A new system for fast and quantitative analysis of heterologous gene expression in plants.

• Large-scale analysis of transcription factor-cis-acting element interactions in plants, or the dissection of complex transcriptional regulatory mechanisms, requires rapid, robust and reliable systems for the quantification of gene expression. • Here, we describe a new system for transient expression analysis of transcription factors, which takes advantage of the fast and easy production and transfection of Physcomitrella patens protoplasts, coupled to flow cytometry quantification of a fluorescent protein (green fluorescent protein). Two small-sized and high-copy Gateway® vectors were specifically designed, although standard binary vectors can also be employed. • As a proof of concept, the regulation of BANYULS (BAN), a key structural gene involved in proanthocyanidin biosynthesis in Arabidopsis thaliana seeds, was used. In P. patens, BAN expression is activated by a complex composed of three proteins (TT2/AtMYB123, TT8/bHLH042 and TTG1), and is inhibited by MYBL2, a transcriptional repressor, as in Arabidopsis. Using this approach, two new regulatory sequences that are necessary and sufficient for specific BAN expression in proanthocyanidin-accumulating cells were identified. • This one hybrid-like plant system was successfully employed to quantitatively assess the transcriptional activity of four regulatory proteins, and to identify their target recognition sites on the BAN promoter.

RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens.

Gene targeting (GT) is a major tool for basic and applied research during which the transforming DNA, which shares sequence homology with a chromosomal target, integrates at the corresponding locus by homologous recombination (HR). In eukaryotes, GT recruits enzymes from the HR-mediated double strand break repair pathway. Different mechanisms of HR have been described which depend on the Rad52 epistasis group of genes, but which specific mechanism is used by the cell for GT remains unclear. In Saccharomyces cerevisiae, the RAD52 protein is essential for GT, and the RAD51 protein plays a minor role. In filamentous fungi and animal cells, however, GT depends on RAD51 and is weakly affected by suppression of RAD52. Genetic evidence also indicates that the non-homologous end-joining pathway of DSB repair has a negative impact on GT efficiencies, but how the balance between these two pathways is controlled is poorly understood. Here, we have examined the role of RAD51 in the only plant that exhibits high GT frequencies, the model bryophyte Physcomitrella patens. Our results show that the two RAD51 proteins have partially redundant functions in the maintenance of genome integrity and resistance to ionizing radiation. Furthermore, we demonstrate that loss of function of the two RAD51 proteins completely abolishes GT and strongly increases illegitimate integration rates in this moss. These findings demonstrate for the first time in plant the critical role of RAD51 in controlling the balance between targeted and random integration events observed upon transgenesis, and confirm that P. patens is a particularly interesting tool for studying GT in higher eukaryotes.

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency.

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.