Heterodera glycines cysts contain an extensive array of endoproteases as well as inhibitors of proteases in H. glycines and Meloidogyne incognita infective juvenile stages

Authors: Masler, Edward; Chitwood, David

Submitted to: Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/3/2016
Publication Date: 3/4/2016
Citation: Masler, E.P., Chitwood, D.J. 2016. Heterodera glycines cysts contain an extensive array of endoproteases as well as inhibitors of proteases in H. glycines and Meloidogyne incognita infective juvenile stages. Nematology. 18(4):489-499.

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 Because several chemical pesticides used to control nematodes have been withdrawn from use, growers face a critical need for the discovery of environmentally and economically sound nematode control agents.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 female nematodes contain a large variety of small, stable molecules that target major metabolic cell enzymes, and that these molecules inhibit the the enzymes differently in different nematode species. These discoveries are significant because they reveal that molecules from nematodes themselves can act as highly specific inhibitors of plant-parasitic nematode metabolism, and have significant potential as natural suppression agents. 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: Heterodera glycines cysts contain proteases, and inhibitors of protease activities in various nematode species. In this investigation, proteases in H. glycines cysts were identified using a commercially available FRET-peptide library comprising 512 peptide pools qualified to detect up to 4 endoprotease types (aspartic, cysteine, metallo- and serine). Native cyst content (nHglCE) digested peptides in over 96% of the pools with all 4 protease types identified. Serine and metalloproteases represented nearly 70% of all proteases detected and were examined further. Trypsin (serine) and matrix metalloprotease (MMP) activities were compared among nHglCE, and H. glycines J2 and Meloidogyne incognita J2 extracts. The relative levels of activity were different for all 3 enzyme sources. Trypsin activity was up to 60-fold greater in M. incognita than in either H. glycines source, while MMP activity was highest in nHglCE and lowest in M. incognita J2. Heat-denatured cyst content (hHglCE) inhibited proteases in all 3 nematode preparations and was generally greater in M. incognita than in H. glycines. Largest differences (5.2-6.4-fold) were observed between M. incognita and nHglCE trypsin and MMP inhibition. In infective juveniles, hHglCE inhibited M. incognita J2 trypsin (IC50 = 0.64 hHglCEeq reaction-1) and MMP (IC50 = 0.54) more potently than either H. glycines trypsin (IC50 = 1.34) or MMP (IC50 = 1.84 ± 0.15). Using 3 MMP substrates (73, 74, 80) revealed clear species differences as well as complex associations between activity and inhibition. MMP73 digestion rates were the same in H. glycines and M. incognita but responses to hHglCE inhibition were different. MMP80 digestion rates were different but inhibition was the same. MMP74 digestion rates and inhibition levels were each different between species. These experiments provide further evidence that the H. glycines cyst should be examined as source of compounds useful for developing nematode control methods.