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Gene regulatory networks in germline development and crop fertility

Principal Supervisor: Professor David Twell - Department of Genetics

Co-supervisor: Ralf Schmid

PhD project title: Gene regulatory networks in germline development and crop fertility

University of Registration: Leicester

Project outline:

Despite the vital role of gametes in plant fertility, seed production and food security, we have limited knowledge of the mechanisms involved in their development. This research aims to uncover the mechanisms that underlie key decisions in plant gamete development and the important role of germline gene networks in plant and crop fertility.

Through screens in the genetic model Arabidopsis thaliana, we have identified several key regulators of male gamete development including the transcription factor DUO1, which is widely conserved and present in important food crop plants such as tomato and rice (Kim et al. 2008; Brownfield et al. 2009). Our work has established a new regulatory framework in plant germline development (Berger and Twell, 2011), and in recent BBSRC-funded work we have discovered a pair of novel zinc finger transcription factors (DAZ1 & DAZ2) that act downstream of DUO1. The DAZ1/DAZ2 proteins are predicted to act by repression of their targets by a mechanism involving the TOPLESS/TOPLESS-RELATED (TPL/TPR) family of co-repressors (Borg et al., 2014).

Project Aims & Description:

This project will explore the evolutionary conservation and mechanisms by which the DUO1-DAZ1/DAZ2 regulatory module coordinates male germ cell division and sperm differentiation in Arabidopsis and in the important crop species, tomato. Genetic and molecular analysis will be combined with comparative transcriptome analysis to uncover gene regulatory networks and their impact on sperm cell differentiation and plant fertility.The project seeks to establish the conservation of DAZ1/DAZ2 function in flowering plants, to identify DAZ1/DAZ2 target genes and to model how the DAZ1/DAZ2 regulon is integrated with the wider gene network under DUO1 control.

The research is expected to deliver novel information and tools of potential value in plant biotechnology and breeding applications such as hybrid seed production and the control of gene flow in transgenic crops.

Possible timeline:

Year 1. Construct novel duo1 and daz1/daz2 gene mutants from crops such as tomato and identify germline targets of the DUO1-DAZ1/DAZ2 regulon based on bioinformatic analysis of existing transcriptomic data.

Year 2. Complete genetic and phenotypic analysis of duo1 and daz1/daz2 gene mutants and establish in vitro/in vivo DNA binding assays for DAZ1/DAZ2.

Year 3. Devise and evaluate a network model for the contribution of DAZ1/DAZ2-targets to male germline development and analyse the contribution of selected targets by knockdown/overexpression studies.

Budget. The expected expenditure on the project will be covered by the £4.5K per year available from the MIBTP for 3 years. Any additional costs will be met by grant funding from a 3-year BBSRC-supported grant award (Value: £450K to DT from November, 2015)

References:

  1. Borg, M., Brownfield, L., Khatab, H., Sidorova, A., Lingaya, M. and Twell, D. (2011) The R2R3 MYB transcription factor DUO1 activates a male germline-specific regulon essential for sperm cell differentiation in Arabidopsis. Plant Cell 23:1-16
  2. Borg, M., Rutley, N., Kagale, S. Hamamura, Y., Gherghinoiu, M., Kumar, S., Sari, U., Esparza-Franco, MA., Sakamoto, W., Rozwadowski, K., Higashiyama, T. and Twell, D. (2014). An EAR-dependent regulatory module promotes male germ cell division and sperm fertility in Arabidopsis. Plant Cell 26:1-17
  3. Brownfield, L., Hafidh, S., Borg, M., Sidorova, A., Mori, T. and Twell, D. (2009) A plant germ cell-specific integrator of cell cycle progression and sperm specification PLoS Genet. 5: e1000430
  4. Kim, H.J., Oh, S-A., Brownfield, L., Ryu, H., Hwang, I., Twell, D*. and Nam, H-G*. (2008) Control of plant male germline proliferation by SCFFBL17 degradation of cell cycle inhibitors. Nature 455, 1134-1137
  5. Berger, F. and Twell, D. (2011) Germline specification and function in plants. Annu Rev Plant Biol 62:461-484.

BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

  • Comparative transcriptomics (microarray & RNA-seq data)
  • HMM-based protein and motif search bioinformatics
  • Integration of ‘omics’ data and network modelling
  • Fluorescence and confocal laser scanning microscopy
  • Protein-interaction (yeast 2-hybrid and bimolecular fluorescence complementation)
  • Plant transformation and transient gene expression assays
  • Gene expression manipulation by analysis of T-DNA knockout lines, overexpression and artificial microRNA based knockdown

Contact: Professor David Twell, University of Leicester