Subtilisin inhibitor like protein ' pp LPI-1' from leaves of pigeonpea (Cajanus cajan, cv. BSMR 736) exhibits inhibition against Helicoverpa armigera gut proteinases

Abstract

Helicoverpa armigera is an orthodox rival of many crop plants affecting agricultural economy. Plant leaves found to accumulate proteinase inhibitors, although this insect pest chooses leaves for laying eggs. Plant defense response at this juncture is not fully explored. In this context, here we are reporting proteinase inhibitor (ppLPI-1) having significant homology with the I13 family from leaves of pigeonpea (cv. BSMR 736). The isolation of ppLPI-1 was carried out from leaves of field-grown pigeonpea under an outbreak of H. armigera. The acetone precipitated ppLPI-1 (125 µg) displayed substantial inhibition potential towards bovine trypsin (56.5 ± 1.8%) and HaGPs (52.6 ± 1.7%) on solution assay. These results were corroborated with dot-blot analysis. The molecular form of ppLPI-1 was characterized by reverse zymography and GXCP. The optimum condition was found to be pH 8 and temperature in the range of 30-40 °C. The protein identification via MASCOT-PMF and NCBI-BLAST search showed substantial homology with an inducible subtilisin inhibitor of Fabaceae comprising Vigna angularis (96%), Canavalia lineata (78%), Cicer arietinum (76%), Glycine max (75%), Medicago truncatula (73%) and Vicia faba (73%) consists of conserved domain of potato inhibitor I family.

Keywords: HaGPs; Helicoverpa armigera; Pigeonpea; Proteinase inhibitor; Subtilisin inhibitor; ppLPI-1.

Fig. 1

a Percent inhibition of trypsin and HaGPs activities by leaves PIs of pigeonpea cv. BSMR 736. Residual enzyme activity was determined using BApNA as the substrate. The results are presented as the mean ± standard deviation (SD), n = 3. b Dot blot assay of HaGPs interactions with the leaves PIs. Different ratios of enzymes (trypsin and HaGPs) and leaves PIs (v/v) were incubated and loaded on X-ray film

Fig. 2

Native-PAGE reverse zymography analysis of leaves PIs of pigeonpea cv. BSMR 736: samples were run on a 10% polyacrylamide gel and stained with a coomassie brilliant blue R-250 and b silver staining. (I) Leaves PIs; (II) seed PIs of pigeonpea c GXCP photograph of leaves PIs (PIs of trypsin and HaGPs). The detailed procedure is mentioned in “Materials and methods” section

Fig. 3

a Total trypsin activity in the presence of crude leaves PIs assayed in different pH conditions. b Total HaGPs activity in presence of crude leaves PIs assayed by performing the reaction at different temperatures. The assays were performed as mentioned in “Materials and methods”. Bar indicate standard deviation from triplicate determination

Fig. 4

An in vitro interaction of leaves PIs of pigeonpea (cv. BSMR 736) with HaGPs. PIs and HaGPs extract were incubated at an equal concentration at 37 °C and visualized by electrophoresis. a Electrophoretic and b quantitative estimation of HaGPs inhibition by pigeonpea leaves PIs after the incubation period. Bar indicate standard deviation from triplicate determination

Fig. 5

a MALDI-TOF-MS spectra (peptide mass fingerprints) of peptides generated from tryptically digested ppLPI-1. b Peptide mass fingerprint (PMF) analysis of ppLPI by MASCOT search tool (version 2.4.1; http://www.matrixscience.com). c Summary of matched peptides of ppLPI-1

Fig. 6

A (a) Amino acid sequence alignments of ppLPI-1 with subtilisin inhibitor previously have been characterized from closely related Fabaceae. The alignment was performed using MUSCLE (Edgar 2004). Accession numbers refer to the following proteins and plant species, respectively: gi|124121 (Vigna angularis), gi|124131 (Vicia faba), gi|357457017 (Medicago truncatula), gi|224447 (Vicia faba), gi|27734408 (Canavalia lineata), gi|502127777 (Cicer arietinum), gi|571489242 (Glycine max), (b) Conserved domain search of ppLPI-1. (c) Phylogram (Dereeper et al. 2008) showing the relationship between the ppLPI-1 with other plant subtilisin inhibitor of Fabaceae. B MEROPS database search results for ppLPI-1 amino acids sequence