Molecular cloning, characterization, and expression of a cDNA encoding an endochitinase gene from the mycoparasite...

Stachybotrys elegans is a mycoparasite of the soilborne plant pathogenic fungus Rhizoctonia solani. The mycoparasitic activity of S. elegans is correlated with the production of cell wall degrading enzymes such as chitinases. This report details the cloning by RACE-PCR and characterization of a full-length cDNA clone, sechi44, that appears to encode an extracellular endochitinase. An analysis of the sechi44 sequence indicates that this gene contains a 1269-bp ORF and encodes a 423-aa polypeptide. The SECHI44 protein has a calculated molecular weight of 44.1kDa and pI of 5.53. Since the SECHI44 protein also appears to encode a signal peptide, an extracellular location for the corresponding protein is predicted. Comparison of SECHI44 sequence with known sequences of fungal endochitinases revealed that SECHI44 is grouped with endochitinases from other mycoparasites. Real-time quantitative RT-PCR analysis showed an elevated level of expression of sechi44 (21-fold) in chitin-rich (induced) as compared to no-carbon (non-induced) culture conditions. In dual culture, the temporal expression of sechi44 increased after 2 days of contact with R. solani, reaching a 10-fold increase after 9 days, followed by a decrease to basic expression level at 12 days. Interestingly, inhibition of sechi44 expression was observed when S. elegans hyphae were in close proximity with R. solani hyphae.