High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library

BMC Plant Biology 2BM2M0e0C2tP,hlaontdBoiollooggyy article x High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library Tanja Egener 2 Jos Granado 2 Marie-Christine Guitton 2 Annette Hohe 2 Hauke Holtorf 2 Jan M Lucht 2 Stefan A Rensing - 2 Katja Schlink 2 Julia Schulte 2 Gabriele Schween 2 Susanne Zimmermann 2 Elke Duwenig elke.duwenig@basf- 1 Bodo Rak 0 Ralf Reski 2 0 Institute of Biology III, Freiburg University , Schanzlestrasse 1, D-79104 Freiburg/Br. , Germany 1 BASF Plant Science GmbH , D-67056 Ludwigshafen , Germany 2 Plant Biotechnology, Freiburg University , Sonnenstrasse 5, D-79104 Freiburg/Br. , Germany Background: The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant. Results: As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants ( = 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics. Conclusions: The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis. - Background The most informative approach to identify a function for a given gene is the precise inactivation or functional alteration of the gene, followed by the analysis of the phenotypic change resulting from this manipulation. Gene targeting based on homologous recombination between a targeting construct with altered or abolished gene function and its cognate endogenous gene has been a highly successful approach for gene function analysis in prokaryotes, lower eukaryotes, and mice. Unfortunately, in higher plants this approach is restricted by the very low ratio of 10-3 to 10-5 targeted relative to illegitimate recombination events. Although a few homologous recombination events between incoming targeting constructs and their cognate genomic sequences have been described, homologous recombination remains very inefficient and gene targeting thus is not routinely possible in higher plants [1,2]. In contrast, gene targeting via homologous recombination occurs with high frequency in the moss Physcomitrella patens[3,4]. After the first demonstration of high-frequency recombination between chromosomal sequences and homologous DNA introduced by transformation [5], gene targeting in Physcomitrella was used successfully to study the function of several genes by creating functional knockouts [69]. The high specificity provided by homologous recombination even allows the specific targeting of single members of multi-gene families [10]. The ease of its genetic manipulation, together with a high degree of conservation of morphological features, biochemical pathways and signal transduction mechanisms between Physcomitrella patens and higher plants [1114] has made the moss an important model system for plant functional genomics. To facilitate a large-scale study of plant gene function using Physcomitrella patens as a model organism, we are developing a collection of Physcomitrella plants with insertion mutations that affect a wide variety of developmental, morphological and physiological characteristics. Transformation with constructs carrying sequences homologous to the genome typically results in 10-fold higher transformation frequencies then the use of non-homologous constructs, and among these transformants a high proportion shows integration of the construct at the homologous genomic locus [3,12]. We argued that compared to a random mutagenesis strategy [15] targeting insertion mutations towards expressed genes would increase the proportion of transformants displaying altered properties, and would decrease the total number of transformants to be screened to find a particular change in phenotype. We therefore developed an efficient transposon-based shuttle mutagenesis system for moss cDNA libraries, and have used pools of insertionmutagenised cDNA clones tagged with a nos-regulated nptII selection cassette for the transformation of Physcomitrella plants (Fig. 1). construction of a cDNA library transposon mutagenesis of the cDNA library transformation of Physcomitrella with the gene disruption cDNA library collection of targeted Physcomitrella mutants Results and Discussion cDNA library To establish a Physcomitrella cDNA library representing most genes expressed during vegetative growth before the onset of differentiation, RNA was extracted from protonemata cultured for different time periods in liquid culture, and a cDNA library in plasmid vectors was established after normalization to decrease redundancy [16]. Mass DNA sequencing and clustering of 57,000 EST sequences yielded 12,000 non-overlapping sequence clusters, and showed a low degree of clone redundancy in the cDNA library used. Sequence analysis of these contigs, together with a large number of additional EST sequences derived from other growth stages and tissues, suggest that the total number of coding sequences for the moss Physcomitrella patens and the flowering plant Arabidopsis thaliana is similar (Rensing et al., submitted), despite a three-fold larger genome size for the moss [12]. Gene-disruption library To create a gene-disruption library of cDNA clones carrying insertion mutations, cDNA clones were subjected to shuttle mutagenesis in E. coli. First, the normalised cDNA pool from the amplified protonema library was subcloned into the minimal vector pUCMinIV (Fig. 2). This plasmid is a 1.7 kb derivative of pUC19 from which we have deleted most non-essential DNA sequences to remove insertion targets within the vector sequence. Pooled minimal vectors with target cDNAs were introduced into a donor E. coli strain carrying an (...truncated)