system was highly efficient for the synthesis of different classes of pharmacological proteins and contains enormous potential for the rapid and abundant biosynthesis of AMPs.
Assuntos
Agrobacterium tumefaciens/metabolismo , Peptídeos Catiônicos Antimicrobianos/biossíntese , Agricultura Molecular/métodos , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Biossíntese de Proteínas , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biotecnologia/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismoRESUMO
Pentalinon andrieuxii is a species used in Mayan traditional medicine due to its biological properties. Recent studies indicate that it produces a pentacyclic triterpene-denominated betulinic acid, which presents various biological activities: antibacterial, antifungal, antiplasmodial, anti-inflammatory, antimalarial, anticancer, leishmanicidal, and antiviral, as well as steroids and sterols with leishmanicidal properties. A recent study also reported the presence of urechitol A and B in the roots; these are secondary metabolites whose biochemical function is as yet unknown. This plant therefore represents a natural source of metabolites with potential application in the pharmaceutical industry. In this chapter, a protocol is described for obtaining transgenic plants, at the reporter gene of the ß-glucuronidase (GUS) via Agrobacterium tumefaciens from hypocotyl and root explants. The protocol established herein could be employed for the manipulation of the genes involved in the biosynthesis of isoprenoids or secondary metabolites of interest. To our knowledge, this is the first report of stable transformation of Pentalinon andrieuxii via Agrobacterium tumefaciens.
Assuntos
Apocynaceae/genética , Técnicas de Cultura de Tecidos/métodos , Transformação Genética , Adaptação Fisiológica , Agrobacterium tumefaciens/metabolismo , Meios de Cultura/química , Genes Reporter , Germinação/efeitos dos fármacos , Glucuronidase/metabolismo , Hipocótilo/crescimento & desenvolvimento , Canamicina/farmacologia , Brotos de Planta/fisiologia , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Sementes/fisiologiaRESUMO
Plant-based platforms are extensively use for the expression of recombinant proteins, including monoclonal antibodies (mAbs). Generally, immunoglobulins (Igs) are sorted to the apoplast, which is often afflicted with intense proteolysis. Here, we describe methods to transiently express mAbs sorted to central vacuole in Nicotiana benthamiana leaves and to characterize the obtained IgG. Central vacuole is an appropriate compartment for the efficient production of Abs, consequently vacuolar sorting should be considered as an alternative strategy to obtain high protein yields.
Assuntos
Anticorpos Monoclonais/análise , Imunoglobulina G/análise , Nicotiana/genética , Vacúolos/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Expressão Gênica , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Espectrometria de Massas/métodos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismo , Vacúolos/metabolismoRESUMO
Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier.
Assuntos
Basidiomycota/metabolismo , Proteínas Fúngicas/genética , Lentinula/genética , Lentinula/metabolismo , Transformação Genética , Basidiomycota/enzimologia , Leveduras , Proteínas Fúngicas/metabolismo , Southern Blotting , Clonagem Molecular , Agrobacterium tumefaciens/metabolismo , Análise de Sequência , Emulsificantes , Eletroforese em Gel de Poliacrilamida , Reação em Cadeia da Polimerase em Tempo Real , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Microscopia de FluorescênciaRESUMO
To cope with toxic levels of H2S, the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens employ the bigR operon to oxidize H2S into sulfite. The bigR operon is regulated by the transcriptional repressor BigR and it encodes a bifunctional sulfur transferase (ST) and sulfur dioxygenase (SDO) enzyme, Blh, required for H2S oxidation and bacterial growth under hypoxia. However, how Blh operates to enhance bacterial survival under hypoxia and how BigR is deactivated to derepress operon transcription is unknown. Here, we show that the ST and SDO activities of Blh are in vitro coupled and necessary to oxidize sulfide into sulfite, and that Blh is critical to maintain the oxygen flux during A. tumefaciens respiration when oxygen becomes limited to cells. We also show that H2S and polysulfides inactivate BigR leading to operon transcription. Moreover, we show that sulfite, which is produced by Blh in the ST and SDO reactions, is toxic to Citrus sinensis and that X. fastidiosa-infected plants accumulate sulfite and higher transcript levels of sulfite detoxification enzymes, suggesting that they are under sulfite stress. These results indicate that BigR acts as a sulfide sensor in the H2S oxidation mechanism that allows pathogens to colonize plant tissues where oxygen is a limiting factor.
Assuntos
Agrobacterium tumefaciens/genética , Dioxigenases/genética , Transferases/genética , Xylella/genética , Agrobacterium tumefaciens/metabolismo , Dioxigenases/química , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/toxicidade , Hipóxia/genética , Hipóxia/metabolismo , Óperon/genética , Oxigênio/metabolismo , Plantas/genética , Plantas/microbiologia , Estresse Fisiológico/genética , Sulfetos/química , Transferases/química , Xylella/metabolismoRESUMO
Mal de Río Cuarto virus (MRCV) is a member of the Fijivirus genus, within the Reoviridae family, that replicates and assembles in cytoplasmic inclusion bodies called viroplasms. In this study, we investigated interactions between ten MRCV proteins by yeast two-hybrid (Y2H) assays and identified interactions of non-structural proteins P6/P6, P9-2/P9-2 and P6/P9-1. P9-1 and P6 are the major and minor components of the viroplasms respectively, whereas P9-2 is an N-glycosylated membrane protein of unknown function. Interactions involving P6 and P9-1 were confirmed by bimolecular fluorescence complementation (BiFC) in rice protoplasts. We demonstrated that a region including a predicted coiled-coil domain within the C-terminal moiety of P6 was necessary for P6/P6 and P6/P9-1 interactions. In turn, a short C-terminal arm was necessary for the previously reported P9-1 self-interaction. Transient expression of these proteins by agroinfiltration of Nicotiana benthamiana leaves showed very low accumulation levels and further in silico analyses allowed us to identify conserved PEST degradation sequences [rich in proline (P), glutamic acid (E), serine (S), and threonine (T)] within P6 and P9-1. The removal of these PEST sequences resulted in a significant increase of the accumulation of both proteins.
Assuntos
Interações Hospedeiro-Patógeno , Corpos de Inclusão/virologia , Folhas de Planta/virologia , Protoplastos/virologia , Reoviridae/genética , Proteínas não Estruturais Virais/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Expressão Gênica , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Oryza/virologia , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteólise , Protoplastos/metabolismo , Protoplastos/ultraestrutura , Reoviridae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Nicotiana/virologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Here, we report a quick and low-cost method to improve plant transformation using Agrobacterium tumefaciens. This method involves the use of physical wounding, ultrasound, and an increase in exposure time to the bacteria. We show how the transformation rate increased from 0 to 14% when an ultrasound pulse of 10 s was used in conjunction with 96 h of bacterial exposure in Eclipta alba explants.