Homologous recombination in plants

Homologous recombination (HR) in plants is of interest both as a tool to perform gene targeting and also HR in a general sense remains a fascinating, complex and poorly understood biological process. In many instances it is desirable to abolish the expression of a gene of interest in order to determine its function. Disadvantages of existing methods are for example, that anti-sense will not lead to a complete loss of function of a given gene.

Gene-tagging using Agrobacterium T-DNA plasmid or transposition requires a huge amount of work before the mutant of interest become available. These methods are currently restricted to few species and to a narrow range of gene disruptions. In fact specific gene modifications cannot be achieved by these methods. In contrast gene targeting (GT) offers a wide range of choices.

Applications of this powerful technique in fundamental and applied studies are numerous. GT involves the direct replacement of an endogenous gene by an in vitro mutated copy involving a mechanism based on HR or gene conversion. HR is very efficient in prokaryotes and yeast while illegitimate recombination is predominant in eukaryotes.

In mammalian systems HR is improved mainly by using embryonic stem cells and negative selection against illegitimate recombination events. Thus, GT has been developed as a routine tool in the past years and has contributed much to the recent success in the understanding the function of some genes in these organisms. Although it has been shown by several laboratories that gene targeting is, in principle, feasible in plants its application is severely hampered by the very low frequencies (10-4 to 10-6) and the complicated nature of molecular events, which have been obtained up to now.

The aim of this work is to capitalise on a number of recent scientific advances to elucidate the mechanism of HR both in somatic and meiotic cells and to develop an effective gene targeting system for plants. In order to optimise the conditions for true GT events, multiple approaches have been combined.

  • Over-expression of recombinases in tobacco
    One approach to make plant cells competent for HR is to modify the expression of the genes involved in this pathway. Multiple proteins are expressed in tobacco plants containing a transgene as a substrate for intrachromosomal recombination.
  • Gene targeting vectors
    The design of GT vectors is essential for successful GT. Vectors containing markers for positive and negative selections have been constructed.
  • Double-strand DNA breaks (DSB)
    Artificially induced DSBs dramatically increase the rate of HR in both mammalian and plant cells. Recently, the induction of DSBs has successfully been used to enable or improve true gene targeting in animal systems. Chimeric nucleases (CN) have been developed to potentially target DSBs to any chromosomal gene, priming them for GT. These recombinant nucleases carry a non-specific DNA cleavage domain (from the restriction enzyme FokI) linked to a precise DNA-binding domain (the DNA recognition domain from zinc finger proteins).

    The ability to control the recognition specificity of the zinc fingers offers the possibility of directing cleavage to specific chromosomal locations. CNs have dramatically increased GT efficiency in human cells by 1000-fold. If such an improvement could be achieved in a plant system it would radically increase the chances of developing a routine GT method. However, developing zinc fingers with a binding specificity for a specific target gene takes considerable time and effort and some nucleases can prove toxic, presumably due to cleavage of non-target sites. A novel idea to overcome some of these problems is currently tested in our laboratory.

  • DNA delivery
    The method of choice is direct gene delivery using particle bombardment.