Distinct expression profiles and different functions of odorant binding proteins in Nilaparvata lugens Stål

Abstract

Background: Odorant binding proteins (OBPs) play important roles in insect olfaction. The brown planthopper (BPH), Nilaparvata lugens Stål (Delphacidae, Auchenorrhyncha, Hemiptera) is one of the most important rice pests. Its monophagy (only feeding on rice), wing form (long and short wing) variation, and annual long distance migration (seeking for rice plants of high nutrition) imply that the olfaction would play a central role in BPH behavior. However, the olfaction related proteins have not been characterized in this insect.

Methodology/principal findings: Full length cDNA of three OBPs were obtained and distinct expression profiles were revealed regarding to tissue, developmental stage, wing form and gender for the first time for the species. The results provide important clues in functional differentiation of these genes. Binding assays with 41 compounds demonstrated that NlugOBP3 had markedly higher binding ability and wider binding spectrum than the other two OBPs. Terpenes and Ketones displayed higher binding while Alkanes showed no binding to the three OBPs. Focused on NlugOBP3, RNA interference experiments showed that NlugOBP3 not only involved in nymph olfaction on rice seedlings, but also had non-olfactory functions, as it was closely related to nymph survival.

Conclusions: NlugOBP3 plays important roles in both olfaction and survival of BPH. It may serve as a potential target for developing behavioral disruptant and/or lethal agent in N. lugens.

Figure 1. Alignment of amino acid sequences of NlugOBPs.

Predicted signal peptides were boxed. Conserved cysteines were highlighted in red and marked with '''' below the alignment, and other conserved residues were highlighted in gray.

Figure 2. Relative mRNA expression level of NlugOBPs in nymphs of different developmental stages, and in adults of different wing forms and genders.

The relative expression level was expressed as mean ± SE (N = 3), with the 1^st instar nymph as the calibrator. N1-N5, nymph of 1^st to 5^th instar; ♂M (macropterous) and ♂B (brachypterous), male adults; ♀M and ♀B, female adults.

Figure 3. Relative mRNA expression level (♀/♂ or M /B) of OBP genes.

(A) For female and male adults, (B) and long and short wing form adults.

Figure 4. Relative expression levels of NlugOBPs in different tissues.

The expression levels of NlugOBPs were quantified by qRT-PCR. ♂An, male antennae; ♀An, female antennae; ♂Ab, male abdomen; ♀Ab, female abdomen; ♂Le, male legs; ♀Le, female legs; ♂Wi, male wings; ♀Wi, female wings.

Figure 5. Expression and purification of three NlugOBPs.

A SDS-PAGE was used to detect the OBP crude extracts from the bacterial pellets before (In-) and after (In+) induction with IPTG, and purified samples after His-tag cleavage by enterokinase (P). The target bands were marked by arrows.

Figure 6. Ligand-binding assay of three NlugOBPs.

(A) Binding curves of 1-NPN (Left) and relative Scatchard plot analyses (Right). (B-D) Competitive binding curves of three OBPs with Alcohols (B), Aldehydes, Esters and Benzoates (C), and Carboxylic acids, Alkane and Terpenes (D). (E) Comparison of binding ability (indicated by 1/Ki values) of three OBPs with 31 compounds. Numbers representing ligands were the same as in Table 3 and Table 4.

Figure 7. Effects of feeding NlugOBP3 dsRNA on NlugOBP3 mRNA level (A), survival rate (B), jumping ability (C), and response to rice seedlings (D-E) of BPH nymphs.

CK, nymph fed with normal diet; dsGFP, fed with diet mixed with dsRNA of green fluorescent protein (0.5 mg/ml); dsOBP3, fed with dsRNA of NlugOBP3 (0.5 mg/ml). Data topped with different letters are significant different as determined using a one-way ANOVA (Duncan’s multiple range test, P<0.05). All error bars represent the SE of the mean obtained from at least three independent replicates.