David J. Cove

Stable transformation of the moss Physcomitrella patens

SummaryWe report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.

Mosses as model systems

Mosses hold many attractions as model organisms for research in plant science. Their position as the simplest of land plants makes them central to the study of plant evolution, particularly in shedding light on how their aquatic predecessors evolved to survive on land. The use of mosses for developmental studies hinges on the ability to observe development in living material at the level of the individual cell. However, more recently techniques for the molecular analysis of mosses have provided tools for new approaches for determining the mechanisms controlling plant development, incorporating both cell and molecular biology.

Molecular Responses to Abscisic Acid and Stress Are Conserved between Moss and Cereals.

Promoter elements from the wheat Em gene have been characterized. These elements are inducible by abscisic acid (ABA) and by osmotic stress. In this study, we demonstrated that the same promoter elements function in a distantly related plant species, the moss Physcomitrella patens. Transient and stable expression of the [beta]-glucuronidase reporter gene was used to determine that the heterologous wheat promoter also responds to osmotic stress and ABA in moss. Mutational analysis of the promoter indicated that the mechanism of gene regulation is conserved in both species. Gel retardation and DNase I footprint analyses were conducted to characterize further the interaction of moss transcription factors with the Em promoter. In addition, the synthesis of stress-related polypeptides in moss was observed. The evolutionary significance of these data and the potential for studying the entire ABA perception-response pathway in moss are discussed.

The Moss Physcomitrella patens, a Model System with Potential for the Study of Plant Reproduction.

The moss Physcomitrella patens has been established as a model system for the study of plant development using a combination of physiological, genetic, and molecular techniques and has been shown to be particularly suitable for the study of morphogenesis at the cellular level. Genetic and molecular techniques devised to study development include mutant isolation and analysis, somatic hybridization of sexually sterile strains, and genetic transformation. Developmental studies of Physcomitrella have so far concentrated on the early stages of gametophyte growth following spore germination or tissue regeneration. Almost no work has been done in this species on the processes involved in sexual reproduction, but it is likely that these processes would be amenable to study and that the techniques that have been devised for studying other developmental processes will also be applicable to the study of sexual reproduction. In this article, we briefly review the current state of knowledge of how development is regulated in this species, the techniques available for developmental genetic studies, and the limited amount of work that has already been done that is relevant to sexual reproduction. A recent review containing background material is given by Cove

Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens

In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates predominantly by homologous recombination with its genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). Overall homology of only 1 kb is sufficient to achieve a 50% yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement following two homologous recombination events. Allele replacement frequency depends on the symmetry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the transforming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single insertions at targeted loci (targeted gene replacements, ‘TGR’) occur with a frequency of 7–20% of all transformants when the minimum requirements for allele replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants.

A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens

Abstract The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time.

Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of the wild-type.

Primary chloronemata growing from germinated spores of the moss Physcomitrella patens adopt one of two preferred polarotropic orientations depending on the wavelength and photon fluence rate of monochromatic light. Growth is mainly parallel to the electrical vector of plane polarised light in blue light and higher fluence rates of red light, and perpendicular to the electrical vector in the green and far-red regions of the spectrum and in low fluence rates of red light. The transition between the two polarotropic orientations, at wavelengths where it can be observed, usually occurs over a narrow range of fluence rates, and at this point the filaments do not grow randomly but tend to adopt in approximately equal numbers one of the preferred directions of growth. The primary chloronemata are positively phototropic in far-red light and in red light of low fluence rates, but tend to grow at right angles to the incident light in high fluence rates of red light. Simultaneous illumination with a high fluence rate of red light and a low fluence rate of far-red light causes a marked increase in the percentage of filaments growing towards the red light source at the expense of those growing at right angles to it, supporting the hypothesis that in red and far-red light, at least, the responses are controlled by the photoequilibrium of a phytochrome pool.

Isopentenyladenine from mutants of the moss, Physcomitrella patens

Abstract The culture media from gametophore over-producing mutants of the moss Physcomitrella patens have been examined for their cytokinin content. Two cytokinins have been detected, one of which has been identified as N6-(Δ2-isopentenyl) adenine (2iP).

The moss Physcomitrella patens: a novel model system for plant development and genomic studies.

The moss Physcomitrella patens has been used as an experimental organism for more than 80 years. Within the last 15 years, its use as a model to explore plant functions has increased enormously. The ability to use gene targeting and RNA interference methods to study gene function, the availability of many tools for comparative and functional genomics (including a sequenced and assembled genome, physical and genetic maps, and >250,000 expressed sequence tags), and a dominant haploid phase that allows direct forward genetic analysis have all led to a surge of new activity. P. patens can be easily cultured and spends the majority of its life cycle in the haploid state, allowing the application of experimental techniques similar to those used in microbes and yeast. Its development is relatively simple, and it generates only a few tissues that contain a limited number of cell types. Although mosses lack vascular tissue, true roots/stems/leaves, and flowers and seeds, many signaling pathways found in angiosperms are intact in moss. For example, the phytohormones auxin, cytokinin, and abscisic acid, as well as the photomorphogenic pigments phytochrome and cryptochrome, are all interwoven into distinct but overlapping pathways and linked to clear developmental phenotypes. In addition, about one quarter of the moss genome contains genes with no known function based on sequence motifs, raising the likelihood of successful discovery efforts to identify new and novel gene functions. The methods outlined in this chapter will enhance the use of the P. patens model system in many laboratories throughout the world. David J. Cove, Pierre-François Perroud, Audra J. Charron, Stuart F. McDaniel, Abha Khandelwal, and Ralph S. Quatrano Department of Biology, Washington University, St. Louis, Missouri 63130 P R O TO CO L S 1 Culturing the Moss Physcomitrella patens, 75 2 Isolation and Regeneration of Protoplasts, 80 3 Somatic Hybridization in P. patens Using PEGinduced Protoplast Fusion, 82 4 Chemical and UV Mutagenesis of Spores and Protonemal Tissue, 84 5 Transformation Using Direct DNA Uptake by Protoplasts, 87 6 Transformation Using T-DNA Mutagenesis, 89 7 Transformation of Gametophytes Using a Biolistic Projectile Delivery System, 91 8 Isolation of DNA, RNA, and Protein from P. patens Gametophytes, 93 This chapter, with full-color images, can be found online at www.cshprotocols.org/emo.

Genetic analysis of the effects of re-transformation of transgenic lines of the mossPhyscomitrella patens

Genetic analysis of the progeny of crosses involving strains of the mossPhyscomitrella patens obtained by re-transforming a stable transgenic line, indicates that the plasmid used for re-transformation inserts at or near the chromosomal location of the related plasmid used to obtain the original transgenic line. The resulting structure may be subject to gene silencing.