Vintherbentsen6953

Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.Viruses are ubiquitous in nature and exist in a variety of habitats. mTOR inhibitor review The advancement in sequencing technologies has revolutionized the understanding of viral biodiversity associated with plant diseases. Deep sequencing combined with metagenomics is a powerful approach that has proven to be revolutionary in the last decade and involves the direct analysis of viral genomes present in a diseased tissue sample. This protocol describes the details of RNA extraction and purification from wild rice plant and their yield, RNA purity, and integrity assessment. As a final step, bioinformatics data analysis including demultiplexing, quality control, de novo transcriptome assembly, taxonomic allocation and read mapping following Illumina HiSeq small and total RNA sequencing are described. Furthermore, the total RNAs extraction protocol and an additional ribosomal rRNAs depletion step which are significantly important for viral genomes construction are provided.Perennial fruit crops are susceptible to many viral pathogens, which often lead to declines in quality and yield. For the production of good quality and virus-free propagation materials, conventional molecular detection methods combining high throughput sequencing technology have been widely applied to virus detection and discovery in fruit trees. Recovery of high-quality RNAs from fruit tree leaf tissues, the critical step for the subsequent molecular analysis, is often complicated by the presence of high levels of RNases and problematic biomolecules. Therefore, the universal extraction methods often require modification according to different properties of various tissues. In this chapter, we provide a set of methods that have been used successfully to isolate total RNAs and small RNAs from various fruit tree leaf tissues and as examples, presented in detail of a modified TRIzol method for total RNAs purification from mulberry (Morus alba L.) leaf tissues and an alternative small RNAs purification protocol using mirVana™ miRNA isolation kit (Ambion/Life Technologies) for some fruit tree leaf tissues. The protocols described here aim to provide examples of what have worked successfully for a range of fruit trees and may be successful for a given sample in the future.Long life cycle and lack of efficient and robust virus inoculation technique are the major technical challenges for studying virus infection in perennial woody plants such as fruit trees. Biolistic technology also called particle bombardment is a physical approach that can directly introduce virions or viral full-length cDNA infectious clones into target cells and tissues by high velocity microcarrier particles. The flexibility and high efficiency of the biolistic inoculation method facilitate research on fruit tree virology and the screening and identification of fruit tree germplasms resistant to viruses. Here, we describe a detailed protocol for the biolistic inoculation of peach with of a cDNA infectious clone of Plum pox virus (PPV) using the Helios gene gun, a biolistic particle delivery system.The first continuous cell line of leafhopper was established over 50 years ago. Since then, leafhopper cell monolayers have been used extensively to assay the infectivity of plant viruses that multiply in their insect vectors and to elucidate the viral determinants for virus transmission via insects. We have established continuous insect cell lines of three rice planthoppers, which have been used to study the mechanisms for replication and spread of rice viruses. The notable advantage of the vector cell monolayer system is that it can reach a uniform infection rate of 100% of the cells in the culture inoculated with diluted viruses, and thus allows for synchronous virus multiplication. The self-propagative nature of leafhopper and planthopper cell lines under favorable conditions ensures the system both dynamic and stable for viral infection. The vector cell monolayer systems and molecular probes, along with reliable traditional methods, certainly facilitate studies on interactions between plant viruses and insect vectors at molecular and cellular levels.Availability of the methods for long-term virus preservation facilitates easy acquirement of viruses, which are needed in many basic and applied virological studies. Cryopreservation is currently considered an ideal means for long-term preservation of plant germplasm. Recent studies have shown that cryopreservation provided an efficient and reliable method for long-term preservation of plant viruses. Here, we describe the detailed procedures of droplet vitrification for long-term preservation of apple stem grooving virus (ASGV), which represents a type of viruses that can invade meristematic cells of the shoot tips, and potato leafroll virus (PLRV), which is a phloem-limited virus that does not infect the apical meristem. Shoot tip cryopreservation provides an advantageous strategy for the long-term preservation of plant viruses.Almost all plants in their natural environment are commonly infected by viruses. These viral infections can cause devastating diseases and result in severe yield and economic losses, making viral diseases an important limiting factor for agricultural production and sustainable development. However, these losses can be effectively reduced through the productions and applications of virus-free plantlets. In vitro culture techniques are the most successful approaches for efficient eradication of various viruses from almost all the most economically important crops. Techniques for producing virus-free plantlets include meristem tip culture, somatic embryogenesis, chemotherapy, thermotherapy, electrotherapy, shoot tip cryotherapy, and micrografting. Among them, meristem tip culture is currently the most widely used. Here, we describe a detailed protocol for producing virus-free plantlets of Chrysanthemum morifolium Ramat using tissue culture techniques.In recent years, plant virus-based vectors have been widely applied to express heterologous proteins for genomic studies and commercial production. Among these versatile RNA viral vectors, the barley yellow striate mosaic virus (BYSMV)-based expression vector system has outstanding capability to express large and multiple heterologous proteins. Here we describe a detailed protocol for expression of heterologous proteins using BYSMV expression systems in monocot plants and insects.As an efficient tool for functional genomics, VIGS (virus-induced gene silencing) has been widely used in reverse and forward genetics to identify genes involved in various biology processes in many plant species. Up to now, at least 50 VIGS vectors based on RNA viruses, DNA viruses or satellites have been developed for either dicots or monocots or both. Silencing specific genes using VIGS vector involves five major steps including, first, choosing an appropriate VIGS vector for the plant; second, selecting a fragment of targeted host gene; third, cloning the fragment into viral VIGS vector; forth, inoculating and infecting the appropriate plant; and fifth, quantifying silencing effects including recording silencing phenotypes and determining silencing efficiency of the target gene. In this chapter, we introduce these steps for VIGS assay in dicots and monocots, by taking a cucumber mosaic virus-based VIGS vector for Nicotiana benthamiana and maize plants as an example. Moreover, we list available VIGS vectors for monocots.Argonaute (AGO) proteins associate with small RNAs (sRNAs) to form an RNA-induced silencing complex (RISC). The ribonuclease (slicer) activity of AGOs is required for the sRNA-complementary target cleavage, which is important for RISC-mediated RNA silencing, especially in plants. Sequencing small RNAs is an obvious choice to understand their expression and downstream effects. It also provides an opportunity to identify novel and polymorphic miRNAs. Recently, we have successfully reconstituted rice (Oryza sativa) AGO1a slicer assays in vitro that are able to recapitulate in vivo miRNA-guided cleavage activity. Here we provide a detailed protocol for the purification of OsAGO1a-sRNA complexes and further slicer assays, small RNA sequencing and bioinformatic analysis. This protocol can be readily adapted for the purification and subsequent analyses of the AGO complexes in other plants.The plant phloem vasculature is crucial for plant growth and development, and is essential for the systemic movement (SM) of plant viruses. Recent transcriptomic studies of the phloem during virus infection have shown the importance of this tissue, yet transcript levels do not provide definitive answers how virus-host interactions favour successful viral SM. Proteomic analyses have been used to identify host-virus protein interactions, uncovering a variety of ways by which viruses utilize host cellular machinery for completion of the viral infection cycle. Despite this new evidence through proteomics, very few phloem centric studies during viral infection have been performed. Here, we describe a protocol for the isolation of phloem tissues and proteins and the subsequent label-free quantitation (LFQ), for identification of proteomic alterations caused by viral infection.In plants, plasmodesmata (PD) are plasmamembrane-lined pores that traverse the cell wall to establish cytoplasmic and endomembrane continuity between neighboring cells. As intercellular channels, PD play pivotal roles in plant growth and development, defense responses, and are also co-opted by viruses to spread cell-to-cell to establish systemic infection. Proteomic analyses of PD-enriched fractions may provide critical insights on plasmodesmal biology and PD-mediated virus-host interactions. However, it is difficult to isolate PD from plant tissues as they are firmly embedded in the cell wall. Here, we describe a protocol for the purification of PD from Nicotiana benthamiana leaves for proteomic analysis.Protein-protein interactions play a crucial role in diverse biological processes. As obligate intracellular parasites, plant viruses live and reproduce in living cells and recruit host proteins through protein-protein interactions to complete their infection process. Elucidation of the protein-protein interaction network between viruses and hosts can advance knowledge in the viral infection process at the molecule level and facilitate the development of novel antiviral technologies. One of the most classic and widely used methods to discover or confirm novel protein interactions in plant cells is the pull-down assay. For plant virology research, this method begins with the expression of a tagged viral protein (such as GST- or His-tagged) as "bait" in model plant species such as Nicotiana benthamiana. The expressed "bait" protein is purified by affinity agarose resin (e.g., glutathione or cobalt chelate) followed by a series of washes. Finally, the "bait"-"prey" protein complexes are subjected to mass spectrometry or immunoblotting analysis.