Abstracts of Talks

s of Talks T1: Large-scale functional genomics of Physcomitrella patens Jan M. Lucht , Tanja Egener , José Granado , Marie-Christine Guitton , Annette Hohe , Hauke Holtorf , Stefan A. Rensing , Katja Schlink , Julia Schulte , Gabriele Schween , Susanne Zimmermann , Elke Duwenig , Bodo Rak , and Ralf Reski 1 1 Plant Biotechnology, Freiburg University, Sonnenstrasse 5, D-79104 Freiburg/Br., Germany 2 BASF Plant Science GmbH, D-67056 Ludwigshafen, Germany 3 Institute of Biology III, Freiburg University, Schänzlestrasse 1, D-79104 Freiburg/Br., Germany. Contact email: [email protected] 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. We have developed a high-throughput system for generating a large number (40,000 to date) of Physcomitrella mutants via two distinct approaches, based on PEG-mediated transformation of protoplasts. The forward route uses transformation with a gene disruption library produced by transposon mutagenesis of a large number of anonymous cDNA clones; it is expected to yield a saturated Physcomitrella mutant collection with insertion mutations in most expressed genes. The reverse route uses knockout DNA constructs specific for defined candidate genes. Quality control with respect to stable integration of the nptII marker gene and ploidy level is performed for each transformant. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Our data show that among the first set of 16,000 transformants, 98% have integrated the nptII cassette into the genome. Phenotypic screens revealed that on average 16% of the transformants differed from the wild-type in a variety of developmental, morphological and physiological characteristics, suggesting a highly efficient mutagenesis. MOSS2002 Talk Abstracts page 16 T2: Comparison of the Physcomitrella gametophyte transcriptome to the Arabidopsis Genome Tomoaki Nishiyama , Tomomichi Fujita , Tadasu Shin-I, Motoaki Seki , Hiroyo Nishide , Ikuo Uchiyama , Asako Kamiya , Piero Carninci , Yoshihide Hayashizaki , Kazuo Shinozaki , Yuji Kohara , and Mitsuyasu Hasebe 1 1 National Institute for Basic Biology, Okazaki 444-8585, Japan. 2 National Institute of Genetics, Mishima, Japan. 3 RIKEN, Wako, Japan. 4 RIKEN Genomic Sciences Center, Yokohama, Japan. contact email: [email protected] The moss, Physcomitrella patens, diverged early in land plant evolution from the vascular plant lineage. To understand the change of genomic content during land plant evolution, genome-wide similarity of Physcomitrella and Arabidopsis was assessed by comparing expressed sequence tag (EST) data of Physcomitrella and sequence data of all the protein in Arabidopsis. We constructed full-length enriched cDNA libraries from, auxin-treated, cytokinin-treated, and non-treated gametophytes of Physcomitrella, and sequenced more than 40,000 clones from both ends. The ESTs are assembled with mRNA sequences of Physcomitrella deposited in GenBank, and 23,000 contigs were obtained. Contigs corresponding to the 5’ and 3’ end of the same clone were paired. Based on these pairs, our clones correspond to 13,000 independent mRNA. We constructed a database of Physcomitrella EST, containing the BLASTX search result of every contigs and clones. Currently, 15 Mb of Physcomitrella non-redundunt EST can be searched with BLAST program in our database. TBLASTN searches against the Physcomitrella database showed that 65% of Arabidopsis genes has a homologue in Physcomitrella with an E value less than or equal to 0.001. Conversely, all the moss contigs (23,000) were used as query in BLASTX searches against nr dataset. Of these, 10,000 showed the highest similarity to a vascular plant gene, and 12,000 found no hit with an E value ≤ 0.001. The remaining 822 contigs had the highest similarity to a gene in an organism other than vascular plants, such as Metazoa and Bacteria, and included genes related to ion transport, DNA damage repair, which may reflect moss specific features. As gametophytes are extremely reduced in Angiosperms, some of the genes functioned in gametophytes may have lost, while most continued to function in sporophytes. MOSS2002 Talk Abstracts page 17 The Physcomitrella EST programme (PEP) Celia Knight , David Cove , Ralph Quatrano 2 and Andrew Cuming 1 1 Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, England 2 Department of Biology, Campus Box 1137, 1 Brookings Drive, Washington University, St. Louis, Mo 63130-4899, USA Contact email: [email protected] The 3 year BBSRC-funded programme, to sequence Physcomitrella ESTs and to offer a service to the plant community for functional analysis of higher plant genes by gene targeting in moss, will complete this year. The achievements of the programme will be discussed; these include linkage of PEP to the NIBB EST collection, resulting in a total of around 60,000 ESTs being available in the public domain; added value to the PEP programme, by construction of a BAC library, by David Lightfoot and colleagues at SIU, Illinois), which is now available for library screening as filter grids (http://www.moss.leeds.ac.uk) and progress towards a phenotypic screen service for identifying altered phenotypes. This talk will draw attention to posters detailing work emanating from PEP resources to be presented at this meeting i.e. Lomas et al. on bioinformatic analysis (poster P10); Panvisavas et al. on analysis of Rho-GTPase genes in Physcomitrella (poster P15); Deeks et al. on analysis of the mago-nashi gene in Physcomitrella (poster P3). The Centre for Plant Sciences at Leeds is one of the largest group of plant scientists in the UK (http://www.plants.leeds.ac.uk). Research specialisms at Leeds are allowing a unique combination of expertise to be linked through PEP and applied to fundamental plant functional genomic analysis. We are focussing on tip growth and incorporating approaches looking at roles in development for the cell wall (see talk by Lee et al., T21); intracellular organelles, initially through peroxisomes in collaboration with Dr Alison Baker and extracellular stimuli, initially through light responses involving GATA transcription factors in collaboration with Prof. Phil Gilmartin. MOSS2002 Talk Abstracts page 18 T4: Molecular analysis of tagged Physcomitrella patens mutants Tanja Egener, José Granado, Hauke Holtorf, Jan M. Lucht, Stefan A. Rensing, Katja Schlink and Ralf Reski University of Freiburg, Plant Biotechnology, Sonnenstr. 5, D-79104 Freiburg, Germany Contact e-mail: [email protected] (www.plant-biotech.net) Functional genomics relies on efficient and fast re-isolation of tagged genomic loci. For Physcomitrella patens three protocols (iPCR, RAGIL, and Trap-cloning) have been established and optimised for the analysis on integration mutants. iPCR is based on restriction and circular religation of genomic DNA, followed by PCR amplification of DNA regions flanking the marker gene. RAGIL, the Rapid Amplification of Genomic Integration Loci is a derivative of RACE protocols. While iPCR and RAGIL rely on PCR-techniques, Trap-cloning is a plasmid rescue approach to clone and screen for marker gene flanking DNA sequences. All protocols have been tested and applied to targeted knockout mutants of Physcomitrella and mutants from our mutant collection and all proved to be applicable for mutant analysis in Physcomitrella. Best results have been obtained by Trap-cloning that has been used successfully to re-isolate large flanking sequences (up to 9kb). To date we re-isolated 21 genomic integration loci, homologous and non-homologous. Mutant analysis of tagged mutants is faster than in Arabidopsis and in contrast to this model plant a gene/function correlation can be rapidly confirmed in Physcomitrella by targeted knockout. This work is a joint project with BASF Plant Science GmbH. MOSS2002 Talk Abstracts page 19 T5: Efficiency of gene targeting in Physcomitrella patens. I. Factors affecting gene targeting efficiency by direct DNA transfer. Y. Kamisugi, A.C. Cuming, C.D. Knight and D.J. Cove Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK Contact e-mail: [email protected] The high efficiency of somatic homologous recombination in Physcomitrella patens is now a widely appreciated phenomenon that is increasingly being exploited as a tool for gene functional analysis. However, the factors affecting the frequency of gene targeting events have not been systematically investigated. These factors include the nature of targeting constructs, the extent of homology between targeted locus and targeting construct, the location and orientation of selectable marker genes within targeting constructs and the means of DNA delivery. As a part of the EC Framework V ‘Pregene’ programme, we are systematically analysing these factors. We have selected two members of small multigene families in P. patens as targets to determine the conditions made by these factors to the efficiency of gene targeting. These are i) a member of the RNA-binding protein ‘Puf-domain’ gene family and ii) a member of the gene family encoding the small rho-1-like GTP binding (rop) protein. By introducing an nptII selectable marker cassette into different site within genomic clones of these genes, we have created a series of targeting constructs of varying homology length and 3’/5’ symmetry. By analysing the nature of the integration events in several hundred stable transformants we are beginning to define, quantitatively, the relationship between homology length, symmetry and targeting efficiency in P. patens. This work is a part of EC framework V, PREGENE project. MOSS2002 Talk Abstracts page 20 T6: A long standing partnership? – W-boxes and their WRKY companions