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

Abstract

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.

Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest

“…Promoters used to drive expression of the transgenes were ubiquist and constitutive. Although successful, the amount of metabolites produced in transgenic tobacco lines is usually relatively low (in the range of a few ng g 21 fresh weight; Lücker et al, 2004;Wei et al, 2004;Farhi et al, 2011) compared with the endogenous production of capsidiol (up to 100 mg g 21 fresh weight; Dokládal et al, 2012) or of diterpenes (up to 75 mg cm 22 depending on the variety; Severson et al, 1984). To be economically viable, the engineering of terpenoids in plants should reach yields comparable to those occurring naturally.…”

The effect of cryptogein with changed abilities to transfer sterols and altered charge distribution on extracellular alkalinization, ROS and NO generation, lipid peroxidation and LOX gene transcription in Nicotiana tabacum

“…As previously described (Dokl adal et al, 2012;Lochman et al, 2005), cryptogein binds these molecules with high affinity, whereas its protein variants can have these abilities altered. For instance, protein L19R(EAEA) binds fatty acids with higher affinity than cryptogein whereas it does not bind sterols (Lochman et al, 2005).…”

“…In water solutions, DHEs form micelles with no or a slight fluorescence due to their self-quenching effect. In the presence of the abundance of stigmasterol micelles, the addition of cryptogein stimulates a rapid increase in fluorescence because of the dilution of DHE in the stigmasterol micelles (Dokl adal et al, 2012). Protein L19R showed a lower (cca 1.6 times) ability to transfer DHE between micelles as compared to X24 ( Fig.…”

“…Cryptogein is composed of 98 amino acid residues with molecular weight 10 kDa (Ricci et al, 1989). This protein induces an intense defence response in tobacco consisting of a hypersensitive reaction (HR) that is characterized by the programmed cell death (PCD) at the site of infection and the development of a systemic acquired resistance (SAR) (Rust erucci et al, 1999;Dokl adal et al, 2012).…”

A Proteomic Approach to Evaluate the Effects of Endogenous Expression of Cryptogein Gene in Crypt-Transgenic Plants of Bacopa monnieri

“…Calcium influxes triggered by these kinases subsequently give rise to calcium-dependant transcriptional changes [14] and activate a wide array of cellular responses, including an NADPH oxidase-dependent oxidative burst [15,16]. Upregulation of several chitinases, peroxidases, peptidyl-prolyl isomerases and proteinase inhibitors in intercellular fluid of cryptogein-treated tobacco leaves were observed using a proteomic approach [17].…”