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Theobroma cacao cystatins impair Moniliophthora perniciosa mycelial growth and are involved in postponing cell death symptoms

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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.

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Abbreviations

BSA:

Bovine serum albumin

PLCPs:

Papain-like cysteine proteases

PCD:

Programmed cell death

NEP:

Necrosis and ethylene-inducing proteins

ORF:

Open reading frame

BApNA:

Nl-alpha-benzoyl-dl-arginine-p-nitroanilide hydrochloride

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Acknowledgments

This research was supported by the ‘Financiadora de Estudos e Projetos’ (FINEP) and the ‘Fundação de Amparo à Pesquisa do Estado da Bahia’ (FAPESB) M. perniciosa proteomic network. F C Alvim was the recipient of a PQI/CAPES graduate fellowship, and C P Pirovani was the recipient of a FAPESB graduate fellowship. We thank Robson José Costa Dias (Laboratório de Genômica, UESC) for technical assistance and Dra. Karina Perez Gramacho (CEPEC, CEPLAC, Brasil) for the support in cacao seedlings inoculations with M. perniciosa basidiospores. We are thankful to Dr. Antonio Figueira (CENA/USP-SP, Brazil) for critical reading of the manuscript. J.C.M. Cascardo is recipient of CNPq research fellowship number 303987/2008-1. R. Margis is recipient of CNPq research fellowship number 302684/2005-0.

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Authors and Affiliations

  1. UESC, DCB, Laboratório de Proteômica, Centro de Biotecnologia e Genética, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus, BA, 45650-000, Brazil

    Carlos Priminho Pirovani, André da Silva Santiago, Lívia Santana dos Santos, Fabienne Micheli, Fátima Cerqueira Alvim & Júlio Cézar de Mattos Cascardo

  2. CIRAD, UMR DAP, Avenue Agropolis TA96/03, 34398, Montpellier Cedex 5, France

    Fabienne Micheli

  3. UFRGS, Centro de Biotecnologia, Laboratório de Genomas e Populações de Plantas, Porto Alegre, Rio Grande do Sul, Brazil

    Rogério Margis

  4. EMBRAPA/CNPMF, Cruz das Almas, Bahia, Brazil

    Abelmon da Silva Gesteira

  5. Departamento de Genética e Evolução-UNICAMP, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil

    Gonçalo Amarante Guimarães Pereira

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  1. Carlos Priminho Pirovani
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  2. André da Silva Santiago
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  3. Lívia Santana dos Santos
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Correspondence to Júlio Cézar de Mattos Cascardo.

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Pirovani, C.P., da Silva Santiago, A., dos Santos, L.S. et al. Theobroma cacao cystatins impair Moniliophthora perniciosa mycelial growth and are involved in postponing cell death symptoms. Planta 232, 1485–1497 (2010). https://doi.org/10.1007/s00425-010-1272-0

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