PURIFICATION OF AN APOPLASTIC SUBTILISIN LIKE PROTEASE WITH CASPASE- 3 LIKE ACTIVITY FROM POTATO P.INFESTANS INFECTED LEAVES

FERNÁNDEZ MB,; GUSTAVO DALEO; MARÍA G. GUEVARA.

Mar del Plata

Encuentro; . VIII Encuentro Latinoamericano y del Caribe de Biotecnología; 2013

In this work, we report for the first time a subtilisin- like protein with caspase- 3 like activity involved in potato- P. infestans (Pi) interaction. Using the activity based protein profiling technology several caspase- like activities had been associated with programmed cell death (PCD) during the last decade. However, only two apoplastic serine proteases with caspase- like activities and related to plant PCD have been purified. These enzymes are not able to cleave DEVD substrates (specific of executioner caspase- 3) although this activity has been frequently implicated in PCD models. In order to purify and identify apoplastic caspase- 3 like protease/s induced during potato- Pi interaction, apoplastic fluid from potato infected leaves for 48h was subjected to ion- exchange and gel filtration chromatography. Caspase- 3 like activity was detected in only one peak that revealed a single band with an apparent mass of 70 kDa by SDS-PAGE. MALDI-TOF-MS analyses revealed that this protein corresponds to a protease belonging to the subtilisin family. These results describe a new apoplastic subtilisin like protease with caspase- 3 like activity involved in potato- Pi interaction, contributing to the knowledge of the family of caspase- like proteases previously considered as incomplete