Functional characterization of Rorippa indica defensin and its efficacy against Lipaphis erysimi.

Poulami Sarkar,Subhrangshu Chatterjee,Jagannath Jana,Samir Ranjan Sikdar

doi:10.1186/s40064-016-2144-2

Abstract

Rorippa indica, a wild crucifer, has been previously reported as the first identified plant in the germplasm of Brassicaceae known to be tolerant towards the mustard aphid Lipaphis erysimi Kaltenbach. We herein report the full-length cloning, expression, purification and characterization of a novel R. indica defensin (RiD) and its efficacy against L. erysimi. Structural analysis through homology modeling of RiD showed longer α-helix and 3rd β-sheet as compared to Brassica juncea defensin (BjD). Recombinant RiD and BjD was purified for studying its efficacy against L. erysimi. In the artificial diet based insect bioassay, the LC50 value of RiD against L. erysimi was found to be 9.099 ± 0.621 µg/mL which is far lower than that of BjD (43.51 ± 0.526 µg/mL). This indicates the possibility of RiD having different interacting partner and having better efficacy against L. erysimi over BjD. In the transient localization studies, RiD signal peptide directed the RiD: yellow fluorescent protein (YFP) fusion protein to the apoplastic regions which indicates that it might play a very important role in inhibiting nutrient uptake by aphids which follow mainly extracellular route to pierce through the cells. Hence, the present study has a significant implication for the future pest management program of B. juncea through the development of aphid tolerant transgenic plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2144-2) contains supplementary material, which is available to authorized users.

Highlights

Brassica juncea (Indian mustard) is one of the most important crops cultivated in India
In the present study, Rorippa defensin (RiD) and Brassica juncea defensin (BjD) were isolated from R. indica and B. juncea respectively
The present study might be promising for the development of aphid tolerance in B. juncea through the generation of transgenics

Summary

Introduction

Brassica juncea (Indian mustard) is one of the most important crops cultivated in India. More than three dozen insect pests are found to be associated with mustard crops in India, of which mustard aphid (Lipaphis erysimi Kalt.) is the most dreaded. These pests are able to reduce the yield up to 96 % (Bakhetia and Sekhon 1989; Singh et al 1982; Dutta et al 2005a) and oil content by 15–32 % (Kanrar et al 2002; Verma and Singh 1987) even after good agricultural practices. Previous study by Bandopadhyay et al (2013) gave a first molecular insight into crucifer defense response against mustard aphid L. erysimi. One of the major identified ESTs was found to be homologous to PDF1.2c (plant defensin) of Arabidopsis (GenBank Accession—JK034054) which has been named as Rorippa defensin (RiD) for the present study

Methods

Results

Discussion

Conclusion