Final Report Summary - RECBREED (Recombination: an old and new tool for plant breeding)
Executive Summary:
The main objective of the RECBREED project was to improve the toolkit of plant breeding. Plant breeders select plants out of variants that show desirable traits. One of the major mechanisms to generate biodiversity is called homologous recombination (HR), a process whereby similar chromosomes exchange segments and reshuffle the parental genes. One particular type of HR, called meiotic recombination, takes place in the cells that generate the male and female gametes (pollen and egg cells). It is a process that is tightly controlled and that is responsible for much of the genetic variation in a species. A major goal of this project was to study the mechanisms controlling meiotic recombination in order to enhance the rate of meiotic recombination. In addition, we attempted to enhance the rate of another type of HR, namely gene targeting (GT), the recombination between a genomic sequence and an introduced DNA fragment. The GT technology is the ideal solution for precise engineering of plant genomes. In both types of recombination, the process is initiated by a DNA double stranded break (DSB), followed by the formation of a duplex between the recombination partners (homology search and strand invasion) and ending with the separation of the recombining partners (resolution of recombination intermediates). We have studied each of these stages through the characterization of the genes involved and the testing of their function.
DSB-induced mutagenesis and gene targeting (GT) was successfully performed in the model plant Arabidopsis and the crop plant tomato. Initially zinc finger nucleases (ZFNs) were used to introduce targeted DSBs. This resulted in footprints (indels consisting of small deletion and insertion) and increased GT frequencies with the Arabidopsis PPO gene and the tomato ALS genes. In a later phase of the project also mutation induction of TAL effector nucleases (TALENs) could be demonstrated. Independently, a new approach of an “in planta” gene targeting technique was developed, based on the separation of the transformation and GT steps. The technique requires less person effort than protocols requiring transformation at the GT step. In planta GT could be demonstrated for the model plant Arabidopsis as well as the crop plant maize. Moreover It could be demonstrated that also by the over expression of a protein that is involved in HR namely AtRAD52-1A GT frequencies can be enhances (10-fold increase in the rate of precise GT). Thus, successful GT in plants could be achieved by different approaches by the RECBREED consortium.
Interesting results on means to influence meiotic recombination were obtained. An early role for heterochromatic centromeric regions and 5S ribosomal gene loci in meiotic chromosome synapsis, together with differing requirements for the Rad51 paralog proteins and the two recombinases Rad51 and Dmc1 could be identified. Differential effects of DNA methylation on meiotic recombination were described for euchromatic and heterochromatic chromosomal regions, and evidence found for roles of additional factors controlling recombination suppression in heterochromatin. A mean to induce genome wide meiotic hyper-recombination phenotypes were identified: The Rad51 paralogs RAD51B and XRCC2 are required to regulate cross over formation; mutants in both genes have more COs than wild type. Thus various means to modulate meiotic recombination were identified.
At the moment analysis of several other factors involved in GT and/or meiotic recombination is still ongoing and it is expected that after the end of the official funding further interesting result especially in respect to crop plants will be obtained by the participants of the RECBREED consortium.
Project Context and Objectives:
The main objective of RECBREED project was to improve plant breeding by enhancing Homologous Recombination (HR rates) in somatic and in meiotic cells. The expected outcomes of such enhancement were to establish an efficient gene targeting (GT) technology for precise engineering of plant genomes and to define means to control the rate of meiotic recombination between homologous or homeologous chromosomes.
The main objectives were fulfilled through the following scientific and technological objectives set for various work packages as follows:
1- Gene targeting and meiotic recombination enhancement via DNA Double strand break DSB induction.
The goal of this work package was to develop techniques to specifically enhance homologous recombination at the initiation stage by controlled induction of Double-Strand Break (DSBs). On one hand this approach was applied for gene targeting stimulation and on the other to enhance meiotic recombination. DSB induction was either targeted at specific sites or stimulated in a genome wide manner. The practical applicability of the approaches was tested in the crop plants, tomato and maize.
DSB-induced mutagenesis and gene targeting (GT) was successfully performed in the model plant Arabidopsis and the crop plant tomato. Initially zinc finger nucleases (ZFNs) were used to introduce DSBs. This resulted in footprints (indels consisting of small deletion and insertion) and increased GT frequencies with the Arabidopsis PPO gene and the tomato ALS genes. During the time frame of the project another powerful genome engineering tool emerged, the so-called TAL effector nucleases (TALENs). TALENs were tested directed for DSB induction in the Arabidopsis Cruciferin gene and the tomato MLH1 and EPSPS genes. Initial experiments show that DSBs were induced as detected by means of footprint analysis.
In order to stimulate meiotic recombination at a specific site ZFNs were selected for DSB induction in 3bp deletion regions in Bur-0 and Tsu-0 ecotypes, which are not present in Col-0. Crossing should result in specific DSBs during meiosis and increased recombination. These ZFNs were expressed under control of the SPO11 promoter. In order to analyze crossover frequencies CAPS and HRM markers were established for Bur-0 and Tsu-0. Unfortunately, the ZFNs were not active on their target sites as found in yeast assays and Arabidopsis transformants, although they showed some toxicity, resulting in partial sterility.
Subsequently, meiotic recombination was analyzed in plants overexpressing SPO11. Since the F1 of Col-0 x Bur-0 had a much extended lifecycle, it was decided to use the Ler-1 ecotype instead. Therefore, Ler-1 CAPS and HRM markers were established and used for analysis of crossover frequencies in F1 plants Ler-1 x Col-0 overexpressing SPO11. Indeed, the results suggested that crossover frequency was changed in several regions on different chromosomes.
Meiocyte-specific promoters for expression in maize were isolated and tested. Promoters of the rice homologs of the Arabidopsis SDS (Solo-dancer), ASY1 and SPO11-1 genes were cloned in GUS or GFP reporter constructs. Since visual quantification was inconclusive, QRT-PCR was performed to determine expression of SPO11 genes under control of OsUbi3, OsSPO11, OsDMC1b and OsASY1 promoters as well as the expression of the endogenous SPO11-1 and 2, ASY1 and DMC1 genes in leaves and anthers. In anthers SPO11 expression from pOsUbi3 gave on average an overexpression of 16 times with respect to the endogenous SPO11-1 levels. However such overexpression was likely to be largely due to expression in the anther wall. For the pDMC1b construct expression of SPO11-1 was similar to that of the endogenous SPO11-1 gene. For both pSPO11-1 and pASY1, the expression levels of SPO11-1 and SPO11-2 were barely detectable.
2- Homology search and heteroduplex formation
Homologous recombination occurs through recognition and invasion of one DNA molecule by another. This invading DNA strand is a coated with Rad51 protein in somatic cells and Rad51 and/or Dmc1 in meiosis, and it is this helical nucleoprotein filament which is the active molecular species in the invasion process. This Workpackage concerned study the roles of the "accessory" protein cofactors (Rad51 paralogs, Brca2, Rad52), and the influence of chromatin structure (using "epiRILS" and Ddm1 mutants) on the efficiency and outcome of this fundamental step of the homologous recombination process. These studies in the model plant Arabidopsis thaliana (Tasks 2.1 2.2) were complemented by work in the crop plant, Maize, on the development and application of recombination-based technologies for crop breeding (Task 2.3).
We characterised the accessory proteins which assist in the formation of the RAD51-DNA nucleofilament and modulate its function. A "missing link" among these, RAD52, was identified as two genes and multiple spliced variants which were used to construct fusion proteins and targeted specifically to nuclear, mitochondrial and chloroplast compartments of Arabidopsis cells. Similarly, plant and subcellular localisation of the five Rad51 paralogs were determined using plants expressing fluorescent protein fusion proteins, expressed both with their own, and with constitutive promoters. Arabidopsis has two Brca2 orthologs and the double mutant was shown be sterile, but to develop normally. Surprisingly, over-expression of one of the Brca2 genes generated severe developemental defects. Roles of these proteins in somatic and meiotic recombination were characterised using knockout mutants, overexpression and RNAi knockdowns. Using DNA methylation mutants and epiRIL lines, genome-wide analyses of the roles of DNA methylation and chromatin structure on rates and distributions of meiotic crossing-ove were carried out. Work with Maize permitted the successful development of an in planta Gene Targeting protocol which, through separation of the transformation and GT steps, requires less person effort than protocols requiring transformation at the GT step.
The analyses carried out in WP2 yielded a number of striking and important results, both scientifically and in terms of potential practical application:
• Meiotic hyper-recombination phenotypes were identified in two mutants, an RNAi line and an overexpression line.
• Identification of and early role for heterochromatic centromeric regions and 5S ribosomal gene loci in meiotic chromosome synapsis, together with differing requirements for the Rad51 paralog proteins and the two recombinases Rad51 and Dmc1.
• An overexpression line shows a 10-fold increase in the rate of precise Gene Targeting.
• Differential roles of DNA methlylation on meiotic recombination were described for euchromatic heterochromatic chromosomal regions, and evidence found for roles of additional factors controlling recombination suppression in heterochromatin.
• We have demonstrated GT in maize using an ‘in planta’ GT protocol which requires less person effort than protocols requiring transformation at the GT step.
• New understanding of the importance and risks of induced DNA breakage in GT protocols.
The work of Workpackage 2 thus fully responded to the projected goals, has led to the developement and application of new techniques and to better understanding of the recombination proteins and chromatin structure in somatic and meiotic cells of plants.
3- Promoting HR via stabilization and resolution of recombination intermediates.
Synapsis of homologous DNA sequences (through the action of the homologous recombination machinery described in WP2), leads to the fomation of mismatch-containing heteroduplex DNA and linked or branched DNA intermediates, such as Holliday junctions. These structures must be processed for the strand exchange reaction to be complete, and the nature of this processing is of great importance with respect to the outcome of the recombination event (gene conversion, crossover or non-crossover). This resolution of recombination intermediates is carried out by a dedicated machinery, including the Structure-specific endonucleases and mismatch repair proteins that are the focus of this WP. Earlier work done by members of the RECBREED consortium, as well as data from the literature, led us to hypothesize that up- or down-regulation of these genes could have a profound effect on the outcome of meiotic recombination or gene targeting between homologous sequences as well between homeologous sequences.
We characterised the roles of the mismatch-repair proteins MLH1 and MSH2 on recombination between homeologous chromosomes. Preliminary studies with overexpressing MLH1 in Arabidopsis did not give the hoped-for increases in crossing-over and so the tomato work focussed on MSH2. RNAi knockdown of MSH2 in hybrids of the related tomato species, S. lycopersicum and S. pimpinellifolium, was used to check for effects on recombination between homeologous chromosomes. Further work with other genetic intervals will be needed to draw concrete conclusions, but no significant change recombination rate was observed in the msh2RNAi plants in the interval tested in this study. We also have characterised the MSH2 genes of maize and constructed RNAi knockdown plants, however the analyses of their effects will be carried out after project end. Our work with structure-specific endonucleases in Arabidopsis focussed on XPF/ERCC1 and MUS81/EME1. Previous work had shown an important role for XFPF/ERCC1 in somatic recombination in Arabidopsis, and we checked here for an implication in meiotic recombination. The results of this study showed no significant differences in meiotic recombination rates in xpf nor in ercc1 mutants. MUS81 is implicated in the generation of (non-interfering) meiotic crossovers in Arabidopsis and we have characterised its partner, EME1. Two EME1 genes were found in Arabidopsis and biochemical studies showed that both form functional complexes with MUS81, and can cleave Holliday junctions in vitro. Overexpression and GFP-tagged protein constructs were expressed in planta for characterisation of these proteins - however we found no stimulation of somatic recombination through overepression. These analyses of effects of overexpression on recombination will continue after the project, particularly as concerns meiotic effects and interactions with other recombination proteins.
In this WP our general goal was to enhance GT and meiotic recombination through the overexpression or down-regulation of structure-specific endonucleases and mismatch repair genes involved in the stabilization and resolution of intermediate structures formed during homologous recombination. Our approach was to ask the fundamental questions in the model system Arabidopsis and then to apply lessons to crops such as tomato and maize. Although the results of this WP were mostly negative and direct manipulation of the recombination resolution machinery of plants has proven to be a real challenge, the work of Workpackage 3 fully responded to the project goals, and has led to the developement and application of new techniques and to better understanding of the recombination proteins in somatic and meiotic cells of plants.
4- Combining individual gene effects for HR enhancement
Each stage in the recombination process is determined by a number of genes which may act in a redundant manner, epistatic manner, additive manner or synergistic manner. The same is true for genes which act during the various stages of the recombination process starting from DSB-induction through homology search and heteroduplex formation and resolution. This WP was aimed at testing such possible additive or synergistic interactions. This WP started relatively late (M24 or M30) as it was meant to be based on results obtained with single genes. Therefore specific goals were not set in advance but some general principles such as overcoming redundant effects, identifying epistatic effects through suppressors of mutants or combining effects that may work in a multiplicative or additive manner. For example it is known that DSBs can be repaired by either of two competing mechanisms, non-homologous end-joining (NHEJ) or homologous recombination. It was thus reasoned (P5) that DSB induction using a ZFN as done in WP1, coupled with a mutation in NHEJ might enhance the chances for gene targeting of a donor homologous to the broken target. Regarding homology search, two homologs of RAD52 were found as part of WP2 (P2). We thought that there might be redundancy in the effect of these genes, therefore P2 we prepared plants with altered expression in both genes to test their effect on meiotic recombination (Collaboration of P2 with P1 and P3). Moreover, we tried to further amplify the enhancing effect of AtRAD52-1 overexpression on GT (reported in WP2) through the combined overexpression with AtRAD52-2. This work is still on-going. In animal cells, the absence of RAD52 protein in mutants of other "accessory" proteins, including Xrcc3 leads to co-lethality. In order to study interactions between the plant genes P3 prepared a double mutant of Atrad52-1 and xrcc3. This mutant did not show any co-lethality, therefore we are currently developing a triple mutant Atrad52-1, Atrad52-2 and xrcc3. Finally, we addressed an interesting problem related to the mode of resolution of Holliday junctions based on findings of P1 in WP3. Indeed, P1 found that mutants of Arabidopsis TOP3α as well as RMI1 are sterile due to recombination-dependent chromosome defects in meiosis I, pointing to an important function of the dissolution pathway in meiotic HR. Since the dissolution pathway is dependent on a double Holliday junction intermediate, as are hypothesized resolution pathways by endonucleases such as MUS81/EME1A, we hypothesized that it should be possible to rescue the somatic and meiotic defects of RTR complex mutants like rmi1 by overexpression of these nucleases. In summary, the scientific context of this WP was determined a posteriori based on the results obtained in the first half of the project. This WP gave rise to several experiments that could not be completed as it started late and involved complex genetic material. However, it stimulated several new collaborations between the partners of this consortium.
The Objectives: In this WP we set to combine the effect of various genes to better understand HR pathways in plants and to enhance GT and meiotic recombination. The specific objectives of WP4 were as follows:
Combining DSB-induction and loss of Non-homologous end joining, using a ZFN protein designed for DSB induction and the Ku80 mutant as NHEJ mutant.
Rescuing sterility of the RTR complex mutants (e.g. TOP3α and RMI1) by overexpression of endonucleases complex of MUS81/EME1A.
Testing co-lethality of a double Atrad52-1 and xrcc3 and triple Atrad52-1/Atrad52-2/xrcc3 mutant.
Overexpression of AtRAD52-1 and AtRAD52-2 to further enhance GT.
As the experiments of the is WP were not finished at the end of the funding period a final assessment of these approaches can only be given in future.
Project Results:
1.3.1 WP1: DSB induction.
Task 1.1 GT by DSB induction with Zn-finger nucleases [P5, P6]
DSB-mediated GT in Arabidopsis thaliana
ZFNs were developed for an artificial GFP locus and the natural PPO locus via the modular assembly method. The GFP ZFNs were shown to be active, producing so-called footprints at the target site. These plants were also used for GT experiments and 3 GT events were obtained. Subsequently, we analysed plants expressing the PPO ZFNs revealing that no footprints could be found. Nevertheless, we used plants, expressing these ZFNs under control of the CaMV 35S promoter, for GT experiments. Floral dip transformation was performed with a repair T-DNA construct and seeds were selected on phosphinothricin to determine the transformation frequency and on butafenacil to identify GT events. Of about 2600 transformants, 8 butafenacil resistant plants were obtained (T1-T8). The same number of transformants of the wild-type control resulted in two butafenacil resistant plants (C1, C2). PCR analysis and Southern blotting showed that plants C1 and C2 and T1 to T7 were true GT events (TGT), but plant T8 was an ectopic gene targeting (EGT) event. Plant lines C1 and T2 did not contain any extra randomly integrated T-DNA copies, whereas all others plant lines contained multiple extra T-DNA copies. Analysis of the next generations of C1 and T2 showed that Mendelian segregation occurred and expected ratios of wild-type, heterozygous and homozygous plants were obtained. Taken together the results showed that TGT plants were obtained and that with the aid of ZFN-mediated DSBs this frequency was 4 times increased.
The PPO ZFNs were probably not very active, since no deletions or insertions (indels) were obtained in the footprint analysis of the genomes of transformed plants. Therefore, new ZFNs were selected via the Oligomerized Pool ENgineering (OPEN) protocol. In this method, libraries of 3 ZF modules each binding a triplet are used for selection of binding to the target half sites. Three target sites for PPO and three for the Cruciferin (CRU) genes were used for the selection. Two pairs of ZF arrays for the CRU gene and one pair for the PPO gene were successfully selected, showing three times or more activity compared to the negative control in bacterial two-hybrid assays. ZF arrays for a second PPO target site were identified via the CoDA method and these sequences were ordered. FokI nuclease domains, which have improved activity and will only form heterodimers, were combined with the selected ZF arrays to form ZFN pairs separated by the 2A ribosomal skipping sequence. The activities of the ZFNs were analysed in planta using next generation sequencing (NGS). The sequencing results showed that none of the newly constructed ZFN pairs had created footprints, whereas footprints were found using plants containing the previously used GFP ZFN pair.
In order to test whether ZFNs were not active indeed, they were analysed in a yeast single strand annealing (SSA) assay. One ZFN pair for the PPO gene showed activity. Since no active ZFNs were obtained for the CRU gene, we tested additional ZF arrays that had been obtained by the OPEN selection in yeast SSA assays. These results showed that many combinations were active. Control tests with the 2A construct that was used for expression of the ZFNs revealed that this was not functional. Expression of the ZFNs from two separate genes (without the 2A sequence) in protoplasts resulted in footprints in the PPO target site, indicating that these ZFNs were active. Subsequent analysis of transgenic plants also resulted in footprints present in the endogenous PPO gene. However the ZFNs for the CRU gene did not show any activity in protoplasts.
Since no active ZFNs for the CRU gene were obtained, we decided to design a new nuclease type, emerged as powerful genome engineering tool during the time frame of the project, the so-called TAL effector nucleases (TALENs) for the CRU gene. Three target sequences were chosen close to the junction of the CRU gene and the RFP 5’ end present in the repair construct. TALENs were cloned and tested in Arabidopsis protoplasts. For TALEN pairs 1/2 and 5/6 footprints were obtained, indicating that DSB had been induced. For TALEN pair 3/4 no footprints were obtained, but the restriction site that was used is not located in the middle of the spacer and therefore small deletions may have been missed in the analysis. Plants were transformed through Agrobacterium-mediated floral dip transformation and these plants will be analysed in the near future for the presence and activity of the TALENs. When active TALENs are obtained, the plants will be used for GT experiments in future projects.
No toxicity of ZFNs and TALENs has been observed. The ZFNs cloned by modular assembly have been expressed in four generations without any obvious negative effect on growth. Third generation plants with the PPO ZFNs cloned by OPEN also did not show any negative effect due to the ZFN expression.
DSB-mediated GT in tomato
The development of an efficient gene targeting procedure for tomato was the main task of P6. As described previously, gene targeting is enhanced when double strand breaks (DSBs) are produced at the genomic target site. Within the framework of Recbreed we aimed to use zinc finger nucleases (ZFNs) to produce targeted DSBs. Another critical aspect for the overall efficiency of a gene targeting procedure is the total number of cells that can be treated to produce to take up the plasmid DNA encoding the ZFNs and thus have a targeted DSB produced in the genome. The higher this number is, the higher the chance to identify gene targeting events. For that purpose we chose to use leaf protoplasts, of which several millions can be isolated and transfected in a single experiment. In order to develop a gene targeting procedure for tomato two features were critical: an efficient protoplast regeneration system and a DSB should be efficiently produced at the target site(s).
Within the first year of Recbreed, protoplasts were prepared from several tomato genotypes and screened for their regeneration capacity. The variety M82 (field processing tomato) was finally identified for its high protoplast plating efficiency (between 30-40%) and its high regeneration capacity (>50%). Moreover M82 tomato is readily amenable to Agrobacterium-mediated transformation, which may be advantageous when testing the effect of genes improving gene targeting identified by other Recbreed partners.
In order to demonstrate the usefulness of the tomato protoplast system for the application of ZFNs, M82 tomato protoplasts were treated with ZFNs and the formation of footprints via non homologous end joining (NHEJ) (insertions or deletions, INDELs) was assessed. By assessing the efficiency of INDEL formation at the target sequence we could determine the relative activity of a ZFN pair. A ZFN pair (ZFN815) targeting the acetolactate synthase (ALS) gene of tobacco was kindly provided by Prof. Daniel Voytas (University of Minnesota, Minneapolis, MN, USA). These ZFNs have been described in the literature (Townsend et al, 2009, Nature 459: 442-445). Sequence analysis showed that the tobacco and tomato ALS genes share a high level of homology, indicating that ZFNs developed for the tobacco ALS genes should also target the tomato ALS1 and ALS2 genes. Tomato protoplasts were transfected with ZFN815 plasmid DNA using polyethylene glycol (PEG) mediated transfection and cultured for 2 days, after which genomic DNA was analyzed for the presence of mutations (INDELs) at the ZFN815 target site. The analysis of two experiments showed that on average 16% of the ALS1 and 5% of the ALS2 sequences contained mutations. These mutations mainly consisted of deletions of 1 to 20 bases or insertions of a few bases at the target site. Protoplasts treated with ZFN815 were grown and eventually regenerated into whole plants which were then individually characterized for the presence of mutations at the ZFN815 cleavage site. More than 15% of the regenerated shoots showed mutations at the ZFN815 cleavage site demonstrating the power of this approach to produce targeted mutations.
Having demonstrated the efficiency of ZFN815 to produce a targeted DSB in the tomato ALS genes, we proceeded with gene targeting experiments. The ALS protein is an essential enzyme for the production of certain amino acids and is the target of sulfonylurea herbicides. Specific single point mutations in the ALS gene result in the production of an ALS protein that is tolerant to the herbicide, making this gene an selectable marker for gene targeting in tomato. Tomato protoplasts were co-transfected with a plasmid encoding for ZFN815 and with a plasmid carrying a modified copy of the ALS gene (DNA donor), including the SNP’s conferring resistance to the herbicide. Using this approach in tomato, several herbicide resistant calli were produced and characterized as gene targeting events by PCR. Unfortunately we also observed an unexpected negative effect of the DNA donor on the protoplast development. Further investigation revealed that this negative effect was due to the down regulation of the expression of the endogenous ALS gene, leading to a suppression of the regeneration of the transfected tomato protoplasts. We were able to partially circumvent this negative effect by adapting the length and amount of the DNA donor. To date the maximum gene targeting efficiency observed is 5 events for 1.106 protoplasts. Several herbicide tolerant calli, resulting from gene targeting events or spontaneous mutation, were regenerated into plants and were used for Southern analysis, confirming the PCR results and revealed that all gene targeting events analysed were true gene targeting events. To our knowledge these gene targeting events are the first ever obtained in the tomato crop.
As described above, we showed that the introduction of a DSB at the target locus significantly enhances the gene targeting efficiency in tomato. However, the production of efficient ZFNs is a cumbersome task that may complicate the targeting of any locus. Therefore we made use of our tomato protoplast protocol to assess the effectiveness of TALENs in tomato. Three pairs of TALENs were designed and introduced on plasmid constructs into tomato protoplasts. By detection of INDEL mutations, all three of these TALEN constructs were shown to be effective in producing DSBs at their respective target sites. We showed that plants containing a null mutation in the MLH1 gene were infertile which is to be expected as this gene is known to be involved in crossover formation during meiotic recombination. We also showed that TALENs targeting the EPSPS gene of tomato were able to produce INDELs at the target sequence, making those reagents useful for attempting gene targeting at the EPSPS locus.
Task 1.2 Induction of meiotic recombination with Zn-finger nucleases [P1, P5]
A thorough bioinformatics analysis was performed in order to determine INDELs as well as SNPs between Arabidopsis cultivars (Col-0, Bur-0 and Tsu-1, respectively) which can be used as molecular markers to survey meiotic recombination frequencies on the one hand and that will allow for ecotype-specific DSB induction mediated by site-specific ZF nucleases. ZF arrays were selected via the OPEN procedure (P5), directed against four 3 bp deletion target sites identified in the ecotypes Bur-0 or Tsu-1 (B16, B36, B59, T32 and T43). For three sites good ZF arrays were obtained, with relative β-gal activity of 3 or more. For the other two sites, which have the same target sequence, only one active array could be obtained. The ZF arrays with the highest activity were cloned in frame with improved FokI nuclease domains, which can only form heterodimers and are more active.
All ZFNs were cloned under the control of the meiosis-specific SPO11-1 promoter. The constructs were transformed into the respective A. thaliana ecotypes by Agrobacterium-mediated transformation and more than 20 stable primary transformants (T1 generation) were selected for each line. In the next generation homozygous single copy lines were selected. During these selection steps partial sterility of the plants was observed, indicating off target effects. Due to the partial sterility we retransformed the ZFNs in their respective ecotypes and again we observed partial sterility for most of the plants.
P5 has not seen ZFN activity for the respective ZFN-2A constructs driven by a constitutive promoter. Additionally, the observed partial sterility of T1 plants under the control of the meiosis-specific SPO11-1 promoter was indicative for unpredicted off-target effects. Moreover, a comparable meiosis-specific experimental set-up with a ZFN that has shown reliable activity driven by inducible and constitutive promoters in previous studies failed in F1 hybrids as no detectable HR induction at the target site was achieved. Therefore we decided to stop the experiments before crossing the homozygous single locus lines into Col-0. Nevertheless, our functional CAPS marker pool that was developed within this framework is still useful for further studies. To differentiate between the chromosomes bearing respective ZFN binding sites (Chr. I, II and III) we identified several polymorphisms in Col-0, Bur-0 and Tsu-1 for CAPS marker analysis. Since the 2nd periodic report we established some of these markers for high resolution melting (HRM) analysis, too. HRM allows a semi high throughput screening for F1 hybrids as well as recombination events in 384 well plates.
Task 1.3 Induction of meiotic recombination by Spo11-1/2 overexpression [P1]
Initiation of homologous recombination (HR) by DSB formation in meiosis is the central function of SPO11. Previously, it was demonstrated that mono- and dicotyledonous plants harbour three distantly related homologues of yeast SPO11: SPO11-1, SPO11-2 and SPO11-3. However, SPO11-1 and SPO11-2 mutant lines that have been described possess a severe meiotic phenotype resulting in almost completely sterile plants (Grelon et al (2001) Embo J 20: 589-600; Hartung & Puchta (2000) Nucleic Acids Res 28: 1548-1554). SPO11 homologs induce meiotic HR by generating DNA double-strand-breaks (DSBs) throughout the genome. The number of SPO11-induced DSBs and the number of cross-over events are thought to be correlated. Our aim was a genome-wide enhancement of recombination by simultaneous overexpression of SPO11-1 and SPO11-2 to test this hypothesis.
Double expression constructs for SPO11-1 and SPO11-2 have been constructed both with the SPO11-1 promoter as well as a constitutive ubiquitin promoter. Subsequently, the respective constructs have been transformed into Arabidopsis (Col-0) via Agrobacterium-mediated transformation. Following the identification of transgenic progeny, stable single locus lines were established by segregation analysis. Homozygous SPO11 lines driven by the SPO11-1 promoter and non-transformed Col-0 as a control line with Bur-0 were crossed. An extremely extended lifecycle in F1 was observed, with an altered rosette phenotype. The first inflorescence did not appear until 3 months without any regularity in one F1 line.
Test crosses between Col-0, Bur-0 and Tsu-1 wild type plants were performed to confirm these results and similar phenotypes were observed in the F1 generation. We have performed backcrosses of these F1 lines with one of the parental ecotype backgrounds to check if the CAPS marker screening is usable under these genetic conditions and it worked well for all combinations. Two of the meiosis-specific SPO11 overexpression lines were used for the backcrosses to perform the analysis of meiotic recombination events in FR generation. For the analysis of meiotic recombination events we used some of the already established CAPS markers from WP1 task 1.2 that allow differentiation of sequence polymorphisms between Bur-0 and Col-0. Additionally, we developed further CAPS markers for task 1.3 and again we established most of them for HRM analysis, too. Meiotic recombination of 20 FR control plants [(Col-0 x Bur-0) x Col-0] as well as 18 and 17 independent FR SPO11 overexpression plants [(SPO11-OE x Bur-0) x SPO11-OE] was analyzed with markers on chromosome II, III and V. Genotyping suggested evidence for changes in the crossover distribution, but this still has to be confirmed statistically. Therefore, we initiated some follow-up studies. The homozygous SPO11 overexpression lines were crossed with Ler-1 instead of Bur-0. We identified Ler-1 as the best crossing partner for Col-0 without having any disadvantage during development in F1 hybrids. We characterized several new CAPS and HRM markers for discrimination between Ler-1 and Col-0 chromosomes. These markers will be used in semi high through-put analysis of backcrosses of the F1 hybrids for meiotic recombination.
Homozygous plant lines expressing SPO11 under control of the Ubiquitin promoter were analysed for intrachromosomal recombination using a recombination reporter substrate to test if homologous recombination is generally enhanced in these lines. The results indicated that SPO11-1/SPO11-2 overexpression did not result in generally enhanced HR frequencies.
Task 1.4 Isolation of a promoter for expression of genes in maize, at the “HR-initiation stage” [P7]
The objective of this task was to isolate promoters that express during meiosis and which were appropriate for expressing genes that modify the frequency of gene targeting or meiotic recombination. These promoters were also intended to be used by P7 in WP2 and WP3 to overexpress Spo11, an HR-initiation candidate for increasing the frequency of meiotic recombination.
Promoter isolation and validation
The previously isolated rice DMC1B promoter was linked to GFP and transformed into maize. Promoter analysis has focused on previously generated pOsDMC1-GUS lines. Surprisingly these lines showed expression that was “constitutive‟ with for example strong expression in the anther wall. Thus this version of the pOsDMC1B promoter was not strictly meiosis-specific, but still the promoter may be useful if it is expressed in meiocytes.
Three other genes were identified with meiosis-specific or preferred expression based on publically available expression data, literature survey and consultation with partners. These were the Arabidopsis genes SDS (Solo-dancer), Arabidopsis ASY1 and SPO11-1. The homologous rice genes have been identified and putative promoter regions isolated. The following promoter reporter fusions have been constructed using 1-2 kb of DNA upstream of the ATG; pOsSpo11-1-GFP; pOsAsy1-GFP, pOsSDS-GFP and pOsSDS-GUS. The promoter-reporter fusions were transformed into maize. Except for pOsDMC1b, all attempts at expression analysis with GFP / GUS visualization on isolated meiocytes were inconclusive, thus we decided to characterize these promoters via qRT-PCR in meiotic tissue samples. As Spo11-expressing lines using these promoters were available, we performed these mRNA expression studies directly on the Spo11 lines that needed to be characterized.
Three Spo11 constructs (pOsAsy1_ZmSpo11-1+pOsAsy1_ZmSpo11-2, pOsSpo11-1_ZmSpo11-1 + pOsSpo11-1_ZmSpo11-2 and pOsDmc1b_ZmSpo11-1) were tested for immature anther expression and leaf expression; as a control a constitutive promoter was also tested (pOsRubi3_ZmSpo11-1+pOsRubi3_ZmSpo11-2). Specific primers targeting endogenous meiotic genes (ZmAsy1, ZmSpo11-1 and ZmDMC1) as well as both synthetic ZmSpo genes (synSpo11-1 and synSpo11-2) were used. As expected, significant expression of synthetic Spo11 genes was only detected in leaves from the pRubi3 and pDMC1b constructs. The level of expression of the synthetic Spo11 genes was about 10% of that expected from the pOsRubi3 promoter suggesting the that Spo11 transcripts may be unstable. No expression was detected in the wild-type A188 control samples nor in pAsy1 and pSpo11-1 constructs, although weak expression levels of endogenous ZmSpo11-1 were detected in these leaf samples. pDMC1-driven expression of transgenes in leaves was very weak, in most cases inferior to endogenous ZmSpo11-1 levels.
In anthers Spo11 expression from pOsRubi3 gave on average an overexpression of 16 times with respect to the endogenous Spo11-1 levels. However such overexpression is likely to be largely due to expression in the anther wall. For the pDMC1b construct expression of synSpo11-1 was similar to that of the endogenous Spo11-1 gene. For both pSpo11-1 and pAsy1, the expression levels of synSpo11-1 and synSpo11-2 were barely detectable.
Effect of Spo11 overexpression on meiotic recombination
To assess the effect of the overexpression of the Spo11 genes, the following strategy was planned: crossing of single loci transgene expressing lines to a divergent inbred line (R), selfing of the F1 progeny of 2 of the best transgene expressing lines and genotyping of the F2 progeny using a genome-wide genotyping chip containing SNPs for at least 4500 genes. Comparison with control F2 plants lacking the transgene will detect if the transgene induces a change in the frequency and/or position of crossovers. Although crosses with line R were made, in the light of the lack of identification of good Spo11 overexpressors in anthers, we decided not to proceed further with this experiment.
1.3.2 WP2: Homology search and heteroduplex formation
Task 2.1 Homology search [P1, P2, P3]
Reporter translational fusions
To better understand the mode of action and the influence of these key actors on the efficiency of the homologous recombination process, we have constructed reporter translational fusions for RAD51 and the RAD51 paralogs with expression of the genes driven by their own promoters, or by the constitutive MAS promoter. Functional validitation of the constructs was confirmed by complementation of the corresponding Arabidopsis mutants and analysis of the plants carrying the Rad51 paralog-GFP fusion proteins has permitted establishment the expression patterns and intracellular localisation of the protein. These studies have also resulted in the identification and characterisation of novel promoters for gene expression.
Roles of the RAD51-family proteins in Meiotic Recombination and Chromosome Synapsis: differing roles at peri-centromeres and chromosome arms.
Studies of the functional roles of these proteins in mitotic and meiotic recombination in Arabidopsis confirmed that all five RAD51 paralogues of Arabidopsis play key roles in recombination and permitted the description of a novel distinction between the dependence of meiotic chromosome synapsis in heterochromatic centromere-proximal and rDNA regions, compared to euchromatic chromosome arms; with the former being mostly DMC1-dependent and the latter requiring RAD51 and the RAD51 paralogs RAD51C and XRCC3. The results of this work provide new insights into the homologous chromosome pairing mechanisms occurring in meiotic prophase: heterochromatic centromeres and 5S rDNA regions pair early and their pairing has different requirements for recombination proteins than does that of the chromosome arms - highlighting the importance of questions concerning the specificities and roles of recombination at different chromosome and/or chromatin regions in the synapsis of homologous chromosomes at meiosis.
Stimulation of Meiotic Recombination in the absence of XRCC2.
The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilisation of the RAD51-DNA nucleofilament. We confirmed that the three "non-meiotic" paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and have shown that the three proteins are not essential for the formation of the RAD51-DNA fibre at DNA breaks in Arabidopsis (immunodetection of radio-induced Rad51 foci). Analyses of meiotic recombination showed increased rates of meiotic crossing-over in xrcc2 and to a lesser extent rad51b but not rad51d mutants. We speculate that XRCC2 and RAD51B affect the balance of the activities of DMC1 and RAD51 in meiosis and in their absence this balance shifts towards cross-over outcomes. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells and this hyper-recombination phenotype is of potential utility and importance in plant breeding for agronomy. Partner P7 (BIOGEMMA) has initiated studies in maize to test for this effect in a major crop plant (see Task 2.3).
The RAD51-S mutant and the roles of RAD51 and Dmc1 in Meiotic Recombination.
The RAD51_GFP fusion protein (see section (i) above) complements the meiotic chromosome fragmentation and sterility of the Arabidopsis rad51 mutant. Further analyses showed however that the fusion protein is not a functional strand-transfer recombinase: conferring a dominant negative phenotype in somatic tissues, while remaining able to fully complement the meiotic defects of rad51 mutants. Presence of this fusion protein thus defines a "separation of function" mutant, similar to the rad51-II3A mutant recently reported in yeast (Cloud et al. Science. 2012. 337:1222). For convenience we have named this mutant RAD51-S. Preliminary results of expression in dmc1 mutant plants confirm the dominant negative effect of the RAD51-GFP fusion in Arabidopsis meiosis, and show that its complementation of the meiotic chromosome fragmentation and sterility of the Arabidopsis rad51 mutant is fully dependent on the presence of DMC1. DMC1 is thus able to repair all meiotic DSB in Arabidopsis and depends upon the presence, but not the strand exchange activity, of RAD51 to do so
These results impose a new vision of the relative roles RAD51 and DMC1 in meiosis and dramatically underscore the importance of the multiple roles of the RAD51 nucleofilament, and of the accessory proteins (such as the RAD51 paralogues) which modulate its function.
Task 2.1b - BRCA2 [P1]
BRCA2 regulates RAD51-filament formation and DNA binding of RAD51, the protein seems to work as a mediator of RAD51 facilitated DNA repair. BRCA2-binding is postulated to lead to replication protein A (RPA) replacement enabling RAD51 to build a nucleoprotein filament with ssDNA. These results suggest a control mechanism of RAD51 by BRCA2, explaining why a loss of BRCA2 function shows similar effects as RAD51 loss. In the Arabidopsis genome two copies of the BRCA2 gene are present and we showed that the brca2 double mutant is viable and, in contrast to mammals, BRCA2 is thus dispensable for development in plants
Studies of meiosis in the double mutant showed that it is sterile, with no viable pollen and male meiosis abrogated at the pachytene stage of prophase. Somatic cells of the double mutant are strongly sensitive to Mitomycin C, indicating a specific defect in somatic DNA crosslink repair. The frequency of intrachromosomal homologous recombination is reduced with and without genotoxic stress, demonstrating the involvement of the protein in HR in somatic cells.
By amplification of the genomic DNA fragment, we have constructed a plasmid for overexpression of BRCA2(IV).The T-DNA construct was transferred into Arabidopsis plants, where however we found aberrant segregations and loss of the selectable marker in the following generation. We have shown that overexpression of this gene results in severe growth defects in Arabidopsis.
Notwithstanding its real interest from the scientific point of view, in the context of this project this result precluded further study of somatic HR rates under conditions of BRCA2 overexpression. Furthermore the growth defects of BRCA2(IV) overexpression preclude its effective use in a biotechnological setting.
Task 2.1c Role of AtRAD52 in GT and meiotic HR [P2]
Identification of Arabidopsis RAD52 homologs.
RAD52 mediates RAD51 loading onto ssDNA ends at DNA breaks, thereby initiating homologous recombination and catalyzing DNA annealing. RAD52 is highly conserved among eukaryotes, but had not been identified in plants. This work reports the discovery of RAD52 homologs present in all plants whose genomes have undergone extensive sequencing. Computational analyses suggest a very early RAD52 gene duplication, followed by later lineage-specific duplications during the evolution of higher plants. Plant RAD52 proteins have high sequence similarity to the oligomerization and DNA binding N-terminal domain of RAD52 proteins.
Remarkably, the two identified Arabidopsis AtRAD52 genes encode four open reading frames (ORFs) through differential splicing, each of which specifically localized to the nucleus, mitochondria or chloroplast. The Arabidopsis AtRAD52-1A ORF provided partial complementation to the yeast rad52 mutant. Arabidopsis mutants and RNAi lines defective in the expression of AtRAD52-1 or AtRAD52-2, showed reduced fertility, sensitivity to Mitomycin-C, and decreased levels of intrachromosomal recombination compared to wild-type. In summary, computational and experimental analyses provide clear evidence for the presence of functional RAD52 DNA-repair homologs in plants.
Overexpression of AtRAD52-1A::EGFP enhances gene-targeting.
Overexpression of AtRAD52-1A is associated with a 10-fold increase in Gene targeting rate. The four GT events originating from the AtRAD52-1A-EGFP line included one true and precise GT event, while all three GT events detected in WT plants were non-precise targeting events. Thus, in AtRAD52-1A-EGFP plants we obtained a rate of 5.43x10-5 bona fide GT while no true GT events were detected in WT plants.
Silencing/overexpression of RAD52 has only minor or no effects on meiotic recombination.
Tests of meiotic recombination rates in absence or overexpression of the RAD52 homologues showed different results for the different AtRAD52 fusion proteins. Overexpression AtRAD52-1A, localized to the mitochondria and the nucleus, led to an increase in meiotic recombination rate of ~14% compared to WT. No change in meiotic recombination rates were however detected in a AtRAD52-1B over- expressor (mitochondrial localisation), nor in AtRAD52-2B overexpressor lines (chloroplast localisation).
RNAi constructs were used to specifically knock-down expression of the two genes and while 35S-AtRAD52-1RNAi gave no change in meiotic recombination rate, 35S-AtRAD52-2RNAi increased meiotic recombination rate by ~18% compared to WT. We are combining the RNAi knockdowns and effects on meiotic recombination will be scored within about a month. Similarly, combination of 35S-AtRAD52-1A::mCFP and 35S-AtRAD52-2A::EGFP lines’ effect on meiotic recombination will be scored within about a month. The effect of combined overexpression of the two AtRAD52 nuclear variants on gene-targeting will also be tested.
Task 2.2 Chromatin modification and Meiotic HR [P4,P2]
Task 2.2a Chromatin modifications and Meiotic HR [P4]
This task aimed to assess whether alteration in epigenetic properties of chromatin would have an influence on the frequencies of meiotic crossovers (COs).
Genome-wide analysis to determine the number of COs on each chromosome for all four populations analyzed (the hypomethylated met1-3, the epiRIL lines epi01 and epi12, and the WT control) showed that loss of DNA methylation is not altering the total level of COs, but rather their distribution along the chromosomes. We hypothesize that this phenomena is due to the tight regulation of meiotic COs, by mechanisms such as CO interference and homeostasis.
The effects of hypomethylation on frequencies of meiotic recombination observed in other chromosomes were only partially reproduced in chromosome 4. Unlike in other chromosomes, in chromosome 4 only 1 out of 4 euchromatic regions showed an increase in recombination frequencies in the met1-3 population, and there was no correlation between the methylation status of the examined fragment and its level of recombination, with the exception of one euchromatic region. This result is similar to our former results obtained in the genome-wide experiment for other chromosomes in the epiRIL populations. Since the different heterochromatin structure of chromosome 4 did not allow an informative analysis of meiotic recombination frequencies in our Col/Ler populations, we generated new Columbia/ Wassilewskija genotyping populations. To generate "knob-less" populations we used the Landsberg and C24 ecotypes as parental lines, both do not have the hk4S. We designed a new set of KASP genotyping assays in order to measure recombination frequencies around the hk4S and centromeric regions as well as on other euchromatic regions on chromosome 4. We included chromosome 2 in our analysis since it is similar to chromosome 4 but lacking the hk4S, thus serving as an intra-genomic control.
We found that when recombination occurs between two similar chromosomes, i.e. lacking the hk4S on chromosome 4, the effect of hypomethylation on recombination frequencies is well reproduced, showing, as before, an increase in recombination frequencies in hypomethylated euchromatic intervals. For the Ws/Col populations which have the hk4S, we also observed an increase in recombination frequencies in hypomethylated euchromatic regions of chromosome 4 with a smaller effect on chromosome 2. Interestingly, we noticed that the recombination frequency was exceptionally high around the centromere in the Ws/Col populations containing the hk4S and we speculate that the presence of the heterochromatic knob (hk4S) is inducing recombination at the adjacent centromere.
Task 2.2b The effect of DDM1 on meiotic recombination [P2]
[This part was not in the original proposal. It was added to task 2.2 because of its direct relation to the topic and to replace the part on MLH1 following negative results with AtMLH1 overexpression in parts of task 3.1]
Meiotic recombination is tightly regulated by cis and trans-acting factors. While DNA methylation and chromatin remodeling affect chromosome structure, their impact on meiotic recombination is not well understood. In order to study the effect of DNA methylation on the landscape of chromosomal recombination we analyzed meiotic recombination in the DECREASED DNA METHYLATION 1 mutant (ddm1). DDM1 is a SWI2/SNF2-like chromatin remodeling protein necessary for DNA methylation and heterochromatin maintenance in Arabidopsis thaliana. The rate of meiotic recombination between markers located in euchromatic regions was significantly higher in both heterozygous (DDM1/ddm1) and homozygous (ddm1/ddm1) backgrounds than in WT. The effect on recombination was similar for both male and female meiocytes. Contrary to expectations, ddm1 had no effect on crossover numbers between markers in heterochromatic pericentric regions that underwent demethylation. These results are surprising because pericentromeric regions are hypermethylated and were expected to be the most affected by demethylation. Thus, DDM1 loss of function may trigger changes that enhance meiotic recombination in euchromatin regions, but are not sufficient to induce the same events in heterochromatic segments. This work uncovers the repressive role of methylation on meiotic recombination in euchromatic regions and suggests the role of additional factors controlling recombination suppression in heterochromatin.
Task 2.3 Enhancing GT and meiotic recombination in maize by specific over-expression of genes involved in homology search [P7]
A. Enhancing GT by expression of homology research genes
Validation of the test GT system.
The test system for GT in maize is based on the reconstitution of a defective nptII gene. The 3’ region of nptII is integrated into the plant genome at the Target Locus (TL), which has a target site for the meganuclease I-SceI allowing the creation of a DSB at the TL when I-SceI is present. The goal of the GT work in RecBreed is to retransform these TL lines with a repair DNA (Donor Locus construct or DL) and DNA encoding I-SceI plus another gene that putatively further stimulates GT. The DL contains sequences homologous to the TL and has the 5’ part of nptII. Thus GT at the TL results in reconstitution of nptII and resistance to kanamycin. In previous work, in the EU FP6 project TAGIP, maize lines were stably transformed with the a DL construct which contained the 5’ nptII region flanked by I-SceI sites and a dexamethasone-inducible I-SceI gene (I-SceI-GR). These DL(I-SceI-GR) lines were crossed to TL lines and progeny containing both the TL and DL loci treated with dexamethasone.
We analysed these DL(I-SceI-GR)+TL+/- I-SceI (constitutive) plants and showed, by PCR, excision of the DL and recombination of the DL and TL by NHEJ. We also developed an assay for somatic GT based on application of kanamycin to plants; green resistant sectors were observed in kanamycin –bleached leaves. Green sectors were excised and shown by PCR to have reconstituted nptII. We also regenerated fully kanamycin-resistant plants, via in-vitro regeneration of kanamycin resistant plantlets. Southern analysis confirmed that “GT” had occurred. For 2 GT events from TL1 (GT1 and GT2) the DL recombined at the TL as predicted for a true GT event at the TL; i.e. reconstitution of nptII and loss of the original TL locus. However, unexpectedly, no DL excision was detected from the DL locus.
GT events were characterised by PCR and by DNA sequencing of the genomic GT loci. PCR of GT descendants showed that in the progeny of the GT1 and GT2 events kanamycin resistance is strictly correlated with the present of a modified TL1 locus. However for the GT3 to GT6 events kanamycin resistance was linked to a modified DL locus, the TL2 locus being unaltered. The DNA sequence of the GT1 and GT2 loci was as predicted for GT event involving a double recombination between the TL1 and DL loci. However the sequence of the other GT events showed that there had been a single crossover between the DL and TL2 locus restoring nptII at the DL1 locus with variable lengths of the TL1 locus being integrated into the DL1 locus via NHEJ in the different GT events. These studies thus confirmed the suspected mechanism of GT via ectopic recombination.
In order to understand why only crosses with the TL1 line gave the expected GT events the TL locus the different TL and DL parental lines were resequenced to ensure that all the I-SceI sites were functional. It transpired that the DL parent used for the crosses with TL1 lacked the I-SceI site adjacent to the 5’ region of nptII (this site had probably been mutated after having been cut by I-SceI-GR). Thus in the TL1 x DL crosses recombination to restore nptII activity is favoured at the TL1 loci because a DSB can only be created next to a defective nptII at the TL1 locus.
Testing the homology search candidate RAD54
P2 has shown that overexpression of RAD54 increases GT in Arabidopsis (Shaked et al (2005), Even-Faitelson et al (2011)). Two strategies have been tested to evaluate the effect of yeast RAD54 using either the native yeast sequence (ScRAD54) or a version optimized for maize expression (SynZmRAD54). In previous reports we described experiments where TL lines were retransformed with a DL construct with I-SceI and with or without RAD54. No kanamycin resistant plants were regenerated. A second strategy involved crossing TL lines to a DL construct expressing I-SceI-GR and synZmRAD54 (optimized for maize expression). In this reporting period the crosses were performed but insufficient time remained to grow these plants, extract embryos and test GT. To determine if RAD54 might stimulate intrachromosomal recombination plants expressing I-SceI or I-SceI+synRAD54 were crossed to a GU-US intrachromosomal reporter line. All the progeny plants containing the reporter + I-SceI or reporter + I-SceI and RAD54 possessed GUS positive sectors by histochemical staining. Controls (WT, reporter or I-SceI or I-SceI+RAD54 alone) had no GUSpositive sectors. However reporter + I-SceI + RAD54 plants showed no significant difference with respect to the number of GUS positive sectors as reporter + I-SceI plants, suggesting that synZmRAD54 does not stimulate intrachromosomal recombination.
B. Enhancing meiotic recombination by expression of homology research genes.
Partner P3 showed that the Arabidopsis Xrcc2 mutant strongly stimulates meiotic recombination. The maize Xrcc2 homolog was identified by reciprocal BLAST analysis. There appears to be only one Xrcc2 gene in maize (GRMZM2G002626). A 300 bp fragment specific to ZmXrcc2 used to create an RNAi construct. In order to preferentially downregulate ZmXrcc2 in meiosis the best candidate meiosis-expressed promoter identified, pDMC1B, was linked to this RNAi fragment. 34 transformed lines were generated and in the first instance crossed to WT (WT as male). No obvious effect on female fertility was observed since all plants produced normal quantities of seed. Insufficient time was available in the project to perform anther expression analyses to select plants that have ZmXccr2 downregulation. These plants would then be crossed to a divergent maize line and the F2 progeny genotyped to assess any effects on meiotic recombination.
1.3.3 WP3: Promoting HR via stabilization and resolution of recombination intermediates
Task 3.1 Heteroduplex stabilization by the mismatch repair machinery [P2]
Mismatch repair genes play diverse and opposite roles. Some of them, like MLH1, promote crossover via stabilization of Holiday junctions, others like MSH2 have an anti-recombination effect in particular when mismatches are present in the Holiday junctions. Our attempts to enhance meiotic recombination via overexpression of AtMLH1 in Arabidopsis have not given the hoped-for results. Therefore, MLH1 was not tested in tomato. Instead we focused our efforts on the suppression of MSH2. When the project was initiated the tomato genome was not available and there was no mutant for MSH2 in tomato. Therefore, we set to identify the MSH2 tomato homolog and we tried to silence it using an RNAi approach in a meiotic-specific manner (under SPO11 promoter). Petunia and Arabidopsis MSH2 were blast against the Tomato database. The sequence of the StMSH2 was obtained. A 513bp fragment including the 3’UTR was amplified from micro-Tom cDNA and subcloned in sense and antisense orientation in pKanibbal under either 35S or SPO11 (meiotic specific) promoter. Each clone was subsequently subcloned into pART27 to confer Kanamycin resistance. We transformed Solanum lycopersicum Var. Micro Tom. Plants showing resistance were grown, RNA was extracted from flowers before meiosis young flower buds and Msh2 was quantified by RT PCR method. From the five lines that were silenced only two were viable (SPO79 and SPO74) the other three were sterile and couldn’t be used for the following experiment. The plants with silenced StMSH2 were crossed to Solanum pimpinellifolium. Seeds from the resulting cross were grown under Kanamycin selection and once again MSH2 transcript was quantified by qRT PCR from young flowers again showing reduction in expression (See details in periodic report). Using genetic markers meiotic recombination was evaluated in the F2 generation of Solanum lycopersicum Var. MicroTom X Solanum pimpinellifolium, SPO11::msh2RNAi-79/74 X Solanum pimpinellifolium.
Figure 3.1: The effect of down-regulation of AtMSH2 on homeologous recombination was tested in a cross between WT tomato, variety Micro-Tom and its relative Solanum pimpinellifolium compared to the same cross where the Micro-Tom parent has been transformed with an siRNA construct that was shown to reduce AtMSH2 transcript level
Marker position WT SPO11::RNAi_msh2
No. of Plants Rec.
Plants Genetic Distance (cM)* No. of Plants Rec.
Plants Genetic Distance (cM)*
Chromosome 9 88 9 5.1 135 13 4.8
So far we have obtained results for only one pair of markers on chromosome 9. We see only a minor and not significant increase in meiotic recombination as a result of AtMSH2 suppression (see Table above). Additional markers are currently being tested, enabling us to reach conclusions.
Task 3.2a – Heteroduplex resolution by XPF-family structure-specific endonucleases [P3]
Role of XPF/ERCC1 in somatic and meiotic recombination
Previous studies by P3 showed that mutation in XPF or ERCC1 affect somatic homologous recombination in plants (Dubest et al., 2002, 2004). To address whether these mutations also affect meiotic recombination, we analysed meiotic recombination rates in Arabidopsis xpf and ercc1 mutants using the cruciferin promoter RFP/GFP-based assay from the group of A. Levy (partner P2). Analyses of progeny of selfed plants showed no significant effect on meiotic recombination rates in Arabidopsis xpf and ercc1 mutant plants, when compared to the corresponding wild-type plants for two intervals on chromosomes III and V (Similar results were obtained for male and female meiotic recombination (See detailed result in the periodic report). Thus, although the XPF-ERCC1 complex plays an important role in somatic recombination, its absence appears to have no significant effect on meiotic recombination rates in the two chromosomal intervals tested.
Effects on somatic recombination of overexpression of XPF and ERCC1
Given the important roles of XPF and ERCC1 in somatic recombination, we have built and validated in planta, fluorescently tagged, fusion protein expression constructs to elucidate these effects on somatic recombination. The reporter translational fusions for the Arabidopsis XPF and the ERCC1 proteins were constructed with the complete genomic sequence (i.e. + introns + exons) expressed by their own promoters (Figure 2A). Stop codons were removed and the sequences fused to the GFP gene to generate the fluorescently tagged translational fusion constructs. The functionality of the fusion proteins has been confirmed by complementation of the UV and MMC hypersensitivity of the corresponding Arabidopsis xpf and ercc1 mutant plants (Figure 2B). Exploiting the GFP fluorescence of the tagged proteins, we have analysed the temporal and spatial expression patterns of the tagged-fusion proteins, which show clear nuclear localisation of the proteins and that the genes are strongly and ubiquitously expressed in roots and flowers (Fig. 3). Preliminary data suggests also that their expression is differentially regulated with respect to the cell cycle. In future studies, the fluorescent tags will permit immunological approaches, such as co-immunoprecipitation, for the characterisation of protein complexes. These transgenic lines have been crossed with the mitotic recombination tester lines from the Puchta lab (Orel et al., 2003; Mannuss et al., 2010). Somatic recombination rates will be analysed in coming months.
Task 3.2b - Mus81/Eme1 [P1]
MUS81 is a highly conserved endonuclease and together with EME1 (also referred to as MMS4 in S. cerevisiae) it is involved in the resolution of 3’ flap structures and Holliday-like DNA junctions. Loss of the protein results in sensitivity to DNA damaging agents in yeast and mammals. Interestingly, the role of the protein in meiosis differs drastically between eukaryotes. Whereas in S. pombe loss of MUS81 results in complete sterility, the mutant is partially fertile in S. cerevisiae and fully fertile in mammals. We identified a homologue of MUS81 in the genome of Arabidopsis thaliana and isolated a full-length cDNA of this gene. Analyzing two independent T-DNA insertion lines of AtMUS81, we found that they are sensitive to the genotoxins MMS and MMC. Both mutants have a deficiency in homologous recombination in somatic cells but only after induction of genotoxic stress. Atmus81 mutants display a moderate decrease in meiotic recombination. It has been hypothesized that AtMUS81 is involved in the generation of a second class of meiotic crossovers that are interference insensitive. We were also able to identify two closely linked homologues of EME1 in the Arabidopsis genome (AtEME1A and AtEME1B). By biochemical analysis we were able to demonstrate that indeed both the AtMUS81/AtEME1A and the AtMUS81/AtEME1B complex are able to process intact Holliday junctions.
As described in Annex I MUS81/EME1 should be cloned under control of a constitutive ubiquitin promoter as well as under control of the meiosis-specific DMC1 promoter. For both promoters constructs were cloned and have been transferred into Arabidopsis plants by Agrobacterium-mediated transformation. The somatic expression of the constructs under control of the ubiquitin promoter was analyzed in homozygous plant lines containing an additional recombination substrate for intrachromosomal recombination to test if HR is generally enhanced in the lines. Furthermore, quantitative RT-PCRs completed the analysis of the somatic expression lines. Further analysis of plants containing the constructs under control of the meiotic DMC1 promoter were put on hold until RMI1 mutant lines containing the same construct (see below) have been processed. 36 of 70 overexpression lines under control of the ubiquitin promoter were characterized as homozygous single locus lines and tested for enhancement of HR by recombination assays. 8 lines exhibited a moderately increased HR rate and were selected for three further recombination assays.
MUS81/EME1A overexpression displayed a wide range of HR enhancement with relatively high standard deviations. These differences were due to the fact that very low numbers of spots were counted on the seedlings. Because of the heterogeneity of the results 4 lines were selected for quantitative RT-PCRs to evaluate the expression of MUS81, EME1A and the 651 recombination construct. 2 of 4 lines showed a 30-fold increase of MUS81 transcripts and an about 130-fold increase of EME1A whereas the other two lines had only slightly different levels to the wild type control. The expression of the recombination substrate was increased between 2- and 11-fold in comparison to wild type. Nevertheless, the expression patterns and the HR enhancement do not correlate with each other. Hence, a silencing effect can be excluded. In addition to the conventional overexpression, MUS81 was also overexpressed in RMI1 mutant lines under control of the DMC1 and the RMI2 promoter – two strong promoters during meiosis. RMI1 together with RECQ4A and TOP3α form the so-called RTR complex in Arabidopsis thaliana, which has been shown to dissolve Holliday junctions by generating non-crossover products via the dissolution pathway of HR. Mutants of each of the three complex partners therefore were shown to have an increased rate of spontaneous somatic homologous recombination events. Interestingly, mutants of Arabidopsis TOP3α as well as RMI1 are sterile due to recombination-dependent chromosome defects in meiosis I, pointing to an important function of the dissolution pathway in meiotic HR.
Since the dissolution pathway is dependent on a double Holliday junction intermediate, as are hypothesized resolution pathways by nucleases, e.g. MUS81 or GEN1, it should be possible to rescue the somatic and meiotic defects of RTR complex mutants like rmi1 by overexpression of these nucleases. To test this hypothesis, two constructs of MUS81 under control of the DMC1 and RMI2 promoters were transferred into two independent Arabidopsis mutant lines of RMI1. Both constructs were transformed into A. thaliana rmi1-1 and rmi1-2 mutant plants. Through BAR resistance segregation, homozygous lines have been established. In addition, further resolvases from different organisms were cloned in a similar manner and also transformed into rmi1 mutant plants and established as homozygous lines. Since efforts shifted more to compare the effects of the expression of different resolvases in sterile rmi1 mutants, results of these experiments are described in WP4.
Figure 3.4. Meiotic MUS81/EME1A expression in Arabidopsis. The two constructs possess either the DMC1 or the RMI2 promoter from Arabidopsis thaliana. For selection the BAR resistance gene was cloned behind the second gene
Task 3.3 Enhancement of meiotic HR and GT using the most promising crossover promotion (CP) gene in maize [P7]
MSH2 downregulation in maize
Bioinformatic searches confirmed that the maize MSH2 gene (accession GRMZM2G056075 on chromosome 7) was the best homolog of AtMSH2. BLAST analyses against maize genomic sequences revealed partial sequences that have some homology to ZmMSH2 on chr1 (GRMZM2G392172; ZmMSH2-like 1) and chr2 (AC193754.3_FG008; ZmMSH2-like2). However alignments suggest that they do not represent additional maize MSH2 copies. Examination of the expression pattern of ZmMSH2 (Gene Atlas data, figure 5) shows that expression was highest in the immature tassel and cob, suggesting a role in meiosis for this gene. A 300bp region of the ZmMSH2 sequence that fell outside the region of homology to the ZmMSH2-like genes was chosen to design an RNAi construct. In order to preferentially downregulate ZmMSH2 in meiosis the best candidate meiosis-expressed promoter identified, pDMC1B, was linked to this RNAi fragment. 36 transformed lines were generated and in the first instance crossed to WT (WT as male). No obvious effect on female fertility was observed since all plants produced normal quantities of seed. Insufficient time was available in the project to perform anther expression analyses to select plants that have ZmMSH2 downregulation. These plants would then be crossed to a divergent maize line and the F2 progeny genotyped to assess any effects on meiotic recombination.
Enhancing GT by expression of crossover promotion genes:
At M30 there was no available strong crossover promotion gene candidate to stimulate GT. Thus it was decided in this reporting period to test whether abiotic stresses could enhance GT using the test GT system developed in Task2.3. In this system the frequency of GT is proportional to the number of green sectors observed on a leaf after kanamycin application. Many reports show that abiotic stress induces DNA repair genes expression and increases significantly homologous recombination rates (Yao and Kovalchulk, Mutation Research, 2011). Several abiotic stress conditions were tested on wild-type maize plantlets: UV, bleomycin (DSB), heat (37°C), 5-azacytidine (demethylation). In these experiments, HR gene expression was assessed in order to select the best stress conditions. To evaluate the effect on GT, these stress conditions were applied to TL+DL plants that were already scored positive in the kanamycin-resistance leaf assay. The clearest results were obtained with 5-Azacytidine and elevated temperature (figure 6). Seed was first imbibed for 25 hours in 30µM 5-Azacytidine. The number of plants with green kanamycin resistant sectors was reduced 3-fold for TL2/ DL plants but not for TL1/DL plants. It might be anticipated that changes in methylation may affect genomic ‘openness’ and accessibility towards the production of DSBs by I-SceI and subsequent recombination. To test the effect of elevated temperature plantlet at the 4 leaf stage were transferred to a 37°C incubator for 8 hours (night), then a kanamycin solution applied to the plant apex. Such a treatment increased the number of plants with green kanamycin-resistant sectors two to three fold. Since the in-vitro optimum of the I-SceI restriction enzyme is 37°C it is likely that this augmentation of GT is directly caused by increased DSB production. However it cannot be excluded that other factors might contribute to this increase (eg genomic structure: activity of other recombination proteins).
1.3.4 WP4: Combining individual gene effects for HR enhancement
Task 4.1a Enhancing GT by combined gene effects [P5]
Combining DSB-induction and loss of Non-homologous end joining
ZFN-mediated footprints in Ku80 mutant: Cycles of DSB formation and repair will eventually lead to mutations. We have shown this recently by using targeted ZFNs (de Pater et al., 2009). It is likely that the spectrum of mutations will be altered in DNA repair mutants and therefore we are testing several repair mutants for ZFN-mediated footprinting. Crossings were done between plants having the GFP-HPT target site and expressing the corresponding ZFNs with the ku80 NHEJ mutant. In the next generation homozygous mutant plants were obtained. These plants were analysed for footprints in the target site. It seems that more footprints are present in the ku80 mutant than in the wild type (Figure 4.1A). This might mean that in NHEJ mutants DSB repair is less accurate and more often results in footprints. We did not observe differences in the length of deletion in comparison with the wild type (Figure 4.1B). In order to test ZFNs designed for DSB formation of natural target sites, ZFNs targeted to the PPO gene and TALENs targeted to the CRU gene were introduced in an Arabidopsis ku80 mutant (Jia et al 2012) via Agrobacterium-mediated transformation. A mixture of two Agrobacterium strains each one harbouring one of the ZFN or TALEN genes from the pairs were used. Since the transformation frequency of this ku80 mutant is at least 5 times lower than that of the wild type, only few transformants were obtained. These plant lines will be analysed for the formation of footprints and compared to the results of the wild-type (see WP1).
Jia Q, Bundock P, Hooykaas PJJ, de Pater S (2012) Agrobacterium tumefaciens T-DNA integration and gene-targeting in Arabidopsis thaliana non-homologous end-joining mutants. J Botany Doi:10.1155/2012/989272.
de Pater, S., Neuteboom, L. W., Pinas, J. E., Hooykaas, P. J. J. and van der Zaal, B. J. (2009). ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotech. J. 7, 821–835.
Task 4.1b The effect of combined overexpression of the two AtRAD52 nuclear variants on gene-targeting [P2]
In WP2 we showed evidence for enhanced GT upon AtRAD52-1 overexpression. We are in the process of testing the combined effect of AtRAD52-1 and AtRAD52-2 overexpression on GT using lines which were and described in WP2. These lines are crossed and F1 co-expressing both genes will be tested for Gene targeting.
Task 4.2 Enhancing Meiotic HR by combined gene effects [P1, P2, P3]
Rescuing sterility of the RTR complex mutants by overexpression of endonucleases (P1)
Mutants of the A. thaliana RMI1 gene show a sterile phenotype due to defects in the separation of meiotic chromosomes. RMI1 together with RECQ4A and TOP3α form the so-called RTR complex in Arabidopsis thaliana, which has been shown to dissolve Holliday junctions by generating non-crossover products via the dissolution pathway of HR. Mutants of each of the three complex partners therefore were shown to have an increased rate of spontaneous somatic homologous recombination events. Interestingly, mutants of Arabidopsis TOP3α as well as RMI1 are sterile due to recombination-dependent chromosome defects in meiosis I, pointing to an important function of the dissolution pathway in meiotic HR.
Since the dissolution pathway is dependent on a double Holliday junction intermediate, as are hypothesized resolution pathways by endonucleases such as MUS81/EME1A, it should be possible to rescue the somatic and meiotic defects of RTR complex mutants like rmi1 by overexpression of these nucleases.
Beside the A. thaliana MUS81/EME1A complex, a number of other HJ resolvases were cloned and transformed into rmi1 mutant plants: A. thaliana GEN1, S. cerevisiae SLX1/SLX4, E. coli RusA and E. coli RuvABC. All constructs were cloned to be expressed under the control of the A. thaliana DMC1 and RMI2 promoters, both of which show a strong meiotic expression. In the case of the eukaryotic resolvases, each gene was cloned between a separate promoter and terminator. The bacterial resolvases were cloned as fusion proteins containing N-terminal NLS sequences. The E. coli RuvABC complex, however, was cloned to be expressed by a single promoter as a polycistronic mRNA, in which each protein contains an NLS sequence and the proteins being separated by T2A ribosomal stutter sequences leading to production of three proteins from a single mRNA (constructs shown in Figure 4.2).
Figure 4.2: Composition of T-DNAs of the cloned resolvase constructs. All T-DNAs are flanked by left and right border sequences (LB, RB). Proximal to the right border, a PPT resistance cassette is located. Each resolvase construct was cloned under the control of a 35S terminator and either a DMC1 or a RMI2 promoter. The resolvases used are AtGEN1 (from A. thaliana cDNA), AtMUS81/AtEME1A (from A, thaliana cDNA), ScSLX1/ScSLX4 (from S. cerevisiae AH 109 gDNA), EcRusA (from E. coli gDNA) and EcRuvA/EcRuvB/EcRuvC (from E. coli gDNA). The bacterial resolvases EcRusA and EcRuvABC additionally contain a NLS sequence. To express three proteins from a single mRNA, the EcRuvABC genes were cloned under the control of a single promoter and separated by T2A ribosomal skipping sequences.
Following transformation of rmi1-1 heterozygous plants with these constructs, in the T1 generation lines were established that carried the T-DNA using PPT resistance. Of these plants, all were genotyped by PCR for the status of the rmi1-1 mutation. All plants carrying a T-DNA and being homozygous for the rmi1-1 mutation were transferred into pots and grown in the greenhouse until formation of mature siliques. Then, fertility of these plants was quantified by counting the mean number of viable seeds per silique. Surprisingly, no fertile plants could be found with any construct (table 1).
In the course of the experiments, it was questioned whether the promoters used, DMC1 and RMI2, were sufficient for this experiment. The DMC1 protein is expressed in meiosis I, but the expression time might be too early for the required action of a resolvase. Microarray expression data of the RMI2 gene indicate a strong meiotic expression; there has been no data showing any function for the RMI2 protein in meiosis, however. We are in the process of establishing new rmi1-1 plant lines in which the resolvases are expressed under the control of meiosis-specific promoters SPO11-1, MS5 and MTG10.3.
Table 1: Overview of Atrmi1-1 resolvase expression lines. For each resolvase expression line transformed into mutant line rmi1-1 IC9C, numbers of positively selected primary transformants in T1 generation (rmi1-1+/-) are given in column 2. After genotyping, the numbers of resolvase-positive rmi1-1-/- plants that were analysed for their fertility are given in column 3. No transformed line showed a deviation in fertility from the sterile rmi1-1 mutant, irrespective of the resolvase construct used.
Line T1 Atrmi1-1-/- % fertile
rmi1-1 IC9C::(DMC1-P)-GEN1 86 19 0%
rmi1-1 IC9C::(RMI2-P)-GEN1 83 18 0%
rmi1-1 IC9C::(DMC1-P)-MUS81/EME1A 97 23 0%
rmi1-1 IC9C::(RMI2-P)- MUS81/EME1A 63 13 0%
rmi1-1 IC9C::(DMC1-P)-ScSLX1/ScSLX4 172 41 0%
rmi1-1 IC9C::(RMI2-P)-ScSLX1/ScSLX4 174 43 0%
rmi1-1 IC9C::(DMC1-P)-EcRusA 152 37 0%
rmi1-1 IC9C::(RMI2-P)-EcRusA 171 41 0%
rmi1-1 IC9C::(DMC1-P)-EcRuvABC 160 37 0%
rmi1-1 IC9C::(RMI2-P)-EcRuvABC 133 30 0%
Task 4.2b Test for colethality in rad52 and xrcc3 double mutant plants. [P2 and P3]
In the context of TASK 2.1c of Workpackage 2, P2 identified and characterised the plant homologues of RAD52, a mediator of RAD51 loading onto ssDNA ends, thereby initiating homologous recombination. A striking phenotype of rad52 mutants in animal cells, is the co-lethality of the absence of RAD52 protein in mutants of other Rad51 "accessory" proteins, including Xrcc3 (Fujimori 2001). P3 identified the Arabidopsis xrcc3 mutant and is working with it in TASK2.1a. In collaboration with P2, P3 thus constructed double atrad52 xrcc3 Arabidopsis plants to verify whether this colethality also exists in plants. The current status of this work is:Heterozygote xrcc3 +/- plants were crossed to rad52-1-/- and rad52-2-/-.
In the F1:
- xrcc3+/-_rad52-1+/- grew normally and although seeds were not counted, visual inspection revealed no major problems of fertility beyond the slight reduction observed in rad52-1 single mutant plants.
- In contrast, fertility seemed more strongly affected in xrcc3+/-_rad52-2+/-. This phenotype will be confirmed soon in the F2 generation.
In the F2 (progeny of xrcc3+/-_rad52-1/2 +/-)
- Two viable xrcc3-/-_rad52-1-/- plants were identified among the F2, excluding the hypothesis of colethality of the double mutant (14 were rad52-1 -/-; 48 plants genotyped). Notwithstanding, the low proportion of these plants in the F2 population is suggestive and further analyses will be carried out.
- Similarly for rad52-2, among 48 plants tested, 14 were rad52-2 -/- and 2 were xrcc3-/- rad52-2-/-.
- All plants are growing normally, have started bolting and we will soon be able to check the fertility rad52-/- xrcc3-/+ (xrcc3 -/- are sterile). .
- In contrast to the F1, xrcc3+/-_rad52-2+/- look fine and fertility not strongly affected.
Conclusion
These results show clearly that the xrcc3-/- rad52-1-/-, and the xrcc3-/- rad52-2-/- double mutants are viable. Arabidopsis xrcc3-/- rad52 double mutants are not synthetically lethal, in contrast to animals. To verify that this is not die to a redundancy of function between the two RAD51 genes, we have crossed the two double mutants (rad52-1-/- xrcc3+/- and rad52-2-/- xrcc3+/-) to make the triple mutant, xrcc3-/- rad52-1-/- rad52-2-/-. Lethality or viability of the triple mutants should be available by the end of June.
Task 4.2c Combined effect of loss of function of RAD52-1 and RAD52-2 on meiotic recombination [P1, P2]
The loss of function of either AtRAD52-1 or AtRAD52-2 has almost no effect on DNA recombination and repair (see WP2). We hypothesized that this might be due to redundancy in their function. For this purpose we are preparing a double mutant in both genes. However, to simplify the various assays and work with a dominant phenotype we have co-expressed AtRAD52-1 RNAi and AtRAD52-2 RNAi lines. These lines are being analysed by P1 for defects in meiosis and were crossed with our fluorescent seed tester (P2). The effect on meiotic recombination will be scored in the near future.
Potential Impact:
The main objective of RECBREED project was to improve plant breeding by enhancing Homologous Recombination (HR rates) in somatic and in meiotic cells. The expected outcomes of such enhancement were to establish an efficient gene targeting (GT) technology for precise engineering of plant genomes and to increase the rate of meiotic recombination between homologous or homeologous chromosomes. These objectives have been achieved
Through the RecBreed project new tools to induce HR were developed in tomato and maize. These can be used to improve plant breeding of these corps, that have great economical value. But the tools are also applicable to other species.
The project developed “new tools from technologies that support both research & development and the production of industrial prototypes for the breeding of crop plants (excluding forest and fruit tree species) using innovative gene technology breeding methods (transgenics/cisgenics/ intragenics)". Indeed, in the case of gene targeting we were able to obtain by trangenic approach ("transgenics") and in the case of enhancement of meiotic recombination, by a transgenic approach one is in principle able to obtain ("intragenics") that carry no transgenic sequences. Within the funding period, not only "research & development" of both processes of the model plant Arabidopsis was performed but also "industrial prototypes for the breeding of crop plants" tomato and maize could be obtained.
RECBREED project succeeded to set up enabling techniques helping to better exploit plant biodiversity. This project addresses fundamental aspects of homologous recombination. It impacts modern plant breeding and is expected to provide long-term benefits. In addition, topics such as the enhancement of meiotic recombination enable to better exploit natural variation within a species as well as from a very broad range of exotic resources .. By enabling more precise and controlled transformation of plants, our work on gene targeting, can facilitate public acceptance and regulatory aspects of work with transgenics, and thus have long-term impact.
Finally, gene targeting enables to exploit the wealth of “omics” data in various ways, for example, through functional validation via targeted gene knockouts. In addition, precise targeting in the original genomic context is less prone to gene silencing than regular transgenesis techniques, GT will thus facilitate exploitation of metabolic data and the stable and efficient production of novel bioactive molecules for industrial or therapeutic purposes.
Scientific Impact
We established feasible gene targeting techniques, that can be applied directly to cultivated plant species. Moreover, we laid the ground for the improvement of breeding by enhancing meiotic recombination. Therefore we are convinced that the impact is indeed as far reaching as discussed below.
An important aspect of this project is that in the EU, public reluctance to consume GMO products has strongly hindered the implementation of biotechnology progress in agriculture while in other countries (USA, China, Japan) this Agro-biotech revolution is taking place. Gene Targeting technology enables precise and controlled alteration of the genome. Single nucleotide changes can be introduced into genes. Such precise and minimal alterations of the genome could enable companies to make new products that would be acceptable to consumers and thus to integrate the post-genomic era biotechnology revolution into agriculture and health. The controlled and precise modifications made possible by this technology should also significantly facilitate the legal aspects of registration of GMOs. On has to keep in mind that many natural varieties of cultivated plant species were produced by screens after application of mutagens like X-rays. Thus, multiple undirected DSB were introduced and the respective (partly major) genomic changes are in most cased uncharacterized in these culitvars. In contrast, a single DSB targeted to the site of interest by a synthetic nuclease result in an NHEJ event that might kock out an ORF by just one or two nucleotide changes. Obviously, here a reevaluation of regulation procedures in platn breeding is required in the long run .
Enhancement of breeding procedures by raising the frequency of homologous or homeologous recombination will facilitate the creation of new cultivars via standard, non-transgenic breeding. Such plants by definition do not differ from classical non-transgenic cultivars. They therefore can be used in the same way in agriculture as non-transgenic cultivars - no GMO specific issues will arise at all.
The industrial partners involved in this project are well aware of these aspects and obtained new tools and new IP through the RECBREED project. The breeding sector is a significant source of income in Europe. Seed sales by European breeders in 2006/2007 represented 4000 M€ or 37% of total worldwide seed sales. New emergent markets such as in Asia will ensure that this market continues to expand together with the continuing adoption of hybrid seed technology, for example in rice and perhaps in the future for wheat. Enhancing meiotic HR or GT provides an edge to European companies and benefits also for farmers who might be provided more rapidly with products with novel traits. It is of strategic importance for Europe that the European Breeding sector remains strong and can provide seed adapted to European conditions and needs.
There are several innovative aspects in this proposal. Enhancing recombination in a controlled way will be used to optimise the traditional breeding process so that diversity can be obtained from smaller breeding populations and so that rare recombination events can be obtained; an especially important aspect will be in rapidly introgressing genetic material from diverse sources (including distant relatives) which requires removal undesirable linked alleles/genes. Thus effort in breeding will be reduced and new varieties with potentially desirable social and environmental attributes will be created and delivered rapidly to the market. It is important to note that plant breeders devote considerable efforts in reducing the time to market of new varieties since the first to the market with an improved variety dominates the market. For example techniques such as the use of counter-seasons to obtain two or more breeding cycles per year and more recently the use of dihaploids to rapidly genetically fix varieties are widely used.
At the technological level, we hope to turn GT into a routine tool in plants. As discussed above, this technology has a very high potential economic impact in the Agro-biotech industry and also the pharma industry via “molecular farming” for protein production in plants. We will test several genes, working at different levels of the DNA recombination process (initiation, invasion, strand exchange, heteroduplex formation and resolution). As a result of this work, the Arabidopsis model has the potential to become a paradigm to study GT in other organisms with naturally low GT rates, such as mammalian species. Yeast, where the most advanced research is done in the field of homologous recombination, is naturally hyper-recombinogenic and is clearly not always a good model for GT in animals and plants. These innovations should result both in scientific publications and in patents.
European added value
One reason to carry this work at the European level was that the research in DNA recombination in plants is far more advanced in Europe than in the USA.. This is expressed by the number of quality publications, the number of research groups involved and the support of granting agencies. The RECBREED project brought together leading groups in the field of DNA recombination in plants and thus further increased the competitiveness of the EU in this field. The GT and improved breeding techniques might have a great impact on implementing the Agro-biotech revolution and the EU is in an excellent position to harvest the fruits from this revolution. Moreover the EU has a special urgent need for precise DNA technologies that are acceptable to the broad public rather than the random type of alterations currently practiced.
Each partner contributed with specific skills and know-how to the project. The research of the consortium was successful due to thet fact that it was a collaborative effort, at the EU level, rather than efforts undertaken separately by individual labs .
At the international level, USA companies (e.g. Dupont or Dow chemical or Monsanto) have in-house researchers carrying research in the field of DNA recombination and precise transgenesis (including ZFN-based technologies and site-specific integration). RECBREED succeeded in connecting leading EU scientists with the leading EU-industry in plant biotech. We are convinved that the project helps to reinforce the position of the EU in this competitive field.
Economical impact
During the last decade the plant breeding industry has changed significantly. Consumer demands on fresh and good quality vegetable products are increasing continuously and therefore the need to look closer into complex traits is required. By investing in new technologies Europe has been able to continuously grow its excellent world economic position in the breeding industry.
The breeding industries (seed companies) are the driving forces in the agricultural production chain and Europe plays a very prominent role in seed business. The international seed trade is a rapidly growing economic activity, and the global turnover of seed exports has doubled from 2000 million euro to 4000 million euro in the years 1990-2007. Four of the five top seed exporting countries in the world are European member states, i.e. France, the Netherlands, Germany and Denmark, with the annual turnover of seed exports by the two biggest exporters, The Netherlands and France, being about 550 million euro each, only slightly lower than that of the USA.
Specifically, in the vegetable breeding industry The Netherlands is by far the number one export country in the world. Over 70% of the world professional vegetable breeders use seeds directly derived from the Dutch breeding companies. Annual vegetable seed market sales are estimated at 2.5 billion Euro, of which the European companies together represent more than 50%, emphasizing the strong position of Europe in this industry.
Impact
Out of the top ten vegetable seed companies, eight have their roots in Europe and five of them are collaborating with KeyGene on their innovative research programs. We expect that in the long run that they will be able to benefit by applying the techniques set up with RECBREED to different crop plants of interest
List of Websites:
Project website address: http://www.recbreed.eu/
Project Coordinator:
Prof. Holger Puchta,
Karlsruhe Institute of Technology (KIT – UNIKARL)
Tel: +49 721 608-48894, Fax: +49 721 608-44874
E-mail: holger.puchta@bio.uka.de;
The main objective of the RECBREED project was to improve the toolkit of plant breeding. Plant breeders select plants out of variants that show desirable traits. One of the major mechanisms to generate biodiversity is called homologous recombination (HR), a process whereby similar chromosomes exchange segments and reshuffle the parental genes. One particular type of HR, called meiotic recombination, takes place in the cells that generate the male and female gametes (pollen and egg cells). It is a process that is tightly controlled and that is responsible for much of the genetic variation in a species. A major goal of this project was to study the mechanisms controlling meiotic recombination in order to enhance the rate of meiotic recombination. In addition, we attempted to enhance the rate of another type of HR, namely gene targeting (GT), the recombination between a genomic sequence and an introduced DNA fragment. The GT technology is the ideal solution for precise engineering of plant genomes. In both types of recombination, the process is initiated by a DNA double stranded break (DSB), followed by the formation of a duplex between the recombination partners (homology search and strand invasion) and ending with the separation of the recombining partners (resolution of recombination intermediates). We have studied each of these stages through the characterization of the genes involved and the testing of their function.
DSB-induced mutagenesis and gene targeting (GT) was successfully performed in the model plant Arabidopsis and the crop plant tomato. Initially zinc finger nucleases (ZFNs) were used to introduce targeted DSBs. This resulted in footprints (indels consisting of small deletion and insertion) and increased GT frequencies with the Arabidopsis PPO gene and the tomato ALS genes. In a later phase of the project also mutation induction of TAL effector nucleases (TALENs) could be demonstrated. Independently, a new approach of an “in planta” gene targeting technique was developed, based on the separation of the transformation and GT steps. The technique requires less person effort than protocols requiring transformation at the GT step. In planta GT could be demonstrated for the model plant Arabidopsis as well as the crop plant maize. Moreover It could be demonstrated that also by the over expression of a protein that is involved in HR namely AtRAD52-1A GT frequencies can be enhances (10-fold increase in the rate of precise GT). Thus, successful GT in plants could be achieved by different approaches by the RECBREED consortium.
Interesting results on means to influence meiotic recombination were obtained. An early role for heterochromatic centromeric regions and 5S ribosomal gene loci in meiotic chromosome synapsis, together with differing requirements for the Rad51 paralog proteins and the two recombinases Rad51 and Dmc1 could be identified. Differential effects of DNA methylation on meiotic recombination were described for euchromatic and heterochromatic chromosomal regions, and evidence found for roles of additional factors controlling recombination suppression in heterochromatin. A mean to induce genome wide meiotic hyper-recombination phenotypes were identified: The Rad51 paralogs RAD51B and XRCC2 are required to regulate cross over formation; mutants in both genes have more COs than wild type. Thus various means to modulate meiotic recombination were identified.
At the moment analysis of several other factors involved in GT and/or meiotic recombination is still ongoing and it is expected that after the end of the official funding further interesting result especially in respect to crop plants will be obtained by the participants of the RECBREED consortium.
Project Context and Objectives:
The main objective of RECBREED project was to improve plant breeding by enhancing Homologous Recombination (HR rates) in somatic and in meiotic cells. The expected outcomes of such enhancement were to establish an efficient gene targeting (GT) technology for precise engineering of plant genomes and to define means to control the rate of meiotic recombination between homologous or homeologous chromosomes.
The main objectives were fulfilled through the following scientific and technological objectives set for various work packages as follows:
1- Gene targeting and meiotic recombination enhancement via DNA Double strand break DSB induction.
The goal of this work package was to develop techniques to specifically enhance homologous recombination at the initiation stage by controlled induction of Double-Strand Break (DSBs). On one hand this approach was applied for gene targeting stimulation and on the other to enhance meiotic recombination. DSB induction was either targeted at specific sites or stimulated in a genome wide manner. The practical applicability of the approaches was tested in the crop plants, tomato and maize.
DSB-induced mutagenesis and gene targeting (GT) was successfully performed in the model plant Arabidopsis and the crop plant tomato. Initially zinc finger nucleases (ZFNs) were used to introduce DSBs. This resulted in footprints (indels consisting of small deletion and insertion) and increased GT frequencies with the Arabidopsis PPO gene and the tomato ALS genes. During the time frame of the project another powerful genome engineering tool emerged, the so-called TAL effector nucleases (TALENs). TALENs were tested directed for DSB induction in the Arabidopsis Cruciferin gene and the tomato MLH1 and EPSPS genes. Initial experiments show that DSBs were induced as detected by means of footprint analysis.
In order to stimulate meiotic recombination at a specific site ZFNs were selected for DSB induction in 3bp deletion regions in Bur-0 and Tsu-0 ecotypes, which are not present in Col-0. Crossing should result in specific DSBs during meiosis and increased recombination. These ZFNs were expressed under control of the SPO11 promoter. In order to analyze crossover frequencies CAPS and HRM markers were established for Bur-0 and Tsu-0. Unfortunately, the ZFNs were not active on their target sites as found in yeast assays and Arabidopsis transformants, although they showed some toxicity, resulting in partial sterility.
Subsequently, meiotic recombination was analyzed in plants overexpressing SPO11. Since the F1 of Col-0 x Bur-0 had a much extended lifecycle, it was decided to use the Ler-1 ecotype instead. Therefore, Ler-1 CAPS and HRM markers were established and used for analysis of crossover frequencies in F1 plants Ler-1 x Col-0 overexpressing SPO11. Indeed, the results suggested that crossover frequency was changed in several regions on different chromosomes.
Meiocyte-specific promoters for expression in maize were isolated and tested. Promoters of the rice homologs of the Arabidopsis SDS (Solo-dancer), ASY1 and SPO11-1 genes were cloned in GUS or GFP reporter constructs. Since visual quantification was inconclusive, QRT-PCR was performed to determine expression of SPO11 genes under control of OsUbi3, OsSPO11, OsDMC1b and OsASY1 promoters as well as the expression of the endogenous SPO11-1 and 2, ASY1 and DMC1 genes in leaves and anthers. In anthers SPO11 expression from pOsUbi3 gave on average an overexpression of 16 times with respect to the endogenous SPO11-1 levels. However such overexpression was likely to be largely due to expression in the anther wall. For the pDMC1b construct expression of SPO11-1 was similar to that of the endogenous SPO11-1 gene. For both pSPO11-1 and pASY1, the expression levels of SPO11-1 and SPO11-2 were barely detectable.
2- Homology search and heteroduplex formation
Homologous recombination occurs through recognition and invasion of one DNA molecule by another. This invading DNA strand is a coated with Rad51 protein in somatic cells and Rad51 and/or Dmc1 in meiosis, and it is this helical nucleoprotein filament which is the active molecular species in the invasion process. This Workpackage concerned study the roles of the "accessory" protein cofactors (Rad51 paralogs, Brca2, Rad52), and the influence of chromatin structure (using "epiRILS" and Ddm1 mutants) on the efficiency and outcome of this fundamental step of the homologous recombination process. These studies in the model plant Arabidopsis thaliana (Tasks 2.1 2.2) were complemented by work in the crop plant, Maize, on the development and application of recombination-based technologies for crop breeding (Task 2.3).
We characterised the accessory proteins which assist in the formation of the RAD51-DNA nucleofilament and modulate its function. A "missing link" among these, RAD52, was identified as two genes and multiple spliced variants which were used to construct fusion proteins and targeted specifically to nuclear, mitochondrial and chloroplast compartments of Arabidopsis cells. Similarly, plant and subcellular localisation of the five Rad51 paralogs were determined using plants expressing fluorescent protein fusion proteins, expressed both with their own, and with constitutive promoters. Arabidopsis has two Brca2 orthologs and the double mutant was shown be sterile, but to develop normally. Surprisingly, over-expression of one of the Brca2 genes generated severe developemental defects. Roles of these proteins in somatic and meiotic recombination were characterised using knockout mutants, overexpression and RNAi knockdowns. Using DNA methylation mutants and epiRIL lines, genome-wide analyses of the roles of DNA methylation and chromatin structure on rates and distributions of meiotic crossing-ove were carried out. Work with Maize permitted the successful development of an in planta Gene Targeting protocol which, through separation of the transformation and GT steps, requires less person effort than protocols requiring transformation at the GT step.
The analyses carried out in WP2 yielded a number of striking and important results, both scientifically and in terms of potential practical application:
• Meiotic hyper-recombination phenotypes were identified in two mutants, an RNAi line and an overexpression line.
• Identification of and early role for heterochromatic centromeric regions and 5S ribosomal gene loci in meiotic chromosome synapsis, together with differing requirements for the Rad51 paralog proteins and the two recombinases Rad51 and Dmc1.
• An overexpression line shows a 10-fold increase in the rate of precise Gene Targeting.
• Differential roles of DNA methlylation on meiotic recombination were described for euchromatic heterochromatic chromosomal regions, and evidence found for roles of additional factors controlling recombination suppression in heterochromatin.
• We have demonstrated GT in maize using an ‘in planta’ GT protocol which requires less person effort than protocols requiring transformation at the GT step.
• New understanding of the importance and risks of induced DNA breakage in GT protocols.
The work of Workpackage 2 thus fully responded to the projected goals, has led to the developement and application of new techniques and to better understanding of the recombination proteins and chromatin structure in somatic and meiotic cells of plants.
3- Promoting HR via stabilization and resolution of recombination intermediates.
Synapsis of homologous DNA sequences (through the action of the homologous recombination machinery described in WP2), leads to the fomation of mismatch-containing heteroduplex DNA and linked or branched DNA intermediates, such as Holliday junctions. These structures must be processed for the strand exchange reaction to be complete, and the nature of this processing is of great importance with respect to the outcome of the recombination event (gene conversion, crossover or non-crossover). This resolution of recombination intermediates is carried out by a dedicated machinery, including the Structure-specific endonucleases and mismatch repair proteins that are the focus of this WP. Earlier work done by members of the RECBREED consortium, as well as data from the literature, led us to hypothesize that up- or down-regulation of these genes could have a profound effect on the outcome of meiotic recombination or gene targeting between homologous sequences as well between homeologous sequences.
We characterised the roles of the mismatch-repair proteins MLH1 and MSH2 on recombination between homeologous chromosomes. Preliminary studies with overexpressing MLH1 in Arabidopsis did not give the hoped-for increases in crossing-over and so the tomato work focussed on MSH2. RNAi knockdown of MSH2 in hybrids of the related tomato species, S. lycopersicum and S. pimpinellifolium, was used to check for effects on recombination between homeologous chromosomes. Further work with other genetic intervals will be needed to draw concrete conclusions, but no significant change recombination rate was observed in the msh2RNAi plants in the interval tested in this study. We also have characterised the MSH2 genes of maize and constructed RNAi knockdown plants, however the analyses of their effects will be carried out after project end. Our work with structure-specific endonucleases in Arabidopsis focussed on XPF/ERCC1 and MUS81/EME1. Previous work had shown an important role for XFPF/ERCC1 in somatic recombination in Arabidopsis, and we checked here for an implication in meiotic recombination. The results of this study showed no significant differences in meiotic recombination rates in xpf nor in ercc1 mutants. MUS81 is implicated in the generation of (non-interfering) meiotic crossovers in Arabidopsis and we have characterised its partner, EME1. Two EME1 genes were found in Arabidopsis and biochemical studies showed that both form functional complexes with MUS81, and can cleave Holliday junctions in vitro. Overexpression and GFP-tagged protein constructs were expressed in planta for characterisation of these proteins - however we found no stimulation of somatic recombination through overepression. These analyses of effects of overexpression on recombination will continue after the project, particularly as concerns meiotic effects and interactions with other recombination proteins.
In this WP our general goal was to enhance GT and meiotic recombination through the overexpression or down-regulation of structure-specific endonucleases and mismatch repair genes involved in the stabilization and resolution of intermediate structures formed during homologous recombination. Our approach was to ask the fundamental questions in the model system Arabidopsis and then to apply lessons to crops such as tomato and maize. Although the results of this WP were mostly negative and direct manipulation of the recombination resolution machinery of plants has proven to be a real challenge, the work of Workpackage 3 fully responded to the project goals, and has led to the developement and application of new techniques and to better understanding of the recombination proteins in somatic and meiotic cells of plants.
4- Combining individual gene effects for HR enhancement
Each stage in the recombination process is determined by a number of genes which may act in a redundant manner, epistatic manner, additive manner or synergistic manner. The same is true for genes which act during the various stages of the recombination process starting from DSB-induction through homology search and heteroduplex formation and resolution. This WP was aimed at testing such possible additive or synergistic interactions. This WP started relatively late (M24 or M30) as it was meant to be based on results obtained with single genes. Therefore specific goals were not set in advance but some general principles such as overcoming redundant effects, identifying epistatic effects through suppressors of mutants or combining effects that may work in a multiplicative or additive manner. For example it is known that DSBs can be repaired by either of two competing mechanisms, non-homologous end-joining (NHEJ) or homologous recombination. It was thus reasoned (P5) that DSB induction using a ZFN as done in WP1, coupled with a mutation in NHEJ might enhance the chances for gene targeting of a donor homologous to the broken target. Regarding homology search, two homologs of RAD52 were found as part of WP2 (P2). We thought that there might be redundancy in the effect of these genes, therefore P2 we prepared plants with altered expression in both genes to test their effect on meiotic recombination (Collaboration of P2 with P1 and P3). Moreover, we tried to further amplify the enhancing effect of AtRAD52-1 overexpression on GT (reported in WP2) through the combined overexpression with AtRAD52-2. This work is still on-going. In animal cells, the absence of RAD52 protein in mutants of other "accessory" proteins, including Xrcc3 leads to co-lethality. In order to study interactions between the plant genes P3 prepared a double mutant of Atrad52-1 and xrcc3. This mutant did not show any co-lethality, therefore we are currently developing a triple mutant Atrad52-1, Atrad52-2 and xrcc3. Finally, we addressed an interesting problem related to the mode of resolution of Holliday junctions based on findings of P1 in WP3. Indeed, P1 found that mutants of Arabidopsis TOP3α as well as RMI1 are sterile due to recombination-dependent chromosome defects in meiosis I, pointing to an important function of the dissolution pathway in meiotic HR. Since the dissolution pathway is dependent on a double Holliday junction intermediate, as are hypothesized resolution pathways by endonucleases such as MUS81/EME1A, we hypothesized that it should be possible to rescue the somatic and meiotic defects of RTR complex mutants like rmi1 by overexpression of these nucleases. In summary, the scientific context of this WP was determined a posteriori based on the results obtained in the first half of the project. This WP gave rise to several experiments that could not be completed as it started late and involved complex genetic material. However, it stimulated several new collaborations between the partners of this consortium.
The Objectives: In this WP we set to combine the effect of various genes to better understand HR pathways in plants and to enhance GT and meiotic recombination. The specific objectives of WP4 were as follows:
Combining DSB-induction and loss of Non-homologous end joining, using a ZFN protein designed for DSB induction and the Ku80 mutant as NHEJ mutant.
Rescuing sterility of the RTR complex mutants (e.g. TOP3α and RMI1) by overexpression of endonucleases complex of MUS81/EME1A.
Testing co-lethality of a double Atrad52-1 and xrcc3 and triple Atrad52-1/Atrad52-2/xrcc3 mutant.
Overexpression of AtRAD52-1 and AtRAD52-2 to further enhance GT.
As the experiments of the is WP were not finished at the end of the funding period a final assessment of these approaches can only be given in future.
Project Results:
1.3.1 WP1: DSB induction.
Task 1.1 GT by DSB induction with Zn-finger nucleases [P5, P6]
DSB-mediated GT in Arabidopsis thaliana
ZFNs were developed for an artificial GFP locus and the natural PPO locus via the modular assembly method. The GFP ZFNs were shown to be active, producing so-called footprints at the target site. These plants were also used for GT experiments and 3 GT events were obtained. Subsequently, we analysed plants expressing the PPO ZFNs revealing that no footprints could be found. Nevertheless, we used plants, expressing these ZFNs under control of the CaMV 35S promoter, for GT experiments. Floral dip transformation was performed with a repair T-DNA construct and seeds were selected on phosphinothricin to determine the transformation frequency and on butafenacil to identify GT events. Of about 2600 transformants, 8 butafenacil resistant plants were obtained (T1-T8). The same number of transformants of the wild-type control resulted in two butafenacil resistant plants (C1, C2). PCR analysis and Southern blotting showed that plants C1 and C2 and T1 to T7 were true GT events (TGT), but plant T8 was an ectopic gene targeting (EGT) event. Plant lines C1 and T2 did not contain any extra randomly integrated T-DNA copies, whereas all others plant lines contained multiple extra T-DNA copies. Analysis of the next generations of C1 and T2 showed that Mendelian segregation occurred and expected ratios of wild-type, heterozygous and homozygous plants were obtained. Taken together the results showed that TGT plants were obtained and that with the aid of ZFN-mediated DSBs this frequency was 4 times increased.
The PPO ZFNs were probably not very active, since no deletions or insertions (indels) were obtained in the footprint analysis of the genomes of transformed plants. Therefore, new ZFNs were selected via the Oligomerized Pool ENgineering (OPEN) protocol. In this method, libraries of 3 ZF modules each binding a triplet are used for selection of binding to the target half sites. Three target sites for PPO and three for the Cruciferin (CRU) genes were used for the selection. Two pairs of ZF arrays for the CRU gene and one pair for the PPO gene were successfully selected, showing three times or more activity compared to the negative control in bacterial two-hybrid assays. ZF arrays for a second PPO target site were identified via the CoDA method and these sequences were ordered. FokI nuclease domains, which have improved activity and will only form heterodimers, were combined with the selected ZF arrays to form ZFN pairs separated by the 2A ribosomal skipping sequence. The activities of the ZFNs were analysed in planta using next generation sequencing (NGS). The sequencing results showed that none of the newly constructed ZFN pairs had created footprints, whereas footprints were found using plants containing the previously used GFP ZFN pair.
In order to test whether ZFNs were not active indeed, they were analysed in a yeast single strand annealing (SSA) assay. One ZFN pair for the PPO gene showed activity. Since no active ZFNs were obtained for the CRU gene, we tested additional ZF arrays that had been obtained by the OPEN selection in yeast SSA assays. These results showed that many combinations were active. Control tests with the 2A construct that was used for expression of the ZFNs revealed that this was not functional. Expression of the ZFNs from two separate genes (without the 2A sequence) in protoplasts resulted in footprints in the PPO target site, indicating that these ZFNs were active. Subsequent analysis of transgenic plants also resulted in footprints present in the endogenous PPO gene. However the ZFNs for the CRU gene did not show any activity in protoplasts.
Since no active ZFNs for the CRU gene were obtained, we decided to design a new nuclease type, emerged as powerful genome engineering tool during the time frame of the project, the so-called TAL effector nucleases (TALENs) for the CRU gene. Three target sequences were chosen close to the junction of the CRU gene and the RFP 5’ end present in the repair construct. TALENs were cloned and tested in Arabidopsis protoplasts. For TALEN pairs 1/2 and 5/6 footprints were obtained, indicating that DSB had been induced. For TALEN pair 3/4 no footprints were obtained, but the restriction site that was used is not located in the middle of the spacer and therefore small deletions may have been missed in the analysis. Plants were transformed through Agrobacterium-mediated floral dip transformation and these plants will be analysed in the near future for the presence and activity of the TALENs. When active TALENs are obtained, the plants will be used for GT experiments in future projects.
No toxicity of ZFNs and TALENs has been observed. The ZFNs cloned by modular assembly have been expressed in four generations without any obvious negative effect on growth. Third generation plants with the PPO ZFNs cloned by OPEN also did not show any negative effect due to the ZFN expression.
DSB-mediated GT in tomato
The development of an efficient gene targeting procedure for tomato was the main task of P6. As described previously, gene targeting is enhanced when double strand breaks (DSBs) are produced at the genomic target site. Within the framework of Recbreed we aimed to use zinc finger nucleases (ZFNs) to produce targeted DSBs. Another critical aspect for the overall efficiency of a gene targeting procedure is the total number of cells that can be treated to produce to take up the plasmid DNA encoding the ZFNs and thus have a targeted DSB produced in the genome. The higher this number is, the higher the chance to identify gene targeting events. For that purpose we chose to use leaf protoplasts, of which several millions can be isolated and transfected in a single experiment. In order to develop a gene targeting procedure for tomato two features were critical: an efficient protoplast regeneration system and a DSB should be efficiently produced at the target site(s).
Within the first year of Recbreed, protoplasts were prepared from several tomato genotypes and screened for their regeneration capacity. The variety M82 (field processing tomato) was finally identified for its high protoplast plating efficiency (between 30-40%) and its high regeneration capacity (>50%). Moreover M82 tomato is readily amenable to Agrobacterium-mediated transformation, which may be advantageous when testing the effect of genes improving gene targeting identified by other Recbreed partners.
In order to demonstrate the usefulness of the tomato protoplast system for the application of ZFNs, M82 tomato protoplasts were treated with ZFNs and the formation of footprints via non homologous end joining (NHEJ) (insertions or deletions, INDELs) was assessed. By assessing the efficiency of INDEL formation at the target sequence we could determine the relative activity of a ZFN pair. A ZFN pair (ZFN815) targeting the acetolactate synthase (ALS) gene of tobacco was kindly provided by Prof. Daniel Voytas (University of Minnesota, Minneapolis, MN, USA). These ZFNs have been described in the literature (Townsend et al, 2009, Nature 459: 442-445). Sequence analysis showed that the tobacco and tomato ALS genes share a high level of homology, indicating that ZFNs developed for the tobacco ALS genes should also target the tomato ALS1 and ALS2 genes. Tomato protoplasts were transfected with ZFN815 plasmid DNA using polyethylene glycol (PEG) mediated transfection and cultured for 2 days, after which genomic DNA was analyzed for the presence of mutations (INDELs) at the ZFN815 target site. The analysis of two experiments showed that on average 16% of the ALS1 and 5% of the ALS2 sequences contained mutations. These mutations mainly consisted of deletions of 1 to 20 bases or insertions of a few bases at the target site. Protoplasts treated with ZFN815 were grown and eventually regenerated into whole plants which were then individually characterized for the presence of mutations at the ZFN815 cleavage site. More than 15% of the regenerated shoots showed mutations at the ZFN815 cleavage site demonstrating the power of this approach to produce targeted mutations.
Having demonstrated the efficiency of ZFN815 to produce a targeted DSB in the tomato ALS genes, we proceeded with gene targeting experiments. The ALS protein is an essential enzyme for the production of certain amino acids and is the target of sulfonylurea herbicides. Specific single point mutations in the ALS gene result in the production of an ALS protein that is tolerant to the herbicide, making this gene an selectable marker for gene targeting in tomato. Tomato protoplasts were co-transfected with a plasmid encoding for ZFN815 and with a plasmid carrying a modified copy of the ALS gene (DNA donor), including the SNP’s conferring resistance to the herbicide. Using this approach in tomato, several herbicide resistant calli were produced and characterized as gene targeting events by PCR. Unfortunately we also observed an unexpected negative effect of the DNA donor on the protoplast development. Further investigation revealed that this negative effect was due to the down regulation of the expression of the endogenous ALS gene, leading to a suppression of the regeneration of the transfected tomato protoplasts. We were able to partially circumvent this negative effect by adapting the length and amount of the DNA donor. To date the maximum gene targeting efficiency observed is 5 events for 1.106 protoplasts. Several herbicide tolerant calli, resulting from gene targeting events or spontaneous mutation, were regenerated into plants and were used for Southern analysis, confirming the PCR results and revealed that all gene targeting events analysed were true gene targeting events. To our knowledge these gene targeting events are the first ever obtained in the tomato crop.
As described above, we showed that the introduction of a DSB at the target locus significantly enhances the gene targeting efficiency in tomato. However, the production of efficient ZFNs is a cumbersome task that may complicate the targeting of any locus. Therefore we made use of our tomato protoplast protocol to assess the effectiveness of TALENs in tomato. Three pairs of TALENs were designed and introduced on plasmid constructs into tomato protoplasts. By detection of INDEL mutations, all three of these TALEN constructs were shown to be effective in producing DSBs at their respective target sites. We showed that plants containing a null mutation in the MLH1 gene were infertile which is to be expected as this gene is known to be involved in crossover formation during meiotic recombination. We also showed that TALENs targeting the EPSPS gene of tomato were able to produce INDELs at the target sequence, making those reagents useful for attempting gene targeting at the EPSPS locus.
Task 1.2 Induction of meiotic recombination with Zn-finger nucleases [P1, P5]
A thorough bioinformatics analysis was performed in order to determine INDELs as well as SNPs between Arabidopsis cultivars (Col-0, Bur-0 and Tsu-1, respectively) which can be used as molecular markers to survey meiotic recombination frequencies on the one hand and that will allow for ecotype-specific DSB induction mediated by site-specific ZF nucleases. ZF arrays were selected via the OPEN procedure (P5), directed against four 3 bp deletion target sites identified in the ecotypes Bur-0 or Tsu-1 (B16, B36, B59, T32 and T43). For three sites good ZF arrays were obtained, with relative β-gal activity of 3 or more. For the other two sites, which have the same target sequence, only one active array could be obtained. The ZF arrays with the highest activity were cloned in frame with improved FokI nuclease domains, which can only form heterodimers and are more active.
All ZFNs were cloned under the control of the meiosis-specific SPO11-1 promoter. The constructs were transformed into the respective A. thaliana ecotypes by Agrobacterium-mediated transformation and more than 20 stable primary transformants (T1 generation) were selected for each line. In the next generation homozygous single copy lines were selected. During these selection steps partial sterility of the plants was observed, indicating off target effects. Due to the partial sterility we retransformed the ZFNs in their respective ecotypes and again we observed partial sterility for most of the plants.
P5 has not seen ZFN activity for the respective ZFN-2A constructs driven by a constitutive promoter. Additionally, the observed partial sterility of T1 plants under the control of the meiosis-specific SPO11-1 promoter was indicative for unpredicted off-target effects. Moreover, a comparable meiosis-specific experimental set-up with a ZFN that has shown reliable activity driven by inducible and constitutive promoters in previous studies failed in F1 hybrids as no detectable HR induction at the target site was achieved. Therefore we decided to stop the experiments before crossing the homozygous single locus lines into Col-0. Nevertheless, our functional CAPS marker pool that was developed within this framework is still useful for further studies. To differentiate between the chromosomes bearing respective ZFN binding sites (Chr. I, II and III) we identified several polymorphisms in Col-0, Bur-0 and Tsu-1 for CAPS marker analysis. Since the 2nd periodic report we established some of these markers for high resolution melting (HRM) analysis, too. HRM allows a semi high throughput screening for F1 hybrids as well as recombination events in 384 well plates.
Task 1.3 Induction of meiotic recombination by Spo11-1/2 overexpression [P1]
Initiation of homologous recombination (HR) by DSB formation in meiosis is the central function of SPO11. Previously, it was demonstrated that mono- and dicotyledonous plants harbour three distantly related homologues of yeast SPO11: SPO11-1, SPO11-2 and SPO11-3. However, SPO11-1 and SPO11-2 mutant lines that have been described possess a severe meiotic phenotype resulting in almost completely sterile plants (Grelon et al (2001) Embo J 20: 589-600; Hartung & Puchta (2000) Nucleic Acids Res 28: 1548-1554). SPO11 homologs induce meiotic HR by generating DNA double-strand-breaks (DSBs) throughout the genome. The number of SPO11-induced DSBs and the number of cross-over events are thought to be correlated. Our aim was a genome-wide enhancement of recombination by simultaneous overexpression of SPO11-1 and SPO11-2 to test this hypothesis.
Double expression constructs for SPO11-1 and SPO11-2 have been constructed both with the SPO11-1 promoter as well as a constitutive ubiquitin promoter. Subsequently, the respective constructs have been transformed into Arabidopsis (Col-0) via Agrobacterium-mediated transformation. Following the identification of transgenic progeny, stable single locus lines were established by segregation analysis. Homozygous SPO11 lines driven by the SPO11-1 promoter and non-transformed Col-0 as a control line with Bur-0 were crossed. An extremely extended lifecycle in F1 was observed, with an altered rosette phenotype. The first inflorescence did not appear until 3 months without any regularity in one F1 line.
Test crosses between Col-0, Bur-0 and Tsu-1 wild type plants were performed to confirm these results and similar phenotypes were observed in the F1 generation. We have performed backcrosses of these F1 lines with one of the parental ecotype backgrounds to check if the CAPS marker screening is usable under these genetic conditions and it worked well for all combinations. Two of the meiosis-specific SPO11 overexpression lines were used for the backcrosses to perform the analysis of meiotic recombination events in FR generation. For the analysis of meiotic recombination events we used some of the already established CAPS markers from WP1 task 1.2 that allow differentiation of sequence polymorphisms between Bur-0 and Col-0. Additionally, we developed further CAPS markers for task 1.3 and again we established most of them for HRM analysis, too. Meiotic recombination of 20 FR control plants [(Col-0 x Bur-0) x Col-0] as well as 18 and 17 independent FR SPO11 overexpression plants [(SPO11-OE x Bur-0) x SPO11-OE] was analyzed with markers on chromosome II, III and V. Genotyping suggested evidence for changes in the crossover distribution, but this still has to be confirmed statistically. Therefore, we initiated some follow-up studies. The homozygous SPO11 overexpression lines were crossed with Ler-1 instead of Bur-0. We identified Ler-1 as the best crossing partner for Col-0 without having any disadvantage during development in F1 hybrids. We characterized several new CAPS and HRM markers for discrimination between Ler-1 and Col-0 chromosomes. These markers will be used in semi high through-put analysis of backcrosses of the F1 hybrids for meiotic recombination.
Homozygous plant lines expressing SPO11 under control of the Ubiquitin promoter were analysed for intrachromosomal recombination using a recombination reporter substrate to test if homologous recombination is generally enhanced in these lines. The results indicated that SPO11-1/SPO11-2 overexpression did not result in generally enhanced HR frequencies.
Task 1.4 Isolation of a promoter for expression of genes in maize, at the “HR-initiation stage” [P7]
The objective of this task was to isolate promoters that express during meiosis and which were appropriate for expressing genes that modify the frequency of gene targeting or meiotic recombination. These promoters were also intended to be used by P7 in WP2 and WP3 to overexpress Spo11, an HR-initiation candidate for increasing the frequency of meiotic recombination.
Promoter isolation and validation
The previously isolated rice DMC1B promoter was linked to GFP and transformed into maize. Promoter analysis has focused on previously generated pOsDMC1-GUS lines. Surprisingly these lines showed expression that was “constitutive‟ with for example strong expression in the anther wall. Thus this version of the pOsDMC1B promoter was not strictly meiosis-specific, but still the promoter may be useful if it is expressed in meiocytes.
Three other genes were identified with meiosis-specific or preferred expression based on publically available expression data, literature survey and consultation with partners. These were the Arabidopsis genes SDS (Solo-dancer), Arabidopsis ASY1 and SPO11-1. The homologous rice genes have been identified and putative promoter regions isolated. The following promoter reporter fusions have been constructed using 1-2 kb of DNA upstream of the ATG; pOsSpo11-1-GFP; pOsAsy1-GFP, pOsSDS-GFP and pOsSDS-GUS. The promoter-reporter fusions were transformed into maize. Except for pOsDMC1b, all attempts at expression analysis with GFP / GUS visualization on isolated meiocytes were inconclusive, thus we decided to characterize these promoters via qRT-PCR in meiotic tissue samples. As Spo11-expressing lines using these promoters were available, we performed these mRNA expression studies directly on the Spo11 lines that needed to be characterized.
Three Spo11 constructs (pOsAsy1_ZmSpo11-1+pOsAsy1_ZmSpo11-2, pOsSpo11-1_ZmSpo11-1 + pOsSpo11-1_ZmSpo11-2 and pOsDmc1b_ZmSpo11-1) were tested for immature anther expression and leaf expression; as a control a constitutive promoter was also tested (pOsRubi3_ZmSpo11-1+pOsRubi3_ZmSpo11-2). Specific primers targeting endogenous meiotic genes (ZmAsy1, ZmSpo11-1 and ZmDMC1) as well as both synthetic ZmSpo genes (synSpo11-1 and synSpo11-2) were used. As expected, significant expression of synthetic Spo11 genes was only detected in leaves from the pRubi3 and pDMC1b constructs. The level of expression of the synthetic Spo11 genes was about 10% of that expected from the pOsRubi3 promoter suggesting the that Spo11 transcripts may be unstable. No expression was detected in the wild-type A188 control samples nor in pAsy1 and pSpo11-1 constructs, although weak expression levels of endogenous ZmSpo11-1 were detected in these leaf samples. pDMC1-driven expression of transgenes in leaves was very weak, in most cases inferior to endogenous ZmSpo11-1 levels.
In anthers Spo11 expression from pOsRubi3 gave on average an overexpression of 16 times with respect to the endogenous Spo11-1 levels. However such overexpression is likely to be largely due to expression in the anther wall. For the pDMC1b construct expression of synSpo11-1 was similar to that of the endogenous Spo11-1 gene. For both pSpo11-1 and pAsy1, the expression levels of synSpo11-1 and synSpo11-2 were barely detectable.
Effect of Spo11 overexpression on meiotic recombination
To assess the effect of the overexpression of the Spo11 genes, the following strategy was planned: crossing of single loci transgene expressing lines to a divergent inbred line (R), selfing of the F1 progeny of 2 of the best transgene expressing lines and genotyping of the F2 progeny using a genome-wide genotyping chip containing SNPs for at least 4500 genes. Comparison with control F2 plants lacking the transgene will detect if the transgene induces a change in the frequency and/or position of crossovers. Although crosses with line R were made, in the light of the lack of identification of good Spo11 overexpressors in anthers, we decided not to proceed further with this experiment.
1.3.2 WP2: Homology search and heteroduplex formation
Task 2.1 Homology search [P1, P2, P3]
Reporter translational fusions
To better understand the mode of action and the influence of these key actors on the efficiency of the homologous recombination process, we have constructed reporter translational fusions for RAD51 and the RAD51 paralogs with expression of the genes driven by their own promoters, or by the constitutive MAS promoter. Functional validitation of the constructs was confirmed by complementation of the corresponding Arabidopsis mutants and analysis of the plants carrying the Rad51 paralog-GFP fusion proteins has permitted establishment the expression patterns and intracellular localisation of the protein. These studies have also resulted in the identification and characterisation of novel promoters for gene expression.
Roles of the RAD51-family proteins in Meiotic Recombination and Chromosome Synapsis: differing roles at peri-centromeres and chromosome arms.
Studies of the functional roles of these proteins in mitotic and meiotic recombination in Arabidopsis confirmed that all five RAD51 paralogues of Arabidopsis play key roles in recombination and permitted the description of a novel distinction between the dependence of meiotic chromosome synapsis in heterochromatic centromere-proximal and rDNA regions, compared to euchromatic chromosome arms; with the former being mostly DMC1-dependent and the latter requiring RAD51 and the RAD51 paralogs RAD51C and XRCC3. The results of this work provide new insights into the homologous chromosome pairing mechanisms occurring in meiotic prophase: heterochromatic centromeres and 5S rDNA regions pair early and their pairing has different requirements for recombination proteins than does that of the chromosome arms - highlighting the importance of questions concerning the specificities and roles of recombination at different chromosome and/or chromatin regions in the synapsis of homologous chromosomes at meiosis.
Stimulation of Meiotic Recombination in the absence of XRCC2.
The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilisation of the RAD51-DNA nucleofilament. We confirmed that the three "non-meiotic" paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and have shown that the three proteins are not essential for the formation of the RAD51-DNA fibre at DNA breaks in Arabidopsis (immunodetection of radio-induced Rad51 foci). Analyses of meiotic recombination showed increased rates of meiotic crossing-over in xrcc2 and to a lesser extent rad51b but not rad51d mutants. We speculate that XRCC2 and RAD51B affect the balance of the activities of DMC1 and RAD51 in meiosis and in their absence this balance shifts towards cross-over outcomes. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells and this hyper-recombination phenotype is of potential utility and importance in plant breeding for agronomy. Partner P7 (BIOGEMMA) has initiated studies in maize to test for this effect in a major crop plant (see Task 2.3).
The RAD51-S mutant and the roles of RAD51 and Dmc1 in Meiotic Recombination.
The RAD51_GFP fusion protein (see section (i) above) complements the meiotic chromosome fragmentation and sterility of the Arabidopsis rad51 mutant. Further analyses showed however that the fusion protein is not a functional strand-transfer recombinase: conferring a dominant negative phenotype in somatic tissues, while remaining able to fully complement the meiotic defects of rad51 mutants. Presence of this fusion protein thus defines a "separation of function" mutant, similar to the rad51-II3A mutant recently reported in yeast (Cloud et al. Science. 2012. 337:1222). For convenience we have named this mutant RAD51-S. Preliminary results of expression in dmc1 mutant plants confirm the dominant negative effect of the RAD51-GFP fusion in Arabidopsis meiosis, and show that its complementation of the meiotic chromosome fragmentation and sterility of the Arabidopsis rad51 mutant is fully dependent on the presence of DMC1. DMC1 is thus able to repair all meiotic DSB in Arabidopsis and depends upon the presence, but not the strand exchange activity, of RAD51 to do so
These results impose a new vision of the relative roles RAD51 and DMC1 in meiosis and dramatically underscore the importance of the multiple roles of the RAD51 nucleofilament, and of the accessory proteins (such as the RAD51 paralogues) which modulate its function.
Task 2.1b - BRCA2 [P1]
BRCA2 regulates RAD51-filament formation and DNA binding of RAD51, the protein seems to work as a mediator of RAD51 facilitated DNA repair. BRCA2-binding is postulated to lead to replication protein A (RPA) replacement enabling RAD51 to build a nucleoprotein filament with ssDNA. These results suggest a control mechanism of RAD51 by BRCA2, explaining why a loss of BRCA2 function shows similar effects as RAD51 loss. In the Arabidopsis genome two copies of the BRCA2 gene are present and we showed that the brca2 double mutant is viable and, in contrast to mammals, BRCA2 is thus dispensable for development in plants
Studies of meiosis in the double mutant showed that it is sterile, with no viable pollen and male meiosis abrogated at the pachytene stage of prophase. Somatic cells of the double mutant are strongly sensitive to Mitomycin C, indicating a specific defect in somatic DNA crosslink repair. The frequency of intrachromosomal homologous recombination is reduced with and without genotoxic stress, demonstrating the involvement of the protein in HR in somatic cells.
By amplification of the genomic DNA fragment, we have constructed a plasmid for overexpression of BRCA2(IV).The T-DNA construct was transferred into Arabidopsis plants, where however we found aberrant segregations and loss of the selectable marker in the following generation. We have shown that overexpression of this gene results in severe growth defects in Arabidopsis.
Notwithstanding its real interest from the scientific point of view, in the context of this project this result precluded further study of somatic HR rates under conditions of BRCA2 overexpression. Furthermore the growth defects of BRCA2(IV) overexpression preclude its effective use in a biotechnological setting.
Task 2.1c Role of AtRAD52 in GT and meiotic HR [P2]
Identification of Arabidopsis RAD52 homologs.
RAD52 mediates RAD51 loading onto ssDNA ends at DNA breaks, thereby initiating homologous recombination and catalyzing DNA annealing. RAD52 is highly conserved among eukaryotes, but had not been identified in plants. This work reports the discovery of RAD52 homologs present in all plants whose genomes have undergone extensive sequencing. Computational analyses suggest a very early RAD52 gene duplication, followed by later lineage-specific duplications during the evolution of higher plants. Plant RAD52 proteins have high sequence similarity to the oligomerization and DNA binding N-terminal domain of RAD52 proteins.
Remarkably, the two identified Arabidopsis AtRAD52 genes encode four open reading frames (ORFs) through differential splicing, each of which specifically localized to the nucleus, mitochondria or chloroplast. The Arabidopsis AtRAD52-1A ORF provided partial complementation to the yeast rad52 mutant. Arabidopsis mutants and RNAi lines defective in the expression of AtRAD52-1 or AtRAD52-2, showed reduced fertility, sensitivity to Mitomycin-C, and decreased levels of intrachromosomal recombination compared to wild-type. In summary, computational and experimental analyses provide clear evidence for the presence of functional RAD52 DNA-repair homologs in plants.
Overexpression of AtRAD52-1A::EGFP enhances gene-targeting.
Overexpression of AtRAD52-1A is associated with a 10-fold increase in Gene targeting rate. The four GT events originating from the AtRAD52-1A-EGFP line included one true and precise GT event, while all three GT events detected in WT plants were non-precise targeting events. Thus, in AtRAD52-1A-EGFP plants we obtained a rate of 5.43x10-5 bona fide GT while no true GT events were detected in WT plants.
Silencing/overexpression of RAD52 has only minor or no effects on meiotic recombination.
Tests of meiotic recombination rates in absence or overexpression of the RAD52 homologues showed different results for the different AtRAD52 fusion proteins. Overexpression AtRAD52-1A, localized to the mitochondria and the nucleus, led to an increase in meiotic recombination rate of ~14% compared to WT. No change in meiotic recombination rates were however detected in a AtRAD52-1B over- expressor (mitochondrial localisation), nor in AtRAD52-2B overexpressor lines (chloroplast localisation).
RNAi constructs were used to specifically knock-down expression of the two genes and while 35S-AtRAD52-1RNAi gave no change in meiotic recombination rate, 35S-AtRAD52-2RNAi increased meiotic recombination rate by ~18% compared to WT. We are combining the RNAi knockdowns and effects on meiotic recombination will be scored within about a month. Similarly, combination of 35S-AtRAD52-1A::mCFP and 35S-AtRAD52-2A::EGFP lines’ effect on meiotic recombination will be scored within about a month. The effect of combined overexpression of the two AtRAD52 nuclear variants on gene-targeting will also be tested.
Task 2.2 Chromatin modification and Meiotic HR [P4,P2]
Task 2.2a Chromatin modifications and Meiotic HR [P4]
This task aimed to assess whether alteration in epigenetic properties of chromatin would have an influence on the frequencies of meiotic crossovers (COs).
Genome-wide analysis to determine the number of COs on each chromosome for all four populations analyzed (the hypomethylated met1-3, the epiRIL lines epi01 and epi12, and the WT control) showed that loss of DNA methylation is not altering the total level of COs, but rather their distribution along the chromosomes. We hypothesize that this phenomena is due to the tight regulation of meiotic COs, by mechanisms such as CO interference and homeostasis.
The effects of hypomethylation on frequencies of meiotic recombination observed in other chromosomes were only partially reproduced in chromosome 4. Unlike in other chromosomes, in chromosome 4 only 1 out of 4 euchromatic regions showed an increase in recombination frequencies in the met1-3 population, and there was no correlation between the methylation status of the examined fragment and its level of recombination, with the exception of one euchromatic region. This result is similar to our former results obtained in the genome-wide experiment for other chromosomes in the epiRIL populations. Since the different heterochromatin structure of chromosome 4 did not allow an informative analysis of meiotic recombination frequencies in our Col/Ler populations, we generated new Columbia/ Wassilewskija genotyping populations. To generate "knob-less" populations we used the Landsberg and C24 ecotypes as parental lines, both do not have the hk4S. We designed a new set of KASP genotyping assays in order to measure recombination frequencies around the hk4S and centromeric regions as well as on other euchromatic regions on chromosome 4. We included chromosome 2 in our analysis since it is similar to chromosome 4 but lacking the hk4S, thus serving as an intra-genomic control.
We found that when recombination occurs between two similar chromosomes, i.e. lacking the hk4S on chromosome 4, the effect of hypomethylation on recombination frequencies is well reproduced, showing, as before, an increase in recombination frequencies in hypomethylated euchromatic intervals. For the Ws/Col populations which have the hk4S, we also observed an increase in recombination frequencies in hypomethylated euchromatic regions of chromosome 4 with a smaller effect on chromosome 2. Interestingly, we noticed that the recombination frequency was exceptionally high around the centromere in the Ws/Col populations containing the hk4S and we speculate that the presence of the heterochromatic knob (hk4S) is inducing recombination at the adjacent centromere.
Task 2.2b The effect of DDM1 on meiotic recombination [P2]
[This part was not in the original proposal. It was added to task 2.2 because of its direct relation to the topic and to replace the part on MLH1 following negative results with AtMLH1 overexpression in parts of task 3.1]
Meiotic recombination is tightly regulated by cis and trans-acting factors. While DNA methylation and chromatin remodeling affect chromosome structure, their impact on meiotic recombination is not well understood. In order to study the effect of DNA methylation on the landscape of chromosomal recombination we analyzed meiotic recombination in the DECREASED DNA METHYLATION 1 mutant (ddm1). DDM1 is a SWI2/SNF2-like chromatin remodeling protein necessary for DNA methylation and heterochromatin maintenance in Arabidopsis thaliana. The rate of meiotic recombination between markers located in euchromatic regions was significantly higher in both heterozygous (DDM1/ddm1) and homozygous (ddm1/ddm1) backgrounds than in WT. The effect on recombination was similar for both male and female meiocytes. Contrary to expectations, ddm1 had no effect on crossover numbers between markers in heterochromatic pericentric regions that underwent demethylation. These results are surprising because pericentromeric regions are hypermethylated and were expected to be the most affected by demethylation. Thus, DDM1 loss of function may trigger changes that enhance meiotic recombination in euchromatin regions, but are not sufficient to induce the same events in heterochromatic segments. This work uncovers the repressive role of methylation on meiotic recombination in euchromatic regions and suggests the role of additional factors controlling recombination suppression in heterochromatin.
Task 2.3 Enhancing GT and meiotic recombination in maize by specific over-expression of genes involved in homology search [P7]
A. Enhancing GT by expression of homology research genes
Validation of the test GT system.
The test system for GT in maize is based on the reconstitution of a defective nptII gene. The 3’ region of nptII is integrated into the plant genome at the Target Locus (TL), which has a target site for the meganuclease I-SceI allowing the creation of a DSB at the TL when I-SceI is present. The goal of the GT work in RecBreed is to retransform these TL lines with a repair DNA (Donor Locus construct or DL) and DNA encoding I-SceI plus another gene that putatively further stimulates GT. The DL contains sequences homologous to the TL and has the 5’ part of nptII. Thus GT at the TL results in reconstitution of nptII and resistance to kanamycin. In previous work, in the EU FP6 project TAGIP, maize lines were stably transformed with the a DL construct which contained the 5’ nptII region flanked by I-SceI sites and a dexamethasone-inducible I-SceI gene (I-SceI-GR). These DL(I-SceI-GR) lines were crossed to TL lines and progeny containing both the TL and DL loci treated with dexamethasone.
We analysed these DL(I-SceI-GR)+TL+/- I-SceI (constitutive) plants and showed, by PCR, excision of the DL and recombination of the DL and TL by NHEJ. We also developed an assay for somatic GT based on application of kanamycin to plants; green resistant sectors were observed in kanamycin –bleached leaves. Green sectors were excised and shown by PCR to have reconstituted nptII. We also regenerated fully kanamycin-resistant plants, via in-vitro regeneration of kanamycin resistant plantlets. Southern analysis confirmed that “GT” had occurred. For 2 GT events from TL1 (GT1 and GT2) the DL recombined at the TL as predicted for a true GT event at the TL; i.e. reconstitution of nptII and loss of the original TL locus. However, unexpectedly, no DL excision was detected from the DL locus.
GT events were characterised by PCR and by DNA sequencing of the genomic GT loci. PCR of GT descendants showed that in the progeny of the GT1 and GT2 events kanamycin resistance is strictly correlated with the present of a modified TL1 locus. However for the GT3 to GT6 events kanamycin resistance was linked to a modified DL locus, the TL2 locus being unaltered. The DNA sequence of the GT1 and GT2 loci was as predicted for GT event involving a double recombination between the TL1 and DL loci. However the sequence of the other GT events showed that there had been a single crossover between the DL and TL2 locus restoring nptII at the DL1 locus with variable lengths of the TL1 locus being integrated into the DL1 locus via NHEJ in the different GT events. These studies thus confirmed the suspected mechanism of GT via ectopic recombination.
In order to understand why only crosses with the TL1 line gave the expected GT events the TL locus the different TL and DL parental lines were resequenced to ensure that all the I-SceI sites were functional. It transpired that the DL parent used for the crosses with TL1 lacked the I-SceI site adjacent to the 5’ region of nptII (this site had probably been mutated after having been cut by I-SceI-GR). Thus in the TL1 x DL crosses recombination to restore nptII activity is favoured at the TL1 loci because a DSB can only be created next to a defective nptII at the TL1 locus.
Testing the homology search candidate RAD54
P2 has shown that overexpression of RAD54 increases GT in Arabidopsis (Shaked et al (2005), Even-Faitelson et al (2011)). Two strategies have been tested to evaluate the effect of yeast RAD54 using either the native yeast sequence (ScRAD54) or a version optimized for maize expression (SynZmRAD54). In previous reports we described experiments where TL lines were retransformed with a DL construct with I-SceI and with or without RAD54. No kanamycin resistant plants were regenerated. A second strategy involved crossing TL lines to a DL construct expressing I-SceI-GR and synZmRAD54 (optimized for maize expression). In this reporting period the crosses were performed but insufficient time remained to grow these plants, extract embryos and test GT. To determine if RAD54 might stimulate intrachromosomal recombination plants expressing I-SceI or I-SceI+synRAD54 were crossed to a GU-US intrachromosomal reporter line. All the progeny plants containing the reporter + I-SceI or reporter + I-SceI and RAD54 possessed GUS positive sectors by histochemical staining. Controls (WT, reporter or I-SceI or I-SceI+RAD54 alone) had no GUSpositive sectors. However reporter + I-SceI + RAD54 plants showed no significant difference with respect to the number of GUS positive sectors as reporter + I-SceI plants, suggesting that synZmRAD54 does not stimulate intrachromosomal recombination.
B. Enhancing meiotic recombination by expression of homology research genes.
Partner P3 showed that the Arabidopsis Xrcc2 mutant strongly stimulates meiotic recombination. The maize Xrcc2 homolog was identified by reciprocal BLAST analysis. There appears to be only one Xrcc2 gene in maize (GRMZM2G002626). A 300 bp fragment specific to ZmXrcc2 used to create an RNAi construct. In order to preferentially downregulate ZmXrcc2 in meiosis the best candidate meiosis-expressed promoter identified, pDMC1B, was linked to this RNAi fragment. 34 transformed lines were generated and in the first instance crossed to WT (WT as male). No obvious effect on female fertility was observed since all plants produced normal quantities of seed. Insufficient time was available in the project to perform anther expression analyses to select plants that have ZmXccr2 downregulation. These plants would then be crossed to a divergent maize line and the F2 progeny genotyped to assess any effects on meiotic recombination.
1.3.3 WP3: Promoting HR via stabilization and resolution of recombination intermediates
Task 3.1 Heteroduplex stabilization by the mismatch repair machinery [P2]
Mismatch repair genes play diverse and opposite roles. Some of them, like MLH1, promote crossover via stabilization of Holiday junctions, others like MSH2 have an anti-recombination effect in particular when mismatches are present in the Holiday junctions. Our attempts to enhance meiotic recombination via overexpression of AtMLH1 in Arabidopsis have not given the hoped-for results. Therefore, MLH1 was not tested in tomato. Instead we focused our efforts on the suppression of MSH2. When the project was initiated the tomato genome was not available and there was no mutant for MSH2 in tomato. Therefore, we set to identify the MSH2 tomato homolog and we tried to silence it using an RNAi approach in a meiotic-specific manner (under SPO11 promoter). Petunia and Arabidopsis MSH2 were blast against the Tomato database. The sequence of the StMSH2 was obtained. A 513bp fragment including the 3’UTR was amplified from micro-Tom cDNA and subcloned in sense and antisense orientation in pKanibbal under either 35S or SPO11 (meiotic specific) promoter. Each clone was subsequently subcloned into pART27 to confer Kanamycin resistance. We transformed Solanum lycopersicum Var. Micro Tom. Plants showing resistance were grown, RNA was extracted from flowers before meiosis young flower buds and Msh2 was quantified by RT PCR method. From the five lines that were silenced only two were viable (SPO79 and SPO74) the other three were sterile and couldn’t be used for the following experiment. The plants with silenced StMSH2 were crossed to Solanum pimpinellifolium. Seeds from the resulting cross were grown under Kanamycin selection and once again MSH2 transcript was quantified by qRT PCR from young flowers again showing reduction in expression (See details in periodic report). Using genetic markers meiotic recombination was evaluated in the F2 generation of Solanum lycopersicum Var. MicroTom X Solanum pimpinellifolium, SPO11::msh2RNAi-79/74 X Solanum pimpinellifolium.
Figure 3.1: The effect of down-regulation of AtMSH2 on homeologous recombination was tested in a cross between WT tomato, variety Micro-Tom and its relative Solanum pimpinellifolium compared to the same cross where the Micro-Tom parent has been transformed with an siRNA construct that was shown to reduce AtMSH2 transcript level
Marker position WT SPO11::RNAi_msh2
No. of Plants Rec.
Plants Genetic Distance (cM)* No. of Plants Rec.
Plants Genetic Distance (cM)*
Chromosome 9 88 9 5.1 135 13 4.8
So far we have obtained results for only one pair of markers on chromosome 9. We see only a minor and not significant increase in meiotic recombination as a result of AtMSH2 suppression (see Table above). Additional markers are currently being tested, enabling us to reach conclusions.
Task 3.2a – Heteroduplex resolution by XPF-family structure-specific endonucleases [P3]
Role of XPF/ERCC1 in somatic and meiotic recombination
Previous studies by P3 showed that mutation in XPF or ERCC1 affect somatic homologous recombination in plants (Dubest et al., 2002, 2004). To address whether these mutations also affect meiotic recombination, we analysed meiotic recombination rates in Arabidopsis xpf and ercc1 mutants using the cruciferin promoter RFP/GFP-based assay from the group of A. Levy (partner P2). Analyses of progeny of selfed plants showed no significant effect on meiotic recombination rates in Arabidopsis xpf and ercc1 mutant plants, when compared to the corresponding wild-type plants for two intervals on chromosomes III and V (Similar results were obtained for male and female meiotic recombination (See detailed result in the periodic report). Thus, although the XPF-ERCC1 complex plays an important role in somatic recombination, its absence appears to have no significant effect on meiotic recombination rates in the two chromosomal intervals tested.
Effects on somatic recombination of overexpression of XPF and ERCC1
Given the important roles of XPF and ERCC1 in somatic recombination, we have built and validated in planta, fluorescently tagged, fusion protein expression constructs to elucidate these effects on somatic recombination. The reporter translational fusions for the Arabidopsis XPF and the ERCC1 proteins were constructed with the complete genomic sequence (i.e. + introns + exons) expressed by their own promoters (Figure 2A). Stop codons were removed and the sequences fused to the GFP gene to generate the fluorescently tagged translational fusion constructs. The functionality of the fusion proteins has been confirmed by complementation of the UV and MMC hypersensitivity of the corresponding Arabidopsis xpf and ercc1 mutant plants (Figure 2B). Exploiting the GFP fluorescence of the tagged proteins, we have analysed the temporal and spatial expression patterns of the tagged-fusion proteins, which show clear nuclear localisation of the proteins and that the genes are strongly and ubiquitously expressed in roots and flowers (Fig. 3). Preliminary data suggests also that their expression is differentially regulated with respect to the cell cycle. In future studies, the fluorescent tags will permit immunological approaches, such as co-immunoprecipitation, for the characterisation of protein complexes. These transgenic lines have been crossed with the mitotic recombination tester lines from the Puchta lab (Orel et al., 2003; Mannuss et al., 2010). Somatic recombination rates will be analysed in coming months.
Task 3.2b - Mus81/Eme1 [P1]
MUS81 is a highly conserved endonuclease and together with EME1 (also referred to as MMS4 in S. cerevisiae) it is involved in the resolution of 3’ flap structures and Holliday-like DNA junctions. Loss of the protein results in sensitivity to DNA damaging agents in yeast and mammals. Interestingly, the role of the protein in meiosis differs drastically between eukaryotes. Whereas in S. pombe loss of MUS81 results in complete sterility, the mutant is partially fertile in S. cerevisiae and fully fertile in mammals. We identified a homologue of MUS81 in the genome of Arabidopsis thaliana and isolated a full-length cDNA of this gene. Analyzing two independent T-DNA insertion lines of AtMUS81, we found that they are sensitive to the genotoxins MMS and MMC. Both mutants have a deficiency in homologous recombination in somatic cells but only after induction of genotoxic stress. Atmus81 mutants display a moderate decrease in meiotic recombination. It has been hypothesized that AtMUS81 is involved in the generation of a second class of meiotic crossovers that are interference insensitive. We were also able to identify two closely linked homologues of EME1 in the Arabidopsis genome (AtEME1A and AtEME1B). By biochemical analysis we were able to demonstrate that indeed both the AtMUS81/AtEME1A and the AtMUS81/AtEME1B complex are able to process intact Holliday junctions.
As described in Annex I MUS81/EME1 should be cloned under control of a constitutive ubiquitin promoter as well as under control of the meiosis-specific DMC1 promoter. For both promoters constructs were cloned and have been transferred into Arabidopsis plants by Agrobacterium-mediated transformation. The somatic expression of the constructs under control of the ubiquitin promoter was analyzed in homozygous plant lines containing an additional recombination substrate for intrachromosomal recombination to test if HR is generally enhanced in the lines. Furthermore, quantitative RT-PCRs completed the analysis of the somatic expression lines. Further analysis of plants containing the constructs under control of the meiotic DMC1 promoter were put on hold until RMI1 mutant lines containing the same construct (see below) have been processed. 36 of 70 overexpression lines under control of the ubiquitin promoter were characterized as homozygous single locus lines and tested for enhancement of HR by recombination assays. 8 lines exhibited a moderately increased HR rate and were selected for three further recombination assays.
MUS81/EME1A overexpression displayed a wide range of HR enhancement with relatively high standard deviations. These differences were due to the fact that very low numbers of spots were counted on the seedlings. Because of the heterogeneity of the results 4 lines were selected for quantitative RT-PCRs to evaluate the expression of MUS81, EME1A and the 651 recombination construct. 2 of 4 lines showed a 30-fold increase of MUS81 transcripts and an about 130-fold increase of EME1A whereas the other two lines had only slightly different levels to the wild type control. The expression of the recombination substrate was increased between 2- and 11-fold in comparison to wild type. Nevertheless, the expression patterns and the HR enhancement do not correlate with each other. Hence, a silencing effect can be excluded. In addition to the conventional overexpression, MUS81 was also overexpressed in RMI1 mutant lines under control of the DMC1 and the RMI2 promoter – two strong promoters during meiosis. RMI1 together with RECQ4A and TOP3α form the so-called RTR complex in Arabidopsis thaliana, which has been shown to dissolve Holliday junctions by generating non-crossover products via the dissolution pathway of HR. Mutants of each of the three complex partners therefore were shown to have an increased rate of spontaneous somatic homologous recombination events. Interestingly, mutants of Arabidopsis TOP3α as well as RMI1 are sterile due to recombination-dependent chromosome defects in meiosis I, pointing to an important function of the dissolution pathway in meiotic HR.
Since the dissolution pathway is dependent on a double Holliday junction intermediate, as are hypothesized resolution pathways by nucleases, e.g. MUS81 or GEN1, it should be possible to rescue the somatic and meiotic defects of RTR complex mutants like rmi1 by overexpression of these nucleases. To test this hypothesis, two constructs of MUS81 under control of the DMC1 and RMI2 promoters were transferred into two independent Arabidopsis mutant lines of RMI1. Both constructs were transformed into A. thaliana rmi1-1 and rmi1-2 mutant plants. Through BAR resistance segregation, homozygous lines have been established. In addition, further resolvases from different organisms were cloned in a similar manner and also transformed into rmi1 mutant plants and established as homozygous lines. Since efforts shifted more to compare the effects of the expression of different resolvases in sterile rmi1 mutants, results of these experiments are described in WP4.
Figure 3.4. Meiotic MUS81/EME1A expression in Arabidopsis. The two constructs possess either the DMC1 or the RMI2 promoter from Arabidopsis thaliana. For selection the BAR resistance gene was cloned behind the second gene
Task 3.3 Enhancement of meiotic HR and GT using the most promising crossover promotion (CP) gene in maize [P7]
MSH2 downregulation in maize
Bioinformatic searches confirmed that the maize MSH2 gene (accession GRMZM2G056075 on chromosome 7) was the best homolog of AtMSH2. BLAST analyses against maize genomic sequences revealed partial sequences that have some homology to ZmMSH2 on chr1 (GRMZM2G392172; ZmMSH2-like 1) and chr2 (AC193754.3_FG008; ZmMSH2-like2). However alignments suggest that they do not represent additional maize MSH2 copies. Examination of the expression pattern of ZmMSH2 (Gene Atlas data, figure 5) shows that expression was highest in the immature tassel and cob, suggesting a role in meiosis for this gene. A 300bp region of the ZmMSH2 sequence that fell outside the region of homology to the ZmMSH2-like genes was chosen to design an RNAi construct. In order to preferentially downregulate ZmMSH2 in meiosis the best candidate meiosis-expressed promoter identified, pDMC1B, was linked to this RNAi fragment. 36 transformed lines were generated and in the first instance crossed to WT (WT as male). No obvious effect on female fertility was observed since all plants produced normal quantities of seed. Insufficient time was available in the project to perform anther expression analyses to select plants that have ZmMSH2 downregulation. These plants would then be crossed to a divergent maize line and the F2 progeny genotyped to assess any effects on meiotic recombination.
Enhancing GT by expression of crossover promotion genes:
At M30 there was no available strong crossover promotion gene candidate to stimulate GT. Thus it was decided in this reporting period to test whether abiotic stresses could enhance GT using the test GT system developed in Task2.3. In this system the frequency of GT is proportional to the number of green sectors observed on a leaf after kanamycin application. Many reports show that abiotic stress induces DNA repair genes expression and increases significantly homologous recombination rates (Yao and Kovalchulk, Mutation Research, 2011). Several abiotic stress conditions were tested on wild-type maize plantlets: UV, bleomycin (DSB), heat (37°C), 5-azacytidine (demethylation). In these experiments, HR gene expression was assessed in order to select the best stress conditions. To evaluate the effect on GT, these stress conditions were applied to TL+DL plants that were already scored positive in the kanamycin-resistance leaf assay. The clearest results were obtained with 5-Azacytidine and elevated temperature (figure 6). Seed was first imbibed for 25 hours in 30µM 5-Azacytidine. The number of plants with green kanamycin resistant sectors was reduced 3-fold for TL2/ DL plants but not for TL1/DL plants. It might be anticipated that changes in methylation may affect genomic ‘openness’ and accessibility towards the production of DSBs by I-SceI and subsequent recombination. To test the effect of elevated temperature plantlet at the 4 leaf stage were transferred to a 37°C incubator for 8 hours (night), then a kanamycin solution applied to the plant apex. Such a treatment increased the number of plants with green kanamycin-resistant sectors two to three fold. Since the in-vitro optimum of the I-SceI restriction enzyme is 37°C it is likely that this augmentation of GT is directly caused by increased DSB production. However it cannot be excluded that other factors might contribute to this increase (eg genomic structure: activity of other recombination proteins).
1.3.4 WP4: Combining individual gene effects for HR enhancement
Task 4.1a Enhancing GT by combined gene effects [P5]
Combining DSB-induction and loss of Non-homologous end joining
ZFN-mediated footprints in Ku80 mutant: Cycles of DSB formation and repair will eventually lead to mutations. We have shown this recently by using targeted ZFNs (de Pater et al., 2009). It is likely that the spectrum of mutations will be altered in DNA repair mutants and therefore we are testing several repair mutants for ZFN-mediated footprinting. Crossings were done between plants having the GFP-HPT target site and expressing the corresponding ZFNs with the ku80 NHEJ mutant. In the next generation homozygous mutant plants were obtained. These plants were analysed for footprints in the target site. It seems that more footprints are present in the ku80 mutant than in the wild type (Figure 4.1A). This might mean that in NHEJ mutants DSB repair is less accurate and more often results in footprints. We did not observe differences in the length of deletion in comparison with the wild type (Figure 4.1B). In order to test ZFNs designed for DSB formation of natural target sites, ZFNs targeted to the PPO gene and TALENs targeted to the CRU gene were introduced in an Arabidopsis ku80 mutant (Jia et al 2012) via Agrobacterium-mediated transformation. A mixture of two Agrobacterium strains each one harbouring one of the ZFN or TALEN genes from the pairs were used. Since the transformation frequency of this ku80 mutant is at least 5 times lower than that of the wild type, only few transformants were obtained. These plant lines will be analysed for the formation of footprints and compared to the results of the wild-type (see WP1).
Jia Q, Bundock P, Hooykaas PJJ, de Pater S (2012) Agrobacterium tumefaciens T-DNA integration and gene-targeting in Arabidopsis thaliana non-homologous end-joining mutants. J Botany Doi:10.1155/2012/989272.
de Pater, S., Neuteboom, L. W., Pinas, J. E., Hooykaas, P. J. J. and van der Zaal, B. J. (2009). ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotech. J. 7, 821–835.
Task 4.1b The effect of combined overexpression of the two AtRAD52 nuclear variants on gene-targeting [P2]
In WP2 we showed evidence for enhanced GT upon AtRAD52-1 overexpression. We are in the process of testing the combined effect of AtRAD52-1 and AtRAD52-2 overexpression on GT using lines which were and described in WP2. These lines are crossed and F1 co-expressing both genes will be tested for Gene targeting.
Task 4.2 Enhancing Meiotic HR by combined gene effects [P1, P2, P3]
Rescuing sterility of the RTR complex mutants by overexpression of endonucleases (P1)
Mutants of the A. thaliana RMI1 gene show a sterile phenotype due to defects in the separation of meiotic chromosomes. RMI1 together with RECQ4A and TOP3α form the so-called RTR complex in Arabidopsis thaliana, which has been shown to dissolve Holliday junctions by generating non-crossover products via the dissolution pathway of HR. Mutants of each of the three complex partners therefore were shown to have an increased rate of spontaneous somatic homologous recombination events. Interestingly, mutants of Arabidopsis TOP3α as well as RMI1 are sterile due to recombination-dependent chromosome defects in meiosis I, pointing to an important function of the dissolution pathway in meiotic HR.
Since the dissolution pathway is dependent on a double Holliday junction intermediate, as are hypothesized resolution pathways by endonucleases such as MUS81/EME1A, it should be possible to rescue the somatic and meiotic defects of RTR complex mutants like rmi1 by overexpression of these nucleases.
Beside the A. thaliana MUS81/EME1A complex, a number of other HJ resolvases were cloned and transformed into rmi1 mutant plants: A. thaliana GEN1, S. cerevisiae SLX1/SLX4, E. coli RusA and E. coli RuvABC. All constructs were cloned to be expressed under the control of the A. thaliana DMC1 and RMI2 promoters, both of which show a strong meiotic expression. In the case of the eukaryotic resolvases, each gene was cloned between a separate promoter and terminator. The bacterial resolvases were cloned as fusion proteins containing N-terminal NLS sequences. The E. coli RuvABC complex, however, was cloned to be expressed by a single promoter as a polycistronic mRNA, in which each protein contains an NLS sequence and the proteins being separated by T2A ribosomal stutter sequences leading to production of three proteins from a single mRNA (constructs shown in Figure 4.2).
Figure 4.2: Composition of T-DNAs of the cloned resolvase constructs. All T-DNAs are flanked by left and right border sequences (LB, RB). Proximal to the right border, a PPT resistance cassette is located. Each resolvase construct was cloned under the control of a 35S terminator and either a DMC1 or a RMI2 promoter. The resolvases used are AtGEN1 (from A. thaliana cDNA), AtMUS81/AtEME1A (from A, thaliana cDNA), ScSLX1/ScSLX4 (from S. cerevisiae AH 109 gDNA), EcRusA (from E. coli gDNA) and EcRuvA/EcRuvB/EcRuvC (from E. coli gDNA). The bacterial resolvases EcRusA and EcRuvABC additionally contain a NLS sequence. To express three proteins from a single mRNA, the EcRuvABC genes were cloned under the control of a single promoter and separated by T2A ribosomal skipping sequences.
Following transformation of rmi1-1 heterozygous plants with these constructs, in the T1 generation lines were established that carried the T-DNA using PPT resistance. Of these plants, all were genotyped by PCR for the status of the rmi1-1 mutation. All plants carrying a T-DNA and being homozygous for the rmi1-1 mutation were transferred into pots and grown in the greenhouse until formation of mature siliques. Then, fertility of these plants was quantified by counting the mean number of viable seeds per silique. Surprisingly, no fertile plants could be found with any construct (table 1).
In the course of the experiments, it was questioned whether the promoters used, DMC1 and RMI2, were sufficient for this experiment. The DMC1 protein is expressed in meiosis I, but the expression time might be too early for the required action of a resolvase. Microarray expression data of the RMI2 gene indicate a strong meiotic expression; there has been no data showing any function for the RMI2 protein in meiosis, however. We are in the process of establishing new rmi1-1 plant lines in which the resolvases are expressed under the control of meiosis-specific promoters SPO11-1, MS5 and MTG10.3.
Table 1: Overview of Atrmi1-1 resolvase expression lines. For each resolvase expression line transformed into mutant line rmi1-1 IC9C, numbers of positively selected primary transformants in T1 generation (rmi1-1+/-) are given in column 2. After genotyping, the numbers of resolvase-positive rmi1-1-/- plants that were analysed for their fertility are given in column 3. No transformed line showed a deviation in fertility from the sterile rmi1-1 mutant, irrespective of the resolvase construct used.
Line T1 Atrmi1-1-/- % fertile
rmi1-1 IC9C::(DMC1-P)-GEN1 86 19 0%
rmi1-1 IC9C::(RMI2-P)-GEN1 83 18 0%
rmi1-1 IC9C::(DMC1-P)-MUS81/EME1A 97 23 0%
rmi1-1 IC9C::(RMI2-P)- MUS81/EME1A 63 13 0%
rmi1-1 IC9C::(DMC1-P)-ScSLX1/ScSLX4 172 41 0%
rmi1-1 IC9C::(RMI2-P)-ScSLX1/ScSLX4 174 43 0%
rmi1-1 IC9C::(DMC1-P)-EcRusA 152 37 0%
rmi1-1 IC9C::(RMI2-P)-EcRusA 171 41 0%
rmi1-1 IC9C::(DMC1-P)-EcRuvABC 160 37 0%
rmi1-1 IC9C::(RMI2-P)-EcRuvABC 133 30 0%
Task 4.2b Test for colethality in rad52 and xrcc3 double mutant plants. [P2 and P3]
In the context of TASK 2.1c of Workpackage 2, P2 identified and characterised the plant homologues of RAD52, a mediator of RAD51 loading onto ssDNA ends, thereby initiating homologous recombination. A striking phenotype of rad52 mutants in animal cells, is the co-lethality of the absence of RAD52 protein in mutants of other Rad51 "accessory" proteins, including Xrcc3 (Fujimori 2001). P3 identified the Arabidopsis xrcc3 mutant and is working with it in TASK2.1a. In collaboration with P2, P3 thus constructed double atrad52 xrcc3 Arabidopsis plants to verify whether this colethality also exists in plants. The current status of this work is:Heterozygote xrcc3 +/- plants were crossed to rad52-1-/- and rad52-2-/-.
In the F1:
- xrcc3+/-_rad52-1+/- grew normally and although seeds were not counted, visual inspection revealed no major problems of fertility beyond the slight reduction observed in rad52-1 single mutant plants.
- In contrast, fertility seemed more strongly affected in xrcc3+/-_rad52-2+/-. This phenotype will be confirmed soon in the F2 generation.
In the F2 (progeny of xrcc3+/-_rad52-1/2 +/-)
- Two viable xrcc3-/-_rad52-1-/- plants were identified among the F2, excluding the hypothesis of colethality of the double mutant (14 were rad52-1 -/-; 48 plants genotyped). Notwithstanding, the low proportion of these plants in the F2 population is suggestive and further analyses will be carried out.
- Similarly for rad52-2, among 48 plants tested, 14 were rad52-2 -/- and 2 were xrcc3-/- rad52-2-/-.
- All plants are growing normally, have started bolting and we will soon be able to check the fertility rad52-/- xrcc3-/+ (xrcc3 -/- are sterile). .
- In contrast to the F1, xrcc3+/-_rad52-2+/- look fine and fertility not strongly affected.
Conclusion
These results show clearly that the xrcc3-/- rad52-1-/-, and the xrcc3-/- rad52-2-/- double mutants are viable. Arabidopsis xrcc3-/- rad52 double mutants are not synthetically lethal, in contrast to animals. To verify that this is not die to a redundancy of function between the two RAD51 genes, we have crossed the two double mutants (rad52-1-/- xrcc3+/- and rad52-2-/- xrcc3+/-) to make the triple mutant, xrcc3-/- rad52-1-/- rad52-2-/-. Lethality or viability of the triple mutants should be available by the end of June.
Task 4.2c Combined effect of loss of function of RAD52-1 and RAD52-2 on meiotic recombination [P1, P2]
The loss of function of either AtRAD52-1 or AtRAD52-2 has almost no effect on DNA recombination and repair (see WP2). We hypothesized that this might be due to redundancy in their function. For this purpose we are preparing a double mutant in both genes. However, to simplify the various assays and work with a dominant phenotype we have co-expressed AtRAD52-1 RNAi and AtRAD52-2 RNAi lines. These lines are being analysed by P1 for defects in meiosis and were crossed with our fluorescent seed tester (P2). The effect on meiotic recombination will be scored in the near future.
Potential Impact:
The main objective of RECBREED project was to improve plant breeding by enhancing Homologous Recombination (HR rates) in somatic and in meiotic cells. The expected outcomes of such enhancement were to establish an efficient gene targeting (GT) technology for precise engineering of plant genomes and to increase the rate of meiotic recombination between homologous or homeologous chromosomes. These objectives have been achieved
Through the RecBreed project new tools to induce HR were developed in tomato and maize. These can be used to improve plant breeding of these corps, that have great economical value. But the tools are also applicable to other species.
The project developed “new tools from technologies that support both research & development and the production of industrial prototypes for the breeding of crop plants (excluding forest and fruit tree species) using innovative gene technology breeding methods (transgenics/cisgenics/ intragenics)". Indeed, in the case of gene targeting we were able to obtain by trangenic approach ("transgenics") and in the case of enhancement of meiotic recombination, by a transgenic approach one is in principle able to obtain ("intragenics") that carry no transgenic sequences. Within the funding period, not only "research & development" of both processes of the model plant Arabidopsis was performed but also "industrial prototypes for the breeding of crop plants" tomato and maize could be obtained.
RECBREED project succeeded to set up enabling techniques helping to better exploit plant biodiversity. This project addresses fundamental aspects of homologous recombination. It impacts modern plant breeding and is expected to provide long-term benefits. In addition, topics such as the enhancement of meiotic recombination enable to better exploit natural variation within a species as well as from a very broad range of exotic resources .. By enabling more precise and controlled transformation of plants, our work on gene targeting, can facilitate public acceptance and regulatory aspects of work with transgenics, and thus have long-term impact.
Finally, gene targeting enables to exploit the wealth of “omics” data in various ways, for example, through functional validation via targeted gene knockouts. In addition, precise targeting in the original genomic context is less prone to gene silencing than regular transgenesis techniques, GT will thus facilitate exploitation of metabolic data and the stable and efficient production of novel bioactive molecules for industrial or therapeutic purposes.
Scientific Impact
We established feasible gene targeting techniques, that can be applied directly to cultivated plant species. Moreover, we laid the ground for the improvement of breeding by enhancing meiotic recombination. Therefore we are convinced that the impact is indeed as far reaching as discussed below.
An important aspect of this project is that in the EU, public reluctance to consume GMO products has strongly hindered the implementation of biotechnology progress in agriculture while in other countries (USA, China, Japan) this Agro-biotech revolution is taking place. Gene Targeting technology enables precise and controlled alteration of the genome. Single nucleotide changes can be introduced into genes. Such precise and minimal alterations of the genome could enable companies to make new products that would be acceptable to consumers and thus to integrate the post-genomic era biotechnology revolution into agriculture and health. The controlled and precise modifications made possible by this technology should also significantly facilitate the legal aspects of registration of GMOs. On has to keep in mind that many natural varieties of cultivated plant species were produced by screens after application of mutagens like X-rays. Thus, multiple undirected DSB were introduced and the respective (partly major) genomic changes are in most cased uncharacterized in these culitvars. In contrast, a single DSB targeted to the site of interest by a synthetic nuclease result in an NHEJ event that might kock out an ORF by just one or two nucleotide changes. Obviously, here a reevaluation of regulation procedures in platn breeding is required in the long run .
Enhancement of breeding procedures by raising the frequency of homologous or homeologous recombination will facilitate the creation of new cultivars via standard, non-transgenic breeding. Such plants by definition do not differ from classical non-transgenic cultivars. They therefore can be used in the same way in agriculture as non-transgenic cultivars - no GMO specific issues will arise at all.
The industrial partners involved in this project are well aware of these aspects and obtained new tools and new IP through the RECBREED project. The breeding sector is a significant source of income in Europe. Seed sales by European breeders in 2006/2007 represented 4000 M€ or 37% of total worldwide seed sales. New emergent markets such as in Asia will ensure that this market continues to expand together with the continuing adoption of hybrid seed technology, for example in rice and perhaps in the future for wheat. Enhancing meiotic HR or GT provides an edge to European companies and benefits also for farmers who might be provided more rapidly with products with novel traits. It is of strategic importance for Europe that the European Breeding sector remains strong and can provide seed adapted to European conditions and needs.
There are several innovative aspects in this proposal. Enhancing recombination in a controlled way will be used to optimise the traditional breeding process so that diversity can be obtained from smaller breeding populations and so that rare recombination events can be obtained; an especially important aspect will be in rapidly introgressing genetic material from diverse sources (including distant relatives) which requires removal undesirable linked alleles/genes. Thus effort in breeding will be reduced and new varieties with potentially desirable social and environmental attributes will be created and delivered rapidly to the market. It is important to note that plant breeders devote considerable efforts in reducing the time to market of new varieties since the first to the market with an improved variety dominates the market. For example techniques such as the use of counter-seasons to obtain two or more breeding cycles per year and more recently the use of dihaploids to rapidly genetically fix varieties are widely used.
At the technological level, we hope to turn GT into a routine tool in plants. As discussed above, this technology has a very high potential economic impact in the Agro-biotech industry and also the pharma industry via “molecular farming” for protein production in plants. We will test several genes, working at different levels of the DNA recombination process (initiation, invasion, strand exchange, heteroduplex formation and resolution). As a result of this work, the Arabidopsis model has the potential to become a paradigm to study GT in other organisms with naturally low GT rates, such as mammalian species. Yeast, where the most advanced research is done in the field of homologous recombination, is naturally hyper-recombinogenic and is clearly not always a good model for GT in animals and plants. These innovations should result both in scientific publications and in patents.
European added value
One reason to carry this work at the European level was that the research in DNA recombination in plants is far more advanced in Europe than in the USA.. This is expressed by the number of quality publications, the number of research groups involved and the support of granting agencies. The RECBREED project brought together leading groups in the field of DNA recombination in plants and thus further increased the competitiveness of the EU in this field. The GT and improved breeding techniques might have a great impact on implementing the Agro-biotech revolution and the EU is in an excellent position to harvest the fruits from this revolution. Moreover the EU has a special urgent need for precise DNA technologies that are acceptable to the broad public rather than the random type of alterations currently practiced.
Each partner contributed with specific skills and know-how to the project. The research of the consortium was successful due to thet fact that it was a collaborative effort, at the EU level, rather than efforts undertaken separately by individual labs .
At the international level, USA companies (e.g. Dupont or Dow chemical or Monsanto) have in-house researchers carrying research in the field of DNA recombination and precise transgenesis (including ZFN-based technologies and site-specific integration). RECBREED succeeded in connecting leading EU scientists with the leading EU-industry in plant biotech. We are convinved that the project helps to reinforce the position of the EU in this competitive field.
Economical impact
During the last decade the plant breeding industry has changed significantly. Consumer demands on fresh and good quality vegetable products are increasing continuously and therefore the need to look closer into complex traits is required. By investing in new technologies Europe has been able to continuously grow its excellent world economic position in the breeding industry.
The breeding industries (seed companies) are the driving forces in the agricultural production chain and Europe plays a very prominent role in seed business. The international seed trade is a rapidly growing economic activity, and the global turnover of seed exports has doubled from 2000 million euro to 4000 million euro in the years 1990-2007. Four of the five top seed exporting countries in the world are European member states, i.e. France, the Netherlands, Germany and Denmark, with the annual turnover of seed exports by the two biggest exporters, The Netherlands and France, being about 550 million euro each, only slightly lower than that of the USA.
Specifically, in the vegetable breeding industry The Netherlands is by far the number one export country in the world. Over 70% of the world professional vegetable breeders use seeds directly derived from the Dutch breeding companies. Annual vegetable seed market sales are estimated at 2.5 billion Euro, of which the European companies together represent more than 50%, emphasizing the strong position of Europe in this industry.
Impact
Out of the top ten vegetable seed companies, eight have their roots in Europe and five of them are collaborating with KeyGene on their innovative research programs. We expect that in the long run that they will be able to benefit by applying the techniques set up with RECBREED to different crop plants of interest
List of Websites:
Project website address: http://www.recbreed.eu/
Project Coordinator:
Prof. Holger Puchta,
Karlsruhe Institute of Technology (KIT – UNIKARL)
Tel: +49 721 608-48894, Fax: +49 721 608-44874
E-mail: holger.puchta@bio.uka.de;