The Rad50 genes of diploid and polyploid wheat species. Analysis of homologue and homoeologue expression and interactions with Mre11.

The MRN complex plays a central role in the DNA repair pathways of eukaryotic cells and takes part in many other processes, including cell cycle checkpoint signalling, meiosis, DNA replication and telomere maintenance. This complex is formed by the interaction of the products of the Mre11, Rad50 and Nbs1 genes. This paper reports the molecular characterization, expression and interactions of the Rad50 gene in several wheat species with different levels of ploidy. The homoeologous Rad50 wheat genes were found to show a high level of conservation. Most of the RAD50 domains and motifs previously described in other species were also present in wheat RAD50; these proteins are therefore likely to have similar functions. Interactions between the RAD50 wheat proteins and their MRE11 counterparts in the MRN complex were observed. The level of expression of Rad50 in each of the species examined was determined and compared with those previously reported for the Mre11 genes. In some cases similar levels of expression were seen, as expected. The expression of the RAD50 homoeologous genes was assessed in two polyploid wheat species using quantitative PCR. In both cases, an overexpression of the Rad50B gene was detected. Although the results indicate the maintenance of function of these species' three homoeologous Rad50 genes, the biased expression of Rad50B might indicate ongoing silencing of one or both other homoeologues in polyploid wheat. To assess the consequences of such silencing on the formation of the MRN complex, the interactions between individual homoeologues of Rad50 and their genomic counterpart Mre11 genes were examined. The results indicate the inexistence of genomic specificity in the interactions between these genes. This would guarantee the formation of an MRN complex in wheat.

The role of AtMUS81 in DNA repair and its genetic interaction with the helicase AtRecQ4A.

The endonuclease MUS81 has been shown in a variety of organisms to be involved in DNA repair in mitotic and meiotic cells. Homologues of the MUS81 gene exist in the genomes of all eukaryotes, pointing to a conserved role of the protein. However, the biological role of MUS81 varies between different eukaryotes. For example, while loss of the gene results in strongly impaired fertility in Saccharomyces cerevisiae and nearly complete sterility in Schizosaccharomyces pombe, it is not essential for meiosis in mammals. We identified a functional homologue (AtMUS81/At4g30870) in the genome of Arabidopsis thaliana and isolated a full-length cDNA of this gene. Analysing two independent T-DNA insertion lines of AtMUS81, we found that they are sensitive to the mutagens MMS and MMC. Both mutants have a deficiency in homologous recombination in somatic cells but only after induction by genotoxic stress. In contrast to yeast, no meiotic defect of AtMUS81 mutants was detectable and the mutants are viable. Crosses with a hyperrecombinogenic mutant of the AtRecQ4A helicase resulted in synthetic lethality in the double mutant. Thus, the nuclease AtMUS81 and the helicase AtRecQ4A seem to be involved in two alternative pathways of resolution of replicative DNA structures in somatic cells.

Molecular characterization of homologues of both subunits A (SPO11) and B of the archaebacterial topoisomerase 6 in plants.

The Spo11 protein is an eukaryotic homologue of the topoisomerase 6 subunit A from archaebacteria. In yeast Spo11p has been found to bind covalently to double-strand breaks (DSBs) during meiosis. Single homologues of the SPO11 gene exist in various eukaryotes, except plants. Previously, we found in the Arabidopsis thaliana genome two ancient paralogs, AtSPO11-1 and 2. Here we report on the molecular characterization of a third one, AtSPO11-3. This puzzling finding might be explained by the fact that we detected additionally--for the first time outside of the archaebacterial kingdom--a homologue of the subunit B of topoisomerase 6, AtTOP6B. Both AtSPO11-3 and AtTOP6B are abundantly expressed in Arabidopsis and EST comparisons indicate the presence of both genes in various plant species. Via two hybrid studies we could demonstrate that full length AtTop6B is able to interact with AtSpo11-2 and 3 but not with AtSpo11-1. Our data suggest that plants possess in contrast to other eukaryotes an additional archaebacterial kind of topoisomerase.

Elimination of selection markers from transgenic plants.

Selection markers, which were necessary for the isolation of transgenic plants, are no longer required in mature plants, especially when they are grown in fields. Regimes to achieve their efficient elimination, mostly through site-specific recombination or transposition, are being developed.

Molecular characterisation of RecQ homologues in Arabidopsis thaliana.

Members of the RecQ family of DNA helicases are involved in processes linked to DNA replication, DNA recombination and gene silencing. RecQ homologues of various animals have been described recently. Here, for the first time for plants, we characterised cDNAs of all in all six different RecQ-like proteins that are expressed to different extents in Arabidopsis thaliana. Surprisingly, three of these proteins are small in size [AtRecQl1, AtRecQl2, AtRecQl3-606, 705 and 713 amino acids (aa), respectively], whereas the two bigger proteins result from a duplication event during plant evolution [AtRecQl4A and AtRecQl4B-1150 and 1182 aa, respectively]. Another homologue (AtRecQsim, 858 aa) most probably arose by insertion of an unrelated sequence within its helicase domain. The presence of these homologues demonstrates the conservation of RecQ family functions in higher eukaryotes. We also detected a small gene (AtWRNexo) encoding 285 aa which, being devoid of any RecQ-like helicase domain, reveals a striking homology to the exonuclease domain of human Werner protein, a prominent RecQ helicase of larger size. By means of the two-hybrid assay we were able to detect an interaction between AtWRNexo and AtRecQl2, indicating that activities that reside in a single protein chain in mammals might in plants be complemented in trans.

Species-specific double-strand break repair and genome evolution in plants.

Even closely related eukaryotic species may differ drastically in genome size. While insertion of retroelements represents a major source of genome enlargement, the mechanism mediating species- specific deletions is fairly obscure. We analyzed the formation of deletions during double-strand break (DSB) repair in Arabidopsis thaliana and tobacco, two dicotyledonous plant species differing >20-fold in genome size. DSBs were induced by the rare cutting restriction endonuclease I-SCE:I and deletions were identified by loss of function of a negative selectable marker gene containing an I-SCE:I site. Whereas the partial use of micro-homologies in junction formation was similar in both species, in tobacco 40% of the deletions were accompanied by insertions. No insertions could be detected in Arabidopsis , where larger deletions were more frequent, indicating a putative inverse correlation between genome size and the average length of deletions. Such a correlation has been postulated before by a theoretical study on the evolution of related insect genomes and our study now identifies a possible molecular cause for the phenomenon, indicating that species-specific differences in DSB repair might indeed influence genome evolution.

Elevated UV-B radiation reduces genome stability in plants.

Long-term depletion of the stratospheric ozone layer contributes to an increase in terrestrial solar ultraviolet-B radiation. This has deleterious effects on living organisms, such as DNA damage. When exposed to elevated ultraviolet-B radiation (UV-B; 280-315 nm), plants display a wide variety of physiological and morphological responses characterized as acclimation and adaptation. Here we show, using special sun simulators, that elevated solar UV-B doses increase the frequency of somatic homologous DNA rearrangements in Arabidopsis and tobacco plants. Increases in recombination are accompanied by a strong induction of photolyase and Rad51 gene expression. These genes are putatively involved in major DNA repair pathways, photoreactivation and recombination repair. In mutant Arabidopsis plants that are deficient in photoreactivating ultraviolet-induced cyclobutane pyrimidine dimers, recombination under elevated UV-B regimes greatly exceeds wild-type levels. Our results show that homologous recombination repair pathways might be involved in eliminating UV-B-induced DNA lesions in plants. Thus, increases in terrestrial solar UV-B radiation as forecasted for the early 21st century may affect genome stability in plants.

Molecular characterisation of two paralogous SPO11 homologues in Arabidopsis thaliana.

The Spo11 protein of yeast has been found to be covalently bound to double-strand breaks in meiosis, demonstrating a unique role of the protein in the formation of these breaks. Homologues of the SPO11 gene have been found in various eukaryotes, indicating that the machinery involved in meiotic recombination is conserved in eukaryotes. Here we report on SPO11 homologues in plants. In contrast to what is known from other eukaryotes, Arabidopsis thaliana carries in its genome at least two SPO11 homologues, AtSPO11-1 and AtSPO11-2. Both genes are not more closely related to each other than to other eukaryotic SPO11 homologues, indicating that they did not arise via a recent duplication event during higher plant evolution. For both genes three different poly-adenylation sites were found. AtSPO11-1 is expressed not only in generative but also to a lesser extent in somatic tissues. We were able to detect in different organs various AtSPO11-1 cDNAs in which introns were differently spliced-a surprising phenomenon also reported for SPO11 homologues in mammals. In the case of AtSPO11-2 we found that the 3' end of the mRNA is overlapping with a mRNA produced by a gene located in inverse orientation next to it. This points to a possible antisense regulation mechanism. Our findings hint to the intriguing possibility that, at least for plants, Spo11-like proteins might have more and possibly other biological functions than originally anticipated for yeast.

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Double-strand break-induced recombination between ectopic homologous sequences in somatic plant cells.

Homologous recombination between ectopic sites is rare in higher eukaryotes. To test whether double-strand breaks (DSBs) can induce ectopic recombination, transgenic tobacco plants harboring two unlinked, nonfunctional homologous parts of a kanamycin resistance gene were produced. To induce homologous recombination between the recipient locus (containing an I-SceI site within homologous sequences) and the donor locus, the rare cutting restriction enzyme I-SceI was transiently expressed via Agrobacterium in these plants. Whereas without I-SceI expression no recombination events were detectable, four independent recombinants could be isolated after transient I-SceI expression, corresponding to approximately one event in 10(5) transformations. After regeneration, the F1 generation of all recombinants showed Mendelian segregation of kanamycin resistance. Molecular analysis of the recombinants revealed that the resistance gene was indeed restored via homologous recombination. Three different kinds of reaction products could be identified. In one recombinant a classical gene conversion without exchange of flanking markers occurred. In the three other cases homologous sequences were transferred only to one end of the break. Whereas in three cases the ectopic donor sequence remained unchanged, in one case rearrangements were found in recipient and donor loci. Thus, ectopic homologous recombination, which seems to be a minor repair pathway for DSBs in plants, is described best by recombination models that postulate independent roles for the break ends during the repair process.

Chromosomal location and genetic mapping of the mismatch repair gene homologs MSH2, MSH3, and MSH6 in rye and wheat

The efficiency of homeologous recombination is influenced by mismatch repair genes in bacteria, yeast, and mammals. To elucidate a possible role of these genes in homeologous pairing and cross-compatibility in plants, gene probes of wheat (Triticum aestivum) specific for the mismatch repair gene homologues MSH2, MSH3, and MSH6 were used to map them to their genomic positions in rye (Secale cereale). Whereas MSH2 was mapped to the short arm of chromosome 1R, MSH3 was mapped to the long arm of chromosome 2R and MSH6 to the long arm of chromosome 5R. Southern blots with nullisomic-tetrasomic (NT) lines of wheat indicated the presence of the sequences on the respective homeologous group of wheat chromosomes. Additionally, an MSH6-specific homologue could also be detected on homoeologous group 3 of wheat. However, in the well-known, highly homoeologous pairing wheat mutant ph1b the MSH6-specific sequence is not within the deleted part of chromosome 5BL, indicating that the pairing phenotype is not due to a loss of one of the mismatch repair genes tested.

Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells.

To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene. After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker. Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break. Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions. This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation. Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site. This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome.

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.

Somatic intrachromosomal homologous recombination events in populations of plant siblings.

Intrachromosomal homologous recombination in whole tobacco plants was analyzed using beta-glucuronidase as non-selectable marker. We found that recombination frequencies were additive for transgenes in allelic positions and could be enhanced by treatment of plants with DNA-damaging agents. We compared the patterns of distribution of recombination events of different transgenic lines of tobacco and Arabidopsis with the respective Poisson distributions. Some lines showed Poisson-like distributions, indicating that recombination at the transgene locus was occurring in a random fashion in the plant population. In other cases, however, the distributions deviated significantly from Poisson distributions indicating that for specific transgene loci and/or configurations recombination events are not randomly distributed in the population. This was due to overrepresentation of plants with especially many as well as especially few recombination events. Analysis of one tobacco line indicated furthermore that the distribution of recombination events could be influenced by treating the seedlings with external factors. Our results suggest that different plant individuals, or parts of them, might exhibit different transient 'states' of recombination competence. A possible model relating 'recombination silencing' and transcription silencing to heterochromatization of the transgene locus is discussed.

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker.

We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.

Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus.

Transferred DNA (T-DNA) is transferred as a single-stranded derivative from Agrobacterium to the plant cell nucleus. This conclusion is drawn from experiments exploiting the different properties of single- and double-stranded DNA to perform extrachromosomal homologous recombination in plant cells. After transfer from Agrobacterium to plant cells, T-DNA molecules recombined much more efficiently if the homologous sequences were of opposite polarity than if they were of the same polarity. This observation reflects the properties of single-stranded DNA; single-stranded DNA molecules of opposite polarity can anneal directly, whereas single-stranded DNA molecules of the same polarity first have to become double stranded to anneal. Judging from the relative amounts of single- to double-stranded T-DNA derivatives undergoing recombination, we infer that the T-DNA derivatives enter the plant nucleus in their single-stranded form.

Intrachromosomal homologous recombination in whole plants.

A system to assay intrachromosomal homologous recombination during the complete life-cycle of a whole higher eukaryote was set up. Arabidopsis thaliana plants were transformed with a recombination substrate carrying a non-selectable and quantitatively detectable marker gene. The recombination substrates contain two overlapping, non-functional deletion mutants of a chimeric beta-glucuronidase (uidA) gene. Upon recombination, as proven by Southern blot analysis, a functional gene is restored and its product can be detected by histochemical staining. Therefore, cells in which recombination events occurred, and their progeny, can be precisely localized in the whole plant. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Meristematic recombination events revealed cell lineage patterns. Overall recombination frequencies typically were in the range 10(-6)-10(-7) events/genome. Recombination frequencies were found to differ in different organs of particular transgenic lines.