title: TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4.
abstract: 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-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity 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 cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches' broom symptoms. This molecular interaction may be the target for future interventions to control witches' broom disease.
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus/BA, Brasil.
authors.forename: Thyago Hermylly Santana
authors.initials: TH
authors.lastname: Cardoso
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus/BA, Brasil.
authors.forename: Ana Camila Oliveira
authors.initials: AC
authors.lastname: Freitas
authors.affiliation: Universidade Estadual do Sudoeste da Bahia, UESB, Jequié-BA, Brazil.
authors.forename: Bruno Silva
authors.initials: BS
authors.lastname: Andrade
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus/BA, Brasil.
authors.forename: Aurizangela Oliveira de
authors.initials: AO
authors.lastname: Sousa
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus/BA, Brasil.
authors.forename: André da Silva
authors.initials: Ada S
authors.lastname: Santiago
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus/BA, Brasil.
authors.forename: Daniela Martins
authors.initials: DM
authors.lastname: Koop
authors.affiliation: Centro de Pesquisa do Cacau (CEPEC), Comissão Executiva do Plano da Lavoura Cacaueira (CEPLAC), Fitopatologia, Ilhéus, BA, Brazil.
authors.forename: Karina Peres
authors.initials: KP
authors.lastname: Gramacho
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus/BA, Brasil.
authors.forename: Fátima Cerqueira
authors.initials: FC
authors.lastname: Alvim
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus/BA, Brasil.|CIRAD, UMR AGAP, Montpellier, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Micheli
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus/BA, Brasil.
authors.forename: Carlos Priminho
authors.initials: CP
authors.lastname: Pirovani
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002099:Cacao / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015891:Cystatins / Q000378:metabolism*; D057056:Cysteine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018745:Genome, Plant; D015151:Immunoblotting; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009336:Necrosis; D010206:Papain / Q000737:chemistry*; D010940:Plant Proteins / Q000378:metabolism*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D015891:Cystatins; D010940:Plant Proteins; D057056:Cysteine Proteases; D010206:Papain