The selenium-binding protein of Theobroma cacao: A thermostable protein involved in the witches' broom disease resistance.

The selenium-binding proteins are known to be inducers of apoptosis in human and animals, and have been studied as target for the treatment of various types of cancer. In plants, SBP expression has been related to abiotic and biotic stress resistance. The SBP from Theobroma cacao (TcSBP) was first identified from a cocoa-Moniliophthora perniciosa cDNA library. The present study provides details on the TcSBP gene and protein structure. Multiple alignments revealed conserved domains between SBP from plants, human and archea. Homology modeling and molecular docking were performed and showed that the TcSBP has affinity to selenite in the active CSSC site. This result was confirmed by circular dichroism of the recombinant TcSBP, which also presented thermostable behavior. RT-qPCR analysis showed that TcSBP was differentially expressed in resistant vs susceptible cacao varieties inoculated by M. perniciosa and its expression was probably due to hormone induction via cis-regulating elements present in its promotor. The presence of the CSSC domain suggested that TcSBP acted by altering oxidation/reduction of proteins during H2O2 production and programmed cell death in the final stages of the witches' broom disease. To our knowledge, this is the first in silico and in vitro analysis of the SBP from cacao.

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.

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. CONCLUSION: 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.