Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence.

• Klíčová slova
• Cryptogein mutants, elicitors, fluidity, membrane order, plant defence, plasma membrane, reactive oxygen species, signalling.,
• MeSH
• Arabidopsis fyziologie   MeSH
• buněčná membrána metabolismus   fyziologie   MeSH
• fluidita membrány fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• fluorescenční spektrometrie MeSH
• konfokální mikroskopie MeSH
• nemoci rostlin MeSH
• odolnost vůči nemocem fyziologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• tabák fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• reaktivní formy kyslíku MeSH

Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although α- and β-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, β-elicitins (possessing 6-7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the α-isoforms (with only 1-3 lysine residues). To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of β-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein's charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins' movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

• Klíčová slova
• cryptogein, dimerization, lipid transfer proteins, lysine residues, movement, resistance,
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• fosfolipidy metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• imunita rostlin imunologie   MeSH
• kyselina chlorogenová analýza   MeSH
• listy rostlin genetika   imunologie   parazitologie   fyziologie   MeSH
• mutace MeSH
• nemoci rostlin imunologie   parazitologie   MeSH
• Phytophthora genetika   metabolismus   patogenita   MeSH
• proteiny genetika   metabolismus   MeSH
• proteomika metody   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rekombinantní proteiny MeSH
• seskviterpeny analýza   MeSH
• steroly metabolismus   MeSH
• tabák genetika   imunologie   parazitologie   fyziologie   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• capsidiol MeSH Prohlížeč
• cryptogein protein, Phytophthora cryptogea MeSH Prohlížeč
• elicitin, Phytophthora MeSH Prohlížeč
• fosfolipidy MeSH
• fungální proteiny MeSH
• kyselina chlorogenová MeSH
• proteiny MeSH
• reaktivní formy kyslíku MeSH
• rekombinantní proteiny MeSH
• seskviterpeny MeSH
• steroly MeSH