224 research outputs found
Plasmodesmata: Channels for Viruses on the Move
No full textThe symplastic communication network established by plasmodesmata (PD) and connected phloem provides an essential pathway for spatiotemporal intercellular signaling in plant development but is also exploited by viruses for moving their genomes between cells in order to infect plants systemically. Virus movement depends on virus-encoded movement proteins (MPs) that target PD and therefore represent important keys to the cellular mechanisms underlying the intercellular trafficking of viruses and other macromolecules. Viruses and their MPs have evolved different mechanisms for intracellular transport and interaction with PD. Some viruses move from cell to cell by interacting with cellular mechanisms that control the size exclusion limit of PD whereas other viruses alter the PD architecture through assembly of specialized transport structures within the channel. Some viruses move between cells in the form of assembled virus particles whereas other viruses may interact with nucleic acid transport mechanisms to move their genomes in a non-encapsidated form. Moreover, whereas several viruses rely on the secretory pathway to target PD, other viruses interact with the cortical endoplasmic reticulum and associated cytoskeleton to spread infection. This chapter provides an introduction into viruses and their role in studying the diverse cellular mechanisms involved in intercellular PD-mediated macromolecular trafficking
In Vivo RNA Labeling Using MS2
No full textThe trafficking and asymmetric distribution of cytoplasmic RNA is a fundamental process during development and signaling across phyla. Plants support the intercellular trafficking of RNA molecules such as gene transcripts, small RNAs, and viral RNA genomes by targeting these RNA molecules to plasmodesmata (PD). Intercellular transport of RNA molecules through PD has fundamental implications in the cell-to-cell and systemic signaling during plant development and in the systemic spread of viral disease. Recent advances in time-lapse microscopy allow researchers to approach dynamic biological processes at the molecular level in living cells and tissues. These advances include the ability to label RNA molecules in vivo and thus to monitor their distribution and trafficking. In a broadly used RNA labeling approach, the MS2 method, the RNA of interest is tagged with a specific stem-loop (SL) RNA sequence derived from the origin of assembly region of the bacteriophage MS2 genome that binds to the bacteriophage coat protein (CP) and which, if fused to a fluorescent protein, allows the visualization of the tagged RNA by fluorescence microscopy. Here we describe a protocol for the in vivo visualization of transiently expressed SL-tagged RNA and discuss key aspects to study RNA localization and trafficking to and through plasmodesmata in Nicotiana benthamiana plants
Studies on the interaction between "Tobacco Mosaic Virus" movement protein and microtubules
No full textDuring the invasion of a susceptible host, Tobacco mosaic virus (TMV) transports its RNA genome from sites of viral synthesis into neighbouring cells, thus potentiating the spread of infection. In many systems, RNA trafficking is known to be a highly coordinated process, often involving the complex interplay of numerous factors. In plants, however, the mechanisms that govern RNA transport are not well understood. Since viruses have a propensity to exploit pre-existing host machinery for their own purposes, TMV has become a popular model system for the study of RNA trafficking in plants. TMV viral RNA (vRNA) is thought to be transported as a ribonucleoprotein (RNP) with the virally encoded movement protein (MP). Localization studies have demonstrated a temporal redistribution of MP from endoplasmic reticulum (ER)derived replication bodies onto microtubules (MTs) at mid to late stage of infection. A functional link between the association of MP with microtubules (MT) and RNA trafficking has been established, and furthermore, this association does not require other viral components or plant-specific factors, thereby indicating a direct interaction between MP and MTs. In vitro, purified MP interacts directly with preformed, dynamically suppressed MTs and has no requirement for MT polymerization or polymer-specific structural forms. Immunohistochemical studies indicate that MP may interact with the extreme C-termini of α- and β-tubulin, possibly forming a stabilizing sheath. Consistent with the proposal that MP functions as a structural microtubuleassociated protein (MAP), MP:MT complexes assembled in vitro are highly stable. A possible functional overlap between the putative RNA and MT binding domains of MP has been identified, and although MP:MT complexes are unlikely to support RNA transport directly, such complexes have been implicated to modulate MT-dependent molecular motor activity. Collectively, in vitro data are in support of the observation that MP associates with MTs to high amounts, leading to the hypothesis that MPmediated RNA transport and the MP:MT interaction are related, yet functionally distinguishable processes. Furthermore, based on the similarity of MP to endogenous MAPs, a new model is proposed in which MP-mediated trafficking may occur in the form of a translationally competent ER-vesicle, concurrent with the deployment of MP to the MT surface
Defense pathways in Arabidopsis restricting compatible tobamoviruses during infection
No full textVirus infected plants often develop strong disease symptoms including leaf deformation, chlorosis, necrosis and growth inhibition. In agriculture systems virus infection can lead to severe yield losses and a better understanding of plant defenses against viruses is therefore desirable in order for develop new strategies against diseases in crops caused by viruses. Specific resistance to viruses has been studied intensively in the past but host tolerance and recovery have received little attention.
Symptomatic virus infections can persist throughout the life of the host. However, in some cases a recovery from symptoms can be observed. Recovery has been associated with host anti-viral RNA-silencing targeting viral nucleic acids for destructing or inactivation. However, it is well established that compatible viruses suppress RNA-silencing in order to establish and maintain an infection and the exact role of RNA-silencing in onset and maintenance of recovery is therefore unclear. To address this question a “recovery-system” for the tobamovirus Oilseed rape mosaic virus (ORMV) was set up in the model plant Arabidopsis thaliana and characterized (chapter 2). Through the use of Arabidopsis mutants we show that specific RNA-silencing pathways are essential for recovery, included some known to be involved in non-autonomous RNA-silencing. Furthermore, mutants with increased RNA-silencing capacity did recover earlier than wild type plants, suggesting that oscillations in RNA-silencing activity could be involved in the onset of recovery.
RNA-silencing is an important anti-viral defense mechanism but also defense pathways regulated by hormones are induced during compatible virus infections. The changes in gene expression observed upon compatible virus infections are similar to those observed for infection with other biotic plant pathogens, but the importance of virus-induced defense responses is not fully understood. Non-viral plant pathogens predominately live in the apoplast and the presence of pathogen-derived “non-self” molecules is sensed through receptors in the plasma membrane, a mechanism referred to as Pattern-Triggered-Immunity (PTI). It is unclear if intercellular pathogens, such as viruses, can induce defense responses in plants through PTI and if PTI is involved in plant defense against viruses. In this thesis we show that mutants of BAK1; a regulator of many receptors involved in PTI, are hypersuceptible to several RNA viruses (chapter 3). Furthermore crude extracts from virus-infected plants contain compounds that can elicit PTI-responses (chapter 3). Taken together this indicates that virus infections induce PTI through an unidentified, likely plant-derived compound.
Studies of compatible virus infections have focused on plant-virus interactions that lead to disease symptom formation. However, virus infections can progress almost or completely symptomless referred to as tolerance. Mechanisms controlling tolerance to viruses in plants have not been described until now. Infections of Arabidopsis with tobamovirus Tobacco mosaic virus (TMV) progress almost symptomless in most ecotypes. Characterization of TMV infections in tolerant and symptomatic Arabidopsis ecotypes revealed that symptom formation is associated with accelerated viral movement and induction of defense responses (chapter 4). Furthermore is symptom formation independent of RNA-silencing and Salicylic Acid (SA) signaling (chapter 4)
imaging of tagged mRNA in plant tissues using the bacterial transcriptional antiterminator BglG
No full textRNA transport and localization represent important post-transcriptional mechanisms to determine the subcellular localization of protein synthesis. Plants have the capacity to transport messenger (m)RNA molecules beyond the cell boundaries through plasmodesmata and over long distances in the phloem. RNA viruses exploit these transport pathways to disseminate their infections and represent important model systems to investigate RNA transport in plants. Here, we present an in vivo plant RNA-labeling system based on the Escherichia coli RNA-binding protein BglG. Using the detection of RNA in mobile RNA particles formed by viral movement protein (MP) as a model, we demonstrate the efficiency and specificity of mRNA detection by the BglG system as compared with MS2 and λN systems. Our observations show that MP mRNA is specifically associated with MP in mobile MP particles but hardly with MP localized at plasmodesmata. MP mRNA is clearly absent from MP accumulating along microtubules. We show that the in vivo BglG labeling of the MP particles depends on the presence of the BglG-binding stem–loop aptamers within the MP mRNA and that the aptamers enhance the coprecipitation of BglG by MP, thus demonstrating the presence of an MP:MP mRNA complex. The BglG system also allowed us to monitor the cell-to-cell transport of the MP mRNA, thus linking the observation of mobile MP mRNA granules with intercellular MP mRNA transport. Given its specificity demonstrated here, the BglG system may be widely applicable for studying mRNA transport and localization in plants.Fil: Peña, Eduardo José. Université de Strasbourg; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Robles Luna, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Heinlein, Manfred. Université de Strasbourg; Franci
TRAP-SEQ of eukaryotic translatomes applied to the detection of polysome-associated long noncoding RNAs
No full textTranslating ribosome affinity purification (TRAP) technology allows the isolation of polysomal complexes and the RNAs associated with at least one 80S ribosome. TRAP consists of the stabilization and affinity purification of polysomes containing a tagged version of a ribosomal protein. Quantitative assessment of the TRAP RNA is achieved by direct sequencing (TRAP-SEQ), which provides accurate quantitation of ribosome-associated RNAs, including long noncoding RNAs (lncRNAs). Here we present an updated procedure for TRAP-SEQ, as well as a primary analysis guide for identification of ribosome-associated lncRNAs. This methodology enables the study of dynamic association of lncRNAs by assessing rapid changes in their transcript levels in polysomes at organ or cell-type level, during development, or in response to endogenous or exogenous stimuli.Fil: Traubenik, Laura Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Blanco, Flavio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Zanetti, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Reynoso, Mauricio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin
Quantification of plant cell coupling with live-cell microscopy
No full textMovement of nutrients and signaling compounds from cell to cell is an essential process for plant growth and development. To understand processes such as carbon allocation, cell communication, and reaction to pathogen attack it is important to know a specific molecule’s capacity to pass a specific cell wall interface. Transport through plasmodesmata, the cell wall channels that directly connect plant cells, is regulated not only by a fixed size exclusion limit, but also by physiological and pathological adaptation. The noninvasive approach described here offers the possibility of precisely determining the plasmodesmata-mediated cell wall permeability for small molecules in living cells.The method is based on photoactivation of the fluorescent tracer caged fluorescein. Non-fluorescent caged fluorescein is applied to a target tissue, where it is taken up passively into all cells. Imaged by confocal microscopy, loaded tracer is activated by UV illumination in a target cell and its spread to neighboring cells monitored. When combined with high-speed acquisition by resonant scanning or spinning disc confocal microscopy, the high signal-to-noise ratio of photoactivation allows collection of three-dimensional (3D) time series. These contain all necessary functional and anatomical data to measure cell coupling in complex tissues noninvasively.<br/
Simultaneous Detection of mRNA and Protein in S. cerevisiae by Single-Molecule FISH and Immunofluorescence
No full textSingle-molecule fluorescent in situ hybridization (smFISH) enables the detection and quantification of endogenous mRNAs within intact fixed cells. This method utilizes tens of singly labeled fluorescent DNA probes hybridized against the mRNA of interest, which can be detected by using standard wide-field fluorescence microscopy. This approach provides the means to generate absolute quantifications of gene expression within single cells, which can be used to link molecular fluctuations to phenotypes. To be able to correlate the expression of an mRNA and a protein of interest in individual cells, we combined smFISH with immunofluorescence (IF) in yeast cells. Here, we present our smFISH-IF protocol to visualize and quantify two cell cycle-controlled mRNAs (CLN2 and ASH1) and the cell cycle marker alpha-tubulin in S. cerevisiae. This protocol, which is performed over 2 days, can be used to visualize up to three colors at the time (i.e., two mRNAs, one protein). Even if the described protocol is designed for S. cerevisiae, we think that the considerations discussed here can be useful to develop and troubleshoot smFISH-IF protocols for other model organisms.</p
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