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The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

Sara Pereira Menezes1, Edson Mario de Andrade Silva1, Eline Matos Lima1, Aurizângela Oliveira de Sousa1, Bruno Silva Andrade2, Livia Santos Lima Lemos3, Karina Peres Gramacho3, Abelmon da Silva Gesteira4, Carlos Priminho Pirovani1 and Fabienne Micheli15*

Author Affiliations

1 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

2 Universidade Estadual do Sudoeste da Bahia (UESB), Av. José Moreira Sobrinho, Jequié, Bahia 45206-190, Brazil

3 Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna, BA, Brazil

4 Departamento de Biologia Molecular, Embrapa Mandioca e Fruticultura, Rua Embrapa, s/n°, CEP44380-000 Cruz das Almas, Bahia, Brazil

5 CIRAD, UMR AGAP, F-34398 Montpellier, France

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BMC Plant Biology 2014, 14:161  doi:10.1186/1471-2229-14-161

Published: 11 June 2014



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.


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.


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.

Nucleases; Gene expression; ROS production; Molecular modeling