Response of Midgut Trypsin- and Chymotrypsin-Like Proteases of Helicoverpa armigera Larvae Upon Feeding With Peanut BBI: Biochemical and Biophysical Characterization of PnBBI.

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In the present study, Bowman-Birk Inhibitor (BBI) was purified from mature seeds of an interspecific advanced hybrid peanut variety (4368-1) using chromatographic techniques. The biochemical and biophysical characteristics such as low molecular mass, presence of several isoinhibitors and higher-ordered dimer/tetramer, predominance of antiparallel β-sheets and random coils in secondary structure, reactive sites against trypsin and chymotrypsin, broad spectrum of stability toward extreme pH and temperature along with MALDI TOF-TOF analysis (ProteomeXchange identifier PXD016933) ascertained the purified biomolecule from peanut as BBI (PnBBI). Surface plasmon resonance competitive binding analysis revealed the bifunctional PnBBI is a trypsin specific inhibitor with 1:2 stoichiometry as compared to chymotrypsin. A concentration-dependent self-association tendency of PnBBI was further confirmed by 'red shift' in the far-UV CD spectra. Furthermore, the insecticidal potential of PnBBI against Helicoverpa armigera was assessed by in vitro assays and in vivo feeding experiments. A significant reduction in larval body weight was observed with concomitant attenuation in the activity of midgut trypsin-like proteases of H. armigera (HaTPs) fed on PnBBI supplemented diet. The one and two-dimensional zymography studies revealed the disappearance of several isoforms of HaTP upon feeding with PnBBI. qRT-PCR analysis further suggests the role of PnBBI in not only inhibiting the activity of midgut trypsin and chymotrypsin-like proteases but also in modulating their expression. Taken together, the results provide a biochemical and molecular basis for introgressed resistance in peanut interspecific advanced hybrid variety against H. armigera.", "claims": [{"id": "claim-method", "statement": "Statistical differences were determined by one-way ANOVA followed by Tukey test at a significance level of P ≤ 0.05 using Sigma plot, version 12.0, Systat Software Inc., San Jose, CA, United States.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:32265951", "locator": "sections[17] \"Statistical Analysis\" paragraph 2", "quote": "Statistical differences were determined by one-way ANOVA followed by Tukey test at a significance level of P ≤ 0.05 using Sigma plot, version 12.0, Systat Software Inc., San Jose, CA, United States."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The protein identification data by (MALDI MS-MS) analysis can be found in ProteomeXchange consortium via PRIDE (Identifier PXD016933).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:32265951", "locator": "sections[32] \"Data Availability Statement\" paragraph 2", "quote": "The protein identification data by (MALDI MS-MS) analysis can be found in ProteomeXchange consortium via PRIDE (Identifier PXD016933)."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": null, "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:32265951"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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