Substrate specificity and antifungal activity of recombinant tobacco classI chitinases

Endochitinases contribute to the defence response of plants against chitin- containing pathogens. The vacuolar class I chitinases consist of an N-termi nal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer wi th 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by compa ring wild-type chitinase A (CHN A) of Nicotiana tabacum with informative re combinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mas s spectrometry and partial sequencing. The enzymes were tested for their su bstrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest < 50% redu ction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduce d by up to 80%. Relative to CHN A, a variant with two spacers in tandem, wh ich binds chitin, showed very low hydrolytic activity towards chitin and No d factors, but comparable lysozyme activity and enhanced antifungal activit y. Neither hydrolytic activity, substrate specificity nor antifungal activi ty were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chit inolytic activity is not essential for inhibition of T. viride growth by CH N A.