Cloning, overexpression and functional characterization of a class III chitinase from Casuarina glauca nodules

This work was based on the study of a defense-related protein involved in actinorhizal symbiosis established between Casuarina glauca and a nitrogen fixing bacterium from the Frankia genus, namely a class III chitinase (CgCHI3). In order to understand its role during the symbiotic process, recombinant CgCHI3 was overexpressed in Escherichia coli and subsequently characterized. In this context, the recombinant protein showed endochitinase, β-1,3-glucanase, β-1,4 glucanase and lysozyme activity, as well as the capacity to inhibit the growth of non-symbiotic bacteria (E. coli strains BL21 and K12, Paracoccus denitrificans and Bacillus subtilis). It exhibited no antifungal activity against Colletotrichum gloeosporioides, Botrytis cinerea, Trichoderma viride and Fusarium oxysporum and did not affect the growth of the symbiotic bacteria Frankia and Rhizobium, neither the performance of Frankia nodulation factors. These results suggest that recombinant CgCHI3 is a multifunctional protein involved in the infection process, specifically in the lysis of the cell wall during intracellular penetration, or in the formation of the infection thread, or any other modification of the cells in order to accommodate the symbiotic bacteria.