Abstract

Wolbachia may be the most abundant and widespread intracellular symbiont as yet described. This unculturable, maternally inherited bacterium is able to invade and maintain itself in numerous arthropod host species by inducing a variety of reproductive alterations such as induction of parthenogenetic development in certain parasitic wasps, overriding chromosomal sex determination to convert infected genetic males into functional females in crustacea species (feminisation), and most commonly induction of cytoplasmic incompatibility (CI), a form of embryonic lethality in crosses between males and females of different Wolbachia infection status. This proposal aims to identify the bacterial and host genes involved in the induction of these reproductive phenotypes through an integrated genomics, proteomics and post-genomics approach. Identification of these genes will be a major breakthrough towards the goal of using them for applied purposes, for example management of arthropod agricultural pests and disease vectors or improvement of beneficial arthropods.

Objectives

The objectives of the project are:

• To identify Wolbachia genes that are involved in cytoplasmic incompatibility, parthenogenesis and feminisation, using an integrated genomics, proteomics and post-genomics approach.
• To identify host genes that are involved in host- Wolbachia interaction, using a post-genomics approach in Wolbachia -infected Drosophila melanogaster.
• To develop a robust genetic transformation system for Wolbachia, a technology that will enable further functional studies and genetic manipulation of the bacterium for applied purposes.

Description of the work

Candidate Wolbachia genes responsible for the induction of cytoplasmic incompatibility, parthenogenesis and feminisation will be identified initially through comparative genomics and proteomics analysis. For comparative genomics, the genome sequence of three Wolbachia strains, each inducing one of the phenotypes, will be determined through a shot-gun sequencing approach. For proteomics analysis, Wolbachia infected strains and their tetracycline-treated derivatives will be used as sources for protein extracts. Further identification of candidate genes responsible for the induction of cytoplasmic incompatibility will be achieved through comparative microarray-based expression profiling of Wolbachia strains which differ genetically in their ability to induce this phenotype.

Candidate host genes involved in the host- Wolbachia interaction will be identified by a combination of comparative microarray-based expression profiling and proteomics using infected and uninfected Drosophila strains.

An efficient genetic transformation system for Wolbachia will be developed using either homologous recombination or transposition based on Tc1/mariner transposable element vectors.

Involvement of the candidate Wolbachia genes in inducing the phenotypes will be tested by a gene knock-out approach. Candidate genes for the induction of cytoplasmic incompatibility will also be tested by gene complementation of Wolbachia mutants. In the possible absence of a Wolbachia transformation system, Wolbachia cytoplasmic incompatibility genes will be tested by using available germ-line transformation technology for Drosophila melanogaster to express these genes in Drosophila melanogaster tissues.

Deliverables

• The complete and annotated genome sequence of three Wolbachia.strains, each responsible for the induction of cytoplasmic incompatibility, parthenogenesis and feminisation.
• A genetic transformation system for Wolbachia.
• Identified and characterised Wolbachia genes involved in the induction of the three phenotypes.
• Identified and characterised host (Drosophila melanogaster) genes.

Contacts

Coordinator

• Kostas BOURTZIS / IMMB-FORTH GREECE
• Charalambos SAVAKIS / IMMB-FORTH GREECE
• Christos OUZOUNIS / IMMB-FORTH GREECE

Participant

• Artemis-Georgia HATZIGEORGIOU / Synaptic Ltd GREECE
• Gilbert MARTIN / Universite de Poitiers UMR CNRS 6556 / FRANCE
• Michael ASHBURNER / University of Cambridge Department of Genetics / UNITED KINGDOM
• Roger GARRETT / University of Copenhagen Institute of Molecular Biology / DENMARK
• Henk BRAIG / University of Wales Bangor School of Biological Sciences / UNITED KINGDOM
• Siv ANDERSSON / Uppsala University Department of Molecular Evolution / SWEDEN
• Uppsala University Department of Molecular Evolution / SWEDEN
• Richard STOUTHAMER / Wageningen University Department of Plant Sciences / NETHERLANDS