RESEARCH ARTICLE TcCYPR04, a Cacao Papain-Like Cysteine- Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4

The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plant-pathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-prote-ases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immuno-blottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected byMoniliophthora perniciosa during the transition from the biotrophic to the sapro-phytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant toM. perniciosa, respectively, but greater inten-sity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant caca