Springer protocols feed by Plant Sciences 
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Real-Time PCR Protocols for the Quantification of the Begomovirus Tomato Yellow Leaf Curl Sardinia Virus in Tomato Plants and in Its Insect Vector
Tomato yellow leaf curl Sardinia virus (TYLCSV) (Geminiviridae) is an important pathogen, transmitted by the whitefly Bemisia tabaci, that severely affects the tomato production in the Mediterranean basin. Here, we describe real-time PCR protocols suitable for relative and absolute quantification of TYLCSV in tomato plants and in whitefly extracts. Using primers and probe specifically designed for TYLCSV, the protocols for relative quantification allow to compare the amount of TYLCSV present in different plant or whitefly samples, normalized to the amount of DNA present in each sample using endogenous tomato or Bemisia gen...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Detection Methods for Rice Viruses by a Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)
Developing a quick and accurate method to diagnose rice viruses in host plants and in vector insects is very important to control virus diseases of rice. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay, one of the most promising molecular diagnostic methods, was established to detect nine viruses, including eight RNA viruses and one DNA virus, in infected rice plants and the viruliferous vector insects. The sensitivities of the assays were either higher than or similar to those of one-step RT-PCR. With a combination of rapid RNA extraction and a RT-LAMP assay, these nine viruses were detected...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Multiplex RT-PCR Method for the Simultaneous Detection of Nine Grapevine Viruses
Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal contro...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
A New Method to Isolate Total dsRNA
When a diseased plant is suspected to be infected with unknown viruses, the approach of isolating double-stranded RNA (dsRNA) from diseased tissues and analyzing the sequence has been useful for detecting the viruses. This procedure owes its success to the majority of plant pathogenic viruses being RNA viruses, which accumulate dsRNAs as copies of their genome or as a replicative intermediate in infected cells. Conventional dsRNA isolation methods (e.g., chromatography using CF-11 cellulose) require a significant amount of plant material and are laborious and time consuming. Therefore, it has been impractical to isolate ds...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
RT-PCR and Real-Time RT-PCR Methods for the Detection of Potato Virus Y in Potato Leaves and Tubers
Potato virus Y (PVY) is a major threat to potato crops around the world. It is an RNA virus of the family Potyviridae, exhibiting many different strains that cause a range of symptoms in potato. ELISA detection of viral proteins has traditionally been used to quantify virus incidence in a crop or seed lot. ELISA, however, cannot reliably detect the virus directly in dormant tubers, requiring several weeks of sprouting tubers to produce detectable levels of virus. Nor can ELISA fully discriminate between the wide range of strains of the virus. Several techniques for directly detecting the viral RNA have been developed which...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Detection of Plant Virus in Meristem by Immunohistochemistry and In Situ Hybridization
Most plant viruses do not infect the shoot apical meristem (SAM) of a host plant, and this virus-free region of meristem tissue has been used to obtain virus-free clones by meristem tip culture. Thus, the validation of viral distribution in meristem tissues is important for ensuring the appropriate excision of virus-free meristem tips. Although immunohistochemical microscopy and in situ hybridization are classical techniques, they allow us to determine the presence or absence of plant viruses in the shoot meristem tissues of a host plant. Briefly, meristem tissues are excised from infected plants, fixed, embedded in paraff...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Detection of Plant Viruses in Mixed Infection by a Macroarray-Assisted Method
The protocol for a simple, sensitive, and specific method using a cDNA macroarray to detect multiple viruses is provided. The method can be used even at the production sites for crops, which need a reliable routine diagnosis for mixed infection of plant viruses. The method consists of three steps: RNA extraction, duplex RT-PCR, and “microtube hybridization” (MTH). Biotinylated cDNA probes are prepared using RT-PCR and used to hybridize a nylon membrane containing target viral cDNAs by MTH. Positive signals can be visualized by colorimetric reaction and judged by eyes. We here demonstrate this method to detect a...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Efficient Double-Stranded RNA Production Methods for Utilization in Plant Virus Control
Double-stranded RNA (dsRNA) is an inducer molecule of the RNA silencing (RNA interference, RNAi) pathway that is present in all higher eukaryotes and controls gene expression at the posttranscriptional level. This mechanism allows the cell to recognize aberrant genetic material in a highly sequence specific manner. This ultimately leads to degradation of the homologous target sequence, rendering the plant cell resistant to subcellular pathogens. Consequently, dsRNA-mediated resistance has been exploited in transgenic plants to convey resistance against viruses. In addition, it has been shown that enzymatically synthesized ...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Simplified Methods for the Construction of RNA and DNA Virus Infectious Clones
Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA ...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Virus-Induced Gene Silencing of N Gene in Tobacco by Apple Latent Spherical Virus Vectors
Virus infections induce an RNA-mediated defense that targets viral RNAs in a nucleotide sequence-specific manner in plants, commonly referred to as virus-induced gene silencing (VIGS). When the virus carries sequences of plant genes, it triggers virus-induced gene silencing (VIGS) and results in the degradation of mRNA of endogenous homologous gene. VIGS has been shown to have great potential as a reverse-genetics tool for studying of gene functions in plants, and it has several advantages over other functional genomics approaches. Here, we describe VIGS of N gene in tobacco cv. Xanthi nc by ALSV vectors containing fragmen...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Construction of Infectious cDNA Clones Derived from the Potyviruses Clover Yellow Vein Virus and Bean Yellow Mosaic Virus
Infectious cDNA clones are now indispensible tools for the genetic analysis of viral factors involved in viral virulence and host resistance. In addition, infectious cDNA-derived virus vectors that express foreign genes in infected plants enable the production of useful proteins at low cost and can confer novel crop traits. We constructed infectious cDNA clones derived from two potyviruses, Clover yellow vein virus and Bean yellow mosaic virus, which infect legume plants and cause disease. Here, we present our procedure for constructing these potyvirus infectious clones. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Microarray Analysis of R-Gene-Mediated Resistance to Viruses
The complex process for host-plant resistance to viruses is precisely regulated by a number of genes and signaling compounds. Thus, global gene expression analysis can provide a powerful tool to grasp the complex molecular network for resistance to viruses. The procedures for comparative global gene expression profiling of virus-resistant and control plants by microarray analysis include RNA extraction, cDNA synthesis, cRNA labeling, hybridization, array scanning, and data mining steps. There are several platforms for the microarray analysis. Commercial services for the steps from cDNA synthesis to array scanning are now w...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
SuperSAGE as an Analytical Tool for Host and Viral Gene Expression
SuperSAGE is a tag-based transcript profiling method, which allows to analyze the expression of thousands of genes at a time. In SuperSAGE, 26 bp tags are extracted from cDNA using the type III restriction enzyme, EcoP15I. In SuperSAGE, the amount of transcripts was represented by tag counts. Taking advantage of uniqueness of the 26 bp tags, host and virus transcripts can be monitored in virus-infected cells. Combining next generation sequencing technology, we established High-throughput SuperSAGE (Ht-SuperSAGE), which allows the analysis of multiple samples with reduced time and cost. In this chapter, we present the proto...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Detection and Characterization of Mycoviruses in Arbuscular Mycorrhizal Fungi by Deep-Sequencing
Fungal viruses (mycoviruses) often have a significant impact not only on phenotypic expression of the host fungus but also on higher order biological interactions, e.g., conferring plant stress tolerance via an endophytic host fungus. Arbuscular mycorrhizal (AM) fungi in the phylum Glomeromycota associate with most land plants and supply mineral nutrients to the host plants. So far, little information about mycoviruses has been obtained in the fungi due to their obligate biotrophic nature. Here we provide a technical breakthrough, “two-step strategy” in combination with deep-sequencing, for virological study in...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
Analysis and Application of Viroid-Specific Small RNAs Generated by Viroid-Inducing RNA Silencing
Viroids are noncoding RNA pathogens inducing severe to mild disease symptoms on agriculturally important crop plants. Viroid replication is entirely dependent on host transcription machinery, and their replication/accumulation in the infected cells can activate RNA silencing—a host defense mechanism that targets the viroid itself. RNA silencing produces in the cell large amounts of viroid-specific small RNAs of 21–24-nucleotides by cleaving (or “dicing”) entire molecules of viroid RNA. However, viroid replication is resistant to the effects of RNA silencing and disrupts the normal regulation of host...
Source: Springer protocols feed by Plant Sciences - October 9, 2014 Category: Biology Source Type: news
