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Isaac N. Nuñez, Tamara F. Matute, Ilenne Del Valle, Anton Kan, Atri Choksi, Drew Endy, Jim Haseloff, Timothy Rudge, and Fernan Federici, Artificial symmetry-breaking for morphogenetic engineering bacterial colonies. ACS Synthetic Biology. DOI: 10.1021/acssynbio.6b00149 • Publication Date (Web): 30 Oct 2016
Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial co-localization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.
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Grant PK, Dalchau N, Brown JR, Federici F, Rudge TJ, Yordanov B, Patange O, Phillips A, Haseloff J. Orthogonal intercellular signaling for programmed spatial behavior. Molecular Systems Biology 12:849-861, (2016).
Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum-sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell-cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3-oxo-C6- and 3-oxo-C12-homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two-channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long-range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.
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Boehm CR, Ueda M, Nishimura Y, Shikanai T, Haseloff J. A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha. Plant Cell Physiol. 57:291-9. (2016)
Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity.
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Bowman JL, Araki T, Arteaga-Vazquez MA, Berger F, Dolan L, Haseloff J, Ishizaki K, Kyozuka J, Lin SS, Nagasaki H, Nakagami H, Nakajima K, Nakamura Y, Ohashi-Ito K, Sawa S, Shimamura M, Solano R, Tsukaya H, Ueda T, Watanabe Y, Yamato KT, Zachgo S, Kohchi T. The Naming of Names: Guidelines for Gene Nomenclature in Marchantia. Plant Cell Physiol. 57:257-61, (2016).
While Marchantia polymorpha has been utilized as a model system to investigate fundamental biological questions for over almost two centuries, there is renewed interest in M. polymorpha as a model genetic organism in the genomics era. Here we outline community guidelines for M. polymorpha gene and transgene nomenclature, and we anticipate that these guidelines will promote consistency and reduce both redundancy and confusion in the scientific literature.
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Rudge T, Brown J, Federici F, Dalchau N, Phillips A, Ajioka J, & Haseloff J. Characterization of intrinsic properties of promoters. ACS Synthetic Biology Jan 5:89-98. (2016)
Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic contexts of the measurement. Furthermore, in vivo measurement methods must accommodate variation in translation, protein folding, and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered to be extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents, and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method to quantitatively describe promoter properties.
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Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, Raitskin O, Leveau A, Farré G, Rogers C, Smith A, Hibberd J, Webb AA, Locke J, Schornack S, Ajioka J, Baulcombe DC, Zipfel C, Kamoun S, Jones JD, Kuhn H, Robatzek S, Van Esse HP, Sanders D, Oldroyd G, Martin C, Field R, O'Connor S, Fox S, Wulff B, Miller B, Breakspear A, Radhakrishnan G, Delaux PM, Loqué D, Granell A, Tissier A, Shih P, Brutnell TP, Quick WP, Rischer H, Fraser PD, Aharoni A, Raines C, South PF, Ané JM, Hamberger BR, Langdale J, Stougaard J, Bouwmeester H, Udvardi M, Murray JA, Ntoukakis V, Schäfer P, Denby K, Edwards KJ, Osbourn A, Haseloff J. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist 208:13-9. (2015).
Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.
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Yordanov B, Dalchau N, Grant PK, Pedersen M, Emmott S, Haseloff J, & Phillips A. A Computational Method for Automated Characterization of Genetic Components. ACS Synthetic Biology 3:578-88 (2014).
The ability to design and construct synthetic biological systems with predictable behavior could enable significant advances in medical treatment, agricultural sustainability, and bioenergy production. However, to reach a stage where such systems can be reliably designed from biological components, integrated experimental and computational techniques that enable robust component characterization are needed. In this paper we present a computational method for the automated characterization of genetic components. Our method exploits a recently developed multichannel experimental protocol and integrates bacterial growth modeling, Bayesian parameter estimation, and model selection, together with data processing steps that are amenable to automation. We implement the method within the Genetic Engineering of Cells modeling and design environment, which enables both characterization and design to be integrated within a common software framework. To demonstrate the application of the method, we quantitatively characterize a synthetic receiver device that responds to the 3-oxohexanoyl-homoserine lactone signal, across a range of experimental conditions.
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Rudge TJ, Steiner PJ, Kan A and Haseloff J. Cell shape-driven instability generates self-organised, fractal patterning of cell layers. ACS Synthetic Biology, 2:705-714, (2013).
As a model system to study physical interactions in multicellular systems, we used layers of Escherichia coli cells, which exhibit little or no intrinsic coordination of growth. This system effectively isolates the effects of cell shape, growth, and division on spatial self-organization. Tracking the development of fluorescence-labeled cellular domains, we observed the emergence of striking fractal patterns with jagged, self-similar shapes. We then used a large-scale, cellular biophysical model to show that local instabilities due to polar cell-shape, repeatedly propagated by uniaxial growth and division, are responsible for generating this fractal geometry. Confirming this result, a mutant of E. coli with spherical shape forms smooth, nonfractal cellular domains. These results demonstrate that even populations of relatively simple bacterial cells can possess emergent properties due to purely physical interactions. Therefore, accurate physico-genetic models of cell growth will be essential for the design and understanding of genetically programmed multicellular systems.
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Federici F, Rudge TJ, Pollak B, Haseloff J, Gutiérrez RA. Synthetic Biology: opportunities for Chilean bioindustry and education. Biological Research 46:383-93, (2013).
In an age of pressing challenges for sustainable production of energy and food, the new field of Synthetic Biology has emerged as a promising approach to engineer biological systems. Synthetic Biology is formulating the design principles to engineer affordable, scalable, predictable and robust functions in biological systems. In addition to efficient transfer of evolved traits from one organism to another, Synthetic Biology offers a new and radical approach to bottom-up engineering of sensors, actuators, dynamical controllers and the biological chassis they are embedded in. Because it abstracts much of the mechanistic details underlying biological component behavior, Synthetic Biology methods and resources can be readily used by interdisciplinary teams to tackle complex problems. In addition, the advent of robust new methods for the assembly of large genetic circuits enables teaching Biology and Bioengineering in a learning-by-making fashion for diverse backgrounds at the graduate, undergraduate and high school levels. Synthetic Biology offers unique opportunities to empower interdisciplinary training, research and industrial development in Chile for a technology that promises a significant role in this century's economy.
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Rudge TJ, Steiner PJ, Phillips A and Haseloff J. Computational modeling of synthetic microbial biofilms. ACS Synthetic Biology, 1:345-352, (2012).
Microbial biofilms are complex, self-organized communities of bacteria, which employ physiological cooperation and spatial organization to increase both their metabolic efficiency and their resistance to changes in their local environment. These properties make biofilms an attractive target for engineering, particularly for the production of chemicals such as pharmaceutical ingredients or biofuels, with the potential to significantly improve yields and lower maintenance costs. Biofilms are also a major cause of persistent infection, and a better understanding of their organization could lead to new strategies for their disruption. Despite this potential, the design of synthetic biofilms remains a major challenge, due to the complex interplay between transcriptional regulation, intercellular signaling, and cell biophysics. Computational modeling could help to address this challenge by predicting the behavior of synthetic biofilms prior to their construction; however, multiscale modeling has so far not been achieved for realistic cell numbers. This paper presents a computational method for modeling synthetic microbial biofilms, which combines three-dimensional biophysical models of individual cells with models of genetic regulation and intercellular signaling. The method is implemented as a software tool (CellModeller), which uses parallel Graphics Processing Unit architectures to scale to more than 30,000 cells, typical of a 100 μm diameter colony, in 30 min of computation time.
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Wenzel, C., Marrison, J., Mattsson, J. Haseloff, J. and Bougourd, S.M. Ectopic divisions in vascular and ground tissues of Arabidopsis thaliana result in distinct leaf venation. J. Exp. Botany 63:5351-5364, (2012).
Leaf venation patterns vary considerably between species and between leaves within a species. A mechanism based on canalization of auxin transport has been suggested as the means by which plastic yet organized venation patterns are generated. This study assessed the plasticity of Arabidopsis thaliana leaf venation in response to ectopic ground or procambial cell divisions and auxin transport inhibition (ATI). Ectopic ground cell divisions resulted in vascular fragments between major veins, whereas ectopic procambial cell divisions resulted in additional, abnormal vessels along major veins, with more severely perturbed lines forming incomplete secondary and higher-order venation. These responses imply limited vascular plasticity in response to unscheduled cell divisions. Surprisingly, a combination of ectopic ground cell divisions and ATI resulted in massive vascular overgrowth. It is hypothesized that the vascular overproduction in auxin transport-inhibited wild-type leaves is limited by simultaneous differentiation of ground cells into mesophyll cells. Ectopic ground cell divisions may negate this effect by providing undifferentiated ground cells that respond to accumulated auxin by differentiation into vascular cells.
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French C, de Mora K, Joshi N, Elfick A, Haseloff J and Ajioka J. Development of biosensors for the detection of arsenic in drinking water. Arsenic in the Environment. Volume 5, series eds. J. Bundschuh and P. Bhattacharya, Taylor & Francis, London, UK. ISBN 978-0-415-69719-4 (2012)
The term “biosensor” refers to a wide variety of devices. The common ele- ment is that a biological component provides highly specific recognition of a certain target analyte, and this detection event is somehow transduced to give an easily detectable, quantifiable response, preferably one that can be easily con- verted to an electrical signal so that the result can be fed to an electronic device for signal processing, data storage, etc. The biological component in many bio- sensors is either an enzyme, as in the glucose-oxidase–based biosensors used for blood glucose monitoring, or an antibody, as in most optical biosensors. Another class of biosensor, sometimes also referred to as a bioreporter, uses living cells as a component. These cells detect the target analyte via some more-or-less specific receptor and generate a detectable response, most commonly by induction of a reporter gene. Many such devices have been reported in the scientific literature, with detection of mercury and arsenic in the environment being particularly common applications. However, very few such devices are commercially avail- able, the best-known examples being the mutagen-detecting devices such as the SOS-Chromotest (Environmental Bio-Detection Products, Inc.) system. Here we discuss the reasons for this gap between promise and delivery, and ways in which the emerging discipline of synthetic biology may lead to a new generation of whole-cell biosensors.
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Federici F, Dupuy L, Laplaze L, Heisler M & Haseloff J. Integrated genetic and computation methods for in planta cytometry. Nature Methods, 9:483-485 (2012).
We present the coupled use of specifically localized fluorescent gene markers and image processing for automated quantitative analysis of cell growth and genetic activity across living plant tissues. We used fluorescent protein markers to identify cells, create seeds and boundaries for the automatic segmentation of cell geometries and ratiometrically measure gene expression cell by cell in Arabidopsis thaliana. In planta cytometry has several benefits. First, the combined use of specific gene markers and active contour segmentation methods allowed robust segmentation and fitting of cellular models to biological features such as nuclei and plasma membranes. Second, active contour methods can be tailored for image analysis of features at a cellular scale, whereas watershed algorithm–based procedures require processing of an entire image. Third, the use of nuclear fluorescent markers also permitted implementation of nuclear ratiometric measurements, enabling quantitative analysis of the relative contribution of multiple genes to cellular processes and correction for variability in marker fluorescence. The use of normalized units of measurement has aided the standardization and description of genes for design of predictable gene circuits in isolated cells and microbes. Our work combines computational techniques and specific cellular markers to facilitate automatic identification and quantitative characterization of cells.
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Aquea F, Federici F, Moscoso C, Vega A, Jullian P, Haseloff J, Arce-Johnson P. A molecular framework for the inhibition of Arabidopsis root growth in response to boron toxicity. Plant Cell Environ. 35:719-734 (2011).
Boron is an essential micronutrient for plants and is taken up in the form of boric acid (BA). Despite this, a high BA concentration is toxic for the plants, inhibiting root growth and is thus a significant problem in semi-arid areas in the world. In this work, we report the molecular basis for the inhibition of root growth caused by boron. We show that application of BA reduces the size of root meristems, correlating with the inhibition of root growth. The decrease in meristem size is caused by a reduction of cell division. Mitotic cell number significantly decreases and the expression level of key core cell cycle regulators is modulated. The modulation of the cell cycle does not appear to act through cytokinin and auxin signalling. A global expression analysis reveals that boron toxicity induces the expression of genes related with abscisic acid (ABA) signalling, ABA response and cell wall modifications, and represses genes that code for water transporters. These results suggest that boron toxicity produces a reduction of water and BA uptake, triggering a hydric stress response that produces root growth inhibition.
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Maizel A, von Wangenheim D, Federici F, Haseloff J, Stelzer EH. High resolution, live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy. Plant Journal 68:377-385 (2011).
Most plant growth occurs post-embryonically and is characterized by the constant and iterative formation of new organs. Non-invasive time-resolved imaging of intact, fully functional organisms allows studies of the dynamics involved in shaping complex organisms. Conventional and confocal fluorescence microscopy suffer from limitations when whole living organisms are imaged at single-cell resolution. We applied light sheet-based fluorescence microscopy to overcome these limitations and study the dynamics of plant growth. We designed a special imaging chamber in which the plant is maintained vertically under controlled illumination with its leaves in the air and its root in the medium. We show that minimally invasive, multi-color, three-dimensional imaging of live Arabidopsis thaliana samples can be achieved at organ, cellular and subcellular scales over periods of time ranging from seconds to days with minimal damage to the sample. We illustrate the capabilities of the method by recording the growth of primary root tips and lateral root primordia over several hours. This allowed us to quantify the contribution of cell elongation to the early morphogenesis of lateral root primordia and uncover the diurnal growth rhythm of lateral roots. We demonstrate the applicability of our approach at varying spatial and temporal scales by following the division of plant cells as well as the movement of single endosomes in live growing root samples. This multi-dimensional approach will have an important impact on plant developmental and cell biology and paves the way to a truly quantitative description of growth processes at several scales.
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Dupuy, L., Mackenzie, J. and Haseloff, J. Coordination of plant cell division and expansion in a simple morphogenetic system. Proc. Natl. Acad. Sci. USA 107:2711-6 (2010).
Morphogenesis in plants arises from the interplay of genetic and physical interactions within a growing network of cells. The physical aspects of cell proliferation and differentiation are genetically regulated, but constrained by mechanical interactions between the cells. Higher plant tissues consist of an elaborate three-dimensional matrix of active cytoplasm and extracellular matrix, where it is difficult to obtain direct measurements of geometry or cell interactions. To properly understand the workings of plant morphogenesis, it is necessary to have biological systems that allow simple and direct observation of these processes. We have adopted a highly simplified plant system to investigate how cell proliferation and expansion is coordinated during morphogenesis. Coleocheate scutata is a microscopic fresh-water green alga with simple anatomical features that allow for accurate quantification of morphogenetic processes. Image analysis techniques were used to extract precise models for cell geometry and physical parameters for growth. This allowed construction of a deformable finite element model for growth of the whole organism, which incorporated cell biophysical properties, viscous expansion of cell walls, and rules for regulation of cell behavior. The study showed that a simple set of autonomous, cell-based rules are sufficient to account for the morphological and dynamic properties of Coleochaete growth. A variety of morphogenetic behavior emerged from the application of these local rules. Cell shape sensing is sufficient to explain the patterns of cell division during growth. This simplifying principle is likely to have application in modeling and design for engineering of higher plant tissues.
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Ubeda-Tomás S, Federici F, Casimiro I, Beemster GTS, Bhalerao R, Swarup R, Doerner P, Haseloff J and Bennett MJ. Gibberellin signalling in the endodermis controls Arabidopsis root meristem size. Current Biology 19:1194-9 (2009).
Plant growth is driven by cell proliferation and elongation. The hormone gibberellin (GA) regulates Arabidopsis root growth by controlling cell elongation, but it is currently unknown whether GA also controls root cell proliferation. Here we show that GA biosynthetic mutants are unable to increase their cell production rate and meristem size after germination. GA signals the degradation of the DELLA growth repressor proteins GAI and RGA, promoting root cell production. Targeting the expression of gai (a non-GA-degradable mutant form of GAI) in the root meristem disrupts cell proliferation. Moreover, expressing gai in dividing endodermal cells was sufficient to block root meristem enlargement. We report a novel function for GA regulating cell proliferation where this signal acts by removing DELLA in a subset of, rather than all, meristem cells. We suggest that the GA-regulated rate of expansion of dividing endodermal cells dictates the equivalent rate in other root tissues. Cells must double in size prior to dividing but cannot do so independently, because they are physically restrained by adjacent tissues with which they share cell walls. Our study highlights the importance of probing regulatory mechanisms linking molecular- and cellular-scale processes with tissue and organ growth responses.
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Møller IS, Gilliham M, Jhab D, Mayo GM, Roy SJ, Coates JC, Haseloff J and Mark Tester, M. Shoot Na+ exclusion and increased salinity tolerance engineered by cell type-specific alteration of Na+ transport in Arabidopsis. Plant Cell 21:2163-78 (2009).
Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants.
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Haseloff J and Ajioka J, Synthetic biology: history, challenges and prospects. Royal Society Interface 4:S389-91 (2009).
Synthetic biology is an emerging field that seeks to employ engineering principles to reprogramme living systems. Biological systems are characterized by highly complex genetic and cellular networks that are locked together by dynamic, parallel and nonlinear feedback interactions that give rise to properties of self-organization, repair and reproduction. These evolved systems pose formidable challenges to rational engineering approaches. Yet, they are capable of assembling functional structures that are many orders of magnitude more complex than the most sophisticated man-made artefacts, and they do this in a renewable fashion, and cheaply.
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Gardner MJ, Baker AJ, Assie JM, Poethig RS, Haseloff JP, Webb AA. GAL4 GFP enhancer trap lines for analysis of stomatal guard cell development and gene expression. J Exp Bot. 60:213-26 (2009).
To facilitate the monitoring of guard cells during development and isolation, a population of 704 GAL4 GFP enhancer trap lines was screened and four single insert lines with guard cell GFP expression and one with developmentally-regulated guard cell GFP expression were identified. The location of the T-DNA inserts, the expression of the flanking genes, and the promoter activity of the genomic DNA upstream of the T-DNA were characterized. The results indicated that the GFP expression pattern in at least one of the lines was due to elements in the intergenic DNA immediately upstream of the T-DNA, rather than due to the activity of the promoters of genes flanking the insert, and provide evidence for the involvement of Dof elements in regulating guard cell gene expression. It is shown further that the GAL4 GFP lines can be used to track the contribution of guard cell material in vitro, and this method was used to assess the purity of guard cell samples obtained using two methods of guard cell isolation.
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Willemsen V, Bauch M, Bennett T, Campilho A, Wolkenfelt H, Xu J, Haseloff J, Scheres B. The NAC domain transcription factors FEZ and SOMBRERO control the orientation of cell division plane in Arabidopsis root stem cells. Developmental Cell. 15:913-22 (2008).
Because plant cells do not migrate, cell division planes are crucial determinants of plant cellular architecture. In Arabidopsis roots, stringent control of cell divisions leads to a virtually invariant division pattern, including those that create new tissue layers. However, the mechanisms that control oriented cell divisions are hitherto poorly understood. Here, we reveal one such mechanism in which FEZ and SOMBRERO (SMB), two plant-specific NAC-domain transcription factors, control the delicately tuned reorientation and timing of cell division in a subset of stem cells. FEZ is expressed in root cap stem cells, where it promotes periclinal, root cap-forming cell divisions. In contrast, SMB negatively regulates FEZ activity, repressing stem cell-like divisions in the root cap daughter cells. FEZ becomes expressed in predivision stem cells, induces oriented cell division, and activates expression of its negative regulator, SMB, thus generating a feedback loop for controlled switches in cell division plane.
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Dupuy L, Mackenzie J, Rudge T, Haseloff J. A system for modelling cell-cell interactions during plant morphogenesis. Annals of Botany 101:1255-1265 (2008).
During the development of multicellular organisms, cells are capable of interacting with each other through a range of biological and physical mechanisms. A description of these networks of cell-cell interactions is essential for an understanding of how cellular activity is co-ordinated in regionalized functional entities such as tissues or organs. The difficulty of experimenting on living tissues has been a major limitation to describing such systems, and computer modelling appears particularly helpful to characterize the behaviour of multicellular systems. The experimental difficulties inherent to the multitude of parallel interactions that underlie cellular morphogenesis have led to the need for computer models. A new generic model of plant cellular morphogenesis is described that expresses interactions amongst cellular entities explicitly: the plant is described as a multi-scale structure, and interactions between distinct entities is established through a topological neighbourhood. Tissues are represented as 2D biphasic systems where the cell wall responds to turgor pressure through a viscous yielding of the cell wall. This principle was used in the development of the CellModeller software, a generic tool dedicated to the analysis and modelling of plant morphogenesis. The system was applied to three contrasting study cases illustrating genetic, hormonal and mechanical factors involved in plant morphogenesis. Plant morphogenesis is fundamentally a cellular process and the CellModeller software, through its underlying generic model, provides an advanced research tool to analyse coupled physical and biological morphogenetic mechanisms.
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Truernit E, Haseloff J. Arabidopsis thaliana outer ovule integument morphogenesis: ectopic expression of KNAT1 reveals a compensation mechanism. BMC Plant Biol. 14;8:35 (2008).
The Arabidopsis outer ovule integument is a simple two-cell layered structure that grows around the developing embryo and develops into the outer layer of the seed coat. As one of the functions of the seed coat is the protection of the plant embryo, the outer ovule integument is an example for a plant organ whose morphogenesis has to be precisely regulated. To better characterise outer ovule integument morphogenesis, we have isolated some marker lines that show GFP expression in this organ. We have used those lines to identify distinct cell types in the outer integument and to demonstrate similarities between leaves and the outer integument. Using confocal microscopy, we showed that cell sizes and shapes differ between the two cell layers of the outer integument. Expression of KNAT1 in the integuments leads to extra cell divisions specifically in the outer layer of the outer integument. This is being compensated for by a decrease of cell volume in this layer, thus showing that mechanisms exist to control proper ovule integument morphogenesis. The Arabidopsis outer ovule integument can be used as a good model system to study the basic principles of plant organ morphogenesis. This work provides new insights into its development and opens new possibilities for the identification of factors involved in the regulation of cell division and elongation during plant organ growth.
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Truernit E, Haseloff J. A simple way to identify non-viable cells within living plant tissue using confocal microscopy. Plant Methods. 23;4:15 (2008).
Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP) fluorescence was possible. The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy.
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Parizot B, Laplaze L, Ricaud L, Boucheron-Dubuisson E, Bayle V, Bonke M, De Smet I, Poethig SR, Helariutta Y, Haseloff J, Chriqui D, Beeckman T, Nussaume L. Diarch symmetry of the vascular bundle in Arabidopsis root encompasses the pericycle and is reflected in distich lateral root initiation. Plant Physiol. 146:140-148 (2008).
The outer tissues of dicotyledonous plant roots (i.e. epidermis, cortex, and endodermis) are clearly organized in distinct concentric layers in contrast to the diarch to polyarch vascular tissues of the central stele. Up to now, the outermost layer of the stele, the pericycle, has always been regarded, in accordance with the outer tissue layers, as one uniform concentric layer. However, considering its lateral root-forming competence, the pericycle is composed of two different cell types, with one subset of cells being associated with the xylem, showing strong competence to initiate cell division, whereas another group of cells, associated with the phloem, appears to remain quiescent. Here, we established, using detailed microscopy and specific Arabidopsis thaliana reporter lines, the existence of two distinct pericycle cell types. Analysis of two enhancer trap reporter lines further suggests that the specification between these two subsets takes place early during development, in relation with the determination of the vascular tissues. A genetic screen resulted in the isolation of mutants perturbed in pericycle differentiation. Detailed phenotypical analyses of two of these mutants, combined with observations made in known vascular mutants, revealed an intimate correlation between vascular organization, pericycle fate, and lateral root initiation potency, and illustrated the independence of pericycle differentiation and lateral root initiation from protoxylem differentiation. Taken together, our data show that the pericycle is a heterogeneous cell layer with two groups of cells set up in the root meristem by the same genetic pathway controlling the diarch organization of the vasculature.
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K. Bernhardt, E.J. Carter, N.S. Chand, J. Lee, Y. Xu, X. Zhu, J.W. Ajioka, J.M. Goncalves, J. Haseloff, G. Micklem, and D. Rowe. New tools for self-organised pattern formation. IET Synthetic Biology 1:29-31 (2007).
Multicellular organisms undergo self-organisation during development. Our aim was to engineer self-organised pattern formation in free-swimming bacteria cells by providing an artificial system for bi-directional communication. E. coli cells would be equipped with genes derived from independent quorum sensing systems from P. aeruginosa and V. fischeri. These systems enable communication between cell populations and can enable regulated switching between competing cell fates. The negotiation of cell fates within bacterial populations can be visualized precisely by the expression of different fluorescent proteins.
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Dodd, A.N., Jakobsen, M.K., Baker, A.J., Telerow, A., Hou, S.W., Laplaze, L., Barrot, L., Poethig, R.S., Haseloff, J. and Webb, A.A.R. Time of day modulates low-temperature Ca2+ signals in Arabidopsis. Plant Journal 48:962-973 (2006).e
We tested the hypothesis that the circadian clock modulates Ca(2+)-based signalling pathways, using low-temperature (LT)-induced Ca(2+) signals. We investigated the relationship between diurnal and circadian modulation of LT-induced increases in cytosolic-free calcium ([Ca(2+)](cyt)), and regulation of [Ca(2+)](cyt)-dependent outputs of the LT-signalling network (RD29A transcript abundance and stomatal closure). We measured [Ca(2+)](cyt) non-invasively using aequorin, and targeted aequorin to the guard cell using a guard cell-specific GAL4-green fluorescent protein enhancer trap line. LT caused transient increases in whole plant and guard cell [Ca(2+)](cyt). In guard cells, the LT-induced [Ca(2+)](cyt) elevation preceded stomatal closure. In whole plants, the magnitude of LT-induced [Ca(2+)](cyt) transients, measured from the entire plant or specifically the guard cell, varied with the time of day: LT-induced [Ca(2+)](cyt) transients were significantly higher during the mid-photoperiod than at the beginning or end. Diurnal variation in LT-induced guard cell [Ca(2+)](cyt) increases was not correlated to diurnal variation in LT-induced stomatal closure. There was circadian modulation of LT-induced whole plant [Ca(2+)](cyt) increases, which were correlated to the circadian pattern of RD29A induction. In order to understand the significance of LT-induced [Ca(2+)](cyt) increases, we used a computer simulation to demonstrate that, in guard cells, LT-induced [Ca(2+)](cyt) increases measured from a population of cells are likely to represent the summation of cold-induced single-cell [Ca(2+)](cyt) oscillations.
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Dupuy, L.X., Mackenzie, J and Haseloff, J. A biomechanical model for the study of plant morphogenesis: Coleochaete orbicularis. Proceedings of the 5th Plant Biomechanics Conference. Stockholm. (2006)
Pattern formation in the morphogenesis in plants is a phenomenon strongly determined by the genetic programs of particular species. However, because organs are groups comprised of specialized cells that are adherent and not capable of migration, physical processes (cell wall expansion, diffusion) are though to be major aspect influencing the plant development. In the present study, Coleocheate orbicularis, a probable ancestor of land plant species has been used as a model species to study the mechanics of cell expansion in multicellular colonies. Image analysis methods have been developed in order to analyze the kinematics of cell expansion and division, and a simple analytical model were used to estimate both viscosity and cell wall division properties. Numerical computation using the finite element method has been used to simulate the growth of entire colonies using parameters derived from live imaging. Comparison with live experiments showed strong similarities between measured and simulated colonies. Also, parameter analysis showed that not only wall mechanical properties, but also division rate and orientation are factors strongly influencing pattern formation.
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Truernit, E., Siemering, K.R., Hodge, S., Grbic V. and Haseloff, J. A map of KNAT gene expression during Arabidopsis root development. Plant Molecular Biology, 60:1-20 (2006).
Homeodomain proteins are key regulators of patterning during the development of animal and plant body plans. Knotted1-like TALE homeodomain proteins have been found to play important roles in the development of the Arabidopsis shoot apical meristem and are part of a complex regulatory network of protein interactions. We have investigated the possible role of the knotted1-like genes KNAT1, KNAT3, KNAT4, and KNAT5 in Arabidopsis root development. Root growth is indeterminate, and the organ shows distinct zones of cell proliferation, elongation and differentiation along its longitudinal axis. Here we show that KNAT1, KNAT3, KNAT4 and KNAT5 show cell type specific expression patterns in the Arabidopsis root. Moreover, they are expressed in different spatially restricted patterns along the longitudinal root axis and in lateral root primordia. Hormones play an important role in maintenance of root growth, and we have studied their effect on KNAT gene expression. We show that KNAT3 expression is repressed by moderate levels of cytokinin. In addition, we show that the subcellular localization of KNAT3 and KNAT4 is regulated, indicating post-translational control of the activities of these transcription factors. The regulated expression of KNAT1, KNAT3, KNAT4 and KNAT5 within the Arabidopsis root suggests a role for these genes in root development. Our data provide the first systematic survey of KNAT gene expression in the Arabidopsis root.
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Moreno, N., Bougourd, S., Haseloff, J. and Feijo, J.A. Imaging Plant Cells in Handbook of Confocal Microscopy, Ed. J.B. Pawley, Springer Science (2006).
Ever since the time of Hooke, plant cells have been the foundation of many of the fundamental discoveries that have shaped cell biology. Underlying all these findings were significant advances in microscopy that helped to push forward our conceptual thinking, for example, supporting the acceptance of the cell theory. In this chapter we have highlighted what is now the leading edge of this technological effort. Imaging is now more than lenses and microscopes. Computers are also essential, both to accumulate and display research images, and to extract and analyse enormous amounts of quantitative information from them. Recent advances range from major hardware (e.g., two-photon equipment) to the development of computer software that now enables us to derive fresh insights from old histological techniques. We have tried to emphasize that all these techniques are only of value when they enable us to describe previously unknown biological features, and indeed many of the most important technical developments were developed only as part of specific research projects. The ever-growing field of genetically-encoded probes, such as GFP, seems likely to trigger more new and important technical adaptations that will enable us to obtain more and better dynamic information from living systems. Plant cell biology continues to bloom, and much of this growth is supported by modern imaging methods.
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Coates, J.C., Laplaze, L. and Haseloff, J. Armadillo-related proteins promote lateral root development in Arabidopsis. Proc. Natl. Acad Sci. USA, 103:1621-1626 (2006).
Armadillo/beta-catenin and related proteins have important functions during animal and Dictyostelium development, regulating cell differentiation, proliferation, and adhesion. Armadillo-repeat-containing proteins also exist in plants, but the majority have unknown roles. The Arabidopsis genes that show greatest sequence homology to Armadillo/beta-catenin are called ARABIDILLO-1 and -2. Here, we demonstrate that ARABIDILLO-1 and -2 promote lateral root development. arabidillo-1/-2 mutants form fewer lateral roots, and ARABIDILLO-1-overexpressing lines produce more lateral roots than wild-type seedlings. ARABIDILLO-yellow fluorescent protein fusions are nuclear. ARABIDILLO proteins contain an F-box motif, and thus may target other proteins for proteasomal degradation. Overexpression of ARABIDILLO-1 protein fragments, including F-box fragments, in wild-type seedlings reduces lateral root formation to the level of the arabidillo-1/-2 mutant. We have shown that plant beta-catenin-related proteins regulate root development. We suggest that ARABIDILLO proteins may target an inhibitor of lateral root development for degradation and propose that Arabidopsis beta-catenin-related proteins define a previously uncharacterized pathway that promotes root branching.
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Truernit E, Haseloff J. A Role for KNAT Class II Genes in Root Development. Plant Signal Behav. 2(1):10-2, (2007)
Homeodomain proteins set up domains of gene expression during the development of animal and plant body plans. In plants, homeodomain proteins of the KNOX class I family have been shown to play a role in shoot apical meristem development. Recently, we have investigated the role of the Arabidopsis thaliana KNOX class II genes KNAT3, KNAT4 and KNAT5 in root development. These genes showed root domain and cell type specific expression patterns, and their expression was regulated by hormones that influence root growth. Moreover, sub-cellular localization of the KNAT proteins exhibited regulation, suggesting that post-transcriptional control contributes to KNOX class II protein activity. Our data provide a survey of KNAT gene expression in the root and indicate that the investigated KNAT genes might play distinct roles during root development.
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Swarup, R., Kramer, E.M., Perry, P., Knox, K., Leyser , H.M.O., Haseloff, J, Beemster, G., Bhalerao, R. and Bennett, M.J. Root gravitropism requires the intercellular transmission of an auxin signal. Nature Cell Biology 7:1056-1065 (2005).
Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.
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Rudge, T. and Haseloff, J. A computational model of cellular morphogenesis in plants. Lecture Notes in Computer Science: Advances in Artificial Life, 3630:78-87 (2005).
Plant morphogenesis is the development of plant form and structure by coordinated cell division and growth. We present a dynamic computational model of plant morphogenesis at cellular level. The model is based on a self- reproducing cell, which has dynamic state parameters and spatial boundary geometry. Cell-cell signalling is simulated by diffusion of morphogens, and genetic regulation by a program or script. Each cell runs an identical script, equivalent to the genome. The model provides a platform to explore coupled interactions between genetic regulation, spatio-mechanical factors, and signal transduction in multicellular organisation. We demonstrate the capacity of the model to capture the key aspects of plant morphogenesis.
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Laplaze, L., Parizot, B., Baker, A., Ricaud, L., Martinière, A., Auguy, F., Franche, C., Nussaume, L, Bogusz, D., and Haseloff, J. GAL4-GFP Enhancer trap lines for genetic manipulation of lateral root development in Arabidopsis thaliana. Journal of Experimental Botany, 56:2433-42 (2005).
Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.
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Kurup, S., Runions, J.C., Kohler, U., Laplaze, L., Hodge, S. and Haseloff, J. Marking cell lineages in living tissues: pericycle cell-file contribution to the lateral root primordia in Arabidopsis thaliana. Plant Journal, 42:444-453 (2005).
We have generated a novel genetic system to visualize cell lineages in living tissues at high resolution. Heat shock was used to trigger the excision of a specific transposon and activation of a fluorescent marker gene. A histone-YFP marker was used to allow identification of cell lineages and easy counting of cells. Constitutive expression of a green fluorescent membrane protein was used to provide a precise outline of all surrounding cells. Marked lineages can be induced from specific cells within the organism by targeted laser irradiation, and the fate of the marked cells can be followed non-invasively. We have used the system to map cell lineages originating from the initials of primary and lateral roots in Arabidopsis. The lineage marking technique enabled us to measure the differential contribution of primary root pericycle cell files to developing lateral root primordia. The majority of cells in an emerging lateral root primordium derive from the central file of pericycle founder cells while off-centre founder cells contribute only a minor proliferation of tissue near the base of the root. The system shows great promise for the detailed study of cell division during morphogenesis.
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Ingouff, M., Haseloff, J. and Berger F. Polycomb Group genes control developmental timing in plant reproduction. Plant Journal, 42:663-674 (2005).
Polycomb (PcG) group proteins form modular complexes, which maintain repressed transcriptional states of target genes across cell divisions. As PcG complexes provide a memory of cell fate, such proteins might control temporal aspects of development. Loss-of-function of any of the FERTILIZATION INDEPENDENT SEED (FIS) PcG genes perturbs endosperm development. In this report we provide a detailed analysis of the phenotype of fis endosperm development using molecular and cellular markers. Wild type (WT) endosperm development undergoes a series of four major developmental phases timed by successive synchronous nuclei division. In fis endosperm the transition from phase 1, marked by a synchronous mode of nuclei divisions to phase 2, corresponding to the establishment of three mitotic domains, is absent. Accordingly, the expression of seven markers of phase 1 and phase 2 is temporally perturbed. In spite of such changes, specific sequences of developmental events still take place as in the WT. Overall, fis mutations are heterochronic mutations that cause a temporal deregulation in the ontogenic sequence of endosperm development.
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Johnson, A., Hibberd, J., Gay, C., Essah, P., Haseloff, J. Tester, M. and Guiderdoni, E. Spatial control of transgene expression in rice (Oryza sativa L.) using the GAL4 enhancer trapping system. Plant Journal, 41:779-789 (2005).
We used enhancer trapping with the GAL4 transcriptional activator from yeast to obtain spatial control of transgene expression in all organs of the model monocotyledonous species rice (Oryza sativa L. cv. Nipponbare). Our T-DNA enhancer trapping cassette consisted of two principle components: (1) the minimal promoter-equipped gal4 gene placed adjacent to the right border, and (2) the green fluorescent protein gene (gfp) fused to the upstream activation sequence element (UAS) to which GAL4 binds and activates expression, so that gfp expression corresponds to gal4 expression. Agrobacterium-mediated integration of the cassette into the rice genome often brings the gal4 gene under transcriptional control of local genomic enhancers and promoters, resulting in gal4/gfp expression patterns ranging in specificity from single-cell types to constitutive expression. We produced more than 13 000 enhancer trap lines with this cassette and screened T(0) adult plants (1982 lines), T(1) seed (2684 lines) and T(1) seedlings (2667 lines) for gfp expression. Approximately 30% of the lines produced GFP, and we identified lines with gfp expression in specific cell types of all major organs of the rice plant. Subsequently, using the GUS reporter gene (uidA), we demonstrated that UAS:geneX constructs can be transactivated in specific cell types where gal4 and gfp are expressed, thus providing an excellent system for the manipulation of gene expression and physiological function in specific cell types of rice.
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Haseloff J. Old botanical techniques for new microscopes. Biotechniques. 34:1174-1182 (2003).
New 3-D visualization techniques allow for the construction of a precise and simplified geometric description of the cellular architecture of embryos and meristems. This description could be further reduced to a simple numerical scheme for describing the 3-D organization of cells in the meristem. For example, each cell’s position, approximate 3- D shape, and location of adjoining cells could be described in a simple notation, and there is a compelling reason for pursuing this. The fate of an individual plant cell in a meristem is governed by local positional information. Each cell is part of a self-reinforcing network of interactions. To understand the normal regulation of meristem development and mutant defects, we need to turn to dynamic models that incorporate some of these intercellular signaling features. These will be computational models and will need to include (i) an engine for processing the predicted interactions between cells and (ii) a scheme for representing the consequences for cell proliferation and differentiation in the meristem. Here I’ve described the novel combination of modern confocal microscopy and computer-based 3-D reconstruction techniques with classical staining and clearing techniques for imaging intact plant tissues rather than thin sections. This allows for the 3-D arrangement of cells in a plant meristem to be captured and potentially converted into a simple numerical form. The application of rules for plant cell interaction to such a model would result in dynamic behavior that could be animated and visualized. In the future, this could be a framework for directly modeling cell-to-cell dynamics in this complex system.
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Svistoonoff, S., Laplaze, L., Auguy, F., Runions, J., Duponnois, R., Haseloff, J., Franche, C. & Bogusz D. cg12 expression is specifically linked to infection of root hairs and cortical cells during Casurina glauca and Allocasurina verticillata Actinorhizal nodule development. Mol. Plant Micro. Interactions 16:600-607 (2003).
cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during mycorrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.
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Ayre, B.G. Kohler, U., Turgeon, R. and Haseloff, J. Optimisation of trans-splicing riboyme efficiency and specificity by in vivo genetic selection. Nuc. Acids Res. 30:1-9 (2002).
Trans-splicing ribozymes are RNA-based catalysts capable of splicing RNA sequences from one transcript specifically into a separate target transcript. In doing so, a chimeric mRNA can be produced, and new gene activities triggered in living cells dependent on the presence of the target mRNA. Based on this ability of trans-splicing ribozymes to deliver new gene activities, a simple and versatile plating assay was developed in Saccharomyces cerevisiae for assessing and optimizing constructs in vivo. Trans-splicing ribozymes were used to splice sequences encoding a GAL4-derived transcription activator into a target transcript from a prevalent viral pathogen. The transcription activator translated from this new mRNA in turn triggered the expression of genes under the regulatory control of GAL4 upstream-activating sequences. Two of the activated genes complemented metabolic deficiencies in the host strain, and allowed growth on selective media. A simple genetic assay based on phenotypic conversion from auxotrophy to prototrophy was established to select efficient and specific trans-splicing ribozymes from a ribozyme library. This simple assay may prove valuable for selecting optimal target sites for therapeutic agents such as ribozymes, antisense RNA and antisense oligodeoxyribonucleotides, and for optimizing the design of the therapeutic agents themselves, in higher eukaryotes.
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Boisnard-Lorig, C., Colon-Carmona, A., Bauch, M.., Hodge, S., Doerner, P., Bancharel, E., Dumas, C., Haseloff, J. and Berger, F. Dynamic analyses of the expression of the histone::yfp fusion protein in Arabidopsis show that syncytial endosperm is divided into mitotic domains. Plant Cell 13:495-509 (2001).
During early seed development, nuclear divisions in the endosperm are not followed by cell division, leading to the development of a syncytium. The simple organization of the Arabidopsis endosperm provides a model in which to study the regulation of the cell cycle in relation to development. To monitor nuclear divisions, we constructed a HISTONE 2B::YELLOW FLUORESCENT PROTEIN gene fusion (H2B::YFP). To validate its use as a vital marker for chromatin in plants, H2B::YFP was expressed constitutively in Arabidopsis. This enabled the observation of mitoses in living root meristems. H2B::YFP was expressed specifically in Arabidopsis syncytial endosperm by using GAL4 transactivation. Monitoring mitotic activity in living syncytial endosperm showed that the syncytium was organized into three domains in which nuclei divide simultaneously with a specific time course. Each mitotic domain has a distinct spatiotemporal pattern of mitotic CYCLIN B1;1 accumulation. The polar spatial organization of the three mitotic domains suggests interactions between developmental mechanisms and the regulation of the cell cycle.
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Jensen, R.B., Lykke-Andersen, K., Frandsen, G.I., Nielsen, H.B., Haseloff, J., Jespersen, H.M. and Shriver, K. Promiscuous and specific phospholipid binding by domains in ZAC, a membrane associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain. Plant Mol. Biol. 44:799-814 (2000).
Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2 domain are separated by a region without homology to other known proteins. Zac promoter/beta-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca2+ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1-174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence that this phosphoinositide is recognized as a signal in plants. A role for ZAC in the regulation of ARF-mediated vesicular transport in plants is discussed.
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Bougourd, S., Marrison, J. and Haseloff, J. An aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phentypes in mature Arabidopsis embryos. Plant Journal, 24:543-550 (2000).
A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high-resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild-type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three-dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short-root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.
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Kiegle E, Moore CA, Haseloff J, Tester MA, Knight MR. Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root. Plant Journal, 23:267-78 (2000).
Little is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer-trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ([Ca2+]cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid [Ca2+]cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.
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Kiegle, E., Gilliham, M., Haseloff, J and Tester, M. Hyperpolarisation-activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots. Plant Journal, 21:225-229 (2000).
Calcium currents across the plasma membrane of plant cells allow transduction of environmental signals as well as nutritive calcium uptake. Using transgenic Arabidopsis plants with cell-specific expression of green fluorescent protein (GFP), we analyzed whole cell calcium currents in epidermal cells of the rapidly growing root apex, mature epidermal cells, cortical and epidermal cells from the elongation zone, and mature pericycle cells. In cells only from the rapidly growing root apex, a hyperpolarization-activated calcium current was identified. This current was irreversibly inhibited by 10 microM Al3+, as well as being inhibited by 1 mM Co2+ and 100 microM verapamil. In no cells could a depolarisation-activated current be attributed to calcium influx. In the growing root apex, the hyperpolarization-activated calcium current may function to allow constitutive uptake of calcium for rapid cell division and elongation.
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Ayre, B.G., Köhler, U., Goodman, H.M. and Haseloff, J. A model system for targeted gene delivery mediated by trans-splicing ribozymes. Proc. Natl. Acad. Sci. USA. 96:3507-3512 (1999).
We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus. The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity in vivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.
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Kohler, U. Ayre, B., Goodman, H. M. and Haseloff, J. Ribozymes for targeted trans-splicing in vivo. J. Mol. Biol. 284:1935-1950 (1999).
Ribozymes are potential tools for genetic manipulation, and various naturally occurring catalytic RNAs have been dissected and used as the basis for the design of new endoribonuclease activities. While such cleaving ribozymes may work well in vitro, they have not proved to be routinely effective in depleting living cells of the chosen target RNA. Recently, trans-splicing ribozymes have been employed to repair mutant mRNAs in vivo. We have designed modified trans-splicing ribozymes with improved biological activity. These allow accurate splicing of a new 3' exon sequence into a chosen site within a target RNA, and in frame fusion of the exon can result in expression of a new gene product. These trans-splicing ribozymes contain catalytic sequences derived from a self-splicing group I intron, which have been adapted to a chosen target mRNA by fusion of a region of extended complementarity to the target RNA and precise alteration of the guide sequences required for substrate recognition. Both modifications are required for improved biological activity of the ribozymes. Whereas cleaving ribozymes must efficiently deplete a chosen mRNA species to be effective in vivo, even inefficient trans-splicing can allow the useful expression of a new gene activity, dependent on the presence of a chosen RNA. We have targeted trans-splicing ribozymes against mRNAs of chloramphenicol acetyltransferase, human immunodeficiency virus, and cucumber mosaic virus, and demonstrated trans-splicing and delivery of a marker gene in Escherichia coli cells. The improved trans-splicing ribozymes may be tailored for virtually any target RNA, and provide a new tool for triggering gene expression in specific cell types.
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Haseloff, J.; Dormand, E.-L. and Brand, A. H. Live imaging with green fluorescent protein. Methods in Molecular Biology, Vol. 122: Confocal Microscopy Methods and Protocols. Paddock, S. Ed. Humana Press. (1999).
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Haseloff, J. GFP variants for multspectral imaging of living cells, in Methods in Cell Biology, Vol. 58, Kay, S. and Sullivan, K. Eds. Academic Press. (1999).
Unlike enzyme markers, green fluorescent protein can be visualized at high resolution in living cells using confocal microscopy. The images are not prone to fixation or staining artifacts, and can be of exceptional clarity. Moreover, the activities of living cells, such as cytoplasmic streaming, are clearly evident during microscopy. Ordinarily, movement within a sample is a nuisance, placing constraints on the use of sometimes lengthy techniques for noise reduction during confocal microscopy, such as frame averaging. However, it is possible to monitor dynamic events by time-lapse confocal microscopy, and this combination of a vital fluorescent reporter with high-resolution optical techniques shows much promise for use in cell biological and physiological experiments. Genetic systems such as that of Arabidopsis provide a large resource of potentially informative mutants, and there has been much recent improvement in techniques for determining the molecular basis of a particular phenotype. The use of fluorescent proteins will provide further tools for examining the biology of mutant cells. The precision with which particular cellular structures can be decorated with GFP and the ease with which subcellular traffic can be monitored indicate that this approach will be very useful for cell biological and physiological observations, particularly for detailed examination of plant mutant phenotypes.
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Haseloff, J. Imaging Living Cells, R. Rizzuto and C. Fasolato Eds. Springer-Verlag. (1998).
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Schuldt, A..J., Adams, J.H.J., Davidson, C.M., Micklem, D.R., Haseloff, J., St.Johnston, D. and Brand, A. Miranda mediates asymmetric protein and RNA localisation in the developing nervous system. Genes & Development 12:1847-1857 (1998).
Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Here we show that Staufen interacts in vivo with a segment of the prospero 3' UTR. Staufen protein and prospero RNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase. Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Furthermore, Miranda colocalizes with Staufen protein and prospero mRNA during neuroblast divisions, and neither Staufen nor prospero RNA are localized in miranda mutants. Thus Miranda, which localizes Prospero protein, also localizes prospero RNA through its interaction with Staufen protein.
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Berger, F., Haseloff, J., Schiefelbein, J. and Dolan, L. Positional information in root epidermis is defined during embryogenesis and acts in domains with strict boundaries. Current Biology 8:421-430 (1998).
Cell position rather than cell lineage governs most aspects of development in plants. However, the nature and the origin of positional information remains elusive. Animal epidermal patterning relies in many cases on positional information provided by cell-cell communication. The epidermal layer of the Arabidopsis root is made of alternating files of two cell types and thus presents a simple pattern to study positional mechanisms. Clonal analysis of the root epidermis in combination with molecular and morphological markers has shown that cell fate is determined by position relative to the underlying cell layer, the cortex. The epidermal pattern appears to be organised during embryogenesis. Fate is not fixed in the developing root, though, as cells that move into a position previously occupied by neighbour cells ablated using laser microsurgery change fate. In contrast, cell fate is not altered when communication with living neighbour cells is impaired. Precise mapping of the influence of the position of extracellular cues on cell fate has shown that domains of positional information are organised with well-defined boundaries. Cell-fate specification in the root epidermis relies on positional information that is organised in stable domains with sharp boundaries. The epidermal pattern is defined during embryogenesis and positional information remains active in the root until the initiation of cell morphogenesis. The origin of some positional cues might be extracellular.
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Berger, F., Linstead, P., Dolan, L. and Haseloff, J. Stomatal patterning on the hypocotyl of Arabidopsis thaliana is controlled by genes involved in the control of root epidermis patterning. Developmental Biology 194:226-234 (1998).
Stomata complexes are epidermal specialized structures typical of the upper aerial part of plants (shoot). In the model plant Arabidopsis thaliana, we show that in the hypocotyl (the) junction between the shoot and the root), stomata are organized according to a clear pattern reminiscent of the root epidermis pattern. Although stomata complexes are typical of the shoot epidermis, their pattern on the hypocotyl is under the control of genes involved in root epidermis patterning. Moreover, we have isolated a GFP marker line for the hypocotyl epidermal cells which do not differentiate stomata complexes. In this line the root and the hypocotyl epidermal patterns are similar. Our data support the existence of interactions between developmental mechanisms involved in the control of the apical/basal polarity and the radial symmetry of the plant body.
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Haseloff, J. and Siemering, K. R. The uses of GFP in plants, in Green Fluorescent Protein: Properties, Applications and Protocols, Chalfie, M. and Kain, S. Eds. John Wiley. (1998).
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Zernicka-Goetz, M., J. Pines, McLean Hunter, S., Dixon, J. P. C., Siemering, K. R., Haseloff, J. and Evans, M. J. Following cell fate in the living mouse embryo. Development 124:1133-1137 (1997).
It has been difficult to follow many of the dramatic changes in cell fate and cell migration during mouse development. This is because there has been no enduring marker that would allow cells to be recognised in the living embryo. We believe that we have overcome this problem by developing a novel form of green fluorescent protein, named MmGFP, that proves to be easily visible and non toxic to mouse cells and does not perturb embryogenesis. We show that synthetic mRNA encoding MmGFP can be injected into blastomeres to follow the fate of their progeny during preimplantation development. We have made a stable embryonic stem cell line that expresses MmGFP and introduced these fluorescent cells into mouse embryos. For the first time, we have been able to follow the fate of embryonic stem cells in living embryos and to observe directly the contribution of these cells to distinct lineages of the postimplantation embryo. This approach should lead to a more complete description of the dynamics of cell fate in the mouse.
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Haseloff, J., Siemering, K. R., Prasher, D.C. and Hodge, S. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA. 94:2122-2127 (1997).
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant Arabidopsis thaliana, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains sequences similar to those required for recognition of normal plant introns. We have modified the codon usage of the gfp gene to mutate the intron and to restore proper expression in Arabidopsis. GFP is mainly localized within the nucleoplasm and cytoplasm of transformed Arabidopsis cells and can give rise to high levels of fluorescence, but it proved difficult to efficiently regenerate transgenic plants from such highly fluorescent cells. However, when GFP is targeted to the endoplasmic reticulum, transformed cells regenerate routinely to give highly fluorescent plants. These modified forms of the gfp gene are useful for directly monitoring gene expression and protein localization and dynamics at high resolution, and as a simply scored genetic marker in living plants.
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Siemering, K. R., Golbik, R., Sever, R. and Haseloff, J. Mutations that supress the thermosensitivity of green fluorescent protein. Current Biology 6:1653-1663 (1996).
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently attracted great interest as the first example of a cloned reporter protein that is intrinsically fluorescent. Although successful in some organisms, heterologous expression of GFP has not always been straight forward. In particular, expression of GFP in cells that require incubation temperatures around 37 degrees C has been problematic. We have carried out a screen for mutant forms of GFP that fluoresce more intensely than the wild-type protein when expressed in E. coli at 37 degrees C. We have characterized a bright mutant (GFPA) with reduced sensitivity to temperature in both bacteria and yeast, and have shown that the amino acids substituted in GFPA act by preventing temperature-dependent misfolding of the GFP apoprotein. We have shown that the excitation and emission spectra of GFPA can be manipulated by site-directed mutagenesis without disturbing its improved folding characteristics, and have produced a thermostable folding mutant (GFP5) that can be efficiently excited using either long-wavelength ultraviolet or blue light. Expression of GFP5 results in greatly improved levels of fluorescence in both microbial and mammalian cells cultured at 37 degrees C. The thermotolerant mutants of GFP greatly improve the sensitivity of the protein as a visible reporter molecule in bacterial, yeast and mammalian cells. The fluorescence spectra of these mutants can be manipulated by further mutagenesis without deleteriously affecting their improved folding characteristics, so it may be possible to engineer a range of spectral variants with improved tolerance to temperature. Such a range of sensitive reporter proteins will greatly improve the prospects for GFP-based applications in cells that require relatively high incubation temperatures.
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Zernicka-Goetz, M., Pines, J., Ryan, K., Siemering, K. R., Haseloff, J., Evans, M. J. and Gurdon J. B. An indelible lineage marker for Xenopus using a mutated green fluorescent protein. Development 122:3719-3724 (1996).
We describe the use of a DNA construct (named GFP.RN3) encoding green fluorescent protein as a lineage marker for Xenopus embryos. This offers the following advantages over other lineage markers so far used in Xenopus. When injected as synthetic mRNA, its protein emits intense fluorescence in living embryos. It is non-toxic, and the fluorescence does not bleach when viewed under 480 nm light. It is surprisingly stable, being strongly visible up to the feeding tadpole stage (5 days), and in some tissues for several weeks after mRNA injection. We also describe a construct that encodes a blue fluorescent protein. We exemplify the use of this GFP.RN3 construct for marking the lineage of individual blastomeres at the 32- to 64-cell stage, and as a marker for single transplanted blastula cells. Both procedures have revealed that the descendants of one embryonic cell can contribute single muscle cells to nearly all segmental myotomes rather than predominantly to any one myotome. An independent aim of our work has been to follow the fate of cells in which an early regulatory gene has been temporarily overexpressed. For this purpose, we co-injected GFP.RN3 mRNA and mRNA for the early Xenopus gene Eomes, and found that a high concentration of Eomes results in ectopic muscle gene activation in only the injected cells. This marker may therefore be of general value in providing long term identification of those cells in which an early gene with ephemeral expression has been overexpressed.
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Haseloff, J. and Amos, B. GFP in plants. Trends in Genetics 11:328-329 & 374, (1995).
There are two major uses for green fluorescence protein (GFP) in plants: monitoring gene expression and protein localization at high resolution, and providing an easily scored genetic marker in living plants. GFP can be used as a replacement for ß-glucuronidase, which is commonly used as a reporter for genetic fusions in plants, It allows direct imaging of the fluorescent gene product in living cells without the need for prolonged and lethal histochemical staining procedures . In addition, GFP expression can be scored easily using a long-wave UV lamp if high levels of fluorescence intensity can be maintained in transformed plants. An assay for gene expression using fluorescence in vivo would be a very useful tool for plant transformation and breeding experiments.
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Terry, B.R.; Matthews, E.K. and Haseloff, J. Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy. BBRC 217:21-27 (1995).
The cDNA for the green fluorescent protein (GFP) of Aequorea victoria has been expressed in transformed cells of Saccharomyces cerevisiae and the recombinant GFP isolated. Protonation and deprotonation of the cloned and purified GFP produced major effects on its spectral absorption characteristics with an increase in pH enhancing the fluorescence emission of the GFP more than twofold. Finally, molecular characterisation of GFP by fluorescence correlation microscopy in a minimal target volume of 1 fL yielded a translational diffusion coefficient (DT) of 8.7 x 10(-7) cm2.sec-1, equivalent to a Stokes radius of 2.82nm for a monodisperse globular protein of 27kDa.
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Robaglia, C., Bruening, G., Haseloff, J. and Gerlach, W.L. Evolution and replication of tobacco ringspot virus satellite RNA mutants. EMBO J. 12:2969-2976 (1993).
The replication properties of linker insertion-deletion mutants of tobacco ringspot virus satellite RNA have been studied by amplification in plants infected with the helper virus. Sequence analysis of the cDNAs corresponding to the replicated forms shows that only one of the original mutated molecules replicates unaltered, and in general new variants accumulate. Depending on the location of the original mutation three types of sequence modifications were observed: (i) deletion of the mutated region followed by sequence duplication, (ii) sequence duplication and deletion outside of the mutated region and (iii) limited rearrangements at the site of mutation. The mutant that replicates without sequence changes accumulates linear multimeric forms suggesting that self-cleavage is affected although the sequence alteration does not involve the hammerhead catalytic domain. Alternative RNA conformations are likely to play a role in the origin of this phenotype and in the formation of sequence duplications. These results demonstrate the great structural flexibility of this satellite RNA.
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Davies, C., Haseloff, J. and Symons, R.H. Structure, self-cleavage, and replication of two viroid-like satellite RNAs (virusoids) of subterranean clover mottle virus. Virology 177:216-224 (1990).
Both the genomic and viroid-like satellite RNAs (virusoids) from four subterranean clover mottle virus isolates described by Francki et al. were analyzed in detail. Restriction endonuclease mapping of cDNAs prepared from the genomic RNAs from all isolates showed that these RNAs are closely related if not identical. The two virusoids, which can occur together in the same isolate or individually, were sequenced and shown to be able to form highly base-paired viroid-like secondary structures. The left-hand portions of these structures are almost entirely homologous but the right-hand portions show little similarity. The plus, but not the minus, virusoid RNAs contain sequences that can form the hammerhead self-cleavage structure of certain other self-cleaving viroid, virusoid, and satellite RNAs. Plus, but not minus, RNA transcripts from cDNA clones self-cleaved essentially to completion at the predicted site during transcription in vitro. Northern blot analysis of infected leaf tissue extracts revealed the presence of an oligomeric series of plus RNAs (of monomer size and greater) but minus RNAs were present only as high molecular weight species of heterogeneous size. These findings are in agreement with the lack of minus RNA self-cleavage in vitro. Hence, these virusoid RNAs appear to replicate by a rolling-circle mechanism in which only the plus RNAs self-cleave to form monomeric RNAs.
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Haseloff, J. and Gerlach, W.L. Sequences required for self-catalysed cleavage of the satellite RNA of tobacco ringspot virus. Gene 82:45-52 (1989).
The satellite RNA of tobacco ringspot virus (sTobRV) undergoes self-catalysed cleavage during replication. A plasmid for in vitro expression of sTobRV has been constructed and used to obtain a library of mutagenized sTobRV sequences. Screening of these mutants has allowed precise definition of the sequences required for (+) and (-) strand cleavage. The sequences and RNA structures associated with cleavage of each strand differ markedly. Cleavage of the (+) strand requires those sequences flanking the site for cleavage to form a 'hammerhead' domain, similar to those found in other satellite and viroid RNA. In contrast, cleavage of the (-) strand requires only a small region of 12 nucleotides (nt) at the site of cleavage, and a sequence of 55 nt positioned elsewhere in the molecule. Comparison with a closely related satellite suggests that a novel RNA structure may be involved in (-) strand cleavage.
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Haseloff, J. and Gerlach, W.L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334:585-591 (1988).
In vitro mutagenesis of sequences required for the self-catalysed cleavage of a plant virus satellite RNA has allowed definition of an RNA segment with endoribonuclease activity. General rules have been deduced for the design of new RNA enzymes capable of highly specific RNA cleavage, and have been successfully tested against a new target sequence.
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Gerlach, W.L., Llewellen, D. and Haseloff, J. Construction of a plant disease resistance gene from the satellite RNA of tobacco ringspot virus. Nature 328:802-805 (1987).
Tobacco ringspot virus (TobRV) is the type member of the nepoviruses1. It consists of 28-nm isometric particles which contain one or the other of the two single-strand genomic RNAs of 4.8 and 7.2 kilobases (kb) (refs 2 and 3). TobRV infects a wide range of dicotyledonous plants and is the causative agent of the budblight disease of soybean. A small RNA which can replicate to high levels and be encapsidated by TobRV in infected plants has been found during serial passages of virus isolates4,5. It is not required for virus propagation and has no detectable sequence homology with the virus genomic RNAs. It is therefore termed the satellite RNA of tobacco ringspot virus (STobRV). It can be considered a parasite of the virus and it ameliorates disease symptoms when present during virus infection of plants. We report here the expression of forms of the STobRV sequence in transgenic tobacco plants. Plants which express full-length STobRV or its complementary sequence as RNA transcripts show phenotypic resistance when infected with TobRV. This is correlated with the amplification of satellite RNA to high levels during virus infection of plants.
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Symons, R.H., Haseloff, J., Visvader, J.E., Keese, P., Murphy, P.J., Gill, D.S., Gordon, K.H.J. and Bruening, G. On the mechanism of replication of viroids, virusoids and satellite RNAs, in Subviral pathogens of plants and animals, viroids and prions. Academic Press. (1985).
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Ahlquist, P., Strauss, E.G., Rice, C.M., Strauss, J.H., Haseloff, J.P. and Zimmern, D. Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses. J. Virology 53:536-542 (1985).
Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex.
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Kiberstis, P.A., Haseloff, J. and Zimmern, D. 2' Phosphomonoester, 3'-5' phosphodiester bond at a unique site in a circular viral RNA. EMBO J. 4:817-827 (1985).
Solanum nodiflorum mottle virus (SNMV) RNA2 is a single-stranded, covalently closed circular molecule. RNase T2 or nuclease P1 digests of this RNA contain a minor nucleotide of unusual chromatographic and electrophoretic mobility. This nucleotide is resistant to further digestion by T2 or P1 ribonucleases, or by alkali, but is sensitive to venom phosphodiesterase digestion. Alkaline phosphatase digestion yields a product which is RNase T2 and P1 sensitive. The products of these various digests show that the minor nucleotide is a ribonuclease-resistant dinucleotide carrying a 2' phosphomonoester group with the core structure C2'p3'p5'A. This dinucleotide is found in a unique RNase T1 product of SNMV RNA2, thus establishing a unique location in the sequence for the 2' phosphomonoester group at residue 49. Identical results have been obtained with a second related virus. The phosphomonoester group probably results from the RNA ligation event by which the molecules were circularised.
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Haseloff, J., Goelet, P., Zimmern, D., Alquist, P., Dasgupta, R. and Kaesberg, P. Striking similarities in amino acid sequence among non-structural proteins encoded by RNA viruses that have dissimilar genomic organization. Proc. Natl. Acad. Sci. USA. 81:4358-4362, (1984).
The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.
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Haseloff, J., Keese, P.K. and Symons, R.H. Comparative structure and properties of virusoids, in Plant Infectious Agents: viruses, viroids, virusoids and satellites, Robertson, H.D., Howell, S.H., Zaitlin, M. and Malmberg, R. Eds., Cold Spring Harbor (1983).
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Haseloff, J., Mohamed, N.A. and Symons, R.H. Viroid RNAs of cadang-cadang disease of coconuts. Nature 299:316-321 (1982).
Cadang-cadang is a serious lethal disease of coconut palms in the Philippines. Infectivity is associated with four viroid RNAs (ccRNAs) which are from 246 to 574 residues in size but do not vary in sequence complexity. They share sequence and structural homology with other viroids. Variant ccRNAs found in nine isolates sequenced may have arisen as by-products or intermediates of viroid replication.
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Mohamed, N.A., Haseloff, J. and Symons, R.H. Characterization of the different electrophoretic forms of the cadang-cadang viroid. J. Gen. Virol. 63:181-188 (1982).
The relationship between symptom development and changes in the fast and slow electrophoretic forms of the two cadang-cadang disease-associated RNAs (ccRNA-1 and ccRNA-2) has been examined. The fast form of each is present in trees for up to 2 years before the appearance of symptoms, indicating an incubation period of about 2 years. Trees showing the first symptoms (on the nuts) contain only the fast form of the ccRNAs; the development of leaf symptoms coincides with the first detection of the slow form. After this time, both slow and fast forms are found in the newly developing fronds for the next 9 to 12 months but then, as symptoms develop, new fronds contain only the slow form. Therefore, at later stages of disease, only the slow form can be detected in all fronds. The fast form of ccRNA-1 and/or ccRNA-2 therefore appears to be the infectious form in nature. The number of nucleotides in the four ccRNAs as determined by polyacrylamide gel electrophoresis under denaturing conditions are: ccRNA-I fast, 250; ccRNA-1 slow, 300; ccRNA-2 fast, 500; ccRNA-2 slow, 600. On the basis of two-dimensional fingerprints of ribonuclease A and T1 digests of the four ccRNAs, it was concluded that the ccRNAs are composed of repeated sequences of ccRNA-I fast, and each of the ccRNA-2 forms is a dimer of the respective ccRNA-1 form.
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Haseloff, J. and Symons, R.H. Comparative sequence and structure of viroid-like RNAs of two plant viruses. Nuc. Acids Res. 10:3681-3691 (1982).
A newly discovered group of spherical plant viruses contains a bipartite genome consisting of a single-strand linear RNA molecule (RNA 1, Mr 1.5 x 10(6) ), and a single-strand, covalently closed circular viroid-like RNA molecule (RNA 2, Mr approximately 125,000). The nucleotide sequences of the RNA 2 of two of these, velvet tobacco mottle virus and solanum nodiflorum mottle virus, have been determined. RNA 2 of solanum nodiflorum mottle virus consists of 377 residues whereas that of velvet tobacco mottle virus consists of two approximately equimolar species, one of 366 residues and the other, with a single nucleotide deletion, of 365 residues. There is 92-95% sequence homology between the RNA 2 species of the two viruses. The predicted secondary structures possess extensive intramolecular base pairing to give rod-like structures similar to those of viroids. The structural similarities between the RNAs 2 of velvet tobacco mottle virus and solanum nodiflorum mottle virus and viroids may reflect functional similarities.
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Haseloff, J. and Symons, R.H. Chrysanthemum stunt viroid: primary sequence and secondary structure. Nuc. Acids Res. 9:2741-2752 (1981).
The sequence of the 356 nucleotide residues of chrysanthemum stunt viroid (CSV) has been determined. Overlapping linear viroid fragments were obtained by partial ribonuclease digestion, radiolabelled in vitro at their 5'-ends, and sequenced using partial enzymic cleavage methods. Of the CSV sequence, 69% is contained in the published sequence of potato spindle tuber viroid (PSTV). Differences in the primary sequence of CSV and PSTV suggest that neither the positive nor putative negative strands of these two viroids code for functional polypeptide products. However, the two viroids can form similar secondary structures, implicating a role for viroid structure in replication.

Patent applications

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Methods and compositions for modulating root growth in plants. Coates, JC., Laplaze, L. and Haseloff, J. WO 2007/060514 A3R4. (2005).
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Modified HAP1 for plant gene expression. Haseloff J, Bauch M, Boisnard-Lorig C, Hodge S, Laplaze L, Runions J & Kurup S. US Patent 2005/0132432. (2005).
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Targeted gene expression in plants using GAL4. Haseloff, J. and Hodge, S. US Patent 6,255,558 (2001).
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Cell ablation using trans-splicing ribozymes. Haseloff, J., Brand, A., Perrimon, N. and Goodman, H.M., US Patents 5,849,548; 5,863,774; 5,866,384; 5,882,907; 6,010,904 and 6,071,730 (1992-2000).
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Jellyfish green fluorescent protein (GFP) expression in plants. Haseloff, J., Hodge, S., Prasher, D.C. and Siemering, K.R. WO 96/27675 (1996).
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Trans-splicing ribozymes. Haseloff, J. and Goodman, H.M., US Patents 5,874,414 and 6,015,794 (1992-2000).
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Ribozymes:hammerhead ribozymes for targeted cleavage of RNAs. Haseloff, J., Gerlach, W.L., Jennings, P. and Cameron, F.H. US Patents 4,024,695; 5,254,678; 5,494,814; 5,543,508; 5,543,508; 5,574,143; 5,589,580; 5,707,835; 5,747,335; 5,766,942; 5,840,874 and 6,127,114 (1989-2000).