Gene targeting in plants

The plant photoreceptor phytochrome plays an important role in the nucleus as a regulator of transcription. Numerous studies imply, however, that phytochromes in both higher and lower plants mediate physiological reactions within the cytoplasm. In particular, the tip cells of moss protonemal filaments use phytochrome to sense light direction, requiring a signaling system that transmits the directional information directly to the microfilaments that direct tip growth. In this work we describe four canonical phytochrome genes in the model moss species Physcomitrella patens, each of which was successfully targeted via homologous recombination and the distinct physiological functions of each gene product thereby identified. One homolog in particular mediates positive phototropism, polarotropism, and chloroplast movement in polarized light. This photoreceptor thus interacts with a cytoplasmic signal͞response system. This is our first step in elucidating the cytoplasmic signaling function of phytochrome at the molecular level.

Section: Methodsmentioning

confidence: 99%

Targeted knockout in Physcomitrella reveals direct actions of phytochrome in the cytoplasm

Mittmann

Brücker

Zeidler

et al. 2004

“…In most of these studies, DNA was introduced into cultured cells by direct gene transfer (10,11) or Agrobacterium-mediated transformation (12-15). In general, GT efficiencies were low, on both a per-cell basis (Ͻ10 Ϫ7 GT events per cell) and per-integration basis (10 Ϫ6 to 10 Ϫ4 GT events per integration), and negative selection to select against random integrations was not successful (16)(17)(18)(19).…”

Section: ϫ5mentioning

confidence: 99%

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Lloyd

Plaisier

Carroll³

et al. 2005

382

249

Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are not currently available. In this study we investigated the efficacy of a targeted-mutagenesis approach based on zinc-finger nucleases (ZFNs). In this procedure, ZFNs are used to generate double-strand breaks at specific genomic sites, and subsequent repair produces mutations at the break site. To determine whether ZFNs can cleave and induce mutations at specific sites within higher plant genomes, we introduced a construct carrying both a ZFN gene, driven by a heat-shock promoter, and its target into the Arabidopsis genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1-52 bp (median of 4 bp), 14 (13%) were simple insertions of 1-4 bp, and 9 (8%) were deletions accompanied by insertions. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFNinduced mutations. These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes.gene targeting ͉ nonhomologous end joining A major focus of plant biotechnology is genetic modification and improvement of crop plants. With this aim, large-scale genome and͞or EST sequencing projects are underway for many important plant species including rice, maize, wheat, soybean, and tomato (www.ncbi.nlm.nih.gov͞genomes͞PLANTS͞ PlantList.html). The enormous amount of genome-sequence information becoming available has intensified the need for methods that can use this sequence information to generate targeted modifications in plant genes. Targeted-mutagenesis methods could be used experimentally to investigate plant gene function or for genetic modification of important crop plants. Such methods are especially important for species, including most crops, that lack readily available mutant collections (1, 2). Furthermore, targeted mutagenesis could facilitate development of genetically modified crops lacking transgenic DNA, including genes conferring resistance to antibiotics. To date, efficient and widely usable methods for targeted modification of higher plant genomes are not available.The most widely used targeted-mutagenesis strategy is gene targeting (GT) by homologous recombination (3-6). Efficient GT procedures have been available for Ͼ20 years in yeast (7) and mouse (8). In these systems, DNA fragments are introduced into cultured cells, and repair by homologous recombination causes the introduced DNA to be incorporated into the homologous locus. Typically, GT events ...

“…T-DNA integration does not take place by homologous recombination, the most common method of foreign DNA integration in prokaryotes and lower eukaryotes, because no extensive homology between the T-DNA and target sequences has been found. T-DNA therefore integrates by illegitimate recombination (16)(17)(18)(19), the predominant mechanism of DNA integration into the genomes of higher plants (20)(21)(22). However, plant factors involved in illegitimate recombination of T-DNA into the plant genome have not yet been identified.…”

Section: T-dna Transformation ͉ Haplo-insufficiencymentioning

confidence: 99%

An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration

Mysore

Nam

Gelvin

2000

153

150

Agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, the T-DNA, to the plant and integrating the T-DNA into the plant genome. Little is known about the T-DNA integration process, and no plant genes involved in integration have yet been identified. We characterized an Arabidopsis mutant generated by T-DNA insertional mutagenesis, rat5, that is resistant to Agrobacterium root transformation. rat5 contains two copies of T-DNA integrated as a tandem direct repeat into the 3 untranslated region of a histone H2A gene, upstream of the polyadenylation signal sequence. Transient and stable ␤-glucuronidase expression data and assessment of the amount of T-DNA integrated into the genomes of wild-type and rat5 Arabidopsis plants indicated that the rat5 mutant is deficient in T-DNA integration. We complemented the rat5 mutation by expressing the RAT5 histone H2A gene in the mutant plant. Overexpression of RAT5 in wild-type plants increased Agrobacterium transformation efficiency. Furthermore, transient expression of a RAT5 gene from the incoming T-DNA was sufficient to complement the rat5 mutant and to increase the transformation efficiency of wild-type Arabidopsis plants.T-DNA transformation ͉ haplo-insufficiency