Transcription of nuclear organellar DNA in a model plant system

Endosymbiotic gene transfer from cytoplasmic organelles (chloroplasts and mitochondria) to the nucleus is an ongoing process in land plants. Although the frequency of organelle DNA migration is high, functional gene transfer is rare because a nuclear promoter is thought necessary for activity in the nucleus. Here we show that a chloroplast promoter, 16S rrn, drives nuclear transcription, suggesting that a transferred organellar gene may become active without obtaining a nuclear promoter. Examining the chromatin status of a known de novo chloroplast integrant indicates that plastid DNA inserts into open chromatin and that this relaxed condition is maintained after integration. Transcription of nuclear organelle DNA integrants was explored at the whole genome level by analyzing RNA-seq data of Oryza sativa subsp. japonica, and utilizing sequence polymorphisms to unequivocally discriminate nuclear organelle DNA transcripts from those of bona fide cytoplasmic organelle DNA. Nuclear copies of organelle DNA that are transcribed show a spectrum of transcriptional activity but at comparatively low levels compared with the majority of other nuclear genes.Dong Wang, Zhipeng Qu, David L. Adelson, Jian-Kang Zhu, and Jeremy N. Timmi

Instability of Plastid DNA in the Nuclear Genome

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes

Single Molecule PCR Reveals Similar Patterns of Non-Homologous DSB Repair in Tobacco and Arabidopsis

DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1–20 bp) deletions occurred at a minority (25–45%) of repair junctions. Approximately ∼1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ

Endosybiotic evolution in action: Real-time observations of chloroplast to nucleus gene transfer

The origin of new genes has long been considered a fundamental question in evolutionary biology. In eukaryotes, a major pathway for the ‘birth’ of new nuclear genes has been transfer of genes from the cytoplasmic organelles (mitochondria and plastids) to the nucleus. While the vast majority of gene transfer occurred shortly after endosymbiosis, the process continues today and is still driving the evolution of nuclear genomes. In tobacco (Nicotiana tabacum) a number of studies have indicated that DNA can transfer from the chloroplast to the nucleus at relatively high frequency. Less has been known, however, about how a newly transferred organelle gene can become activated in this new genetic environment. In a recent report we observed, in real-time, the activation of a plastid reporter gene newly transferred to the nucleus. A key observation from this study was that non-homologous repair is an important generator of novel sequence combinations which, in rare instances, can result in the nuclear activation of plastid genes. In addition, the activation of relocated genes can be aided by the fortuitous presence of plastid sequences able to promote nuclear expression

Novel Insertion Sequence Elements Associated with Genetic Heterogeneity and Phenotype Conversion in Ralstonia solanacearum

Three insertion sequences (IS) elements were isolated from the phytopathogen Ralstonia solanacearum. Southern hybridization using these IS elements as probes revealed hybridization profiles that varied greatly between different strains of the pathogen. During a spontaneous phenotype conversion event, the promoter of the phcA gene was interrupted by one of these IS elements

Overall segregation of kanamycin resistance in self-fertilised progeny of kr2.1-2.10.

<p>The total number of kanamycin resistant and sensitive seedlings from all seed capsules of kr2.1-2.10, excluding those which deviated significantly (<i>P</i><0.01) from 3∶1 kr∶ks, are shown. <i>P</i> values correspond to deviation from 3∶1 kr∶ks.</p><p>NS <i>P</i>>0.01, ** <i>P</i><0.01, *** <i>P</i><0.001.</p>a<p>In this case the one seed capsule which deviated significantly from 3∶1 kr∶ks was included in the analysis (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000323#pgen-1000323-t001" target="_blank">Table 1</a>).</p