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High-Affinity Copper Transport and Snq2 Export Permease of Saccharomyces cerevisiae Modulate Cytotoxicity of PR-10 from Theobroma cacao

¹UESC, Centro de Biotecnologia e Genética, Laboratório de Biologia de Fungos, Rodovia Ilhéus-Itabuna, km 16, 45650-000 Ilhéus-BA-Brasil; ²UESC, Centro de Biotecnologia e Genética, Laboratório de Biologia Molecular, Rodovia Ilhéus-Itabuna, km 16, 45650-000 Ilhéus-BA-Brasil; ³CEPLAC/CEPEC, Cocoa Research Center, 45600-970 Itabuna-BA-Brasil; ⁴CIRAD, UMR DAP, Avenue Agropolis TA96/03, 34398 Montpellier cedex 5, France

A pathogenesis-related (PR) protein from Theobroma cacao (TcPR-10) was identified from a cacao--Moniliophthora perniciosa interaction cDNA library. Nucleotide and amino acid sequences showed homology with other PR-10 proteins having P loop motif and Betv1 domain. Recombinant TcPR-10 showed in vitro and in vivo ribonuclease activity, and antifungal activity against the basidiomycete cacao pathogen M. perniciosa and the yeast Saccharomyces cerevisiae. Fluorescein isothiocyanate-labeled TcPR-10 was internalized by M. perniciosa hyphae and S. cerevisiae cells and inhibited growth of both fungi. Energy and temperature-dependent internalization of the TcPR-10 suggested an active importation into the fungal cells. Chronical exposure to TcPR-10 of 29 yeast mutants with single gene defects in DNA repair, general membrane transport, metal transport, and antioxidant defenses was tested. Two yeast mutants were hyperresistant compared with their respective isogenic wild type: ctr3Δ mutant, lacking the high-affinity plasma membrane copper transporter and mac1Δ, the copper-sensing transcription factor involved in regulation of high-affinity copper transport. Acute exposure of exponentially growing yeast cells revealed that TcPR-10 resistance is also enhanced in the Snq2 export permease-lacking mutant which has reduced intracellular presence of TcPR-10.