Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera.

Marri Swathi,Aparna Dutta-Gupta,Nalini Mallikarjuna,Prashant K Mishra,Vanka Swaroop,Vadthya Lokya,Kollipara Padmasree

doi:10.3389/fphys.2016.00388

Abstract

Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus.

Highlights

Pigeonpea (Cajanus cajan) is an important pulse crop which occupies a major share in contributing to protein rich diet, in the vegetarian population
Several earlier reports indicated that the proteinase inhibitors (PIs) prepared from crude protein extracts of wild relatives of pigeonpea significantly inhibited the activity of H. armigera gut trypsin-like proteases (HGPs) (Chougule et al, 2003; Parde et al, 2012; Swathi et al, 2015)
In the present study, trypsin-specific PIs (CpPI 63) active against HGPs were purified from mature dry seeds of C. platycarpus using different chromatography methods and characterized to complement the earlier findings

Summary

Introduction

Pigeonpea (Cajanus cajan) is an important pulse crop which occupies a major share in contributing to protein rich diet, in the vegetarian population. In spite of the existence of such a broad spectrum of defense compounds, preharvest devastation due to insect pests in developing seeds and post-harvest losses caused by storage pests in mature seeds are severe in pigeonpea (Giri and Kachole, 1998; Sharma, 2016). Helicoverpa armigera is one among the most devastating insect pests on pigeonpea. It has become a major challenge to control H. armigera In this context, agronomic practices and natural enemies along with host plant resistance and natural plant products which are relatively harmless to the non-target beneficial organisms as well as human beings were considered to offer a potentially reasonable means of controlling insect pests (Andow, 2008)

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