Submitted to: Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2016
Publication Date: 1/3/2017
Citation: Masler, E.P., Chitwood, D.J. 2017. Evaluation of proteases and protease inhibitors in Heterodera glycines cysts obtained from laboratory and field populations. Nematology. 19(1):109-120.
Interpretive Summary: Plant-parasitic nematodes attack all crops of agricultural importance, causing over $10 billion in losses annually to U.S. farmers. Because several chemical pesticides used to control nematodes have been withdrawn from use, growers face a critical need for the discovery of environmentally and economically sound nematode control agents. One approach to discovering new means of controlling nematodes is to identify ways to inhibit their metabolism and infectivity using naturally derived compounds. We discovered that soybean cyst nematodes from both greenhouse culture and field populations contain natural enzyme inhibitors. These discoveries are significant because they reveal that nematode derived molecules have significant potential as natural suppression agents since they can target plant-parasitic nematode activities needed to survive. Consequently, this information will be used by researchers in the agrochemical and agricultural biotechnology industries who are developing safe, selective methods for nematode control.
Technical Abstract: Proteases and proteases inhibitors were evaluated in a number of preparations of Heterodera glycines cysts obtained from glasshouse cultures (GH) and field (LR) populations. Using a FRET-peptide library comprising 512 peptide substrate pools that detect 4 endoprotease types (aspartic, cysteine, metallo- and serine), we found that the relative distributions of 6 endoproteases within the 4 catalytic types were similar among GH and LR preparations. However, levels of mean protease activity (Vmax s-1 µgcyst protein-1) across all 512 pools varied nearly 8-fold (P < 0.05) among the preparations. This variation was not related to cyst source. These qualitative (type distribution) and quantitative relationships persisted when analysis was restricted to the top 40 percent (activity) pools. Analysis of the top 4 percent of substrate pools did reveal some substrate cleavage site preferences between the GH and LR proteases. GH and LR preparations also differed in digestion rates (P < 0.05) of trypsin and matrix metalloprotease (MMP) substrates, with LR rates 2-fold greater than GH rates for each protease. In contrast, inhibition of trypsin activity, measured using Meloidogyne incognita extracts, was 1.6-fold greater (P < 0.05) with GH cyst content than LR content. Inhibition of MMP activity was the same (>60%) with each preparation. Fractionation of GH and LR protease inhibitors by RP-HPLC (CH3CN/0.1% TFA system) yielded similar activity profiles. MMP inhibitors eluted at 35-40% CH3CN, and trypsin inhibitors eluted at both 5% and 35-40% CH3CN, suggesting the possibility of small molecular weight as well as peptide inhibitors. These discoveries illustrate the importance of examining H. glycines cysts as a source of materials for novel nematode controls.