Affinity-tags are removed from Cladosporium fulvum effector proteins expressed in the tomato leaf apoplast

Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mould on tomato (Solanum esculentum). The fungus grows exclusively in the tomato leaf apoplast where it secretes several small (<15 kDa) cysteine-rich proteins that are thought to play a role in disease establishment. To investigate the role of these proteins, and to identify their in planta targets, a targeted proteomics approach was undertaken. C. fulvum proteins were expressed as recombinant fusion proteins carrying various affinity-tags at either their C- or N-terminus. Although these fusion proteins were correctly expressed and secreted into the leaf apoplast, detection of affinity-tagged C. fulvum proteins failed, and affinity purification did not result in the recovery of these proteins. However, when using C. fulvum effector protein-specific antibodies, specific signals were obtained for the different proteins. It is concluded that the stability of the in planta expressed recombinant fusion proteins is insufficient, which results in removal of the affinity-tag from the fusion proteins, irrespective of the C- or N-terminal fusion or the nature of the affinity-tag. Similar phenomena were observed when the fusion proteins were expressed in other Solanaceous species, but not when expressed in Arabidopsis thaliana.