High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library
© Egener et al; licensee BioMed Central Ltd. 2002
Received: 12 April 2002
Accepted: 18 July 2002
Published: 18 July 2002
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.
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.
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.
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 , gene targeting in Physcomitrella was used successfully to study the function of several genes by creating functional knockouts [6–9]. The high specificity provided by homologous recombination even allows the specific targeting of single members of multi-gene families .
Results and Discussion
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 . 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 .
Pools of plasmid DNA prepared from transposon-mutagenised cDNAs were used for large-scale PEG-mediated transformation of moss protoplasts grown in semi-continuous bioreactor cultures [6, 18, 19]. Before transformation, the plasmid DNA was linearised by digestion with a rare-cutting restriction enzyme, SdaI, that cuts in the minimal vector just outside of the cDNA inserts (Fig. 2). Regeneration of protoplasts was done on supplemented Knop medium for 2 weeks without selection, followed by two cycles of G418 selection to eliminate unstable transformants . More than 98% of surviving plants were stable transformants carrying the nptII selection cassette used for the cDNA disruption, as demonstrated by PCR-detection of the nptII coding sequence or a third selection step . The cellular DNA content of all transformants was checked by flow cytometry ; 7.7% of the transformants tested (1242 of 16203) were polyploid. Currently, we have produced more than 22,000 moss transformants; the current production capacity is about 3,000 new transformants per month. This will allow us to establish a large collection of Physcomitrella plants transformed with gene-disruption library constructs; our aim is to obtain a collection of plants carrying mutations in the majority of expressed moss genes.
Phenotypic characterisation of 16,203 gene-disruption library transformants
percentage of deviating plants1
Deviations from wild-type for any morphological characteristic
- Plant structure (less compact than wild-type, fluffy)
- Color (darker or lighter than wild-type)
- Coverage of plant by gametophores (coverage less than 95%; wild-type: completely covered)
- Leaf shape (width/length ratio or symmetry different from wild type)
- Cell shape (irregular patterns, or cell size different than wild-type)
- Uniformity of leaves (more than 10% deviating on one plant, wild-type: all uniform)
Taken together, the collection of Physcomitrella transformants that we are establishing by a cDNA based gene-disruption library transformation approach shows a high degree of phenotypic diversity. The fraction of plants with altered morphological or developmental features – 16.2% – exceeds that which has been previously described for a shuttle mutagenesis strategy using genomic clones of moss DNA (3.9%) . This difference might be caused by preferential, homologous recombination-driven disruption of expressed moss genes after the cDNA mutagenesis approach. The frequency of altered phenotypes in our collection of moss transformants also is considerably higher than the less than 2% reported for confirmed Arabidopsis knockout mutants , which might be attributed to a lower degree of gene redundancy in Physcomitrella (Rensing et al., submitted). The small number of genomic loci tagged by insertion of gene-disruption library constructs in plants from our mutant collection should allow the recovery of insertion sites and the analysis of moss genes affected in transformants with altered phenotypes. Indeed, we have re-isolated genomic sequences surrounding nptII- insertion sites e. g. by PCR-based methods. The presence of introns in some of these sequences suggests that these were derived from the genomic target locus, into which transgene sequences had inserted by homologous recombination (data not shown). We therefore expect that the collection of moss mutants being established here will help to identify novel genes previously unknown in plants [7, 12] and will allow to rapidly link DNA sequence and functional information. Given the high degree of genetic and physiological conservation between moss and higher plants, this collection of gene disruption library transformants will be a valuable tool not only for gene function studies in the moss Physcomitrella, but for plant functional genomics in general.
Materials and Methods
Plasmid constructions and microbiological techniques followed standard procedures [24, 25], details have been described in patent WO 01/38509 and can be obtained under http://ep.espacenet.com from the European Patent Office or upon request from us. Briefly, Physcomitrella patens cDNA libraries subcloned into the minimalised vector pUCMinIV (encoding ampicillin resistance) were transformed into the donor E. coli strain R2117. This strain carries a plasmid-encoded IPTG-inducible transposase gene (tnpA), and a conjugative plasmid comprising the transfer region from plasmid R388 , a chloramphenicol resistance marker and a nos-promoter-nptII -nos terminator expression cassette flanked by the transposon Tn1000 border repeat sequences required for transposition . The nptII cassette was derived from pBIN19 . To increase the efficiency of subsequent plant selection, a point mutation present in the nptII coding sequence  was reverted to the wild-type sequence. Ampicillin-resistant R2117 donor strain transformants with moss cDNA clones were pooled, treated with IPTG to induce transposase activity, and cocultivated with R1037 recipient cells in the presence of IPTG. E. coli strain R1037 carries an IPTG-inducible tnpR resolvase gene and a streptomycin resistance locus, both encoded on the chromosome. Tn1000 transposition in the donor results in the formation of cointegrates between conjugative plasmid and moss cDNA clones, which then can be transferred by conjugation into the recipient where resolution occurs. Recipient cells with mutagenised moss cDNA clones were selected by their simultaneous resistance against ampicillin and streptomycin, and plasmid DNA prepared from pooled cells was used then to transform P. patens.
Plant growth conditions, media and transformation
Physcomitrella patens (Hedw.) B.S.G. was cultured in liquid or on solid modified Knop medium as described . For protoplast isolation, protonema was grown in semi-continuous bioreactor cultures supplemented with 2.5 mM ammonium tartrate [18, 19]. Transformations  were performed with 3 × 105 cells and 50 μg of linearised plasmid DNA. After transformation, plants were grown on Knop medium supplemented with MS-microelements , 4 mg/l myo-inositol, 2.8 mg/l choline chloride, 1 mg/l nicotinic acid, 0.5 mg/l thiamine-HCl, 0.25 mg/l pyridoxine, 0.01 mg/l biotin, 0.25 mg/l p-aminobenzoic acid, 1.9 mg/l Ca-D-pantothenate, 0.015 mg/l riboflavine, 6.76 mg/l adenine, 3.84 mg/l Na-palmitinic acid, 250 mg/l peptone, 920 mg/l ammoniumtartrate and 50 g/l glucose, to facilitate survival of metabolic mutants. Stable transformants were identified by a first selection step on 25 μg/ml G418 for 2 weeks, a non-selective release step of 2 weeks, and further 2 weeks of G418 selection.
The presence of transgenes in G418 resistant moss transformants was confirmed by PCR with nptII specific primers, or a third selection step as described . For Southern blot analyses, 1 μg of genomic DNA isolated by a modified CTAB method  was digested for 5–6 hours with 20 U of restriction enzymes purchased from MBI Fermentas or New England Biolabs. After electrophoresis on 0,7% agarose gels the DNA was transferred onto positively charged nylon membrane (Roche). Fragments carrying nptII -sequences were detected using hybridisation and blocking solutions as well as Anti-digoxigenin-AP conjugate from Roche but CDP-Star from Promega. The DIG-labelled nptII probe was generated by PCR using the random primed labelling mix from Roche and Taq polymerase from Promega.
Availability of materials and mutant plants
Materials, single plant lines described in this communication, as well as sequence information for Physcomitrella EST clones with similarity to defined genes of interest will be available for non-profit research after completion of an appropriate material transfer agreement with BASF Plant Science GmbH and Freiburg University. Also, inquiries for sequence information for Physcomitrella EST clones with similarity to defined genes of interest are encouraged. Requests should be directed to the corresponding author (RR).
We thank Prof. Josef Honerkamp (Freiburg) for helpful discussions about data analysis, and Dr. Fritz Thümmler, vertis Biotechnologie (Bergmoos, Germany) for cloning of cDNA libraries. The expert assistance of our technical staff is greatly appreciated. This is a joint project between Freiburg University and BASF Plant Science GmbH.
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