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Intracellular membrane traffic; plant transgene expression technology
My laboratory investigates the molecular mechanisms that facilitate the fundamental processes of growth, division, and spatial pattering of plant cells. This has begun to reveal how plant cells have evolved distinctive intracellular transport pathways to perform these tasks. In other work, we have also developed genetic technologies that allow transgene activity to be controlled in space and time in a variety of plant species.
We are interested in the intracellular transport pathways that connect the organelles of the plant endomembrane system. These pathways are responsible for the biogenesis of the cell wall, cell plate, plasma membrane, and diverse vacuoles which are among the most biologically interesting and commercially important structures in plants. They feed, clothe, and house the majority of the world’s population, are important targets of biotechnology, and are increasingly recognised as central to wider aspects of plant development and stress adaptation in areas such as the establishment of polarity and the responses to pathogen attack.
Experimental observation and genome analyses indicate that the trafficking pathways have been independently elaborated in the plant and animal lineages, presumably to meet the different demands of cellular specialisation and physiological response in each group of organisms.
Our approach is to use forward and reverse genetics in combination with confocal laser-scanning microscopy of fluorescent protein markers to define the membrane trafficking pathways of plant cells and to identify the molecular mechanisms that control them.
We focus on GTPases of the Rab family. Individual Rab GTPase subclasses are proposed to act in particular intracellular transport steps to ensure that each transport vesicle is targeted accurately to the appropriate cellular compartment. The 57 Rab GTPases of Arabidopsis have been classified into 18 putative subclasses (Rutherford S. and Moore I., 2002, The Arabidopsis Rab GTPases: another enigma variation. Curr. Op. Cell Biol., 5, 518-528). Our work has led to the development of tools to investigate the mechanisms of plant intracellular membrane traffic by fluorescence microscopy [1,3,5]. These tools have allowed us to identify some of the Rab GTPases that act in the major transport steps between the ER, Golgi, PM and vacuoles [e.g. 2, 4, 5]. Research has now shifted to those members if the family that appear to be plant-specific and to the elucidation of their effectors. Some of this work has illuminated the early endosomal system and diversification of function within ancient Rab GTPase families (Betts, H., 2004, DPhil Thesis, University of Oxford; Chow et al., 2008).
Membrane trafficking mutants in Arabidopsis
In an alternative approach to identify important and plant-specific membrane trafficking genes, we have conducted large-scale screens for membrane trafficking mutants in Arabidopsis. These are based on the use of fluorescent protein markers to reveal membrane trafficking defects. Work is underway to characterise these defects and to clone the mutant loci. The first of these has provided insight into the evolution of the ARF-GEF family in secretion and endocytosis in polarised cells and into the targets of the drug brefeldinA in Arabidopsis (Teh and Moore 2007 Nature 448: 493-496).
TRANSGENE EXPRESSION TECHNOLOGIES
We have also developed the pOp/LhG4 and pOp/LhGR systems for spatial and temporal control of transgene expression in plants. These are based on a chimaeric transcription factor, LhG4, comprising a high-affinity DNA-binding mutant of the E. coli lac repressor fused to a transcription activation domain from the yeast Gal4 protein. This molecule activates transcription from the pOp promoter which is otherwise physiologically silent in transgenic plants. We are nearing the end of a programme that has generated a collection of lines expressing LhG4 under a series of defined promoters and enhancer traps and these can be used in conjunction with the pOp promoter to express genes of interest in many tissue- and cell-specific patterns. This system is of particular value if a gene of interest needs to be studied in a variety of selected cell types and especially where the expression of the transgene is likely to compromise plant viability or fertility. In recent years this system has been used by our collaborators and others to investigate various aspects of Arabidopsis biology including embryogenesis, cytokinin metabolism, and meristem control.
We have also fused the ligand-binding domain of the rat glucocorticoid receptor to LhG4 to generate a steroid-inducible molecule, LhGR, which provides temporal control over pOp promoter expression. The pOp/LhGR system in Arabidopsis exhibits lower levels of uninduced expression and none of the inhibitory side-effects that affect other inducible expression systems in plants. We have begun to generate lines that express LhGR under control of tissue-specific promoters so that genes of interest can be activated at defined times in specific cell types.
Publications (while at this department)
Kirchhelle, C., Chow, C.-M., Foucart, C., Neto, H., Stierhof, Y.-D., Kalde, M., Walton, C., Fricker, M., Smith, Richard S., Jérusalem, A., Irani, I., and Moore, I. (2016). The Specification of Geometric Edges by a Plant Rab GTPase Is an Essential Cell-Patterning Principle During Organogenesis in Arabidopsis. Developmental Cell 36, 386-400
Visscher AM, Belfield EJ, Vlad D, Irani N, Moore I, Harberd NP (2015) Overexpressing the Multiple-Stress Responsive Gene At1g74450 Reduces Plant Height and Male Fertility in Arabidopsis thaliana. PLoS ONE 10(10): e0140368. doi:10.1371/journal.pone.0140368
Au, K.K.C, Perez-Gomez, J, Neto, H, Muller, C, Meyer, A.J, Fricker, M.D, Moore, I. (2012) A perturbation in glutathione biosynthesis disrupts endoplasmic reticulum morphology and secretory membrane traffic in Arabidopsis thaliana Plant Journal. 71 (6): pp 881-894.
Boutte, Y, Frescatada-Rosa, M, Men, S, Chow, C.-M, Ebine, K, Gustavsson, A, Johansson, L, Ueda, T, Moore, I, Jurgens, G, Grebe, M. (2010) Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis EMBO Journal. 29 (3): pp 546-558.
da, Costa D.S, Pereira, S, Moore, I, Pissarra, J. (2010) Dissecting cardosin B trafficking pathways in heterologous systems Planta. 232 (6): pp 1517-1530.
Camacho, L, Smertenko, A.P, Perez-Gomez, J, Hussey, P.J, Moore, I. (2009) Arabidopsis Rab-E GTPases exhibit a novel interaction with a plasma-membrane phosphatidylinositol-4-phosphate 5-kinase Journal of Cell Science. 122 (23): pp 4383-4392.
Johansen, J.N, Chow, C.-M, Moore, I, Hawes, C. (2009) AtRAB-H1b and AtRAB-H1c GTPases, homologues of the yeast Ypt6, target reporter proteins to the Golgi when expressed in Nicotiana tabacum and Arabidopsis thaliana Journal of Experimental Botany. 60 (11): pp 3179-3193.
Moore, I, Murphy, A. (2009) Validating the location of fluorescent protein fusions in the endomembrane system Plant Cell. 21 (6): pp 1632-1636.
Pinheiro, H, Samalova, M, Geidner, N, Chory, J, Martinez, A, Moore, I. (2009) Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway Journal of Cell Science. 122 (20): pp 3749-3758.
Chow, CM, Neto, H, Foucart, C, Moore, I. (2008) Rab-A2 and Rab-A3 GTPases define a trans-golgi endosomal membrane domain in Arabidopsis that contributes substantially to the cell plate. Plant Cell. 20 (1): pp 101-23.
Duarte, P, Pissarra, J, Moore, I. (2008) Processing and trafficking of a single isoform of the aspartic proteinase cardosin a on the vacuolar pathway Planta. 227 (6): pp 1255-1268.
Samalova, M, Fricker, M, Moore, I. (2008) Quantitative and Qualitative Analysis of Plant Membrane Traffic Using Fluorescent Proteins Methods in Cell Biology. 85: pp 353-380.
Woollard, A.A, Moore, I. (2008) The functions of Rab GTPases in plant membrane traffic Current Opinion in Plant Biology..
Perez-Gomez, J, Moore, I. (2007) Plant Endocytosis: It Is Clathrin after All Current Biology. 17 (6):.
Teh, O.-K, Moore, I. (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature. 448 (7152): pp 493-496.
Fricker, M, Runions, J, Moore, I. (2006) Quantitative fluorescence microscopy: From art to science Annual Review of Plant Biology. 57: pp 79-107.
Moore, I, Samalova, M, Kurup, S. (2006) Transactivated and chemically inducible gene expression in plants Plant Journal. 45 (4): pp 651-683.
Samalova, M, Fricker, M, Moore, I. (2006) Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins Traffic. 7 (12): pp 1701-1723.
Baroux, C, Blanvillainh, R, Betts, H, Batoko, H, Craft, J, Martinez, A, Gallois, P, Moore, I. (2005) Predictable activation of tissue-specific expression from a single gene locus using the pOp/LhG4 transactivation system in Arabidopsis Plant Biotechnology Journal. 3 (1): pp 91-101.
Craft, J, Samalova, M, Baroux, C, Townley, H, Martinez, A, Jepson, I, Tsiantis, M, Moore, I. (2005) New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis Plant Journal. 41 (6): pp 899-918.
Rutherford, S, Brandizzi, F, Townley, H, Craft, J, Wang, Y, Jepson, I, Martinez, A, Moore, I. (2005) Improved transcriptional activators and their use in mis-expression traps in Arabidopsis Plant Journal. 43 (5): pp 769-788.
Samalova, M, Brzobohaty, B, Moore, I. (2005) pOp6/LhGR: A stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco Plant Journal. 41 (6): pp 919-935.
Wielopolska, A, Townley, H, Moore, I, Waterhouse, P, Helliwell, C. (2005) A high-throughput inducible RNAi vector for plants Plant Biotechnology Journal. 3 (6): pp 583-590.
Zheng, H, Camacho, L, Wee, E, Batoko, H, Legen, J, Leaver, C.J, Malho, R, Hussey, P.J, Moore, I. (2005) A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis Plant Cell. 17 (7): pp 2020-2036.
Kotzer, A.M, Brandizzi, F, Neumann, U, Paris, N, Moore, I, Hawes, C. (2004) AtRabF2b (Ara7) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells Journal of Cell Science. 117 (26): pp 6377-6389.
Swarup, R, Kargul, J, Marchant, A, Zadik, D, Rahman, A, Mills, R, Yemm, A, May, S, Williams, L, Millner, P, Tsurumi, S, Moore, I, Napier, R, Kerr, I.D, Bennett, M.J. (2004) Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1 Plant Cell. 16 (11): pp 3069-3083.
Zheng, H, Kunst, L, Hawes, C, Moore, I. (2004) A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus Plant Journal. 37 (3): pp 398-414
Betts, H, Moore, I. (2003) Plant Cell Polarity: The Ins-and-Outs of Sterol Transport Current Biology. 13 (19):.
Brandizzi, F, Saint-Jore, C, Moore, I, Hawes, C. (2003) The relationship between endomembranes and the plant cytoskeleton Cell Biology International. 27 (3): pp 177-179.
Moore, I. (2002) Gravitropism: Lateral thinking in auxin transport Current Biology. 12 (13):.
Rutherford, S, Moore, I. (2002) The Arabidopsis Rab GTPase family: Another enigma variation Current Opinion in Plant Biology. 5 (6): pp 518-528.
Saint-Jore, C.M, Evins, J, Batoko, H, Brandizzi, F, Moore, I, Hawes, C. (2002) Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks Plant Journal. 29 (5): pp 661-678.
Baroux, C, Blanvillain, R, Moore, I.R, Gallois, P. (2001) Transactivation of BARNASE under the AtLTP1 promoter affects the basal pole of the embryo and shoot development of the adult plant in Arabidopsis Plant Journal. 28 (5): pp 503-515.
Batoko, H, Moore, I. (2001) Plant cytokinesis: KNOLLE joins the club Current Biology. 11 (11):.
Batoko, H, Zheng, H.-Q, Hawes, C, Moore, I. (2000) A Rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for normal Golgi movement in plants Plant Cell. 12 (11): pp 2201-2217.
Galweiler, L, Conlan, R.S, Mader, P, Palme, K, Moore, I. (2000) The DNA-binding activity of Gal4 is inhibited by methylation of the Gal4 binding site in plant chromatin Plant Journal. 23 (1): pp 143-157.
Moore, I, Galweiler, L, Grosskopf, D, Schell, J, Palme, K. (1998) A transcription activation system for regulated gene expression in transgenic plants Proceedings of the National Academy of Sciences of the United States of America. 95 (1): pp 376-381.
Moore, I, Diefenthal, T, Zarsky, V, Schell, J, Palme, K. (1997) A homolog of the mammalian GTPase Rab2 is present in Arabidopsis and is expressed predominantly in pollen grains and seedlings Proceedings of the National Academy of Sciences of the United States of America. 94 (2): pp 762-767.
Moore, I. (1996) Methods for jacks-of-many-trades Trends in Plant Science. 1 (4): pp 130-131
Moore, I, Schell, J, Palme, K. (1995) Subclass-specific sequence motifs identified in Rab GTPases Trends in Biochemical Sciences. 20 (1): pp 10-12.
Brzobohaty, B, Moore, I, Palme, K. (1994) Cytokinin metabolism: implications for regulation of plant growth and development Plant Molecular Biology. 26 (5): pp 1483-1497.