植物生理

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 ...

The small size of most plant virus genomes and their very limited coding capacities requires that plant viruses are dependent on proteins expressed by the host plant for all stages of their life cycle. Identification of these host proteins is essential if we are to understand in any meaningful way t ...

Direct gene transfer into tobacco leaf protoplasts via electroporation is used in many laboratories for transient-expression studies. Protoplasts in general are a convenient model to study events that occur in planta rapidly. Transient expression provides answers in a matter of da ...

A main objective of electroporation is to transform cells by stable incorporation and expression of foreign genetic material. Incorporation may result in transformation with marker genes, as well as desired traits from a breeder’s point of view (e.g., male sterility, virus resistance, in ...

Plant cell walls present a barrier to DNA uptake not present in mammalian cells or plant protoplasts. Transformation methods that force DNA through this network of cellulose fibers include particle gun technology (ballistics; 1), imbibition of dried seeds (2), and use of silicon carbide “wh ...

Protoplast fusion and subsequent in vitro plant regeneration, leading to somatic hybridization, offer opportunities for transferring entire genomes from one plant into another, regardless of the interspecific crossing barriers. In contrast to techniques for plant transfor ...

The complex nature of plant metabolism often can seriously hinder the functional analysis of many proteins; and this has led some researchers to attempt to express such proteins in less sophisticated organisms. Unfortunately, the obvious choice, Escherichia coli, is less than ideal with ...