Schaftoside Interacts With NlCDK1 Protein: A Mechanism of Rice Resistance to Brown Planthopper, Nilaparvata lugens

Abstract

Brown planthopper (BPH) Nilaparvata lugens Stål is a serious insect pest of rice in Asian countries. Active compounds have close relationship with rice resistance against BPH. In this study, HPLC, MS/MS, and NMR techniques were used to identify active compounds in total flavonoids of rice. As a result, a BPH resistance-associated compound, Peak 1 in HPLC chromatogram of rice flavonoids, was isolated and identified as schaftoside. Feeding experiment with artificial diet indicated that schaftoside played its role in a dose dependent manner, under the concentration of 0.10 and 0.15 mg mL^-1, schaftoside showed a significant inhibitory effect on BPH survival (p < 0.05), in comparison with the control. The fluorescent spectra showed that schaftoside has a strong ability to bind with NlCDK1, a CDK1 kinase of BPH. The apparent association constant K_A for NlCDK1 binding with schaftoside is 6.436 × 10³ L/mol. Docking model suggested that binding of schaftoside might affect the activation of NlCDK1 as a protein kinase, mainly through interacting with amino acid residues Glu12, Thr14 and Val17 in the ATP binding element GXGXXGXV (Gly11 to Val18). Western blot using anti-phospho-CDK1 (pThr14) antibody confirmed that schaftoside treatment suppressed the phosphorylation on Thr-14 site of NlCDK1, thus inhibited its activation as a kinase. Therefore, this study revealed the schaftoside-NlCDK1 interaction mode, and unraveled a novel mechanism of rice resistance against BPH.

Keywords: CDK1 protein; brown planthopper; flavonoids; interaction mechanism; rice; schaftoside; varietal resistance.

FIGURE 1

Content of total flavonoids in the leaf sheathes of different rice varieties. Data were presented as means ± SD (n = 5). Data were analyzed using one-way ANOVA and post-hoc analysis with Tukey’s HSD test, and relative content with the same letter were not significantly different (P < 0.05). DW, dried weight.

FIGURE 2

Determination of Peak 1 related compound in rice flavonoids by HPLC. (A) HPLC fingerprints of flavonoids extract from different rice varieties. (B) A calibration curve constructed by plotting the peak-areas to the relative concentrations of Peak 1 related compound. (C) Relative content of Peak 1 related compound in flavonoids extract from different rice varieties, calculated according to the calibration curve in (B). Data was analyzed by ANOVA with Tukey’s HSD tests and relative content with the same letter were not significantly different (P < 0.05). Data were presented as means ± SD (n = 5).

FIGURE 3

MS/MS of the pseudomolecular ions [M-H]^- of Peak 1 related compound.

FIGURE 4

NMR spectra of Peak 1 related compound schaftoside. (A) ¹H NMR spectra; (B) ¹⁴C NMR spectra.

FIGURE 5

HPLC chromatographs of total flavonoids and the standard sample of schaftoside. Showing the Peak 1 related compound in the flavonoids of RHT rice variety (upper) has the same retention time with the standard sample of schaftoside (lower).

FIGURE 6

Effects of schaftoside treatment on BPH. The results were expressed as the means ± SD from 10 independent experiments with 10 glass tubes (10 nymphs in each tube) examined for each treatment. The survival rates were analyzed by one-way ANOVA and Tukey HSD at each sampling time, and the asterisks on the bars indicated significant differences between the control (0 mg/mL) and the corresponding treatment group (^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 7

SDS–PAGE gel analysis of recombinant protein NlCDK1. The expression of NlCDK1 expressed in E. coli strain BL21(DE3)/pET32a was induced by 0.5 mM IPTG. The samples were analyzed in 12% SDS–PAGE and stained with Coomassie Brilliant Blue G-250. M: molecular weight markers; Lane 1: total cell lysate of BL21(DE3)/pET32a prior to induction; lane 2: protein of total cells after IPTG induction for 12 h; lane 3: purified and denatured NlCDK1 protein in inclusion bodies; lane 4: purified and refolded NlCDK1 protein.

FIGURE 8

Fluorescence spectra and plots of log[(F₀-F)/F] versus log[Q] for NlCDK1 binding with schaftoside. (A) C (schaftoside)/(μmol L^-1): a, 0; b, 0.5; c, 1.0; d, 2.0;e, 3.0; f, 5.0; g, 7.0; h, 10.0; i, 13.0; j, 17.0; k, 21.0; l, 28.0; m, 35.0; n, 45.0; o, 55.0;p, 67.0; q, 79.0; r, 93.0; s, 107.0; t, 123.0; u, 139.0. (B) C (NlCDK1) = 0.24 × 10^-6 mol L^-1; pH = 7.4; ex = 281 nm. The increasing concentration of schaftoside (B) was in accordance with that in the fluorescence spectra (A) when it was titrating from a to l; log[(F₀-F)/F] = logK_A + nlog[Q] was presented as y = 1.0493x–1.1914.

FIGURE 9

Binding mode of schaftoside to NlCDK1 binding sites. (A,C,D) Schaftoside binding with NlCDK1. (B) The same amino acid residues located close to the binding regions of NlCDK1 as shown in (A), without binding schaftoside. Carbon atoms in NlCDK1 and schaftoside are shown in blue and green, respectively. Colors of other atoms are as follows: oxygen (red), nitrogen (light blue). In (C,D), gray color represented for non-binding regions of NlCDK1 (local spacial structure).

FIGURE 10

Western blot detects the phosphorylation at Thr-14 of NlCDK1 kinase.