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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Mycology and Nematology Genetic Diversity and Biology Laboratory » Research » Publications at this Location » Publication #310276

Title: Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome

Author
item Masler, Edward

Submitted to: Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2014
Publication Date: 1/1/2015
Citation: Masler, E.P. 2015. Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome. Nematology. 17(1):91-102.

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 Because several chemical pesticides used to control nematodes have been withdrawn from use, growers face a critical need for the discovery of environmentally and economically sound nematode control agents.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 small, stable molecules that target a major metabolic center of the cell called the proteasome, and that these molecules can inhibit the three core enzymes in the proteasome. These discoveries are significant because they reveal the new concept that molecules from nematodes themselves can act as highly specific inhibitors of plant-parasitic nematode metabolism, and present 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: Proteases from Heterodera glycines and Meloidogyne incognita juveniles were inhibited by heat-stable content of H. glycines female cysts (HglCE), and by the plant polyphenol epigallocatechin gallate (EGCG). General protease activities detected using the nematode peptide KSAYMRFa were inhibited by EGCG (IC50 1.19mM, H. glycines; 0.34mM, M. incognita) but not by HglCE. HglCE and EGCG each inhibited chymotrypsin-like (CT-L) activity detected using the proteasome substrate LLVY. EGCG IC50’s were 0.47mM (H. glycines) and 0.15mM (M. incognita). HglCE IC50’s were 0.158 and 0.005 HglCEeq/µl for H. glycines and M. incognita, respectively. Across all substrate-inhibitor combinations, M. incognita proteases were inhibited more robustly (P < 0.05) than those from H. glycines. Species differences were most pronounced with the CT-L-HglCE combination. In addition to CT-L protease, two other enzymes characteristic of the proteasome, post-glutamate peptide hydrolyzing (PGPH) and trypsin-like (T-L) proteases, were detected in M. incognita. HglCE inhibited all three proteases. HglCE also inhibited CT-L, PGPH, and T-L proteases within the catalytic subunits from yeast (Saccharomyces cerevisae) and human proteasomes. Addition of 0.04% SDS to reactions increased (P < 0.05) the activities of M. incognita CT-L (28.0 ± 3.1 Vmax/min/µg) and PGPH (12.4 ± 1.2 Vmax/min/µg) proteases 12- and 4.3-fold respectively, a response characteristic of proteasome proteases. T-L activity was low (0.31 ± 0.25 Vmax/min/µg) and was not increased by SDS. The proteasome inhibitors MG-132 and aclacinomycin A each inhibited M. incognita CT-L and PGPH activities more than 80 % (P < 0.05) at 20-100µM, while HglCE, equivalent to 0.04 cyst/µl, inhibited the same proteases by 84 ± 3% and 68 ± 3% (P < 0.05), respectively. T-L activities in M. incognita extracts and pure proteasome subunits were completely inhibited by HglCE. CT-L activity in native preparations of H. glycines cysts was low (Vmax/min/µg < 1), and not affected by HglCE.