Investigation of a phytaspase activity from Nicotiana benthamiana: purification, identification, and characterization

Phytaspases are specific plant proteases related to stress development and plant defense responses to adverse factors, such as insect or agrobacterial attacks. Phytaspases belong to the subtilisin-like proteases (subtilases), and they are distinct from other subtilases due to their strict substrate specificity and unusual life cycle. Phytaspases are localized in the apoplast under normal circumstances, sharing this feature with the majority of other subtilases. However, phytaspases move into the cell interior under stress development via a clathrin-mediated mechanism, and among subtilases, such behavior has been described only for phytaspases. The unique specificity in the structure of phytaspases enables in silico genomic analysis to predict candidates for phytaspases among the numerous plant subtilases. For example, three subtilases have been expected to be phytaspases for the plant Nicotiana benthamiana . Here, we demonstrate that N. benthamiana possesses a phytaspase-like activity that alters localization upon stress induction, and we isolate and purify the enzyme corresponding to this activity from leaf apoplast washes. Mass spectrometric analysis is used to identify the purified enzyme and establish which protein corresponds to this activity. Additionally, we characterize the substrate preferences of the N. benthamiana phytaspase. • Phytaspase-like VEIDase activity is detected in Nicotiana benthamiana leaves. • Methyl viologen treatment causes changes in stress-related enzyme localization. • Phytaspase activity in N. benthamiana leaves corresponds to NbSBT1.9 plant protease. • N. benthamiana phytaspase is a post-translationally modified monomeric subtilase. • The best washing liquid to obtain apoplast subtilases is double distilled water.