Dataset Information

Protocol for a Binary Choice Feeding Assay Using Adult, Axenic Drosophila.

ABSTRACT: This protocol is designed to prepare adult axenic Drosophila before monitoring their behavior in a two-choice feeding assay, where flies are confronted with an axenic versus a dead or alive bacteria-contaminated feeding solution. Several aspects of the procedure, including raising and aging flies in axenic conditions, starving adult flies, and composing feeding solutions, are detailed. The bacterium used in this protocol, Erwinia carotovora carotovora-15 ²¹⁴¹ (Ecc-15 ²¹⁴¹ ), is commonly used to decipher the mechanisms controlling host-pathogen interactions in the Drosophila model. For complete details on the use and execution of this protocol, please refer to Charroux et al. (2020).

SUBMITTER: Charroux B

PROVIDER: S-EPMC7756975 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Protocol for a Binary Choice Feeding Assay Using Adult, Axenic Drosophila.

Charroux Bernard B Royet Julien J

STAR protocols 20200925 3

This protocol is designed to prepare adult axenic Drosophila before monitoring their behavior in a two-choice feeding assay, where flies are confronted with an axenic versus a dead or alive bacteria-contaminated feeding solution. Several aspects of the procedure, including raising and aging flies in axenic conditions, starving adult flies, and composing feeding solutions, are detailed. The bacterium used in this protocol, Erwinia carotovora carotovora-15 2141</su ...[more]

PMID: 33377013

Similar Datasets

Project description:A precise mapping of pathogen-host interactions is essential for comprehensive understanding of the processes of infection and pathogenesis. The most frequently used techniques for interactomics are the yeast two-hybrid binary methodologies, which do not recapitulate the pathogen life cycle, and the tandem affinity purification mass spectrometry co-complex methodologies, which cannot distinguish direct from indirect interactions. New technologies are thus needed to improve the mapping of pathogen-host interactions. In the current study, we detected binary interactions between influenza A virus polymerase and host proteins during the course of an actual viral infection, using a new strategy based on trans-complementation of the Gluc1 and Gluc2 fragments of Gaussia princeps luciferase. Infectious recombinant influenza viruses that encode a Gluc1-tagged polymerase subunit were engineered to infect cultured cells transiently expressing a selected set of Gluc2-tagged cellular proteins involved in nucleocytoplasmic trafficking pathways. A random set and a literature-curated set of Gluc2-tagged cellular proteins were tested in parallel. Our assay allowed the sensitive and accurate recovery of previously described interactions, and it revealed 30% of positive, novel viral-host protein-protein interactions within the exploratory set. In addition to cellular proteins involved in the nuclear import pathway, components of the nuclear pore complex such as NUP62 and mRNA export factors such as NXF1, RMB15B, and DDX19B were identified for the first time as interactors of the viral polymerase. Gene silencing experiments further showed that NUP62 is required for efficient viral replication. Our findings give new insights regarding the subversion of host nucleocytoplasmic trafficking pathways by influenza A viruses. They also demonstrate the potential of our infectious protein complementation assay for high-throughput exploration of influenza virus interactomics in infected cells. With more infectious reverse genetics systems becoming available, this strategy should be widely applicable to numerous pathogens.

Dataset Information

Protocol for a Binary Choice Feeding Assay Using Adult, Axenic Drosophila.

Publications

Protocol for a Binary Choice Feeding Assay Using Adult, Axenic <i>Drosophila</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets