Colm Carraher
Plant & Food Research
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Publication
Featured researches published by Colm Carraher.
Insect Biochemistry and Molecular Biology | 2008
Renee Smart; Aidan Kiely; Morgan Beale; Ernesto Vargas; Colm Carraher; Andrew V. Kralicek; David L. Christie; Chen Chen; Richard D. Newcomb; Coral G. Warr
Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions. Immunocytochemistry experiments in Drosophila S2 cells imply that Or22a has seven transmembrane domains but that its membrane topology is opposite to that of GPCRs, with a cytoplasmic N-terminus and extracellular C-terminus. To investigate Or signalling mechanisms, we expressed Or43b in Sf9 and HEK293 cells, and show that inhibitors of heterotrimeric G proteins (GDP-beta-S), adenylate cyclase (SQ22536), guanylyl cyclase (ODQ), cyclic nucleotide phosphodiesterases (IBMX) and phospholipase C (U73122) have negligible impact on Or43b responses. Whole cell patching of Or43b/Or83b-transfected HEK293 cells revealed the opening of plasma membrane cation channels on addition of ligand. The response was blocked by lanthanum and by 2-APB, but not by Ruthenium red or SKF96365. Based on these data, we conclude that Drosophila Ors comprise a novel family of seven transmembrane receptors that in HEK293 cells signal by opening cation channels, through a mechanism that is largely independent of G proteins.
Chemical Senses | 2009
Melissa D. Jordan; Alisha Anderson; Doreen Begum; Colm Carraher; Astrid Authier; Sean D.G. Marshall; Aidan Kiely; Laurence N. Gatehouse; David R. Greenwood; David L. Christie; Andrew V. Kralicek; Stephen C. Trowell; Richard D. Newcomb
Moths recognize a wide range of volatile compounds, which they use to locate mates, food sources, and oviposition sites. These compounds are recognized by odorant receptors (OR) located within the dendritic membrane of sensory neurons that extend into the lymph of sensilla, covering the surface of insect antennae. We have identified 3 genes encoding ORs from the tortricid moth, Epiphyas postvittana, a pest of horticulture. Like Drosophila melanogaster ORs, they contain 7 transmembrane helices with an intracellular N-terminus, an orientation in the plasma membrane opposite to that of classical GPCRs. EpOR2 is orthologous to the coreceptor Or83b from D. melanogaster. EpOR1 and EpOR3 both recognize a range of terpenoids and benzoates produced by plants. Of the compounds tested, EpOR1 shows the best sensitivity to methyl salicylate [EC(50) = 1.8 x 10(-12) M], a common constituent of floral scents and an important signaling compound produced by plants when under attack from insects and pathogens. EpOR3 best recognizes the monoterpene citral to low concentrations [EC(50) = 1.1 x 10(-13) M]. Citral produces the largest amplitude electrophysiological responses in E. postvittana antennae and elicits repellent activity against ovipositing female moths. Orthologues of EpOR3 were found across 6 families within the Lepidoptera, suggesting that the ability to recognize citral may underpin an important behavior.
PLOS Genetics | 2012
Jérôme Albre; Marjorie A. Liénard; Tamara Sirey; Silvia Schmidt; Leah Tooman; Colm Carraher; David R. Greenwood; Christer Löfstedt; Richard D. Newcomb
Chemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation.
Insect Biochemistry and Molecular Biology | 2013
Pablo F. German; Selene van der Poel; Colm Carraher; Andrew V. Kralicek; Richard D. Newcomb
Insect olfactory receptors (ORs) are a novel family of ligand-gated cation channels that can respond to volatile organic compounds at low concentrations. They are involved in the detection of odorants associated with mate recognition, food localisation and predator avoidance. These receptors form a complex that is currently thought to contain at least two subunit members: the non-canonical Orco ion channel subunit and a ligand-binding receptor subunit. The integral membrane proteins SNMP1 and 2 are also associated with olfactory function, with SNMP1 required for cis-vaccinyl acetate reception in Drosophila melanogaster. In order to investigate protein-protein interactions among these membrane proteins we measured intermolecular Förster/Fluorescence Resonance Energy Transfer (FRET) in live insect cells by acceptor photobleaching. Fusion proteins containing Cyan Fluorescent Protein or Yellow Fluorescent Protein were produced using baculovirus-mediated expression in High Five™ cells. The majority of the recombinant products were of the expected size for the fusion proteins and located within intracellular membranes. We were able to show FRET efficiencies providing evidence for homomeric and heteromeric interactions of the ligand-binding OR, Or22a, and Orco (Or22a-Or22a, Or22a-Orco, Orco-Orco). There was no evidence for an interaction between SNMP1 and Orco or between SNMP2 and Orco or Or22a. However, fusion proteins of SNMP1 and Or22a did show an interaction by FRET, suggesting SNMP1 may interact with at least some insect olfactory receptor complexes. In summary, this study supports previously observed homomeric and heteromeric interactions between Orco and the ligand-binding OR, Or22a, and identifies a novel interaction between Or22a and SNMP1.
Insect Biochemistry and Molecular Biology | 2014
Jacob A. Corcoran; Melissa D. Jordan; Colm Carraher; Richard D. Newcomb
The development of rapid and reliable assays to characterize insect odorant receptors (ORs) and pheromone receptors (PRs) remains a challenge for the field. Typically ORs and PRs are functionally characterized either in vivo in transgenic Drosophila or in vitro through expression in Xenopus oocytes. While these approaches have succeeded, they are not well suited for high-throughput screening campaigns, primarily due to inherent characteristics that limit their ability to screen large quantities of compounds in a short period of time. The development of a practical, robust and consistent in vitro assay for functional studies on ORs and PRs would allow for high-throughput screening for ligands, as well as for compounds that could be used as novel olfactory-based pest management tools. Here we describe a novel method of utilizing human embryonic kidney cells (HEK293) transfected with inducible receptor constructs for the functional characterization of ORs in 96-well plates using a fluorescent spectrophotometer. Using EposOrco and EposOR3 from the pest moth, Epiphyas postvittana as an example, we generated HEK293 cell lines with robust and consistent responses to ligands in functional assays. Single-cell sorting of cell lines by FACS facilitated the selection of isogenic cell lines with maximal responses, and the addition of epitope tags on the N-termini allowed the detection of recombinant proteins in homogenates by western blot and in cells by immunocytochemistry. We thoroughly describe the methods used to generate these OR-expressing cell lines, demonstrating that they have all the necessary features required for use in high-throughput screening platforms.
Journal of Economic Entomology | 2008
Robert Edwards; Colm Carraher; Joanne Poulton; Manoharie Sandanayaka; Jacqui H. Todd; Shirley S. Dobson; Nicola Mauchline; Garry Hill; Cathy McKenna; Richard D. Newcomb
Abstract Three species of armored scale (Hemiptera: Diaspididae) are found on kiwifruit (Actinidia spp.) in New Zealand orchards: latania scale, Hemiberlesia lataniae (Signoret); greedy scale, Hemiberlesia rapax (Comstock); and oleander scale, Aspidiotus nerii (Bouché). Each of them is a quarantine pest in some of the markets to which New Zealand kiwifruit are exported. Adult females of the three species can be distinguished morphologically; however, the task is laborious when large numbers must be identified. Furthermore, it is not possible to distinguish among the immature stages. A DNA-based diagnostic using a multiplex polymerase chain reaction (PCR) method based on differences in the cytochrome oxidase I and II genes was developed to distinguish the three species. The test relies on the rapid isolation of amplifiable DNA by using a protease (prepGEM), followed by multiplex PCR using primers that distinguish the species at three or more nucleotide positions within cytochrome oxidase I and II, resulting in PCR products of characteristic size for each species. The test was validated in a double-blind experiment and then used to determine the relative distribution and abundance of the three species on leaves and fruit of ‘Hayward’ and ‘Hort16A’ kiwifruit across the dominant growing regions throughout New Zealand during the 2007 season. In total, 3,418 scale insects were identified to species level: 1,904 (56%) were latania scale; 1,473 (43%) were greedy scale; and 41 (1%) were oleander scale. Since the last survey in 1988, latania scale has displaced greedy scale as the dominant species of armored scale on Hayward kiwifruit in the North Island and was found for the first time in the South Island. Only a single latania scale was found on Hort16A fruit, consistent with previous reports of reduced rates of settlement on the fruit of this cultivar by latania scale compared with greedy scale.
Protein Expression and Purification | 2009
Yang Xun; Pierre Tremouilhac; Colm Carraher; Christoph Gelhaus; Kiyoshi Ozawa; Gottfried Otting; Nicholas E. Dixon; Matthias Leippe; Joachim Grötzinger; Andrew J. Dingley; Andrew V. Kralicek
Amoebapore A is a pore-forming protein produced by the pathogenic parasite Entamoeba histolytica, which causes human amoebic dysentery. The pore-forming activity of amoebapore A is regulated by pH-dependent dimerization, a prerequisite for membrane insertion and pore formation. Understanding of these important processes has been hampered by the cytotoxicity of amoebapore A, which prevents the production of this protein in cell-based expression systems. In this study, a protocol for the cell-free production of active recombinant amoebapore A is presented. Protein yields of approximately 500 microg/ml of cell-free reaction were achieved. Recombinant amoebapore A was purified using a three-step procedure. To facilitate the structural characterization of the dimeric and pore forms, we adapted the cell-free system to isotope label amoebapore A for NMR studies. The preliminary assignment of a 2D 1H-15N HSQC spectrum of a uniformly 13C/15N-labeled sample was achieved using a combinatorial selective 15N-labeling approach coupled with available 1H(N) chemical shift data, resulting in the unambiguous assignment of resonances from 55 of the 77 residues. To confirm these results and obtain the full sequence-specific assignments of the 2D 1H-15N HSQC spectrum, a 3D HNCA spectrum was recorded. These assignment data will be used to aid the characterization of amoebapore A dimer formation and membrane insertion.
Biosensors and Bioelectronics | 2018
Murugathas Thanihaichelvan; Leo A. Browning; Marissa P. Dierkes; Roger Martinez Reyes; Andrew V. Kralicek; Colm Carraher; Colleen Marlow; N. O. V. Plank
Easily fabricated random network carbon nanotube field-effect transistors (CNT-FETs) have benefitted from improved separation techniques to deliver CNTs with current formulations providing at least 99% semiconducting tube content. Amongst the most promising applications of this device platform are electronic biosensors, where the network conduction is affected through tethered probes such as aptamers which act as molecular scale electrostatic gates. However, the prevailing assumption that these biosensor devices would be optimized if metallic tubes were entirely eliminated has not been examined. Here, we show that metallic-semiconducting junctions in aptasensors are sensing hotspots and that their impact on sensing is heightened by the CNT networks proximity to percolation. First, we use a biased conducting AFM tip to gate a CNT-FET at the nanoscale and demonstrate that the strongest device response occurs when gating at metallic-semiconducting junctions. Second, we resolve the target sensitivity of an aptasensor as a function of tube density and show heightened sensitivity at densities close to the percolation threshold. We find the strongest sensing response where the 1% of metallic tubes generate a high density of metallic-semiconducting junctions but cannot form a percolated metallic path across the network. These findings highlight the critical role of metallic tubes in CNT-FET biosensor devices and demonstrate that network composition is an important variable to boost the performance of electronic biosensors.
Data in Brief | 2018
Murugathas Thanihaichelvan; Leo A. Browning; Marissa P. Dierkes; Roger Martinez Reyes; Andrew V. Kralicek; Colm Carraher; Colleen Marlow; N. O. V. Plank
This article presents the raw and analyzed data from a set of experiments performed to study the role of junctions on the electrostatic gating of carbon nanotube (CNT) network field effect transistor (FET) aptasensors. It consists of the raw data used for the calculation of junction and bundle densities and describes the calculation of metallic content of the bundles. In addition, the data set consists of the electrical measurement data in a liquid gated environment for 119 different devices with four different CNT densities and summarizes their electrical properties. The data presented in this article are related to research article titled “Metallic-semiconducting junctions create sensing hot-spots in carbon nanotube FET aptasensors near percolation” (doi:10.1016/j.bios.2018.09.021) [1].
Insect Biochemistry and Molecular Biology | 2015
Colm Carraher; Julie E. Dalziel; Melissa D. Jordan; David L. Christie; Richard D. Newcomb; Andrew V. Kralicek
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MacDiarmid Institute for Advanced Materials and Nanotechnology
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