Novel methodology for improving cell biology of yeast for the investigation of interactions between Blumeria graminis and hosts.
S. YU (1), A. Henderson (1), A. Dawson (1), R. Woodruff (1), E. Lockyer (1), E. Read (1), G. Sritharan (1), M. Ryan (1), M. Sgroi (1), P. Ngou (1), Y. Liu (1), Y. Xiang (1), R. Zhang (1) (1) Imperial College London, United Kingdom

Candidate Secreted Effector Proteins (CSEPs) and their binding targets play a vital role in plant immunity. The aims of this study were to investigate the binding targets of barley powdery mildew CSEPs by using yeast two-hybrid (Y2H) screening and to discover which CSEPs can inhibit the growth of baker yeast. In order to perform Y2H screening successfully, yeast transformation efficiency has to be improved. The endocytic factors involved in yeast transformation was studied to determine novel high efficiency yeast transformation protocols, in particular modified LiAc/SS-DNA/PEG method. The cell growth signalling was shown to influence yeast endocytosis. When cell growth signalling is “on”, yeast endocytosis and vacuolar trafficking are promoted. Several factors in cell growth signalling pathway were shown to lead to an increased yeast transformation efficiency up to 100 times in comparison with the current published highest transformation efficiency. This indicates that there is a possibility of obtaining high yeast transformation efficiency from manipulating cell growth signalling and suggests that it is possible to achieve yeast transformation efficiencies up to 107 CFU per µg plasmid DNA and per 108 cells with a 14 Kb plasmid DNA, which eventually would facilitate more rapid and more efficient yeast two-hybrid screening. As for yeast growth inhibition, we have also tested whether CSEPs library inhibits yeast growth in inhibition screens. We adapted high-throughput LR-clonase reaction and yeast transformation protocols and obtained a yeast strain for each N-terminally GFP-tagged CSEP with an inducible expression system. This library allows for screening all the yeast strains simultaneously for growth performances under different conditions. This may lead to the identification of conserved eukaryotic cell targets of some of the powdery mildew effectors.    

Abstract Number: P7-221
Session Type: Poster