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.

• MeSH
• bílkoviny řas * MeSH
• cholesterol metabolismus   MeSH
• ergosterol analogy a deriváty   metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• fungální proteiny chemie   metabolismus   MeSH
• kinetika MeSH
• liposomy MeSH
• Phytophthora metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bílkoviny řas * MeSH
• cholesterol MeSH
• cryptogein protein, Phytophthora cryptogea MeSH Prohlížeč
• dehydroergosterol MeSH Prohlížeč
• ergosterol MeSH
• fungální proteiny MeSH
• liposomy MeSH
• transportní proteiny MeSH

We prepared a series of cryptogein mutants, an elicitor from Phytophthora cryptogea, with altered abilities to bind sterols and fatty acids. The induction of the early events, i.e., synthesis of active oxygen species and pH changes, in suspension tobacco cells by these mutated proteins was proportional to their ability to bind sterols but not fatty acids. Although the cryptogein-sterol complex was suggested to be a form triggering a defense reaction in tobacco, some proteins unable to bind sterols induced the synthesis of active oxygen species and pH changes. The modeling experiments showed that conformational changes after the introduction of bulky residues into the omega loop of cryptogein resemble those induced by sterol binding. These changes may be necessary for the ability to trigger the early events by elicitins. However, the ability to stimulate necrosis in suspension tobacco cells and the expression of defense proteins in tobacco plants were linked neither to the lipid binding capacity nor to the capacity to provoke the early events. On the basis of these experiments and previous results, we propose that elicitins could stimulate two signal pathways. The first one induces necroses and the expression of pathogen-related proteins, includes tyrosine protein kinases and mitogen-activated protein kinases, and depends on the overall structure and charge distribution. The second type of interaction is mediated by phospholipase C and protein kinase C. It triggers the synthesis of active oxygen species and pH changes. This interaction depends on the ability of elicitins to bind sterols.

• MeSH
• bílkoviny řas chemická syntéza   genetika   metabolismus   toxicita   MeSH
• cirkulární dichroismus MeSH
• ergosterol analogy a deriváty   metabolismus   MeSH
• fenylalaninamoniaklyasa biosyntéza   MeSH
• fungální proteiny MeSH
• kvantitativní vztahy mezi strukturou a aktivitou MeSH
• mastné kyseliny metabolismus   MeSH
• metabolismus lipidů MeSH
• mutageneze cílená * MeSH
• mykotoxiny chemická syntéza   genetika   toxicita   MeSH
• NADPH-oxidasy biosyntéza   MeSH
• nemoci rostlin mikrobiologie   MeSH
• Phytophthora genetika   patogenita   MeSH
• počítačová simulace MeSH
• proteiny MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rekombinantní proteiny chemická syntéza   genetika   toxicita   MeSH
• rostlinné proteiny biosyntéza   MeSH
• tabák cytologie   enzymologie   metabolismus   mikrobiologie   MeSH
• vazba proteinů genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bílkoviny řas MeSH
• cryptogein protein, Phytophthora cryptogea MeSH Prohlížeč
• dehydroergosterol MeSH Prohlížeč
• elicitin, Phytophthora MeSH Prohlížeč
• ergosterol MeSH
• fenylalaninamoniaklyasa MeSH
• fungální proteiny MeSH
• mastné kyseliny MeSH
• mykotoxiny MeSH
• NADPH-oxidasy MeSH
• pathogenesis-related proteins, plant MeSH Prohlížeč
• proteiny MeSH
• reaktivní formy kyslíku MeSH
• rekombinantní proteiny MeSH
• rostlinné proteiny MeSH

Cryptogein, a protein from oomycete Phytophthora cryptogea, induces a hypersensitive cell death in Nicotiana tabacum. We prepared a new series of cryptogein mutant proteins with altered abilities to bind sterols and with altered charge distribution in the proteins. The effect of the mutations on the cryptogein ability to induce plant defence mechanisms associated with hypersensitive cell death were examined. Our results with new mutants support the previous findings that the sterol binding does not influence synthesis of ROS, cytosol acidification and development of leaf necrosis as these events seem to be more likely affected by the charge distribution and the overall protein structure. This hypothesis was also applicable on other mechanisms involved in the execution of plant cell death such as the NO generation, the stimulation of lipid peroxidation (determination of malondialdehyde and hydroxy fatty acids levels) and LOX gene transcription. In addition, the ability to bind sterols was found to serve not only for pathogen utilisation in its own metabolism but also to have an important function for the destabilization of plant membrane facilitating the pathogen spread inside the plant tissue as well as intensively contributing to the development of plant cell death. Considering the insertion of charged amino acid residues in the protein structure, the change localized in the protein surface affected its biological activity more effectively than that change inside the protein cavity. Moreover, the insertion of negative charged amino acids influenced mainly the events involved in the early phase of defence reaction, while the positive residues affected especially the necrotic activity of cryptogein.

• Klíčová slova
• Cryptogein, Hydroxy fatty acids analysis, Hypersensitive response, Lipid peroxidation, Mutant proteins, Nicotiana tabaccum,
• MeSH
• buněčná membrána metabolismus   MeSH
• buněčná smrt MeSH
• extracelulární prostor MeSH
• fungální proteiny metabolismus   MeSH
• genetická transkripce MeSH
• koncentrace vodíkových iontů MeSH
• listy rostlin genetika   metabolismus   mikrobiologie   MeSH
• mastné kyseliny metabolismus   MeSH
• nemoci rostlin mikrobiologie   MeSH
• oxid dusnatý metabolismus   MeSH
• peroxidace lipidů MeSH
• Phytophthora patogenita   fyziologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• steroly metabolismus   MeSH
• tabák genetika   metabolismus   mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cryptogein protein, Phytophthora cryptogea MeSH Prohlížeč
• fungální proteiny MeSH
• mastné kyseliny MeSH
• oxid dusnatý MeSH
• reaktivní formy kyslíku MeSH
• rostlinné proteiny MeSH
• steroly MeSH

Elicitins are low-molecular-weight proteins representing the elicitor family secreted by many species of the oomycete Phytophthora. Elicitins induce a hypersensitive reaction in tobacco, a process that is triggered by binding of elicitin to the high-affinity site on the plasma membrane. Specific interaction of cryptogein with the binding sites on tobacco plasma membranes was studied using the piezoelectric biosensor in real time in a flow-through mode. Cryptogeins (wild-type and mutant forms) were covalently immobilized on the sensing surface, and membrane vesicles containing receptors were in solution. Kinetic characterization of the interaction provided values of kinetic rate association (k(a))=5.74 . 10(6)M(1)s(-1) and kinetic rate dissociation (k(d))=6.8710(-4)s(-1) constants, respectively. The kinetic equilibrium dissociation constant was calculated as K(D)=12.0 nM. The piezoelectric biosensor appeared to be a convenient tool for studying interactions of receptors embedded in membrane vesicles.

• MeSH
• bílkoviny řas analýza   metabolismus   MeSH
• biosenzitivní techniky metody   MeSH
• buněčná membrána metabolismus   MeSH
• fungální proteiny MeSH
• membránové proteiny analýza   metabolismus   MeSH
• Phytophthora metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• tabák metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bílkoviny řas MeSH
• cryptogein protein, Phytophthora cryptogea MeSH Prohlížeč
• fungální proteiny MeSH
• membránové proteiny MeSH
• rostlinné proteiny MeSH

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.

• MeSH
• bílkoviny řas chemie   MeSH
• fungální proteiny MeSH
• konformace proteinů MeSH
• kvantitativní vztahy mezi strukturou a aktivitou * MeSH
• ligandy MeSH
• mastné kyseliny chemie   MeSH
• mutace MeSH
• substituce aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bílkoviny řas MeSH
• cryptogein protein, Phytophthora cryptogea MeSH Prohlížeč
• fungální proteiny MeSH
• ligandy MeSH
• mastné kyseliny 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

The level of resistance induced in different tomato genotypes after β-CRY treatment correlated with the upregulation of defence genes, but not sterol binding and involved ethylene and jasmonic acid signalling. Elicitins, a family of small proteins secreted by Phytophthora and Pythium spp., are the most well-known microbe-associated molecular patterns of oomycetes, a lineage of fungus-like organisms that include many economically significant crop pathogens. The responses of tomato plants to elicitin INF1 produced by Phytophthora infestans have been studied extensively. Here, we present studies on the responses of three tomato genotypes to β-cryptogein (β-CRY), a potent elicitin secreted by Phytophthora cryptogea that induces hypersensitive response (HR) cell death in tobacco plants and confers greater resistance to oomycete infection than acidic elicitins like INF1. We also studied β-CRY mutants impaired in sterol binding (Val84Phe) and interaction with the binding site on tobacco plasma membrane (Leu41Phe), because sterol binding was suggested to be important in INF1-induced resistance. Treatment with β-CRY or the Val84Phe mutant induced resistance to powdery mildew caused by the pathogen Pseudoidium neolycopersici, but not the HR cell death observed in tobacco and potato plants. The level of resistance induced in different tomato genotypes correlated with the upregulation of defence genes including defensins, β-1,3-glucanases, heveins, chitinases, osmotins, and PR1 proteins. Treatment with the Leu41Phe mutant did not induce this upregulation, suggesting similar elicitin recognition in tomato and tobacco. However, here β-CRY activated ethylene and jasmonic acid signalling, but not salicylic acid signalling, demonstrating that elicitins activate different downstream signalling processes in different plant species. This could potentially be exploited to enhance the resistance of Phytophthora-susceptible crops.

• Klíčová slova
• Defence-related genes, Elicitins, Pseudoidium neolycopersici, Resistance, Signalling, Sterol binding,
• MeSH
• cyklopentany metabolismus   MeSH
• ethyleny metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• interakce hostitele a patogenu MeSH
• kyselina salicylová metabolismus   MeSH
• listy rostlin metabolismus   mikrobiologie   MeSH
• nemoci rostlin mikrobiologie   MeSH
• oxylipiny metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• Phytophthora MeSH
• Pythium MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• signální transdukce * MeSH
• Solanum lycopersicum metabolismus   mikrobiologie   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cryptogein protein, Phytophthora cryptogea MeSH Prohlížeč
• cyklopentany MeSH
• ethylene MeSH Prohlížeč
• ethyleny MeSH
• fungální proteiny MeSH
• jasmonic acid MeSH Prohlížeč
• kyselina salicylová MeSH
• oxylipiny MeSH
• peroxid vodíku MeSH
• reaktivní formy kyslíku MeSH
• regulátory růstu rostlin MeSH

Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins.

• Klíčová slova
• ATPase, Cryptogein, Glutamine synthetase, NADP-malic enzyme, Rab8, Resistance,
• MeSH
• buněčná membrána metabolismus   MeSH
• chromatografie kapalinová MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fungální proteiny genetika   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• PAMP struktury metabolismus   MeSH
• Phytophthora fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tabák genetika   metabolismus   mikrobiologie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• trypsin chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fungální proteiny MeSH
• membránové proteiny MeSH
• PAMP struktury MeSH
• rostlinné proteiny MeSH
• trypsin MeSH

Some phytopathogenic fungi within Phytophthora species are unable to synthesize sterols and therefore must pick them up from the membranes of their host-plant, using an unknown mechanism. These pseudo-fungi secrete elicitins which are small hydrophilic cystein-rich proteins. The results show that elicitins studied interact with dehydroergosterol in the same way, but with some time-dependent differences. Elicitins have one binding site with a similar strong affinity for dehydroergosterol. Using a non-steroid hydrophobic fluorescent probe, we showed that phytosterols are able to similarly bind to elicitins. Moreover, elicitins catalyze sterol transfer between phospholipidic artificial membranes. Our results afford the first evidence for a molecular activity of elicitins which appears to be extracellular sterol carrier proteins. This property should contribute to an understanding of the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions, since elicitins trigger defence reactions in plants.

• MeSH
• bílkoviny řas * MeSH
• biologický transport fyziologie   MeSH
• ergosterol analogy a deriváty   metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• fosfolipidy metabolismus   MeSH
• fungální proteiny metabolismus   fyziologie   MeSH
• fytosteroly metabolismus   MeSH
• kinetika MeSH
• naftalensulfonany metabolismus   MeSH
• Phytophthora chemie   MeSH
• rostliny mikrobiologie   MeSH
• transportní proteiny metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• vazebná místa fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 2-(4-toluidino)-6-naphthalenesulfonic acid MeSH Prohlížeč
• Alpha-elicitin capsicein protein, Phytophthora capsici MeSH Prohlížeč
• bílkoviny řas * MeSH
• cryptogein protein, Phytophthora cryptogea MeSH Prohlížeč
• dehydroergosterol MeSH Prohlížeč
• ergosterol MeSH
• fluorescenční barviva MeSH
• fosfolipidy MeSH
• fungální proteiny MeSH
• fytosteroly MeSH
• naftalensulfonany MeSH
• transportní proteiny MeSH

Elicitins are proteinaceous elicitors that induce the hypersensitive response and plant resistance against diverse phytopathogens. Elicitin recognition by membrane receptors or high-affinity sites activates a variety of fast responses including the production of reactive oxygen species (ROS) and nitric oxide (NO), leading to induction of plant defense genes. Beta-cryptogein (CRY) is a basic β-elicitin secreted by the oomycete Phytophthora cryptogea that shows high necrotic activity in some plant species, whereas infestin 1 (INF1) secreted by the oomycete P. infestans belongs to acidic α-elicitins with a significantly weaker capacity to induce necrosis. We compared several mutated forms of β-CRY and INF1 with a modulated capacity to trigger ROS and NO production, bind plant sterols and induce cell death responses in cell cultures of Nicotiana tabacum L. cv. Xanthi. We evidenced a key role of the lysine residue in position 13 in basic elicitins for their biological activity and enhancement of necrotic effects of acidic INF1 by the replacement of the valine residue in position 84 by larger phenylalanine. Studied elicitins activated in differing intensity signaling pathways of ROS, NO and phytohormones jasmonic acid, ethylene and salicylic acid, known to be involved in triggering of hypersensitive response and establishment of systemic resistance.

• Klíčová slova
• Cryptogein, Elicitins, Infestin, Nitric oxide, Plant defense responses, Reactive oxygen species,
• MeSH
• bílkoviny řas genetika   MeSH
• dusík * MeSH
• fungální proteiny metabolismus   MeSH
• kyslík MeSH
• Phytophthora * MeSH
• reaktivní formy kyslíku MeSH
• rostliny metabolismus   MeSH
• sekvence aminokyselin MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas MeSH
• dusík * MeSH
• fungální proteiny MeSH
• kyslík MeSH
• reaktivní formy kyslíku MeSH