Dissecting the role of Cladosporium fulvum (secreted) proteases and protease inhibitors

In order to facilitate infection, fungal pathogens produce various types of secreted proteases likely to target and perturb important plant proteins that are involved in controlling basal defense. In addition, they secrete several protease inhibitors such as Avr9 of Cladosporium fulvum that, based on its structure, is predicted to be a carboxy peptidase inhibitor. Protease inhibitors are potentially able to deactivate or detoxify host target proteases and, therefore, might play an important role during infection. In this study we are investigating the role of C. fulvum protease and protease inhibitors in disease establishment by using both functional genomics and biochemical approaches. We mined the genome of C. fulvum and found numerous proteases and protease inhibitors of which many are secreted. Expression analyses of these genes were performed using RNA extracted from fungal mycelium grown in vitro on liquid media under different conditions as well as from inoculated susceptible tomato plants. Interestingly, many of these genes are highly expressed only in vitro and/or in planta and based on their expression profiles we selected a number of candidates for further functional analyses. We will generate knock-out mutants of the selected proteases and protease inhibitors to identify their role in virulence. In addition, biochemical approaches will be used to pull-down the host target proteins of some presumably important candidate proteins such as for example the Avr9 protein