Physiological and proteomic approaches to evaluate the role of sterol binding in elicitin-induced resistance

Cryptogein is a proteinaceous elicitor secreted by Phytophthora cryptogea that can induce resistance to P. parasitica in tobacco plants. On the basis of previous computer modelling experiments, by site-directed mutagenesis a series of cryptogein variants was prepared with altered abilities to bind sterols, phospholipids or both. The sterol binding and phospholipid transfer activities corresponded well with the previously reported structural data. Induction of the synthesis of reactive oxygen species (ROS) in tobacco cells in suspension and proteomic analysis of intercellular fluid changes in tobacco leaves triggered by these mutant proteins were not proportional to their ability to bind or transfer sterols and phospholipids. However, changes in the intercellular proteome corresponded to transcription levels of defence genes and resistance to P. parasitica and structure-prediction of mutants did not reveal any significant changes in protein structure. These results suggest, contrary to previous proposals, that the sterol-binding ability of cryptogein and its mutants, and the associated conformational change in the ω-loop, might not be principal factors in either ROS production or resistance induction. Nevertheless, the results support the importance of the ω-loop for the interaction of the protein with the high affinity binding site on the plasma membrane.