title: Theobroma cacao cystatins impair Moniliophthora perniciosa mycelial growth and are involved in postponing cell death symptoms.
abstract: Three cystatin open reading frames named TcCys1, TcCys2 and TcCys3 were identified in cDNA libraries from compatible interactions between Theobroma cacao (cacao) and Moniliophthora perniciosa. In addition, an ORF named TcCys4 was identified in the cDNA library of the incompatible interaction. The cDNAs encoded conceptual proteins with 209, 127, 124, and 205 amino acid residues, with a deduced molecular weight of 24.3, 14.1, 14.3 and 22.8 kDa, respectively. His-tagged recombinant proteins were purified from Escherichia coli expression, and showed inhibitory activities against M. perniciosa. The four recombinant cystatins exhibited K(i) values against papain in the range of 152-221 nM. Recombinant TcCYS3 and TcCYS4 immobilized in CNBr-Sepharose were efficient to capture M. perniciosa proteases from culture media. Polyclonal antibodies raised against the recombinant TcCYS4 detected that the endogenous protein was more abundant in young cacao tissues, when compared with mature tissues. A ~85 kDa cacao multicystatin induced by M. perniciosa inoculation, MpNEP (necrosis and ethylene-inducing protein) and M. perniciosa culture supernatant infiltration were detected by anti-TcCYS4 antibodies in cacao young tissues. A direct role of the cacao cystatins in the defense against this phytopathogen was proposed, as well as its involvement in the development of symptoms of programmed cell death.
authors.affiliation: UESC, DCB, Laboratório de Proteômica, Centro de Biotecnologia e Genética, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus, BA, 45650-000, Brazil.
authors.forename: Carlos Priminho
authors.initials: CP
authors.lastname: Pirovani
authors.forename: André
authors.initials: A
authors.lastname: da Silva Santiago
authors.forename: Lívia Santana
authors.initials: LS
authors.lastname: dos Santos
authors.forename: Fabienne
authors.initials: F
authors.lastname: Micheli
authors.forename: Rogério
authors.initials: R
authors.lastname: Margis
authors.forename: Abelmon
authors.initials: A
authors.lastname: da Silva Gesteira
authors.forename: Fátima Cerqueira
authors.initials: FC
authors.lastname: Alvim
authors.forename: Gonçalo Amarante Guimarães
authors.initials: GA
authors.lastname: Pereira
authors.forename: Júlio Cézar
authors.initials: JC
authors.lastname: de Mattos Cascardo
mesh_terms: D001483:Base Sequence; D002099:Cacao / Q000737:chemistry* / Q000235:genetics; D016923:Cell Death / Q000187:drug effects*; D015891:Cystatins / Q000494:pharmacology*; D017931:DNA Primers; D018076:DNA, Complementary; D025282:Mycelium / Q000187:drug effects* / Q000254:growth & development; D016366:Open Reading Frames; D010802:Phylogeny
chemical_list: D015891:Cystatins; D017931:DNA Primers; D018076:DNA, Complementary