Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella.

KEY MESSAGE: In Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency. Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA-DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p < 0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.

Differential requirements for RAD51 in Physcomitrella patens and Arabidopsis thaliana development and DNA damage repair.

RAD51, the eukaryotic homolog of the bacterial RecA recombinase, plays a central role in homologous recombination (HR) in yeast and animals. Loss of RAD51 function causes lethality in vertebrates but not in other animals or in the flowering plant Arabidopsis thaliana, suggesting that RAD51 is vital for highly developed organisms but not for others. Here, we found that loss of RAD51 function in the moss Physcomitrella patens, a plant of less complexity, caused a significant vegetative phenotype, indicating an important function for RAD51 in this organism. Moreover, loss of RAD51 caused marked hypersensitivity to the double-strand break-inducing agent bleomycin in P. patens but not in Arabidopsis. Therefore, HR is used for somatic DNA damage repair in P. patens but not in Arabidopsis. These data imply fundamental differences in the use of recombination pathways between plants. Moreover, these data demonstrate that the importance of RAD51 for viability is independent of taxonomic position or complexity of an organism. The involvement of HR in DNA damage repair in the slowly evolving species P. patens but not in fast-evolving Arabidopsis suggests that the choice of the recombination pathway is related to the speed of evolution in plants.

Phytotoxicity and innate immune responses induced by Nep1-like proteins.

We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)-like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses.

The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.

The model bryophyte Physcomitrella patens exhibits high frequencies of gene targeting when transformed with DNA constructs containing sequences homologous with genomic loci. 'Targeted gene replacement' (TGR) resulting from homologous recombination (HR) between each end of a targeting construct and the targeted locus occurs when either single or multiple targeting vectors are delivered. In the latter instance simultaneous, multiple, independent integration of different transgenes occurs at the targeted loci. In both single gene and 'batch' transformations, DNA can also be found to undergo 'targeted insertion' (TI), integrating at one end of the targeted locus by HR with one flanking sequence of the vector accompanied by an apparent non-homologous end-joining (NHEJ) event at the other. Untargeted integration at nonhomologous sites also occurs, but at a lower frequency. Molecular analysis of TI at a single locus shows that this occurs as a consequence of concatenation of the transforming DNA, in planta, prior to integration, followed by HR between a single site in the genomic target and two of its repeated homologues in the concatenated vector. This reinforces the view that HR is the major pathway by which transforming DNA is integrated in Physcomitrella.

An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.

The moss Physcomitrella patens is the only land plant known to date with highly efficient homologous recombination in its nuclear DNA, making it a unique model for plant functional genomics approaches. For high-throughput production of knockout plants, a robust transformation system based on polyethylene glycol-mediated transfection of protoplasts was developed and optimised. Both the DNA conformation and pre-culture of plants used for protoplast isolation significantly affected transformation efficiencies. Employing a newly developed PCR high-throughput method, the gene-targeting efficiency in more than 1000 plants transformed with different cDNA-based knockout constructs was determined and analysed with regard to the length and intron/exon structure of the homologous gene locus. Different targeting constructs, each containing an identical selectable marker gene, were applied as batch DNA in a single transformation experiment and resulted in double-knockout plants. Thus, the fast and efficient generation of multiple targeted gene-knockouts is now feasible in Physcomitrella.

Effects of nutrients, cell density and culture techniques on protoplast regeneration and early protonema development in a moss, Physcomitrella patens.

To regenerate auxotrophic mutants of Physcomitrella patens, two media of increasing complexity were developed. The survival rate of protoplasts was around 30% higher on full medium when compared to standard minimal medium. Protoplast survival was higher in a medium containing 2.5 mmol/L ammonium tartrate compared to a medium with 5 mmol/L of this compound. Solid medium had a positive effect on protoplast survival compared to either liquid medium or solid medium overlaid with cellophane; the maximum survival rate being 31.6%. However, the number of surviving protoplasts without any cell division during the first ten days increased on solid medium. Density and survival rate of protoplasts were positively correlated, but the formation of long protonema filaments decreased markedly. The effect of different protoplast densities could be explained partly by physiologically active compounds excreted into the medium.

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

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.

Functional knockout of the adenosine 5'-phosphosulfate reductase gene in Physcomitrella patens revives an old route of sulfate assimilation.

The reduction of adenosine 5'-phosphosulfate (APS) to sulfite catalyzed by adenosine 5'-phosphosulfate reductase is considered to be the key step of sulfate assimilation in higher plants. However, analogous to enteric bacteria, an alternative pathway of sulfate reduction via phosphoadenosine 5'-phosphosulfate (PAPS) was proposed. To date, the presence of the corresponding enzyme, PAPS reductase, could be neither confirmed nor excluded in plants. To find possible alternative routes of sulfate assimilation we disrupted the adenosine 5'-phosphosulfate reductase single copy gene in Physcomitrella patens by homologous recombination. This resulted in complete loss of the correct transcript and enzymatic activity. Surprisingly, the knockout plants grew on sulfate as the sole sulfur source, and the concentration of thiols in the knockouts did not differ from the wild type plants. However, when exposed to a sublethal concentration of cadmium, the knockouts were more sensitive than wild type plants. When fed [(35)S]sulfate, the knockouts incorporated (35)S in thiols; the flux through sulfate reduction was approximately 50% lower than in the wild type plants. PAPS reductase activity could not be measured with thioredoxin as reductant, but a cDNA and a gene coding for this enzyme were detected in P. patens. The moss Physcomitrella patens is thus the first plant species wherein PAPS reductase was confirmed on the molecular level and also the first organism wherein both APS- and PAPS-dependent sulfate assimilation co-exist.