http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_1 A variety of techniques have been used to examine plant viral genomes, the functions of virus-encoded proteins, plant responses induced by virus infection and plant–virus interactions. This overview considers these technologies and how they have been used to identify novel viral and plant proteins or genes involved in disease and resistance responses, as well as defense signaling. These approaches include analysis of spatial and temporal responses by plants to infection, and techniques that allow the expression of viral genes transiently or transgenically in planta, the expression of plant and foreign genes from virus vectors, the silencing of plants genes, imaging of live, infected cells, and the detection of interactions between viral proteins and plant gene products, both in planta and in various in vitro or in vivo systems. These methods and some of the discoveries made using these approaches are discussed. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_10 The technique described was developed for the separation of begomovirus DNA. DNA products resulting from and during geminiviral replication are characterized by the application of strand-specific separation and identification by strand-specific DNA probing of Southern blots. The mapping of the initiation site of complementary-strand DNA synthesis, by this technique is also presented. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_11 The Begomovirus genus is the largest genus of the Geminiviridae family and comprises the whitefly transmitted geminiviruses that infect dicotyledonous plants. They can be either mono or bipartite. In this chapter, we describe the cloning of begomovirus replication modules and the subsequent functional characterization of geminivirus replication origins. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_12 Viroids, as a consequence of not encoding any protein, are extremely dependent on their hosts. Replication of these minimal genomes, composed exclusively by a circular RNA of 246–401 nt, occurs in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism with three steps: (1) synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases recruited and redirected to transcribe RNA templates, (2) cleavage to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3) circularization through an RNA ligase or autocatalytically. This consistent but still fragmentary picture has emerged from a combination of studies with in vitro systems (analysis of RNA preparations from infected plants, transcription assays with nuclear and chloroplastic fractions, characterization of enzymes and ribozymes mediating cleavage and ligation of viroid strands, dissection of 5′ terminal groups of viroid strands, and in situ hybridization and microscopy of subcellular fractions and tissues), and in vivo systems (tissue infiltration studies, protoplasts, studies in planta and use of transgenic plants expressing viroid RNAs). 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_13 The interaction between viral polymerases and their cognate RNAs is vital to regulate the timing and abundance of viral replication products. Despite this, only minimal detailed information is available for the interaction between viral polymerases and cognate RNAs. We study the biochemical interactions using two viral polymerases that could serve as models for other plus-strand RNA viruses: the replicase from the tripartite brome mosaic virus (BMV), and the recombinant RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV). Replicase binding sites in the BMV RNAs were mapped using a template competition assay. The minimal length of RNA required for RNA binding by the HCV RdRp was determined using fluorescence spectroscopy. Lastly, regions of the HCV RdRp that contact the RNA were determined by a method coupling reversible protein-RNA crosslinking, affinity purification, and mass spectrometry. These analyses of RdRp-RNA interaction will be presented as three topics in this chapter. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_14 The ability to combine nucleic acid hybridisation or immunospecific reactions with structural and ultrastructural analysis of virus-infected tissues has provided the opportunity to resolve the spatial details of infection with respect to the production of virus-specific products and the nature of the host response. These technologies may seem lengthy and complex but offer high rewards in terms of revealing the details of host—virus interactions not otherwise accessible. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_15 Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) play crucial roles in establishing general host defense mechanisms against viral infections in plants and the development of disease symptoms. Understanding these fundamental processes requires the sensitive and specific detection of small RNA species. However, because of the small size of miRNAs and siRNAs, their detection is technically demanding. Here, we describe methods for robust and sensitive detection of small RNAs by Northern blot analysis and in situ hybridization. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_16 During their infection in plants, viruses can form double stranded (ds) RNA structures. These dsRNAs can be recognized by plants as “aberrant” signals and short interfering RNA (siRNA) molecules of 19–25 nt will be produced with sequences derived from the viral source. Knowledge about antiviral siRNA profiles including siRNA size, distribution, polarity, etc. provides valuable insights to plant-virus interactions. In this chapter, we describe a simple method for cloning siRNA from virus-infected plants. This protocol includes isolation of small RNAs, their ligation to a pair of 5′ and 3′ adapters, RT-PCR/PCR amplification, and subsequent concatamerization before pGEM-T cloning and sequencing. Concatamers containing as many as 15 small RNA inserts can be produced. This protocol has successfully been apphed to leaf materials of monocots and dicots infected with poty-, carmo-, and sobemo-viruses. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_17 Single-stranded RNA plant viruses not only code for viral proteins within their RNA genomes, they often maintain elaborate RNA secondary structures. These structures can be integral to a variety of viral processes, such as viral translation, genome replication, subgenomic mRNA transcription, and genome encapsidation. RNA secondary structures may function to recruit and bind trans-acting protein factors, or may become part of higher order tertiary RNA structures, which themselves may be functionally relevant. To fully understand such viral RNA elements and their mechanisms of action, it is necessary to first determine their secondary structures. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_18 Analysis of viral RNA encapsidation assay provides a rapid means of assaying which of the progeny RNA are competent for packaging into stable mature virions. Generally, a parallel analysis of total RNA and RNA obtained from purified virions is advisable for accurate interpretation of the results. In this, we describe a series of in vivo assays in which viral RNA encapsidation can be verified. These include whole plants inoculated either mechanically or by Agroinfiltration and protoplasts. The encapsidation assay described here is for an extensively studied plant RNA virus, brome mosaic virus, and can be reliably applied to other viral systems as well as with appropriate buffers. In principle, the encapsidation assay requires purification of virions from either symptomatic leaves or transfected plant protoplasts followed by RNA isolation. The procedure involves grinding the infected tissue in an appropriate buffer followed by a low speed centrifugation step to remove the cell debris. The supernatant is then emulsified with an organic solvent such as chloroform to remove chlorophyll and cellular material. After a low seed centrifugation, the supernatant is subjected to high speed centrifugation to concentrate the virus as a pellet. Depending on the purity required, the partially purified virus preparation is further subjected to sucrose density gradient centrifugation. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_19 Replication of the viral RNA genome performed by the viral replicase is the central process during the viral infection cycle (Nagy and Pogany, see earlier chapter four). Most RNA viruses assign one or more proteins translated from their own genomes for assembling the viral replicase complex, which consists of the viral RNA, viral proteins, and several subverted host proteins embedded in cellular membranes. Understanding the various biochemical activities of the replication proteins can lead to target identification for human intervention to control viral infections or the damage to the host cells. The replicase proteins of tomato bushy stunt virus (TBSV) are selected as model system to study the dynamics of interactions between viral replicase proteins using surface plasmon resonance (SPR) analysis. The SPR assay provides real-time protein interaction data by measuring the change in refractive index at the surface of the sensor chip due to the change in mass resulting from the interaction between the immobilized protein and the protein that is being passed over the immobilized chip surface. SPR-based biosensor BIAcore X was used to carry out TBSV replicase protein interaction studies. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_2 Coat proteins (CPs) of all plant viruses have an early function in disassembly of parental virus and a late function in assembly of progeny virus. Depending on the virus, however, CPs may play a role in many steps of the infection cycle in between these early and late functions. It has been shown that CPs can play a role in translation of viral RNA, targeting of the viral genome to its site of replication, cell-to-cell and/or systemic movement of the virus, symptomatology and virulence of the infection, activation of Rgene-mediated host defenses, suppression of RNA silencing, interference with suppression of RNA silencing, and determination of the specificity of virus transmission by vectors. These functions are reviewed in this chapter. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_20 RNA–protein interactions control viral RNA replication, transcription, translation, and particle assembly. Progress toward understanding the functional significance of RNA–protein complexes in the viral life cycle is hindered by the lack of high resolution structural information. Challenges to acquiring structural data include RNA's inherent instability and conformational plasticity, coupled with the comparatively high cost of generating large quantities of RNA for biophysical experiments. The potential for successful structure determination is increased by conducting biochemical experiments that outline interacting domains and identify key residues. These approaches are aimed at defining and characterizing RNA and protein substrates that are suitable for high resolution structural analysis. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_21 Most plant viruses move between plant cells with the help of their movement proteins (MPs). MPs are multifunctional proteins, and one of their functions is almost invariably binding to nucleic acids. Presumably, the MP—nucleic acid interaction is directly involved in formation of nucleoprotein complexes that function as intermediates in the cell-to-cell transport of many plant viruses. Thus, when studying a viral MP, it is important to determine whether or not it binds nucleic acids, and to characterize the hallmark parameters of such binding, i.e., preference for single- or double-stranded nucleic acids and binding cooperativity and sequence specificity. Here, we present two major experimental approaches, native gel mobility shift assay and ultra violet (UV) light cross-linking, for detection and characterization of MP binding to DNA and RNA molecules. We also describe protocols for purification of recombinant viral MPs over-expressed in bacteria and production of different DNA and RNA probes for these binding assays. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_22 Movement proteins (MPs) are virally encoded factors that mediate transport of viral nucleic acid between plant cells. Many MPs are able to move between cells themselves. This feature serves as the basis for evaluation of the transport activity of individual MPs. MPs are transiently expressed as a fusion to autofluo-rescent proteins such as green fluorescent protein (GFP) in individual epidermal cells of leaves by biolistic delivery. Expressing cells can be directly monitored for subcellular localization and cell-to-cell movement of the MP:GFP fusion protein into neighboring cells by confocal scanning microscopy. During the time frame of transient expression, numerous cells are evaluated at several time points, and the accumulated data are depicted in a graph termed “movement profile.” Thus, a movement profile will provide information on the correlation between subcellular localization of the MP in the expressing cell and the efficiency of cell-to-cell transport, the time course and efficiency of targeting of the MP to plasmodesmata, and the translocation efficiency of the MP into neighboring cells. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_23 RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and animals. To counteract RNA silencing, viruses evolved silencing suppressors that interfere with siRNA guided RNA silencing pathway. We used the heterologous Drosophila in vitro embryo RNA to analyze the molecular mechanism of suppression of silencing suppressors. We found that different silencing suppressors inhibit the RNA silencing via binding to siRNAs. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, suppressors uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. Here, we provide the protocol for the detailed analysis of p19 silencing suppressors of tombusviruses in the heterologous Drosophila in vitro system. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_24 Posttranslational modification of proteins is a key regulatory mechanism in a variety of cellular processes. This chapter outlines the concepts and methods used to investigate protein phosphorylation and its physiological relevance during plant virus infection. Rather than providing an exhaustive review of the experimental protocols for protein phosphorylation analysis, we focus on methods that can be used to study phosphorylation of viral proteins. We address the following points: how to determine that a viral protein of interest is phosphorylated; how to map the phosphorylation sites; how to identify the protein kinase(s) involved. Finally, we describe a number of useful strategies to evaluate the biological significance of phosphorylation. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_25 Replication of the genome of positive-strand RNA plant viruses takes place in membrane-bound complexes that contain viral replicase proteins, viral RNA, and host proteins. Many viral replicase proteins play a crucial role in the assembly of replication complexes at intracellular membranes. They are integral membrane proteins that interact directly with the membranes and bring other proteins and the viral RNA to the complex via protein—protein or protein—RNA interactions. In this chapter, we describe subcellular fractionation methods that determine whether viral proteins are integral membrane proteins in planta. Differential centrifugation techniques are used to produce membrane-enriched fractions, which can then be analyzed for the presence of viral replicase proteins by immunoblotting. Confirmation of the membrane-association is obtained by membrane flotation assays and treatment of membrane-enriched fractions with high salt or high pH followed by detection of the viral proteins. Because many plant viruses replicate in association with the endoplasmic reticulum (ER), we also discuss two techniques to specifically analyze the interaction of viral proteins with these membranes. These techniques are continuous sucrose-gradient fractionation in the presence or absence of 3 mM Mg2+ and glycosylation assays. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_26 In terms of functional genomics research, Nicotiana benthamiana, more so than other model plants, is highly amenable to high-throughput methods, especially those employing virus-induced gene silencing and agroinfiltration. Furthermore, through recent and ongoing sequencing projects, there are now upward of 18,000 unique N. benthamiana ESTs to support functional genomics research. Despite these advances, the cell biology of N. benthamiana itself, and in the context of virus infection, lags behind that of other model systems. Therefore, to meet the challenges of diverse cell biology studies that will be derived from ongoing functional genomics projects, a series of methods relevant to the characterization of membrane and protein dynamics in virus-infected cells are provided here. The data presented here were derived from our studies with plant rhabdoviruses. However, the employed techniques should be broadly applicable within the field of plant virology. We report here on the use of a novel series of binary vectors for the transient or stable expression of autofluorescent protein fusions in plants. Use of these vectors in conjunction with advanced microscopy techniques such as fluorescent recovery after photobleaching and total internal fluorescence microscopy, has revealed novel insight into the membrane and protein dynamics of virus-infected cells. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_27 This chapter introduces an efficient and accurate site-directed mutagenesis protocol, which allows the color selection of mutants through the simultaneous activation or deactivation of the α-peptide of β-galactosidase. It uses doublestranded plasmid DNA as the mutational template. This protocol can efficiently create mutations of large inserts at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. Thus, constructs containing whole open-reading frames and whole viral genomes can be subjected to site-directed mutagenesis and used for subsequent functional studies. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_28 Maize streak virus (MSV) genome has four open reading frames. C1 and C2 encoded by the complementary sense are required for virus replication, while V1 and V2 encoded by virion sense are required for infectivity. V1 encodes movement protein (MP), while V2 encodes coat protein (CP). Deletion or mutation of MSV CP does not prevent virus replication in single cells or protoplasts but leads to a loss of infectivity in the inoculated plant suggesting that MSV CP is required for virus movement. Towards understanding the role of MSV CP and MP in virus movement, the interaction of MSV CP and MP with viral DNA was investigated using the South-western assay. Wild type and truncated MSV CPs and MP were expressed in E. coli and the expressed CPs and MP were used to investigate interaction with single-stranded (ss) and double-stranded (ds) DNA. The results showed MSV MP does not bind DNA in the assay while MSV CP bound ss and ds viral and uidA DNA in a sequence non-specific manner. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_29 Yeast two-hybrid systems are powerful tools to identify novel protein–protein interactions and have been extensively used to study viral protein interactions. The most commonly used systems are GAL4-based and LexA-based systems. Over the last decade, a range of modifications and improvements have been made to the original yeast two-hybrid system to expand the scope of molecular interaction assays and to eliminate false positives. Detailed protocols are provided for yeast strain storage, yeast transformation, yeast mating, preparation of growth and selection medium, quantitative reporter gene assays (α- and β-galactosidase liquid assays) and detection of fusion protein by Western blot. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_3 Plant viruses spread from the initially infected cells to the rest of the plant in several distinct stages. First, the virus (in the form of virions or nucleic acid protein complexes) moves intracellularly from the sites of replication to plasmodesmata (PD, plant-specific intercellular membranous channels), the virus then transverses the PD to spread intercellularly (cell-to-cell movement). Long-distance movement of virus occurs through phloem sieve tubes. The processes of plant virus movement are controlled by specific viral movement proteins (MPs). No extensive sequence similarity has been found in MPs belonging to different plant virus taxonomic groups. Moreover, different MPs were shown to use different pathways and mechanisms for virus transport. Some viral transport systems require a single MP while others require additional virus-encoded proteins to transport viral genomes. In this review, we focus on the functions and properties of different classes of MPs encoded by RNA containing plant viruses. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_30 Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to study the three-dimensional structure of proteins and nucleic acids at atomic resolution. Since the NMR data can be recorded in solution, conditions such as pH, salt concentration, and temperature can be adjusted so as to closely mimic the biomacromolecules natural milieu. In addition to structure determination, NMR applications can investigate time-dependent phenomena, such as dynamic features of the biomacromolecules, reaction kinetics, molecular recognition, or protein folding. The advent of higher magnetic field strengths, new technical developments, and the use of either uniform or selective isotopic labeling techniques, currently allows NMR users the opportunity to investigate the tertiary structure of biomacromolecules of ∼50 kDa. This chapter will outline the basic protocol for structure determination of proteins by NMR spectroscopy. In general, there are four main stages: (i) preparation of a homogeneous protein sample, (ii) the recording of the NMR data sets, (iii) assignment of the spectra to each NMR observable atom in the protein, and (iv) generation of structures using computer software and the correctly assigned NMR data. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-102-4_31 This chapter describes techniques for in vivo imaging of fluorescent fusion proteins in living cells by confocal laser scanning microscopy (CLSM). Methods are provided for (i) producing the constructs for transient expression from plasmids or virus-based vectors, (ii) introduction of constructs to plant epidermal cells; (iii) imaging of the expressed proteins by CLSM and image processing, and (iv) studying the expression in the presence of agents that affect the integrity or function of cytoskeletal elements. Notes are provided to aid comprehension and indicate problems. 2008-04-01T04:00:00Z