Host sensing with T. atroviride G protein-coupled receptors

Trichoderma atroviride is a mycoparasitic fungus commercially applied as biological control agent against several fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and killing of the host through production of specialised infection structures, cell wall lysing enzymes, and antifungal metabolites. Investigations on the underlying signal transduction pathways of T. atroviride and T. virens revealed that alpha subunits of heterotrimeric G proteins, which are responsible for transducing signals from transmembrane G protein- coupled receptors (GPCRs) to a variety of intracellular targets, are involved in host recognition and activation of the mycoparasitic response. Besides their role in the regulation of general growth properties and conidiation, Tga1 and Tga3, the subgroup I and III G alpha subunits of T. atroviride, were shown to directly affect mycoparasitism-related processes like chitinase gene transcription, antifungal metabolite production, and formation of infection structures. Extracellular signals such as diffusible host-derived molecules or lectins present on the host surface were previously shown to induce enzyme production and infection structure formation (coiling, appresoria) in Trichoderma, but until now the involved receptors transducing these host-derived signals to intracellular signalling pathways have not been identified. Based on the fundamental role of G protein signalling in mycoparasitism, the aim of the proposed project is the isolation of GPCRs from T. atroviride and the elucidation of their functions by generating respective knockout mutants. Furthermore, by applying the yeast Two-Hybrid system direct interactions of the isolated GPCRs with the Trichoderma G protein alpha and beta subunits Tga1, Tga2, Tga3, and Tgb1 shall be identified and for those receptors which are mycoparasitism-related additional interaction partners shall be screen for. As the knowledge on Trichoderma signalling pathways involved in transducing host-derived signals is very limited, the isolation and characterisation of the responsible receptors being on the top of the respective signalling cascade and the identification of their interaction partners would be a milestone for a better understanding of the processes underlying mycoparasitism in this important biocontrol fungus. As Trichoderma also interacts with the plant, the expected results could lead to establishment of Trichoderma as a model for reciprocal interactions between the three participants Trichoderma-host-plant.

Aim of this project was the analysis of G protein-coupled receptors (GPCRs) of the mycoparasitic fungus Trichoderma atroviride. To this end, the genomic repertoire of genes encoding GPCRs of T. Atroviride was analysed and selected GPCRs were functionally characterised. These experiments led to the identification of Gpr1 as the first receptor of a mycoparasitic fungus which is essential for recognizing phytopathogenic host fungi and for governing mycoparasitism-related processes. Beneficial fungi of the genus Trichoderma are commercially applied as biocontrol agents to guard plants against fungal diseases, thereby representing a promising alternative to chemical fungicides. Besides directly interacting with the plant itself in a beneficial way, Trichoderma is able to kill phytopathogenic fungi by a process called mycoparasitism. During the mycoparasitic interaction, Trichoderma specifically recognizes the host fungus and transduces host-derived signals via intracellular signalling pathways resulting in the activation of mycoparasitism-related processes such as the formation of infection structures, the secretion of hydrolytic enzymes, and the production of antifungal metabolites (antibiotics, toxins). Investigations on these signaling pathways of T. atroviride revealed an essential role of heterotrimeric G protein and cAMP signaling during the interaction with and attack of the host fungus. To extend our knowledge on G protein signaling, the genomic repertoire of T. atroviride was analyzed resulting in the identification and systematization of more than 50 putative GPCRs. In order to find out if these receptors are involved in recognizing the presence of host fungi, selected GPCR-encoding genes were functionally characterized by analyzing their expression and generating respective knock-down mutants of Trichoderma. Among the GPCRs analyzed concerning their role in mycoparasitism, only knock-down of gpr1 led to avirulent mutants unable to recognize and attach to host hyphae. Furthermore, gpr1 knock-down mutants were deficient in producing antifungal metabolites and in responding to the presence of living host fungi with the production of hydrolytic enzymes such as chitinases and proteases. Further analyses suggested that the Gpr1 receptor is relevant for transducing host-derived signals via the cAMP pathway resulting in mycoparasitism-related infection structure formation. Although gpr1 mutants show defects similar to those of mutants missing the Tga1 or Tga3 G-alpha proteins, no direct interaction between Gpr1 and any of the three G-alpha proteins of T. atroviride could be proven; instead we found novel putative Gpr1 interactors. The results on receptors governing host recognition and mycoparasitism during Trichoderma biocontrol obtained in the frame of this project help to understand the mode of action of this fungus and, in a long-term perspective, may contribute to improvement of Trichoderma as biocontrol agent.