We have previously reported the identification and initial characterization of a novel plant-like family 19 chitinase (As-
p50) that is secreted into the perivitelline fluid surrounding infective larvae of Ascaris suum prior to hatching (Jeng et al 2002, Mol Biochem Parasitol 124: 11-21). We have subsequently identified a number of putative orthologues of this chitinase in the nematode EST databases and three putative ortholgues in the published genome sequence of C. elegans (cosmid clones C08B6.4, R10D12.15 and T05H4.7). Previously family 19 glycosyl hydrolases had only been reported from plants and a few bacteria.We have initiated a study using C. elegans as a model to evaluate the biological functions of family 19 chitinases in nematodes and in particular their possible involvement in fungal resistance, as is the case in plants. We have expressed recombinant C08B6.4 and R10D12.15 in Rosetta Gami (DE3) cells and studies are in progress to examine their substrate specificity and antifungal properties. A
c08b6.4:gfp reporter construct with GFP expression driven by the minimal 3 kb upstream of the ATG is transiently expressed by hypodermal cells of 3-fold stage larvae and hatched L1s with a timing similar to that of As-
p50, suggesting that both C08B6.4 and As-
p50 may play roles in hatching, formation of the L1 cuticle and/or the initial molt. In addition this construct is also expressed in four anterior neurons. We have identified these neurons as AIAs and AUAs. In contrast, a full-length
c08b6.4:gfp translational fusion is also expressed in the spermatatheca, gland cells and marginal cells of the pharynx. Taken together these results suggest that C08B6.4 may also be involved in formation and/or remodeling of the egg shell following fertilization, molting of the pharynx and in nutrition and fungal resistance. We have investigated gene expression by semi-quantitative PCR and found that C08B6.4 and especially R10D12.15 but not T05H4.7 are significantly upregulated in response to fungal infection. We also observed upregulation of these two genes in
daf-2 gene deletion mutants. This latter observation confirms the results of a larger previous study that employed microarrays (Murphy et al, 2003. Nature 424: 277-283) and in that case a number of other antimicrobial proteins were also upregulated.We are now continuing our studies to examine the potential functions of these novel plant-like chitinases.