Vladimír Mikeš

Are elicitins cryptograms in plant-Oomycete communications?

Abstract. Stimulation of plant natural defenses is an important challenge in phytoprotection prospects. In that context, elicitins, which are small proteins secreted by Phytophthora and Pythium species, have been shown to induce a hypersensitive-like reaction in tobacco plants. Moreover, these plants become resistant to their pathogens, and thus this interaction constitutes an excellent model to investigate the signaling pathways leading to plant resistance. However, most plants are not reactive to elicitins, although they possess the functional signaling pathways involved in tobacco responses to elicitin. The understanding of factors involved in this reactivity is needed to develop agronomic applications. In this review, it is proposed that elicitins could interact with regulating cell wall proteins before they reach the plasma membrane. Consequently, the plant reactivity or nonreactivity status could result from the equilibrium reached during this interaction. The possibility of overexpressing the elicitins directly from genomic DNA in Pichia pastoris allows site-directed mutagenesis experiments and structure/function studies. The recent discovery of the sterol carrier activity of elicitins brings a new insight on their molecular activity. This constitutes a crucial property, since the formation of a sterol-elicitin complex is required to trigger the biological responses of tobacco cells and plants. Only the elicitins loaded with a sterol are able to bind to their plasmalemma receptor, which is assumed to be an allosteric calcium channel. Moreover, Phytophthora and Pythium do not synthesize the sterols required for their growth and their fructification, and elicitins may act as shuttles trapping the sterols from the host plants. Sequence analysis of elicitin genes from several Phytophthora species sheds unexpected light on the phylogenetic relationships among the genus, and suggests that the expression of elicitins is under tight regulatory control. Finally, general involvement of these lipid transfer proteins in the biology of Pythiaceae, and in plant defense responses, is discussed. A possible scheme for the coevolution between Phytophthora and tobacco plants is approached.

A lipid transfer protein binds to a receptor involved in the control of plant defence responses

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.

The fungal elicitor cryptogein is a sterol carrier protein

Cryptogein is a protein secreted by the phytopathogenic pseudo‐fungus, Phytophthora cryptogea. It is a basic 10 kDa hydrophilic protein having a hydrophobic pocket and three disulfide bridges. These common features with sterol carrier proteins led us to investigate its possible sterol transfer activity using the fluorescent sterol, dehydroergosterol. The results show that cryptogein has one binding site with strong affinity for dehydroergosterol. Moreover, this protein catalyzes the transfer of sterols between phospholipidic artificial membranes. This is the first evidence for the existence of an extracellular sterol carrier protein and for a molecular activity of cryptogein. This property should contribute to an understanding of the role of cryptogein in plant‐microorganism interactions.

Fatty acids bind to the fungal elicitor cryptogein and compete with sterols

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid–cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

Elicitation of tobacco cells with ergosterol activates a signal pathway including mobilization of internal calcium

Abstract Ergosterol interacts with tobacco suspension ( Nicotiana tabacum ) cells and triggers pH changes of extracellular medium, oxidative burst and synthesis of phytoalexins. Compared with the responses induced by cryptogein, a proteinaceous elicitor from Phytophthora sp., oxidative burst and ΔpH changes were weaker whereas phytoalexin accumulation was higher with ergosterol. Cryptogein stimulated an apparent continuous uptake of external calcium within 40 min, whereas no net uptake of external calcium occurred upon the addition of ergosterol. However, the elicitation with both cryptogein and ergosterol resulted in an increase of the fluorescence of calcium green 1 in cytosol. The use of several inhibitors of calcium channels (La 3+ , TMB-8, verapamil, ruthenium red, nifedipine) and a protein-kinase inhibitor (staurosporin) suggests that the elicitation with ergosterol includes the mobilization of internal calcium stores mediated by inositol 1,4,5-trisphosphate and serine/threonine protein kinases.

Ergosterol treatment leads to the expression of a specific set of defence-related genes in tobacco

Ergosterol is the main sterol of most fungi. Production of reactive oxygen species after the treatment of tobacco and tomato cells by nano-molar concentrations of ergosterol was previously observed as well as the activation of some stress activated mitogen-activated protein kinases on alfalfa cells. In this paper, the expression of some defence-related genes after the ergosterol treatment of tobacco Nicotiana tabacum plants is reported. The gene expression of pathogenesis related proteins of families PR1, PR3, PR5 and proteinase inhibitors of class I and II together with enzymes participating in the defence response, such as phenylalanine-ammonia lyase and sesquiterpene cyclase, were monitored by RT-qPCR. In addition, the concentrations of salicylic acid, an important signalling molecule, increased in time due to the enzyme activation. On the other hand, ergosterol did not provoke tissue necrosis and the possible cross-talk between the signalling pathways of salicylate and jasmonate was observed. Collected data shows that ergosterol is able to activate the expression of a number of defence genes and could increase resistance against pathogens.

Cercospora beticola toxins. Use of fluorescent cyanine dye to study their effects on tobacco cell suspensions

Abstract The fluorescent dye 3,3′-diethylthiadicarbocyanine iodide [diS-C 2 -(5)] was used to observe plasmalemma transmembrane potential variations of tobacco cells treated with uncoupler (FCCP), respiratory inhibitors (azide and cyanide), and H + -ATPase inhibitors (DCCD and a carbanilate derivative). These chemicals induced an increase in fluorescence, indicating a dissipation of the transmembrane potential. The [diS-C 2 -(5)] was also used to study the effects of two Cercospora beticola toxins on tobacco cells. Changes in fluorescence of [diS-C 2 -(5)] suggested that these two toxins caused a dissipation of the transmembrane potential with a different magnitude whereas kinetics of their association with membranes were comparable.

Variations in rDNA ITS of Czech Armillaria species determined by PCR and HPLC.

We analysed 40 isolates of species Armillaria. borealis, A. cepistipes, A. gallica, A. mellea, A. ostoyae and A. tabescens, mostly collected in the Czech Republic, by PCR-RFLP of the ITS rRNA genes using the restriction endonucleases AluI, HinfI and MboI. Restriction fragments were analysed by ion-exchange high performance liquid chromatography which proved to be more useful informative, and less time-consuming than classical electrophoresis on agarose gel. The HPLC method enabled detection of some heterozygous strains. HinfI discriminated between all six species. Ten isolates were sequenced to confirm changes in restriction sites found by restriction analysis. Cluster analysis based on the restrictions patterns of restriction endonucleases AluI and HinfI divided the analysed species into three groups. The first and the most distant group contained all A. mellea isolates, the second group was formed by A. tabescens and the third group contained species A. borealis, A. cepistipes, A. gallica and A. ostoyae. The A. tabescens group was very homogenous regardless of the origin of isolates (Czech Republic, France and Finland).

Binding of fatty acids to β-cryptogein: Quantitative structure-activity relationships and design of selective protein mutants

Binding of fatty acids to cryptogein, the proteinaceous elicitor from Phytophthora, was studied by using molecular docking and quantitative structure-activity relationships analysis. Fatty acids bind to the groove located inside the cavity of cryptogein. The structure-activity model was constructed for the set of 27 different saturated and unsaturated fatty acids explaining 87% (81% cross-validated) of the quantitative variance in their binding affinity. The difference in binding between saturated and unsaturated fatty acids was described in the model by three electronic descriptors: the energy of the lowest unoccupied molecular orbital, the energy of the highest occupied molecular orbital, and the heat of formation. The presence of double bonds in the ligand generally resulted in stronger binding. The difference in binding within the group of saturated fatty acids was explained by two steric descriptors, i.e., ellipsoidal volume and inertia moment of length, and one hydrophobicity descriptor, i.e., lipophility. The developed model predicted strong binding for two biologically important molecules, geranylgeranyol and farnesol playing an important role in plant signaling as lipid anchors of some membrane proteins. Elicitin mutants selectively binding only one type of ligand were designed for future experimental studies.

Nonspecific Elicitation of Defense Reaction in Suspension Tobacco Cells by Elicitors from Armillaria

Ergosterol and chitin oligomers were detected in water extracts fromArmillaria gallica, A. cepistipes, A. tabescens, A. ostoyae andA. mellea containing as active components elicitors able to trigger early events of defense reaction in suspension tobacco cells. More virulent strains ofA. ostoyae andA. mellea had the same ability of elicitation as weak pathogensA. gallica, A. cepistipes, A. tabescens. The elicitation of the defense reaction early events by chitin oligomers was markedly enhanced by ergosterol probably due to the activation of several signal pathways.