Delic, M. (2007). “Silencing” von drei G-Protein gekoppelten Rezeptoren des Biokontrollpilzes Trichoderma atroviride und Charakterisierung der Transformanten [Diploma Thesis, Technische Universität Wien]. reposiTUm. https://resolver.obvsg.at/urn:nbn:at:at-ubtuw:1-17387
Trichoderma-Stämme werden in der Landwirtschaft als Biokontrollorganismen gegen pflanzenpathogene Pilze wie R. solani, P.<br />ultimum, S. sclerotiorum, B. cinerea und F. graminearum eingesetzt. Sie werden durch ihr chemotrophes Wachstum, wirtsspezifische Erkennung, Formation von Appressorien-ähnlichen Strukturen, Produktion von lytischen Enzymen und Antibiotika charakterisiert. G-Protein gekoppelte Rezeptoren, welche in der Signalübertragung von externen Stimuli beteiligt sind, könnten wichtig für die Wirtserkennung wichtig sein. Bis jetzt konnten drei G-Protein gekoppelte Rezeptoren von T. atroviride (gpr1, gpr2, gpr3), welche der Klasse V cAMP Rezeptoren angehören, kloniert und charakterisiert werden.<br />In dieser Arbeit wurden diese drei Rezeptoren durch Silencing herunterreguliert, die Transformanten durch PCR identifiziert und die Silencingrate durch Real-Time PCR bestimmt. Im Anschluss wurden Konfrontationsversuche der Transformanten sowie des Wildtyps gegen verschiedene Pathogene und eine davon unabhängige Gesamtproteinbestimmung durchgeführt.<br />
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Trichoderma species are used as biocontrol agents in agriculture against a range of plant pathogenic fungi, such as Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Rhizoctonia solani or Pythium ultimum. The characteristics that definde a biological agent such as Trichoderma spp., are chemotrophic growth, host-specific recognition, formation of appressoria-like struktures (infection structures), production of lytic enzymes (e.g. chitinases, glucanases, proteases) and antibiotics (e.g. gliovirin, peptaibols). The cooperation of all these processes leads to growth inhibition and finally to the lysis of the host. The G-protein coupled receptors, which are involved in the signaling of extern stimuli, might be important for the initial host identification. Until now three G-protein coupled receptors from T.<br />atroviride, gpr1, gpr2 and gpr3, which are members of the class V cAMP sensing receptors, were cloned and characterized.<br />As numerous experiments to knock-out these receptors failed, a new strategy had to be found. For this diploma thesis, silencing was the method of choice. After constuction of stem-loop-stem RNA the inverted repeat of each receptor gene was ligated into the silencing vector pSILENT2 and finally transformed into the genome of the biocontrol fungus T. atroviride P1. As a result of a protoplast transformation, several colonies were identified as trasformants with PCR by using specific primers. After silencing the genes gpr1, gpr2 and gpr3, the decrease of gene expression was analyzed by real-time PCR in comparison to a reference gene. Finally, the identified transformants were characerized by the confrontation with pathogenic fungi B. cinerea, S.<br />sclerotiorum, A. nidulans, R. solani and P. ultimum and separate cultivation in liquid media with different carbon sources for protein analysis. During these experiments a phenotypic difference of the gpr1 transformants to the wildtype T. atroviride was assessed. These transformants showed also a different behaviour to the wildtype durng the confrontation experiments with pathogens.<br />