title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca(2+) and Mg(2+) dependent-DNase activity and antifungal action on Moniliophthora perniciosa.
abstract: BACKGROUND
  The production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b.
  
  
  RESULTS
  TcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae.
  
  
  CONCLUSIONS
  To our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively.
authors.forename: Sara
authors.initials: S
authors.lastname: Pereira Menezes
authors.forename: Edson Mario
authors.initials: EM
authors.lastname: de Andrade Silva
authors.forename: Eline
authors.initials: E
authors.lastname: Matos Lima
authors.forename: Aurizângela
authors.initials: A
authors.lastname: Oliveira de Sousa
authors.forename: Bruno
authors.initials: B
authors.lastname: Silva Andrade
authors.forename: Livia
authors.initials: L
authors.lastname: Santos Lima Lemos
authors.forename: Karina
authors.initials: K
authors.lastname: Peres Gramacho
authors.forename: Abelmon
authors.initials: A
authors.lastname: da Silva Gesteira
authors.forename: Carlos Priminho
authors.initials: CP
authors.lastname: Pirovani
authors.affiliation: Departamento de Ciências Biológicas (DCB), Centro de Biotecnologia e Genética (CBG), Universidade Estadual de Santa Cruz (UESC), Rodovia Ilhéus-Itabuna, km 16, 45662-900 Ilhéus, BA, Brazil. fabienne.micheli@cirad.fr.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Micheli
mesh_terms: D000363:Agaricales / Q000187:drug effects / Q000502:physiology*; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000378:metabolism; D001483:Base Sequence; D001499:Bayes Theorem; D002099:Cacao / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology*; D002118:Calcium / Q000494:pharmacology*; D002688:Chitinases / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D060467:Disease Resistance / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005838:Genotype; D008274:Magnesium / Q000494:pharmacology*; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010935:Plant Diseases / Q000235:genetics / Q000276:immunology / Q000382:microbiology; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012260:Ribonucleases / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D000935:Antifungal Agents; D010940:Plant Proteins; D011994:Recombinant Proteins; D003851:Deoxyribonucleases; D012260:Ribonucleases; D002688:Chitinases; D008274:Magnesium; D002118:Calcium