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Cloning and expression analysis of an endo-1,3-β-d-glucosidase from Phytophthora cinnamomi

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Abstract

Phytophthora is considered one of the most destructive genus for many agricultural plant species worldwide, with a strong environmental and economic impact. Phytophthora cinnamomi is a highly aggressive Phytophthora species associated with the forest decline and responsible for the ink disease in chestnut trees (Castanea sativa Miller), a culture which is extremely important in Europe. This pathogenicity occurs due to the action of several enzymes like the hydrolysis of 1,3-β-glucans at specific sites by the enzyme endo-1,3-β-d-glucosidase. The aim of this work to analyze the heterologous expression in two microorganisms, Escherichia coli and Pichia pastoris, of an endo-1,3-β-d-glucosidase encoded by the gene ENDO1 (AM259651) from P. cinnamomi. Different plasmids were used to clone the gene on each organism and the real-time quantitative polymerase chain reaction was used to determine its level of expression. Homologous expression was also analyzed during growth in different carbon sources (glucose, cellulose, and sawdust) and time-course experiments were used for endo-1,3-β-d-glucosidase production. The highest expression of the endo-1,3-β-d-glucosidase gene occurred in glucose after 8 h of induction. In vivo infection of C. sativa by P. cinnamomi revealed an increase in endo-1,3-β-d-glucosidase expression after 12 h. At 24 h its expression decreased and at 48 h there was again a slight increase in expression, and more experiments in order to further explain this fact are underway.

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Authors and Affiliations

  1. Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253, Bragança, Portugal

    Rodrigo Costa & Altino Choupina

  2. Departamento de Microbiología y Genética, Edificio Departamental, Plaza de los Drs. de la Reina s/n, 37007, Salamanca, Spain

    Rodrigo Costa & Angel Domínguez

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  1. Rodrigo Costa
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  2. Angel Domínguez
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  3. Altino Choupina
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Correspondence to Altino Choupina.

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The authors declare that there are no conflicts of interest. Also, are indebted to the careful and constructive criticisms of the reviewers.

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Costa, R., Domínguez, A. & Choupina, A. Cloning and expression analysis of an endo-1,3-β-d-glucosidase from Phytophthora cinnamomi. Mol Biol Rep 47, 935–942 (2020). https://doi.org/10.1007/s11033-019-05185-9

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