Investigating mechanisms of hypovirulence and pathogenicity in the ascomycete plant pathogen Cryphonectria parasitica

The ascomycete plant pathogen, Cryphonectria parasitica , is the causative agent of chestnut blight in the American chestnut, Castanea dentata, which faced near eradication as it succumbed to what is now known as one of the top three deadliest forest diseases in the world. The later discovery of a hypovirulent (attenuated virulence) strain gave hope of recovery through mechanisms of biological control. C. parasitica has since developed into an extensive model system for studying the molecular mechanisms of plant pathogenic fungi and fungal virology. This work encompasses aspects of molecular tool development, hypovirus regulation and plant-pathogen interactions. We have improved utility of the only regulated promoter system available for use in C. parasitica with the addition of a multiple cloning site and a sister clone conferring neomycin resistance. This expression vector can now be utilized for regulated expression of any gene of interest and has potential for use in other fungal systems. Utilization of this expression vector has allowed for regulated expression of the CHV1/EP713 hypovirus. It is now possible to investigate the pleiotropic effects caused by hypovirus encoded proteins in the absence of viral RNA accumulation. In an attempt to identify potential virulence factors, we have identified two potential LysM effector proteins in C. parasitica, LM12 and LM83. The knockout of LM83 is lethal, indicating this may be an essential protein. The LysM protein LM12 has been identified to be a secreted, chitin-binding protein that results in increased fungal virulence on chestnut. Potential perception of fungal invasion occurs through recognition of LM12, which differs from previous characterizations of LysM effectors in biotrophic and hemibiotrophic fungi, suggesting differing roles for LysM effector proteins in necrotrophic pathogens. Additional characterization of a non-secreted LysM protein, LM69, has revealed the potential of additional roles of LysMs in general fungal development. These studies cover varying aspects of the hypovirus-fungus-plant model system of C. parasitica, contributing to the continuing efforts to utilize mycoviruses as biological control agents for modulation of fungal infections and further our understanding of the mechanisms of virulence in plant-pathogenic fungi.