Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens

In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates predominantly by homologous recombination with its genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). Overall homology of only 1 kb is sufficient to achieve a 50% yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement following two homologous recombination events. Allele replacement frequency depends on the symmetry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the transforming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single insertions at targeted loci (targeted gene replacements, ‘TGR’) occur with a frequency of 7–20% of all transformants when the minimum requirements for allele replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and developmen

Giant peroxisomes in a moss (Physcomitrella patens) peroxisomal biogenesis factor 11 mutant

Peroxisomal biogenesis factor 11 (PEX11) proteins are found in yeasts, mammals and plants, and play a role in peroxisome morphology and regulation of peroxisome division. The moss Physcomitrella patens has six PEX11 isoforms which fall into two subfamilies, similar to those found in monocots and dicots. We carried out targeted gene disruption of the Phypa_PEX11-1 gene and compared the morphological and cellular phenotypes of the wild-type and mutant strains. The mutant grew more slowly and the development of gametophores was retarded. Mutant chloronemal filaments contained large cellular structures which excluded all other cellular organelles. Expression of fluorescent reporter proteins revealed that the mutant strain had greatly enlarged peroxisomes up to 10 μm in diameter. Expression of a vacuolar membrane marker confirmed that the enlarged structures were not vacuoles, or peroxisomes sequestered within vacuoles as a result of pexophagy. Phypa_PEX11 targeted to peroxisome membranes could rescue the knock out phenotype and interacted with Fission1 on the peroxisome membrane. Moss PEX11 functions in peroxisome division similar to PEX11 in other organisms but the mutant phenotype is more extreme and environmentally determined, making P. patens a powerful system in which to address mechanisms of peroxisome proliferation and division

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development

The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency

Up-regulated genes annotated as DNA helicases.

<p>Up-regulated genes annotated as DNA helicases.</p

Differentially regulated genes annotated as cell-cycle associatedFold change.

<p>Differentially regulated genes annotated as cell-cycle associatedFold change.</p