RESUMEN

Several species of the Agrobacterium genus represent unique bacterial pathogens able to genetically transform plants, by transferring and integrating a segment of their own DNA (T-DNA, transferred DNA) in their host genome. Whereas in nature this process results in uncontrolled growth of the infected plant cells (tumors), this capability of Agrobacterium has been widely used as a crucial tool to generate transgenic plants, for research and biotechnology. The virulence of Agrobacterium relies on a series of virulence genes, mostly encoded on a large plasmid (Ti-plasmid, tumor inducing plasmid), involved in the different steps of the DNA transfer to the host cell genome: activation of bacterial virulence, synthesis and export of the T-DNA and its associated proteins, intracellular trafficking of the T-DNA and effector proteins in the host cell, and integration of the T-DNA in the host genomic DNA. Multiple interactions between these bacterial encoded proteins and host factors occur during the infection process, which determine the outcome of the infection. Here, we review our current knowledge of the mechanisms by which bacterial and plant factors control Agrobacterium virulence and host plant susceptibility.

Asunto(s)

Agrobacterium tumefaciens , Bacterias , Virulencia/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Plantas Modificadas Genéticamente/genética , Plásmidos , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo

RESUMEN

Agrobacterium T-DNA (transfer DNA) integration into the plant genome relies mostly on host proteins involved in the DNA damage repair pathways. However, conflicting results have been obtained using plants with mutated or down-regulated genes involved in these pathways. Here, we chose a different approach by following the expression of a series of genes, encoding proteins involved in the DNA damage response, during early stages of Agrobacterium infection in tobacco. First, we identified tobacco homologs of Arabidopsis genes induced upon DNA damage and demonstrated that their expression was activated by bleomycin, a DNA-break causing agent. Then, we showed that Agrobacterium infection induces the expression of several of these genes markers of the host DNA damage response, with different patterns of transcriptional response. This induction largely depends on Agrobacterium virulence factors, but not on the T-DNA, suggesting that the DNA damage response activation may rely on Agrobacterium-encoded virulence proteins. Our results suggest that Agrobacterium modulates the plant DNA damage response machinery, which might facilitate the integration of the bacterial T-DNA into the DNA breaks in the host genome.

Asunto(s)

Agrobacterium tumefaciens/genética , Proteínas Bacterianas/metabolismo , Daño del ADN , Regulación de la Expresión Génica de las Plantas , Nicotiana/genética , Factores de Virulencia/metabolismo , Agrobacterium tumefaciens/aislamiento & purificación , Agrobacterium tumefaciens/metabolismo , Agrobacterium tumefaciens/patogenicidad , Proteínas Bacterianas/genética , Genes de Plantas , Nicotiana/metabolismo , Nicotiana/microbiología , Transformación Genética , Factores de Virulencia/genética

RESUMEN

Since its inception in the late 1980s, the delivery of exogenous nucleic acids into living cells via high-velocity microprojectiles (biolistic, or microparticle bombardment) has been an invaluable tool for both agricultural and fundamental plant research. Here, we review the technical aspects and the major applications of the biolistic method for studies involving transient gene expression in plant cells. These studies cover multiple areas of plant research, including gene expression, protein subcellular localization and cell-to-cell movement, plant virology, silencing, and the more recently developed targeted genome editing via transient expression of customized endonucleases.

Asunto(s)

Biolística/métodos , Regulación de la Expresión Génica de las Plantas , Plantas/genética , Edición Génica , Regiones Promotoras Genéticas/genética , Transgenes

RESUMEN

Seeds are central to plant life cycle and to human nutrition, functioning as the major supplier of human population energy intake. To understand better the roles of enzymic writers and erasers of the epigenetic marks, in particular, histone ubiquitylation and the corresponding histone modifiers, involved in control of seed development, we identified the otubain-like cysteine protease OTU1 as a histone deubiquitinase involved in transcriptional repression of the DA1 and DA2 genes known to regulate seed and organ size in Arabidopsis. Loss-of-function mutants of OTU1 accumulate H2B monoubiquitylation and such euchromatic marks as H3 trimethylation and hyperacetylation in the DA1 and DA2 chromatin. These data advance our knowledge about epigenetic regulation of the DA1 and DA2 genes by recognizing OTU1 as a member of a putative repressor complex that negatively regulates their transcription.

RESUMEN

Genetic transformation of host plants by Agrobacterium tumefaciens and related species represents a unique model for natural horizontal gene transfer. Almost five decades of studying the molecular interactions between Agrobacterium and its host cells have yielded countless fundamental insights into bacterial and plant biology, even though several steps of the DNA transfer process remain poorly understood. Agrobacterium spp. may utilize different pathways for transferring DNA, which likely reflects the very wide host range of Agrobacterium. Furthermore, closely related bacterial species, such as rhizobia, are able to transfer DNA to host plant cells when they are provided with Agrobacterium DNA transfer machinery and T-DNA. Homologs of Agrobacterium virulence genes are found in many bacterial genomes, but only one non-Agrobacterium bacterial strain, Rhizobium etli CFN42, harbors a complete set of virulence genes and can mediate plant genetic transformation when carrying a T-DNA-containing plasmid.

Asunto(s)

Agrobacterium tumefaciens , Plantas , Bacterias , Proteínas Bacterianas , ADN Bacteriano , Transferencia de Gen Horizontal , Virulencia

RESUMEN

Besides the massive gene transfer from organelles to the nuclear genomes, which occurred during the early evolution of eukaryote lineages, the importance of horizontal gene transfer (HGT) in eukaryotes remains controversial. Yet, increasing amounts of genomic data reveal many cases of bacterium-to-eukaryote HGT that likely represent a significant force in adaptive evolution of eukaryotic species. However, DNA transfer involved in genetic transformation of plants by Agrobacterium species has traditionally been considered as the unique example of natural DNA transfer and integration into eukaryotic genomes. Recent discoveries indicate that the repertoire of donor bacterial species and of recipient eukaryotic hosts potentially are much wider than previously thought, including donor bacterial species, such as plant symbiotic nitrogen-fixing bacteria (e.g., Rhizobium etli) and animal bacterial pathogens (e.g., Bartonella henselae, Helicobacter pylori), and recipient species from virtually all eukaryotic clades. Here, we review the molecular pathways and potential mechanisms of these trans-kingdom HGT events and discuss their utilization in biotechnology and research.

Asunto(s)

Agrobacterium/genética , Agrobacterium/patogenicidad , Eucariontes/clasificación , Eucariontes/genética , Transferencia de Gen Horizontal , Transformación Genética , Animales , Plantas/genética , Plantas/microbiología

RESUMEN

T-DNA transfer from Agrobacterium to its host plant genome relies on multiple interactions between plant proteins and bacterial effectors. One such plant protein is the Arabidopsis VirE2 interacting protein (AtVIP1), a transcription factor that binds Agrobacterium tumefaciens C58 VirE2, potentially acting as an adaptor between VirE2 and several other host factors. It remains unknown, however, whether the same VirE2 protein has evolved to interact with multiple VIP1 homologues in the same host, and whether VirE2 homologues encoded by different bacterial strains/species recognize AtVIP1 or its homologues. Here, we addressed these questions by systematic analysis (using the yeast two-hybrid and co-immunoprecipitation approaches) of interactions between VirE2 proteins encoded by four major representatives of known bacterial species/strains with functional T-DNA transfer machineries and eight VIP1 homologues from Arabidopsis and tobacco. We also analysed the determinants of the VirE2 sequence involved in these interactions. These experiments showed that the VirE2 interaction is degenerate: the same VirE2 protein has evolved to interact with multiple VIP1 homologues in the same host, and different and mutually independent VirE2 domains are involved in interactions with different VIP1 homologues. Furthermore, the VIP1 functionality related to the interaction with VirE2 is independent of its function as a transcriptional regulator. These observations suggest that the ability of VirE2 to interact with VIP1 homologues is deeply ingrained into the process of Agrobacterium infection. Indeed, mutations that abolished VirE2 interaction with AtVIP1 produced no statistically significant effects on interactions with VIP1 homologues or on the efficiency of genetic transformation.

Asunto(s)

Agrobacterium tumefaciens/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Núcleo Celular/metabolismo , Filogenia , Unión Proteica , Elementos de Respuesta , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Nicotiana/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

Recently, Rhizobium etli, in addition to Agrobacterium spp., has emerged as a prokaryotic species whose genome encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we show that the signal phenolic molecule acetosyringone (AS) induces R. etli vir gene expression both in an R. etli background and in an Agrobacterium tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter transfer DNA (T-DNA). Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium but not from nonhost plants. The early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a than of T-DNA transfer of pTiC58 of A. tumefaciensIMPORTANCE The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by the genome of a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into the plant cell genome. In this study, we explored the transcriptional regulation and induction of virulence genes in R. etli and show similarities to and differences from those of their A. tumefaciens counterparts, contributing to an understanding and a comparison of these two systems. Whereas most vir genes in R. etli follow an induction pattern similar to that of A. tumefaciens vir genes, a few significant differences may at least in part explain the variations in T-DNA transfer efficiency.

Asunto(s)

Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Rhizobium etli/metabolismo , Activación Transcripcional/fisiología , Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/genética , Fabaceae/microbiología , Regiones Promotoras Genéticas , Rhizobium etli/genética , Nicotiana/microbiología , Virulencia

RESUMEN

Historically, the members of the Agrobacterium genus have been considered the only bacterial species naturally able to transfer and integrate DNA into the genomes of their eukaryotic hosts. Yet, increasing evidence suggests that this ability to genetically transform eukaryotic host cells might be more widespread in the bacterial world. Indeed, analyses of accumulating genomic data reveal cases of horizontal gene transfer from bacteria to eukaryotes and suggest that it represents a significant force in adaptive evolution of eukaryotic species. Specifically, recent reports indicate that bacteria other than Agrobacterium, such as Bartonella henselae (a zoonotic pathogen), Rhizobium etli (a plant-symbiotic bacterium related to Agrobacterium), or even Escherichia coli, have the ability to genetically transform their host cells under laboratory conditions. This DNA transfer relies on type IV secretion systems (T4SSs), the molecular machines that transport macromolecules during conjugative plasmid transfer and also during transport of proteins and/or DNA to the eukaryotic recipient cells. In this review article, we explore the extent of possible transfer of genetic information from bacteria to eukaryotic cells as well as the evolutionary implications and potential applications of this transfer.

Asunto(s)

Bacterias/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Eucariontes/genética , Transferencia de Gen Horizontal , Transformación Genética , Sistemas de Secreción Tipo IV/metabolismo

RESUMEN

Different strains and species of the soil phytopathogen Agrobacterium possess the ability to transfer and integrate a segment of DNA (T-DNA) into the genome of their eukaryotic hosts, which is mainly mediated by a set of virulence (vir) genes located on the bacterial Ti-plasmid that also contains the T-DNA. To date, Agrobacterium is considered to be unique in its capacity to mediate genetic transformation of eukaryotes. However, close homologs of the vir genes are encoded by the p42a plasmid of Rhizobium etli; this microorganism is related to Agrobacterium, but known only as a symbiotic bacterium that forms nitrogen-fixing nodules in several species of beans. Here, we show that R. etli can mediate functional DNA transfer and stable genetic transformation of plant cells, when provided with a plasmid containing a T-DNA segment. Thus, R. etli represents another bacterial species, besides Agrobacterium, that encodes a protein machinery for DNA transfer to eukaryotic cells and their subsequent genetic modification.

Asunto(s)

Bacterias/genética , Transferencia de Gen Horizontal , Rhizobium etli/genética , Proteínas Bacterianas/genética , ADN Bacteriano/genética , ADN de Plantas/genética , Genes Reporteros , Mutación , Plásmidos/genética , Virulencia

RESUMEN

During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

Asunto(s)

Agrobacterium/genética , Proteínas Bacterianas/genética , Proteínas F-Box/genética , Factores Reguladores del Interferón/genética , Plásmidos Inductores de Tumor en Plantas/genética , Proteínas Virales/genética , Agrobacterium/clasificación , Agrobacterium/metabolismo , Secuencia de Aminoácidos , Arginina/análogos & derivados , Arginina/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas F-Box/metabolismo , Factores Reguladores del Interferón/clasificación , Factores Reguladores del Interferón/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Filogenia , Plásmidos Inductores de Tumor en Plantas/metabolismo , Plantas Modificadas Genéticamente , Unión Proteica , Homología de Secuencia de Aminoácido , Nicotiana/genética , Nicotiana/metabolismo , Proteínas Virales/clasificación , Proteínas Virales/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo

RESUMEN

Several genes in the Agrobacterium tumefaciens transferred (T)-DNA encode proteins that are involved in developmental alterations, leading to the formation of tumours in infected plants. We investigated the role of the protein encoded by the Atu6002 gene, the function of which is completely unknown. Atu6002 expression occurs in Agrobacterium-induced tumours, and is also activated on activation of plant cell division by growth hormones. Within the expressing plant cells, the Atu6002 protein is targeted to the plasma membrane. Interestingly, constitutive ectopic expression of Atu6002 in transgenic tobacco plants leads to a severe developmental phenotype characterized by stunted growth, shorter internodes, lanceolate leaves, increased branching and modified flower morphology. These Atu6002-expressing plants also display impaired response to auxin. However, auxin cellular uptake and polar transport are not significantly inhibited in these plants, suggesting that Atu6002 interferes with auxin perception or signalling pathways.

Asunto(s)

Agrobacterium/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , Interacciones Huésped-Patógeno , Ácidos Indolacéticos/metabolismo , Transporte Biológico , Flores/anatomía & histología , Flores/crecimiento & desarrollo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fenotipo , Células Vegetales/metabolismo , Hojas de la Planta/anatomía & histología , Hojas de la Planta/crecimiento & desarrollo , Tumores de Planta/microbiología , Transducción de Señal , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Nicotiana/crecimiento & desarrollo

RESUMEN

Agrobacterium is a phytopathogenic bacterium that induces crown gall disease in many plant species by transferring and integrating a segment of its own DNA (T-DNA) into its host genome. Whereas Agrobacterium usually does not trigger an extensive defense response in its host plants, it induces the expression of several defense-related genes and activates plant stress reactions. In the complex interplay between Agrobacterium and its host plant, Agrobacterium has evolved to take advantage of these plant defense pathways for its own purpose of advancement of the infection process. For example, Agrobacterium utilizes the host stress response transcriptional regulator VIP1 to facilitate nuclear import and proteasomal uncoating of its T-DNA during genetic transformation of the host cell. In Arabidopsis, the VIP1 gene expression is repressed by WRKY17, a negative regulator of basal resistance to Pseudomonas. Thus, we examined whether WRKY17 is also involved in plant susceptibility to genetic transformation by Agrobacterium. Using reverse genetics, we showed that a wrky17 mutant displays higher expression of the VIP1 gene in roots, but not in shoots. In a root infection assay, the wrky17 mutant plants were hyper-susceptible to Agrobacterium compared to wild type plants. WRKY17, therefore, may act as a positive regulator of Arabidopsis resistance to Agrobacterium. This notion is important for understanding the complex regulation of Agrobacterium-mediated genetic transformation; thus, although this paper reports a relatively small set of data that we do not plan to pursue further in our lab, we believe it might be useful for the broad community of plant pathologists and plant biotechnologists.

RESUMEN

The genetic transformation of plants mediated by Agrobacterium tumefaciens represents an essential tool for both fundamental and applied research in plant biology. For a successful infection, culminating in the integration of its transferred DNA (T-DNA) into the host genome, Agrobacterium relies on multiple interactions with host-plant factors. Extensive studies have unraveled many of such interactions at all major steps of the infection process: activation of the bacterial virulence genes, cell-cell contact and macromolecular translocation from Agrobacterium to host cell cytoplasm, intracellular transit of T-DNA and associated proteins (T-complex) to the host cell nucleus, disassembly of the T-complex, T-DNA integration, and expression of the transferred genes. During all these processes, Agrobacterium has evolved to control and even utilize several pathways of host-plant defense response. Studies of these Agrobacterium-host interactions substantially enhance our understanding of many fundamental cellular biological processes and allow improvements in the use of Agrobacterium as a gene transfer tool for biotechnology.

Asunto(s)

Agrobacterium tumefaciens/genética , ADN Bacteriano/genética , Plantas Modificadas Genéticamente , Transformación Genética , Transporte Activo de Núcleo Celular , Transporte Biológico , Biotecnología/métodos , Comunicación Celular , Cromatina/metabolismo , Citoplasma/metabolismo , ADN de Plantas/genética , Técnicas de Transferencia de Gen , Plantas/genética , Plantas/microbiología , Transducción de Señal

RESUMEN

VIP1 (VirE2 interacting protein 1), initially discovered as a host protein involved in Agrobacterium-plant cell DNA transfer, is a transcription factor of the basic leucine-zipper (bZIP) domain family that regulates several defence-related genes in Arabidopsis. We have developed assays to assess VIP1 binding to its DNA target in vitro and transcriptional activation efficiency in planta. Several point mutations in the VIP1 response element VRE affected the VIP1 activity, and a strong correlation between VIP1-VRE binding and transcriptional activation levels was observed. Promoter activation by VIP1 was influenced by bacterial and plant proteins known to interact with VIP1 during Agrobacterium infection, i.e., VirE2, VirF and VIP2. VirF, an F-box protein, strongly decreased VIP1 transcriptional activation ability, but not its binding to VRE in vitro, most likely by triggering proteasomal degradation of VIP1. Finally, activation of a VRE-containing promoter was observed in dividing cells, probably resulting from activation of endogenous VIP1.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Transcripción Genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , División Celular , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica/genética , Elementos de Respuesta/genética

RESUMEN

Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

Asunto(s)

Biolística/instrumentación , ADN/administración & dosificación , ADN/genética , Epidermis de la Planta/citología , Epidermis de la Planta/genética , Hojas de la Planta/citología , Hojas de la Planta/genética , Precipitación Química , ADN/química , Expresión Génica , Oro/química , Factores de Tiempo

RESUMEN

One the most intriguing, yet least studied, aspects of the bacterium-host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium. During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA--as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins--into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity.

Asunto(s)

Agrobacterium/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Sustancias Macromoleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transformación Genética/fisiología , Transporte Activo de Núcleo Celular , Proteínas de Arabidopsis/metabolismo , Western Blotting , Cartilla de ADN/genética , Proteínas de Unión al ADN/metabolismo , Interacciones Huésped-Patógeno/fisiología , Canales Iónicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nicotiana

RESUMEN

VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

Asunto(s)

Agrobacterium/citología , Proteínas Bacterianas/metabolismo , Beta vulgaris/microbiología , ADN Bacteriano/genética , Espacio Extracelular/metabolismo , Nicotiana/microbiología , Transformación Genética , Agrobacterium/genética , Agrobacterium/metabolismo , Agrobacterium/fisiología , Beta vulgaris/genética , Interacciones Huésped-Patógeno/genética , Espacio Intracelular/metabolismo , Transporte de Proteínas , Nicotiana/genética

RESUMEN

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins' interactions, the fluorescent signal is reconstituted. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions. Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques. Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK), is demonstrated using this technique.

Asunto(s)

Proteínas Inmovilizadas/química , Mediciones Luminiscentes/métodos , Proteínas/química , Proteínas de Movimiento Viral en Plantas/química , Mapas de Interacción de Proteínas , Solubilidad

RESUMEN

Covalent modifications of histones, such as acetylation, methylation and ubiquitination, are central for regulation of gene expression. Heterochromatic gene silencing, for example, is associated with hypoacetylation, methylation and demethylation, and deubiquitination of specific amino acid residues in histone molecules. Many of these changes can be effected by histone-modifying repressor complexes that include histone lysine demethylases, such as KDM1 in animals and KDM1C in plants. However, whereas KDM1-containing repressor complexes have been implicated in histone demethylation, methylation and deacetylation, whether or not they can also mediate histone deubiquitination remains unknown. We identify an Arabidopsis otubain-like deubiquitinase OTLD1 which directly interacts with the Arabidopsis KDM1C in planta, and use one target gene to exemplify that both OTLD1 and KDM1C are involved in transcriptional gene repression via histone deubiquitination and demethylation. We also show that OTLD1 binds plant chromatin and has enzymatic histone deubiquitinase activity, specific for the H2B histone. Thus, we suggest that, during gene repression, lysine demethylases can directly interact and function in a protein complex with histone deubiquitinases.

Asunto(s)

Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismo , Genes de Plantas , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cromatina/genética , Cromatina/metabolismo , Cartilla de ADN/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Histonas/química , Histonas/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Ubiquitinación