A tool for understanding homologous recombination in plants

Abstract

Attempts for establishing an efficient gene targeting (GT) system in seed plants have hitherto not been successful. In contrast, GT based on homologous recombination is highly efficient in Physcomitrella, making this moss a novel tool in reverse genetics. However, why homologous and illegitimate recombination are differently regulated between Physcomitrella and seed plants is still enigmatic. Here we update the state of the art of GT in Physcomitrella and discuss approaches to unravel this enigma. Identification of molecular factors significantly enhancing GT and their subsequent transfer to crop plants will have a great impact on plant biotechnology by enabling precise genetic engineering. Physcomitrella appears to be the most useful model system in this context.

Principles of Targeted Mutagenesis in the Moss Physcomitrella Patens

“…(3) Linearisation of replacement vectors at the edges of the targeting sequences are required for optimal targeting frequencies. These values are comparable to those reported in recent independent gene disruption studies (Hohe and Reski, 2003). Such a targeting efficiency can only be compared with that observed in budding yeast and signifies that Physcomitrella ranks number one for the efficiency of GT among multicellular eukaryotes (Schaefer, 2001).…”

“…RTF observed in these experiments raised up to 1 in 1000 regenerating colonies and targeted transformation was associated with the insertion of direct tandem repeats of the vector. This work opened the way to the direct generation of knock-outs in Physcomitrella (for recent reviews see Puchta, 1998;Schaefer and Zrÿd, 2001;Puchta, 2002;Schaefer, 2002;Hohe and Reski, 2003).…”

“…The current model proposes that the ratio of targeted to random integration upon transgenesis correlates with the major pathway of double strand break (DSB) repair used by the cells: most DSB are repaired by a non-homologous end joining pathway in plants and animal cells whereas yeast repairs DSB essentially by a homologous recombination pathway. Yet the factors that determine this ratio are currently poorly understood and have been discussed extensively in several reviews (Mengiste and Paszkowski, 1999;Paques and Haber, 1999;Puchta, 2002;Hohe and Reski, 2003); this topic will not be discussed here. Mouse embryonic stem (ES) cells provide the only model system in animal biology proficient for GT approaches and this accounts for its exponential development over the last fifteen years (Müller, 1999).…”

Comparative analysis of the SBP-box gene families in P. patens and seed plants

“…As most important difference to flowering plant molecular genetic models, P. patens offers the possibility of efficient gene targeting via homologous recombination (reviewed by Schaefer, 2002) as well as the possibility to make double or triple gene knockouts in one step (Hohe and Reski, 2003). Moreover, P. patens can fully complete its life cycle when grown in vitro and is thus easily accessible for manipulation in different environmental growth conditions.…”

Expression of Bacillus thuringiensis insecticidal protein gene in transgenic oil palm

“…It is assumed that it was at this time that cells are most vulnerable to the invasion of foreign DNA. Illegitimate recombination between the vulnerable DNA and transgenes may have resulted in stable integration (Tzfira and Citovsky, 2002;Hohe and Reski, 2003). Therefore, factors that induce rapid cell division could play major role in ensuring higher transformation events.…”