scholarly journals Functional characterization of odor responses and gene expression changes in olfactory co-receptor mutants in Drosophila

2021 ◽  
Author(s):  
Alina Vulpe ◽  
Pratyajit Mohapatra ◽  
Karen Menuz
Keyword(s):  
Gene Expression ◽  
Olfactory System ◽  
Transcriptional Profiling ◽  
Functional Characterization ◽  
Olfactory Receptors ◽  
Odorant Receptors ◽  
Olfactory Neurons ◽  
Individual Odor ◽  
Receptor Neurons

Two large families of olfactory receptors, the Odorant Receptors (ORs) and the Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odor binding tuning OR receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco to function. The situation for IRs is more complex. Different tuning IR receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a co-receptor, but its function has not been systematically examined. This is particularly important as recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. Here, we report the effects of Drosophila olfactory co-receptor mutants on odor detection by coeloconic olfactory neurons and determine their broader impact on gene expression through RNASeq analysis. We demonstrate that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses, whereas in ac1 sensilla, amine responses persist in the absence of Ir76b or Ir25a, but are lost in a double-mutant. Such responses do not require Ir8a. Conversely, acid-sensing ORNs require Ir8a, but not Ir76b or Ir25a. Using antennal transcriptional profiling, we find that the expression of acid-sensing IR receptors is significantly reduced in Ir8a mutants, but is unaffected by the loss of Ir25a or Ir76b. Similarly, select OR tuning receptors are also downregulated in Orco2 mutants. In contrast, expression of amine-sensing IR receptors is mostly unchanged in Ir25a and Ir76b mutants. Together, our data reveal new aspects of co-receptor function in the olfactory system.

scholarly journals Ir76b is a Co-receptor for Amine Responses in Drosophila Olfactory Neurons

2021 ◽  
Vol 15 ◽  
Author(s):  
Alina Vulpe ◽  
Karen Menuz
Keyword(s):  
Olfactory Receptor ◽  
Olfactory Receptors ◽  
Olfactory Receptor Neurons ◽  
Odorant Receptors ◽  
Olfactory Neurons ◽  
Bona Fide ◽  
Receptor Neurons ◽  
Large Families ◽  
Odorant Binding

Two large families of olfactory receptors, the Odorant Receptors (ORs) and Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odorant binding “tuning” OrX receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco. The situation for IRs is more complex. Different tuning IrX receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a co-receptor, but its function has not been systematically examined. Surprisingly, recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. Here, we demonstrate that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses. In contrast, amine responses persist in the absence of Ir76b or Ir25a in ac1 sensilla but are lost in a double mutant. We show that responses mediated by acid-sensing neurons do not require Ir76b, despite their expression of this co-receptor. Our study also demonstrates that one population of coeloconic olfactory neurons exhibits Ir76b/Ir25a-dependent and Orco-dependent responses to distinct odorants. Together, our data establish the role of Ir76b as a bona fide co-receptor, which acts in partnership with Ir25a. Given that these co-receptors are among the most highly conserved olfactory receptors and are often co-expressed in chemosensory neurons, our data suggest Ir76b and Ir25a also work in tandem in other insects.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Functional Characterization of a Novel Promoter Element Required for an Innate Immune Response in Drosophila

2003 ◽  
Vol 23 (22) ◽  
pp. 8272-8281 ◽  
Author(s):  
Hanna Uvell ◽  
Ylva Engström
Keyword(s):  
Gene Expression ◽  
Antimicrobial Peptide ◽  
Functional Characterization ◽  
Binding Activity ◽  
Innate Immune ◽  
Site Directed Mutagenesis ◽  
Promoter Element ◽  
Peptide Gene ◽  
Inducible Genes

ABSTRACT Innate immune reactions are crucial processes of metazoans to protect the organism against overgrowth of faster replicating microorganisms. Drosophila melanogaster is a precious model for genetic and molecular studies of the innate immune system. In response to infection, the concerted action of a battery of antimicrobial peptides ensures efficient killing of the microbes. The induced gene expression relies on translocation of the Drosophila Rel transcription factors Relish, Dif, and Dorsal to the nucleus where they bind to κB-like motifs in the promoters of the inducible genes. We have identified another putative promoter element, called region 1 (R1), in a number of antimicrobial peptide genes. Site-directed mutagenesis of the R1 site diminished Cecropin A1 (CecA1) expression in transgenic Drosophila larvae and flies. Infection of flies induced a nuclear R1-binding activity that was unrelated to the κB-binding activity in the same extracts. Although the R1 motif was required for Rel protein-mediated CecA1 expression in cotransfection experiments, our data argue against it being a direct target for the Drosophila Rel proteins. We propose that the R1 and κB motifs are targets for distinct regulatory complexes that act in concert to promote high levels of antimicrobial peptide gene expression in response to infection.

Novel biochemical and functional insights into nuclear Ca2+ transport through IP3Rs and RyRs in osteoblasts

2000 ◽  
Vol 278 (5) ◽  
pp. F784-F791 ◽  
Author(s):  
Olugbenga A. Adebanjo ◽  
Gopa Biswas ◽  
Baljit S. Moonga ◽  
Hindupur K. Anandatheerthavarada ◽  
Li Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Ryanodine Receptors ◽  
Functional Characterization ◽  
Inositol Trisphosphate Receptors ◽  
Nuclear Membranes ◽  
Sds Page ◽  
Direct Regulation

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab34) and anti-IP3R (Ab40) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab40, anti-RyR-1, anti-RyR-2 (Ab129), and anti-RyR-3 (Ab180). Only anti-RyR-1 and Ab40 showed bands corresponding, respectively, to full-length RyR-1 (∼500 kDa) and IP3R-1 (∼250 kDa). Band intensity was reduced by just ∼20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+ concentration ([Ca2+]np) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2+ via Ca2+-ATPase activation (1 mM ATP and ∼100 nM Ca2+). Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3 or cADP ribose resulted in a rapid and sustained [Ca2+]np elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+ influx in osteoblasts through nuclear membrane-resident IP3Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+pathway.

Sign in / Sign up

Export Citation Format

Share Document