A process is provided of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell. The process may be practiced ex vivo or in vivo. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. The present invention is distinguished from prior art interference in gene expression by antisense or triple-strand methods.

Genetic Inhibition by Double-Stranded RNA

Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.

/pdf/agrobacterium-mediated-plant-transformation-the-biology-4ez5vf6vl1.pdf

Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Epigenetics is the study of heritable changes in gene expression that occur without a change in DNA sequence. Epigenetic phenomena have major economic and medical relevance, and several, such as imprinting and paramutation, violate Mendelian principles. Recent discoveries link the recognition of nucleic acid sequence homology to the targeting of DNA methylation, chromosome remodeling, and RNA turnover. Although epigenetic mechanisms help to protect cells from parasitic elements, this defense can complicate the genetic manipulation of plants and animals. Essential for normal development, epigenetic controls become misdirected in cancer cells and other human disease syndromes.

https://science.sciencemag.org/content/sci/286/5439/481.full.pdf

Epigenetics: Regulation Through Repression

Arabidopsis SGS2 and SGS3 Genes Are Required for Posttranscriptional Gene Silencing and Natural Virus Resistance

Pigments are present in all living matter and provide attractive colors and play basic roles in the development of organisms. Human beings, like most animals, come in contact with their surroundings through color, and things can or cannot be acceptable based on their color characteristics. This review presents the basic information about pigments focusing attention on the natural ones; it emphasizes the principal plant pigments: carotenoids, anthocyanins, and betalains. Special considerations are given to their salient characteristics; to their biosynthesis, taking into account the biochemical and molecular biology information generated in their elucidation; and to the processing and stability properties of these compounds as food colorants.

Natural Pigments: Carotenoids, Anthocyanins, and Betalains — Characteristics, Biosynthesis, Processing, and Stability

Flower pigmentation patterns were scored in 185 senseChalcone synthase (Chs) transgenotes and 85 antisenseChs transgenotes; upon first flowering, 139 (75%) of sense transgenotes were found to be phenotypically altered, as were 70 (82%) of the antisense transgenotes. The observed patterns document the range of phenotypic variations that occur, as well as confirm and extend the finding that senseChs constructs produce several types of morphologybased based flower pigmentation patterns that antisenseChs constructs do not. Long-term monitoring for epigenetic variations in one population of 44 senseChs transgenotes showed that 43 (98%) were capable of producing a cosuppression phenotype. The primary determinant of sense-specific patterns of cosuppression ofChs was found to be the repetitiveness and organization pattern of the transgene, not ‘position effects’ by, or ‘readthrough’ from, flanking plant DNA sequences. The degree of cosuppression observed in progeny of transgenotes carrying multiple, dispersed copies as compared to that observed with a single copy of the transgene suggests that sense cosuppression ofChs is subject to a transgene dosage effect.

Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences

By comparing the effects of strong and weak promoters that drive sense chalcone synthase (Chs) transgenes in large populations of independently transformed plants, we show here that a strong transgene promoter is required for high-frequency cosuppression of Chs genes and for production of the full range of cosuppression phenotypes. In addition, sense Chs transgenes driven by a cauliflower mosaic virus 35S promoter possessing a single copy of the upstream activator region (UAR) were found to produce a significantly lower degree of cosuppression than they did when the transgene promoter possessed two or four copies of the UAR. It has been shown elsewhere that 35S promoter strength increases with increasing UAR copy number. Frameshift mutations producing early nonsense codons in the Chs transgene were found to reduce the frequency and the degree of cosuppression. These results suggest that promoter strength and transcript stability determine the degree of cosuppression, supporting the hypothesis that sense cosuppression is a response to the accumulation of transcripts at high concentrations. This conclusion was shown to apply to single-copy transgenes but not necessarily to inversely repeated transgenes. The results presented here also have significance for efficient engineering of cosuppression phenotypes for use in research and agriculture.

The Frequency and Degree of Cosuppression by Sense Chalcone Synthase Transgenes Are Dependent on Transgene Promoter Strength and Are Reduced by Premature Nonsense Codons in the Transgene Coding Sequence.

Summary
A single-copy sense Chalcone synthase (Chs) transgene driven by a strong promoter and producing a fully translatable transcript was converted to an allelic antisense Chs transgene by Cre-lox-mediated DNA recombination in petunia. The sense Chs allele suppressed flower pigmentation in a simple pattern determined by cells at the junctions between adjacent petals, as is typical of single-copy sense Chs transgenes of this type, whereas the antisense Chs allele produced a different pattern of Chs suppression with white petal edges and reduced pigmentation throughout the petal limbs, as is typical of antisense Chs transgenes. In plants carrying a lox-flanked Chs transgene, the presence of Cre protein can cause both sense-specific and antisense-specific patterns to be superimposed in the same flower, suggesting that sense and antisense suppression by single-copy transgenes are mediated by different mechanisms or occur in different cellular or developmental compartments. The presence of Cre also causes the production of numerous, non-clonal white spots, suggesting that the turnover state is not cell-autonomous.

Distinct patterns of pigment suppression are produced by allelic sense and antisense chalcone synthase transgenes in petunia flowers

Plant transgenes may participate in two types of homology-based gene silencing. One requires transcript homology, is post-transcriptional, and is referred to as cosuppression; the other requires promoter homology, is transcriptional, and is similar to paramutation. This paper uses flower color transgenes to address the hierarchical operation of both mechanisms in plants carrying two transgene copies. It is shown that cosuppression of homologous, endogenous flower color genes by single-copy transgenes requires that the transgene be driven by a strong promoter and that the degree of cosuppression is highly sensitive to increasing transgene dosage. Together, these observations suggest that cosuppression should be a sensitive reporter of epigenetic changes in transgene transcription, such as might be caused by paramutation-like interactions between transgene loci. Intercrosses bringing together two homologous transgene loci, one a known epimutable reporter and the other a transgene inverted repeat, result in complete loss of cosuppression in some outcross progeny and a qualitative change in morphology-based patterns of cosuppression in other outcross progeny. This paramutation-like behavior suggests that the transgenes may be altered at the transcriptional level, eliminating cosuppression altogether or changing the spatial pattern of transgene transcription to produce a new pattern of cosuppression.

Qiudeng Que

Papers

Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences

The Frequency and Degree of Cosuppression by Sense Chalcone Synthase Transgenes Are Dependent on Transgene Promoter Strength and Are Reduced by Premature Nonsense Codons in the Transgene Coding Sequence.

Distinct patterns of pigment suppression are produced by allelic sense and antisense chalcone synthase transgenes in petunia flowers

Homology-based control of gene expression patterns in transgenic petunia flowers.

Do unintended antisense transcripts contribute to sense cosuppression in plants