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

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 8h of induction. In vivo infection of C. sativa by P. cinnamomi revealed an increase in endo-1,3-β-D-glucosidase expression after 12h. At 24h its expression decreased and at 48h there was again a slight increase in expression, and more experiments in order to further explain this fact are underway.