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

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.

Keywords: Aphid tolerance; Brassica juncea defensin; Insect pest management; Mustard aphid; Rorippa indica defensin.

Fig. 1

Aphid population on R. indica and B. juncea. The total number of live aphids were recorded over 7 days post infestation at an interval of 24 h. Maximum colonization was observed in case of B. juncea. Whereas a decrease in population of aphids was seen in R. indica after 4 dpi. Bars represent standard error (SE) where number of independent experiments (n) = 3. The significant changes (P ≤ 0.05), marked by asterisk were analyzed by Student’s t test

Fig. 2

a Semi quantitative PCR analysis of PDF1.2c homolog in R. indica (RiD) and B. juncea (BjD) at different time points. b Real time expression profile of PDF1.2c homolog in R. indica and B. juncea upon aphid infestation. Bars represent standard error (SE) of three biological replicates (n). The significant changes (P ≤ 0.01) were marked by asterisk (analyzed by Student’s t test)

Fig. 3

In silico promoter analysis showing important cis acting elements. A 393 bp region was analyzed for upstream elements (ARE: cis-acting regulatory element essential for the anaerobic induction, ABRE: abscisic acid response element, DOF Core: DNA-binding domain with one finger transcription factors, GT1 Motif: light responsive element, I-Box: part of a light responsive element, MeJ: methyl jasmonate inducing region, MYB Core: MYB transcription factor binding region, MYB1AT: dehydration responsive element, TATA Box: core promoter element around −30 of transcription start, W-Box: binding sites for WRKY transcription factors)

Fig. 4

Sequence alignment of RiD (GenBank accession—KP893333) with that of other plant defensins of Brassicaceae, viz. B. juncea (GenBank accession—KU513489), Raphanus sativus (GenBank accession—U18557), Arabidopsis (GenBank accession—NM106233.3), Sinapis alba (GenBank accession—AY998243) and Brassica rapa (GenBank accession—XM009106448)

Fig. 5

Homology model structure of RiD (a) and BjD (c) showing one α-helix and three β-sheet region. Superimposed homology model structure of RiD (cyan) and BjD (green) (b). Color by potential of solvent accessible surface of RiD (d) and BjD (e) have been calculated using APBS. The bars indicates color by potential [positive (blue) and negative (red)]. Electrostatic iso-surfaces of RiD (f) and BiD (g) are also shown

Fig. 6

Purification of RiD and BjD. a Lane 1 Protein marker, Lane 2 purified RiD, Lane 3 purified BjD, Lane 4 Western blot using anti-His antibody (1:5000) against purified RiD, Lane 5 Western blot using anti-His antibody (1:5000) against purified BjD. Arrow indicates purified proteins. b Determination of RiD molecular mass by MALDI-TOF/TOF–MS spectrometry. A sharp peak confirms the quality of purification of RiD

Fig. 7

Localization studies of RiD:YFP and YFP only (as control) in onion epidermal cells. Confocal laser sections show YFP fluorescence in onion epidermal cells expressing RiD:YFP in the apoplastic regions and diffused fluorescence in the cells expressing only YFP. a Fluorescence, b magnified fluorescence, c merged, d bright field. (Magnification: ×20). Bar 75 µm

Fig. 8

Insect bioassay in artificial diet supplemented with RiD and BjD on second instar nymphs of L. erysimi. Insect survivability graph at different concentrations of a RiD (0, 5, 10, 15, 20, 25 µg/mL) and b BjD (0, 10, 20, 30, 40, 50 µg/mL) were recorded over 72 h