Characterisation of the effects on proteases of Heterodera glycines and Meloidogyne incognita second-stage juveniles by inhibitors obtained from cysts of H. glycines

Authors: Masler, Edward

Submitted to: Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/12/2017
Publication Date: 2/19/2018
Citation: Masler, E.P. 2018. Characterisation of the effects on proteases of Heterodera glycines and Meloidogyne incognita second-stage juveniles by inhibitors obtained from cysts of H. glycines. Nematology. 20(1):461-470. https://doi.org/10.1163/15685411-00003151.
DOI: https://doi.org/10.1163/15685411-00003151

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 Because several chemical pesticides used to control nematodes have been withdrawn from use, growers face a critical need for the discovery of environmentally and economically sound nematode control agents.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 nematode females contain natural enzyme inhibitors that are have specific effects on different plant-parasitic nematode species. 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: The protease inhibitor component of Heterodera glycines adult female cyst content was explored using a battery of peptide substrates and H. glycines and Meloidogyne incognita J2 as enzyme sources. Protease inhibitors were prepared by heat-denaturing cyst content to provide heated H. glycines cyst-egg extract (hHglCE), which was used in all inhibition exploration. Eight substrates targeting four endoprotease groups (aspartic, cysteine, metallo- and serine proteases) revealed that protease inhibition by hHglCE varied significantly between H. glycines and M. incognita with 7 of the 8 substrates. Only cysteine protease activity was inhibited equally between H. glycines and M. incognita. Aspartic protease activity was inhibited more strongly (P < 0.05) in H. glycines and serine protease activity was inhibited more strongly (P < 0.05) in M. incognita. Inhibition of 5 matrix metalloprotease (MMP) substrates was alternately stronger in H. glycines (2 substrates) and M. incognita (3 substrates). These variations were particularly intriguing given the potential association of these MMP proteases with developing embryos. Inhibition of digestion of nematode FMRFamide-like peptides (FLPs) showed less variation between nematode species than the targeted substrates, but inhibition did vary significantly across substrates within each species. Digestion of the FLP KSAYMRFa was the least affected by hHglCE in either H. glycines or M. incognita. Fractionation of hHglCE by RP-HPLC clearly demonstrated the presence of high (Fr#5) and low (Fr#14) polarity inhibitor components. Potency of inhibition of M. incognita serine protease activity by these fractions, based upon IC50 values (1.68 and 2.78 hHglCEeq per reaction, respectively) was reduced significantly (P < 0.005) from unfractionated hHglCE (IC50 = 0.61), suggesting inhibitor dilution, loss of component synergy, or both, due to fractionation.