Abstract
Proteinase/Protease inhibitors (PIs) from higher plants play an important role in defense and confer resistance against various insect pests and pathogens. 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.
Highlights
Proteolysis is a key physiological process catalyzed by specific proteases in plants, animals and microorganisms (Lopez-Otin and Bond, 2009)
CNBr activated Sepharose 4B, Sephadex G-50 fine, BAPNA, GLUPHEPA, soybean trypsinCI, tricine, gelatin, phenylmethylsulfonyl fluoride (PMSF), Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), Np-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), Trizol reagent and Coomassie brilliant blue R-250 were purchased from Sigma Aldrich, United States
The 20-60% (NH4)2SO4 fraction of peanut crude proteinase inhibitor (PnCPI) extract that showed prominent Trypsin inhibitor (TI) activity was subjected to Sepharose 4B trypsin affinity chromatography
Summary
Proteolysis is a key physiological process catalyzed by specific proteases in plants, animals and microorganisms (Lopez-Otin and Bond, 2009). Several studies demonstrated the use of PIs in the management of agriculturally important insect pests (Shamsi et al, 2016; War et al, 2018; Clemente et al, 2019) In this regard, serine PIs such as Kunitz and Bowman-Birk inhibitors (BBIs) are receiving considerable attention as a part of Integrated Pest Management (IPM) since lepidopteran insect pests depend predominantly on trypsin- and chymotrypsin-like digestive proteases for attaining energy (Sharma et al, 2000; Srinivasan et al, 2006; Swathi et al, 2016). BBIs are ∼8 kDa proteins with two reactive sites specific for trypsin and chymotrypsin which are stabilized by seven intramolecular disulfide bonds (Macedo et al, 2015) These canonical serine PIs follows the standard mechanism of inhibition by interacting with the active site of proteases by tight binding reaction (Laskowski and Qasim, 2000; Bateman and James, 2011)
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