Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride

Gα subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus

"Silencing" von drei G-Protein gekoppelten Rezeptoren des Biokontrollpilzes Trichoderma atroviride und Charakterisierung der Transformanten

Zsfassung in engl. SpracheTrichoderma-Stämme werden in der Landwirtschaft als Biokontrollorganismen gegen pflanzenpathogene Pilze wie R. solani, P.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.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.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.atroviride, gpr1, gpr2 and gpr3, which are members of the class V cAMP sensing receptors, were cloned and characterized.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.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.5

Microbial Cell / Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

Oxidative folding of secretory proteins in the endoplasmic reticulum (ER) is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS) caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG) in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant) proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity