植物学技术

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 p ...

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 me ...

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 sequenc ...

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 te ...

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 mutat ...

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 imp ...

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 clo ...

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) introducti ...

The generation of infectious clones is routinely the first step for reverse genetic studies of RNA plant virus gene and sequence function. The procedure given here, details the creation of cDNA clones of tobacco mosaic virus, from which infectious transcripts can be generated in vitro with T7 R ...

Reverse genetic approach is widely used in virology as it makes possible direct identification of viral gene function and uses RNA genomes as vectors. Production of infectious cDNA clones is an essential step in developing a reverse genetic system for an RNA virus. Here, we present rapid method f ...

To characterize a virus at the molecular and biological levels, it is necessary to produce an infectious clone. For most of the Geminiviridae, cloning of the genome is relatively easy because of their small genomes and the presence of the virus double-stranded (replicative) DNA form in infected ...

Some DNA fragments are difficult to clone in Escherichia coli by standard methods. It has been speculated that unintended transcription and translation result in expression of proteins that are toxic to the bacteria. This problem is frequently observed during assembly of infectious fu ...

Double-stranded DNA (dsDNA) viruses of plants are believed to be plant pararetroviruses. Their genome is replicated by reverse transcription of a larger than unit-length terminally redundant RNA transcript of the viral genomic DNA using the virus-encoded replicase. In order to produ ...

Apple latent spherical virus (ALSV) expressing green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP) was constructed and used to analyze the local and systemic movement of the virus in infected plants. In Chenopodium quinoa plants inoculated with GFP-ALSV, the infection foci fir ...

Plant-virus interaction studies, for long, plagued by asynchronous/failed infections, have improved since the usage of Agrobacterium as a delivery agent for viral genomes. Popularly known as “agroinoculation,” this method has revolutionized plant virology studies, leading to ...

The Geminiviridae family is a large family of plant viruses that has single-stranded DNA genomes and infects a large variety of crop species. In this chapter, we describe a biolistic inoculation protocol that has been successfully used to propagate new species of geminivirus in permissive h ...

Since their conception in the late 1990s, microarray techniques have become a tool of choice for monitoring pangenomic gene expression. Although there are a large number of variations on the basic methodology the general approach remains standard and involves the comparison of a “test” RNA ...

The central step in virus infection cycle is replication, which depends on viral and host factors. Model hosts, such as yeast, can be very valuable to identify host factors and study the functional interactions of host factors with viral proteins and/or the virus nucleic acids. The advantages of us ...

Plant viruses encode movement proteins (MPs) which play important roles in spreading their infectious materials throughout host plants. This infection is facilitated by cell-to-cell trafficking of MPs through specialized channels termed plasmodesmata, which involves spe ...

Host factors are crucial determinants of viral pathogenicity. Identifying host factors and their contributions to virus infections may lead to the development of novel antiviral strategies. The recently developed virus-induced gene silencing (VIGS) approach offers a rapid mea ...