RESUMO
In previous work, we experimentally demonstrated the possibility of using RNA aptamers to inhibit endogenous protein expression and their function within plant cells In the current work, we show that our proposed method is suitable for inhibiting the functions of exogenous, foreign proteins delivered into the plant via various mechanisms, including pathogen proteins. Stringent experimentation produced robust RNA aptamers that are able to bind to the recombinant HopU1 effector protein of P. syringae bacteria. This research uses genetic engineering methods to constitutively express/transcribe HopU1 RNA aptamers in transgenic A. thaliana. Our findings support the hypothesis that HopU1 aptamers can actively interfere with the function of the HopU1 protein and thereby increase resistance to phytopathogens of the genus P. syringae pv. tomato DC 3000.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plantas Geneticamente Modificadas/genética , Pseudomonas syringae/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/genéticaRESUMO
Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector-coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two-hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain-like cysteine protease (PLCP) 'Responsive to Dehydration 21A' (RD21A), which has been shown to function in the plant defense response. Activity-based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re-localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two-hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector-mediated re-localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense-inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cisteína Proteases/metabolismo , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Doenças das Plantas/parasitologia , Tylenchoidea/fisiologia , Animais , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Beta vulgaris/parasitologia , Núcleo Celular/metabolismo , Parede Celular/metabolismo , Cisteína Proteases/genética , Citoplasma/metabolismo , Feminino , Proteínas de Helminto/genética , Doenças das Plantas/imunologia , Imunidade Vegetal , Transporte Proteico , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/metabolismoRESUMO
The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes (AtCYP19-1/ROC3, AtFKBP65/ROF2, and AtCYP57) in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H) system for proteinâ»protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1) and putative protein phosphatase (At2g30020; ÐТ2), whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1) and RmlC-like cupin (At5g39120). The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1) and clathrin adaptor complex-related protein (At5g05010). Identified interactors confirm our previous findings that immunophilins ROC3, ROF2, and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.
Assuntos
Arabidopsis/metabolismo , Imunofilinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Imunofilinas/genética , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil.
RESUMO
The scope of RNA-aptamers application is becoming wider and has expanded beyond solely medical use. We propose the use of RNA-aptamers in plants to suppress the functions of individual proteins, thereby achieving resistance to various biotic and abiotic stresses. In current work we experimentally demonstrate the possibility of inhibiting protein activity in N. bentamiana plants by quenching the fluorescence level of GFP (green fluorescent protein) as a result of specifically selected RNA-aptamer binding action.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Plantas/fisiologia , Nicotiana/metabolismoRESUMO
We have created and applied to Arabidopsis thaliana a new system of two vectors. The first vector (pEnLox) is intended for activation tagging and contains a multimerized transcriptional enhancer from the cauliflower mosaic virus (CaMV) 35S gene in T-DNA flanked by two loxP-sites and the second vector (pCre) contains the cre gene. Using pEnLox we have generated more than a hundred mutants resistant to the herbicide ammonium glufosinate, and about ten helper-lines resistant to the antibiotic hygromycin obtained with the use of pCre vector and also more than ten double mutants resistant to both selective markers. In at least 3 cases among 40 mutant lines that have been analyzed we observed constitutive ectopic expression of the genes adjacent to the T-DNA insertion that causes development of the mutant phenotype. Also, reversion of the mutants to the wild-type phenotype after removing the CaMV enhancer has been demonstrated. The system presented here provides a new and easier way to analyze A. thaliana gain-of-function mutants.
Assuntos
Arabidopsis/genética , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , Caulimovirus/genética , Genes de Plantas , Integrases/metabolismo , Mutagênese Insercional , Mutação/genética , Fenótipo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Transformação GenéticaRESUMO
The exact localization of an insertion in the genome of transgenic plants obtained by Agrobacterium-mediated transformation is an integral part of most experiments aimed at studying these types of mutants. There are several methods for isolating unknown nucleotide sequences of genomic DNA which flank the borders of T-DNA integrated in the genome of plants. However, all the methods based on PCR have limitations which in some cases do not permit the desired objective to be achieved. We have developed a new technique for isolating flanking sequence tags (FSTs) via modified inverse PCR. This method is highly efficient and simple, but also retains the advantages of previously well-documented approaches.
Assuntos
Arabidopsis/genética , DNA de Plantas/isolamento & purificação , Etiquetas de Sequências Expressas/metabolismo , Reação em Cadeia da Polimerase/métodos , Primers do DNA , DNA Bacteriano , Mutagênese Insercional , Plantas Geneticamente ModificadasRESUMO
Cell walls are essential components of plant cells which perform a variety of important functions for the different cell types, tissues and organs of a plant. Besides mechanical function providing cell shape, cell walls participate in intercellular communication, defense during plant-microbe interactions, and plant growth. The plant cell wall consists predominantly of polysaccharides with the addition of structural glycoproteins, phenolic esters, minerals, lignin, and associated enzymes. Alterations in the cell wall composition created through either changes in biosynthesis of specific constituents or their post-synthetic modifications in the apoplast compromise cell wall integrity and frequently induce plant compensatory responses as a result of these alterations. Here we report that post-synthetic removal of fucose residues specifically from arabinogalactan proteins in the Arabidopsis plant cell wall induces differential expression of fucosyltransferases and leads to the root and hypocotyl elongation changes. These results demonstrate that the post-synthetic modification of cell wall components presents a valuable approach to investigate the potential signaling pathways induced during plant responses to such modifications that usually occur during plant development and stress responses.
Assuntos
Aspergillus nidulans/enzimologia , Fucosiltransferases/metabolismo , Mucoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Arabidopsis/genética , Proteínas de Arabidopsis , Aspergillus nidulans/genética , Parede Celular/genética , Parede Celular/metabolismo , Ativação Enzimática , Fucosiltransferases/genética , Expressão Gênica , Regulação da Expressão Gênica , Mucoproteínas/genética , Mucoproteínas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas Recombinantes , alfa-L-Fucosidase/metabolismo , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Parede Celular/fisiologia , Cloroplastos/fisiologia , Mutação/genética , Doenças das Plantas/imunologia , Folhas de Planta/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular , Parede Celular/microbiologia , DNA de Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Genes Recessivos , Interações Hospedeiro-Patógeno , Imunidade Celular/imunologia , Fotossíntese , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Plastídeos/microbiologia , Plastídeos/fisiologia , Pseudomonas syringae/patogenicidade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Plant immunophilins are a broadly conserved family of proteins, which carry out a variety of cellular functions. In this study, we investigated three immunophilin genes involved in the Arabidopsis thaliana response to Pseudomonas syringae infection: a cytoplasmic localized AtCYP19, a cytoplasmic and nuclear localized AtCYP57, and one nucleus directed FKBP known as AtFKBP65. Arabidopsis knock-out mutations in these immunophilins result in an increased susceptibility to P. syringae, whereas overexpression of these genes alters the transcription profile of pathogen-related defense genes and led to enhanced resistance. Histochemical analysis revealed local gene expression of AtCYP19, AtCYP57, and AtFKBP65 in response to pathogen infection. AtCYP19 was shown to be involved in reactive oxygen species production, and both AtCYP57 and AtFKBP65 provided callose accumulation in plant cell wall. Identification of the involvement of these genes in biotic stress response brings a new set of data that will advance plant immune system research and can be widely used for further investigation in this area.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Aromatase/genética , Imunidade Vegetal/genética , Proteínas de Ligação a Tacrolimo/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Aromatase/metabolismo , Regulação da Expressão Gênica de Plantas , Glucanos/genética , Glucanos/metabolismo , Mutação , Pseudomonas syringae , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Several transcription factors are presently known to regulate the response to cold stress. Here we describe a new positive regulator, ICE2, which is a transcription factor of the bHLH family that participates in the response to deep freezing through the cold acclimation-dependent pathway in Arabidopsis thaliana plants. An overexpression of ICE2 (as we named the At1g12860 locus) in transgenic Arabidopsis plants results in increased tolerance to deep freezing stress after cold acclimation. The seeds of transgenic lines that overexpressed ICE2 were characterized by decreased levels of carbohydrate and increased levels of lipids. The analysis of expression of CBF1 gene (also known as DREB1B), which have been shown to be required for the complete development of cold acclimation response in Arabidopsis indicated a difference between expression of the CBF1 gene in transgenic plants and the wild-type control plants, Col-0. These results suggested that the CBF1 transcription factor, known as one of the regulators of the cold stress response, has a dominant role in providing freezing tolerance in transgenic plants characterized by overexpression of ICE2.