Dr Ian Moore
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.
1. INTRACELLULAR TRANSPORT
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).
2. 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.