The plant proteolytic machinery involves over 700 proteases that control the (in)activation of proteins. Surprisingly, although every protein is regulated by proteolysis, only very few natural substrates of plant proteases have been identified, and the diverse roles of e.g. secreted proteases are still poorly understood. These questions have become even more urgent since glyco-engineered plants are increasingly used for the production of recombinant proteins (RPs) to produce therapeutic antibodies, enzymes and hormones. Notably, transient expression by infiltrating leaves with Agrobacterium cultures (agroinfiltration) is cheaper, safer, more scalable and faster than any other protein expression system. However, glycosylated RPs are secreted into the apoplast where they are cleaved and degraded by plant proteases, many of which accumulate upon agroinfiltration. Therefore, secreted plant proteases cause heavy yield losses and obstruct the true potential of molecular farming in plants.The aim of this project is to comprehensively understand the roles of proteases in the plant apoplast and to use this knowledge to improve recombinant (glyco)protein production in plants by targeted protease depletion.To achieve this aim, my four objectives are to:1) identify the major apoplastic proteases and their natural substrates and unravel proteolytic pathways;2) annotate biological roles to secreted proteases in cellular homeostasis and microbial colonization;3) understand substrate selection and develop software to predict cleavage sites in apoplastic substrates;4) improve RP production by targeted (multi)protease depletion.My expertise on secreted plant proteases puts me in an excellent position to develop a model system for proteolysis in plants and solve a key problem in a new, growing industry in Europe. This project will add a new, exciting direction to my research program and connect basic and applied science.