Expresní systém kvasinky Pichia pastoris se ve velkém měřítku používá na produkci rekombinantních proteinů již několik desetiletí. Pichia pastoris v sobě spojuje výhody eukaryotních i prokaryotních expresních systému. V rámci této práce byly v bioreaktoru produkovány mutantní formy proteinu cryptogeinu. Bylo vyprodukováno a purifikováno pět proteinů: X24, L41F, V84F, V84F/L41F a K13V s průměrnými výtěžky 40-60 mg proteinu na litr bazálního media. Buněčné hustoty na konci fermentace dosahovali 250-400 g/l.

Expression system Pichia pastoris has been used for decades to production of recombinant proteins in large scale. Pichia pastoris combines the advantages of eukaryotic and prokaryotic expression systems. In this work mutant forms of protein cryptogein were produced in the fermenter. Five proteins: X24, L41F, V84F, V84F/L41F and K13V were produced in the bioreactor and purified with average yields of 40-60 mg protein per liter of basal media. Cell density was reached 250-400 g/l at the end of fermentation.

• Klíčová slova
• růstová křivka, fermentor,
• MeSH
• bioreaktory MeSH
• fermentace * fyziologie   genetika   MeSH
• fungální proteiny * biosyntéza   genetika   MeSH
• kultivační média * MeSH
• mikrobiologické techniky MeSH
• Pichia genetika   metabolismus   růst a vývoj   MeSH
• proteomika * metody   MeSH
• rekombinantní proteiny * biosyntéza   genetika   MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• bílkoviny řas genetika   chemická syntéza   MeSH
• ergosterol analogy a deriváty   MeSH
• finanční podpora výzkumu jako téma MeSH
• mutageneze cílená MeSH
• Phytophthora genetika   patogenita   MeSH
• tabák mikrobiologie   MeSH

MAIN CONCLUSION: 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.

• 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

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

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.

• 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