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Chromatin Beacons: Global Sampling of Chromatin Physical Properties Using Chromatin Charting Lines

The extent to which physical properties and intranuclear locations of chromatin can influence transcription output remains unclear and poorly quantified. Because the scale and resolution at which structural parameters can be queried are usually so different from the scale that tr ...

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Transposable Elements as Catalysts for Chromosome Rearrangements

Barbara McClintock first showed that transposable elements in maize can induce major chromosomal rearrangements, including duplications, deletions, inversions, and translocations. More recently, researchers have made significant progress in elucidating the mechan ...

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Recent Advances in Plant Transformation

Plant genetic engineering has become one of the most important molecular tools in the modern molecular breeding of crops. Over the last decade, significant progress has been made in the development of new and efficient transformation methods in plants. Despite a variety of available DNA del ...

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Engineering the Plastid Genome of Nicotiana sylvestris, a Diploid Model Species for Plastid Genetics

The plastids of higher plants have their own ∼120–160-kb genome that is present in 1,000–10,000 copies per cell. Engineering of the plastid genome (ptDNA) is based on homologous recombination between the plastid genome and cloned ptDNA sequences in the vector. A uniform population of enginee ...

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Homologous Recombination in Plants: An Antireview

Homologous recombination (HR) is a central cellular process involved in many aspects of genome maintenance such as DNA repair, replication, telomere maintenance, and meiotic chromosomal segregation. HR is highly conserved among eukaryotes, contributing to genome stability as w ...

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Chromosome Painting for Plant Biotechnology

Fluorescence in situ hybridization (FISH) is an invaluable tool for chromosome analysis and engineering. The ability to visually localize endogenous genes, transposable elements, transgenes, naturally occurring organellar DNA insertions – essentially any unique sequen ...

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Vectors and Methods for Hairpin RNA and Artificial microRNA-Mediated Gene Silencing in Plants

In plant cells, DICER-LIKE4 processes perfectly double-stranded RNA (dsRNA) into short interfering (si) RNAs, and DICER-LIKE1 generates micro (mi) RNAs from primary miRNA transcripts (pri-miRNA) that form fold-back structures of imperfectly dsRNA. Both si and miRNAs direct the endo ...

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Modeling Antiviral Resistance in Plants

The goal of this chapter is to describe in simple terms how the use of ordinary differential equation (ODE) modeling, in conjunction with experimentation, can be utilized to improve our understanding of the dynamics of gene silencing and virus resistance in plants.

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Isolation and Detection of Small RNAs from Plant Tissues

In plants, several classes of non-coding small RNA (sRNA) have been shown to be important regulators of gene expression in a wide variety of biological processes. The two main classes of sRNA, the small-interfering RNA (siRNA) and microRNA (miRNA) classes, are well documented and several exper ...

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Isolation and Analysis of Small RNAs from Virus-Infected Plants

In this chapter, we detail some of the methods available to the researcher for isolating and analyzing virus-derived small RNAs (vsRNAs). These methods have been successfully used for four plant viruses: Cucumber mosaic virus (CMV), including the CMV Y-Satellite, Turnip mosaic virus (TuMV ...

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Use of Hairpin RNA Constructs for Engineering Plant Virus Resistance

Genetically engineered resistance to protect plants against virus infections can be based on protein- and RNA-mediated defense mechanisms. RNA silencing that leads to high-level virus resistance is triggered by virus-specific double-stranded (ds)RNA. The most efficient means ...

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Artificial MicroRNAs for Plant Virus Resistance

RNA silencing is a natural plant defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants by the expression of long stretches of viral sequences in perfectly paired double-stranded or s ...

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Isolation and Biochemical Analysis of Plant Small RNAs

Small RNAs, defined as noncoding 20–30-nt-long RNAs, are instrumental regulators of cellular processes in most eukaryotes. In this chapter we describe three methods for extracting small RNA from cells: a general method, one plant specific and a third particular to conifers. Further, proto ...

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Plant Viroids: Isolation, Characterization/Detection, and Analysis

When Diener discovered Potato spindle tuber viroid in 1971 (Diener, Virology 45:411–428, 1971), only a limited number of techniques were available for plant virus detection and purification. Biological assays using indicator hosts showing characteristic symptoms of infection a ...

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Isolation and Analysis of Plant RNA-Dependent RNA Polymerases

RNA-dependent RNA polymerases (RDRPs) are encoded by RNA viruses as well as eukaryotic organisms such as plants. The function of these cellular RDRPs has been associated with the synthesis of short interfering RNAs (siRNAs), which are essential regulators of genomic integrity and plant v ...

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Virus-Induced Gene Silencing in Soybean

Understanding the biology of a system requires the association of gene functions to phenotypes and vice versa. Although a large number of resources and genomic tools are available for studies in the crop plant soybean, investigations related to gene function have been lacking. This is large ...

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Structural and Functional Analysis of CMV Satellite RNAs in RNA Silencing

Viroids and satellite RNAs, which are the smallest infectious agents in plants, have noncoding RNA genomes and characteristic secondary structures. Some satellite RNAs (satRNAs) cause disease symptoms that are different from those induced by their helper virus. This phenomenon has ...

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A Historical Overview of RNAi in Plants

RNA interference, or RNAi, is arguably one of the most significant discoveries in biology in the last several decades. First recognized in plants (where it was called post-transcriptional gene silencing, PTGS) RNAi is a gene down-regulation mechanism since demonstrated to exist in all euk ...

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Rolling Circle Amplification-Mediated Long Hairpin RNA Library Construction in Plants

Gene silencing has been used widely in gene function studies and crop plant modification. Long hairpin RNA (lhRNA) results in high efficiency of gene silencing; however, constructing multiple lhRNA vectors using traditional approaches is both time consuming and costly. Also, most of the e ...

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Chimeric, Infectious, and Stable Virus Transcripts to Study RNA Silencing in Dark Green Islands

Dark green islands (DGIs) form in some plant virus infections as areas that are resistant to infection. This resistance has been demonstrated to act through a sequence-specific, RNA degradation mechanism termed RNAi. In addition to the virus that induces the initial DGI, a second, infectious ...

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