Double-Strand Break (DSB) Induction

The goal of this part of the project is to develop techniques to specifically enhance homologous recombination at the initiation stage by controlled induction of targeted DNA breaks (DSB). This approach will be applied for gene targeting stimulation and also to enhance meiotic recombination.

 

GT by DSB induction with Zn-finger nucleases

Transgenes are integrated at random positions in the plant genome by non-homologous recombination. As this may lead to position effects and mutation there is an urgent need for methods by which genes can be targeted to specific positions in the genome. This can be achieved by homologous recombination. In plants, however, the frequency of integration by homologous recombination (gene targeting) is much lower than that by non-homologous recombination. Gene targeting (GT) is enhanced when double strand breaks (DSBs) are produced at the target site in the genome. Within the framework of RECBREED zinc finger nucleases (ZFN) are used to produce targeted DSBs in order to obtain targeted mutagenesis and enhance gene targeting.

 

Induction of meiotic recombination by Spo11-1/2 overexpression

The highly conserved SPO11 protein mediates the initiation of homologous recombination by DSB formation in meiosis. Plants harbor three distantly related homologues of SPO11. The AtSpo11-1 and AtSpo11-2 proteins form an endonuclease complex required for DSB induction, Via overexpression meiotic recombination should be enhanced.

 

Isolation of a promoter for expression of genes in maize, at the “HR-initiation stage”

In order to be able to express the most promising HR-related genes in maize promoters that drive meiosis-specific or preferred expression are needed.

 

Main significant results for the first 12 months

• Increased GT in A. thaliana by DSB induction via ZFNs.
• A tomato line was selected with high protoplast plating and regeneration efficiency for use in GT experiments.
• Bioinformatic analysis resulted in the choice of several ZFN target sites for which ZFN proteins are currently prepared.
• Plant lines were established expressing both Spo11-1 and Spo11-2 from either the Spo11-1 promoter or the Ubiquitin promoter.
• Rice homologs of three A. thaliana meiosis-specific genes were identified and their promoter regions cloned in front of reporter genes.

 

Homology search and heteroduplex formation

The Homology search concerns the use of expression of key recombination proteins to modulate the initial interaction between recombining homologous DNA sequences. This workpackage consists of three tasks: homology search, chromatin effect and enhancement of recombination in the crop plant, Maize.

First the research is concentrating on Rad51 protein family, Brca2 and Rad52 proteins. During the first 12 months construction and testing of the expression constructs was performed.

Wild-type and the respective mutant plants with these constructs were achieved, thus confirming the functionality of the fusion proteins by complementation of their corresponding mutant lines (figure 1).

 

Figure 1 – Confirmation of functionality of the “own promoter” fusion

proteins by complementation of the corresponding mutant lines

 

Secondly the goal is to assess whether alternation in epigenetic properties of chromatin influence frequencies of meiotic crossovers.
Finally a GT assay in maize has been developed. This assay is based on the repair of a defective nptII gene giving kanamycin-resistant cells. This assay is being used to test whether overexpression of putative GT stimulating proteins can further enhance GT frequencies.

 

Main significant results for the first 12 months

• Use of expression of key recombination proteins in order to modulate the initial interaction between recombining homologous DNA sequences. Constructions and validation of expression constructs for the Rad51 family proteins and identification and analysis of the Arabidopsis Rad52 orthologs was performed.
• Establishment of lines and the first encouraging tests of the effects of methylation status on meiotic recombination rates.
• A fully satisfactory progress has been made with the validation of the maize GT assay and initiation of transformation experiments for testing the effects of overexpression of the ISce1 and Rad54 genes on GT.

 

Promoting HR via stabilization and resolution of recombination intermediates

For both gene targeting and meiotic recombination, synapsed DNA recombination structures (heteroduplex DNA), formed through the action of Rad51 and other proteins, must be processed and resolved for completing the recombination process. The research here, aims at studying the proteins that contribute to the stabilization and resolution of recombination intermediate heteroduplex structures, such as Holiday junctions or Holliday-like junctions. In a second step, the goal is to determine whether manipulation of these proteins can contribute to enhance HR in Arabidopsis and in, tomato and maize .

 

Main significant results for the first 12 months

• All expression constructs were clone, sequenced and subsequently transformed in Arabidopsis plants. Several homozygous expression lines have been established .
• Fluorescently-tagged translational fusion constructs for the Arabidopsis XPF and the ERCC1 proteins have been built and transformed into wild-type and mutant plants. Constructs expressing the fusion proteins by their own promoters or by the constitutive MAS promoter have been built. The functionality of the fusion proteins has been confirmed and analysis of the temporal and spatial expression patterns of the tagged-fusion proteins have started.

 

Potential impact

Through understanding genetic recombination processes in plants, and the ability to modulate them, it is expected that this project will impact both conventional plant breeding and also result in a practicable gene targeting technique for the controlled modification of important plants. Changing climates and increasing populations worldwide make both of these points of great importance for agriculture. Furthermore, public reluctance in the EU concerning GMO products has strongly hindered the implementation of biotechnology in agriculture, while in other countries (USA, China, Japan) this Agro-biotech revolution is taking place. The controlled and precise modifications made possible by Gene Targeting technology offer a way forward and the knowledge and material developed in the RECBREED project should make a real contribution to this.