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How do ESF1 peptides regulate the Arabidopsis embryo apical-basal axis?

Principal Supervisor: Dr. Jose Gutierrez-Marcos - School of Life Sciences

Co-supervisor: Dr. Jozef Lewandowski - Department of Chemistry

PhD project title: How do ESF1 peptides regulate the Arabidopsis embryo apical-basal axis?

University of Registration: Warwick

Project outline:

Although much is known about the role of non-peptide hormones in regulating growth and development in animals and plants, relatively little is known about the significant role played by small signalling peptides in regulating such processes. Recently, an increasingly large number of genes encoding small, secreted peptides in animals and plants have been implicated in mediating cell–cell interactions. For example, there are demonstrated roles for peptide signals in defence responses, cell proliferation and differentiation, maintenance of stem cell identity, and in reproduction. Although both genetic and biochemical methods have thus far been used to identify and characterise such peptides, we remain ignorant of their biophysical properties and how their physical structure relates to function and performance. This project aims to define the molecular mechanisms by which a new class of peptide ligands (ESF1) regulate the formation of the apical-basal axis of plant embryos.

Specific objectives:

  1. Characterization of the transcriptional network that regulate ESF1 expression. This objective will identify the transcription factors implicated in the spatio-temporal expression of the peptides.
  2. Identification of the membrane receptors implicated in ESF1 recognition. This objective will use mass spectrometry to identify the proteins that interact with ESF1 ligands and create synthetic peptides to test their structure-function relationship.
  3. Characterization of the signalling cascade regulated by ESF1 peptides. This objective will employ CRISPR/Cas9 targeted genome editing to engineer mutations in the kinase cascade know to be implicated in ESF1 function.

Key experimental skills involved:

The student will carry out a series of experiments designed to determine the peptide structure and the interactions of ESF1 with transmembrane receptors. NMR will be used to determine protein size, shape and oligomeric state for ESF1-receptor oligomers and complexes formed. These methods would provide information of the dynamics of these proteins and their interactions with receptor proteins. Complementing these techniques, novel synthetic design of peptide ligands and receptors will be employed for in vivo and in vitro functional assays.

References:

  1. L. M. Costa, E. Marshall, M. Tesfaye, K. A. T. Silverstein, M. Mori, Y. Umetsu, S. L. Otterbach, R. Papareddy, H. G. Dickinson, K. Boutiller, K. A. VandenBosch, S. Ohki, J. F. Gutierrez-Marcos (2014) 'Central Cell-Derived Peptides Regulate Early Embryo Patterning in Flowering Plants', Science, 3244 (6180), 168 – 172
  2. Lewandowski, J.R., et al., Protein dynamics. Direct observation of hierarchical protein dynamics. Science, 2015. 348(6234): p. 578-81

BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

  • Plant genetic engineering
  • CRISPR/cas9 genome engineering
  • Confocal microscopy
  • Protein expression and purification
  • Immunoprecipitation
  • Protein identification by MS analysis
  • NMR structural determination

Contact: Dr Jose Gutierrez Marcos, University of Warwick