Rice dwarf virus upregulates the odorant receptor co‐receptor of Nephotettix cincticeps to enhance perception of 2‐heptanol emitted from virus‐infected rice and promote its transmission

Abstract

BACKGROUND

Insect odorant receptor co‐receptors (Orcos) are pivotal for insect perception of host plant volatiles. We previously found that 2‐heptanol—released from rice dwarf virus (RDV)‐infected rice plants—repels viruliferous green rice leafhoppers (GRLHs, Nephotettix cincticeps), redirecting them to feed on RDV‐free plants and thereby facilitating viral spread. However, whether Orco in N. cincticeps (NcinOrco) influences the detection of this repellent volatile and contributes to viral transmission remains unknown.

RESULTS

Here, we found that NcinOrco contains seven transmembrane domains and is phylogenetically conserved. Expression profiling revealed that NcinOrco exhibits antennal‐predominant expression (~5‐fold) that is significantly upregulated upon RDV acquisition (~2‐fold). Crucially, RNAi‐mediated silencing of NcinOrco abolished the repellent behavioral response of viruliferous GRLHs (VG) to 2‐heptanol (mean number of VG to paraffin oil versus 2‐heptanol: dsGFP, 9.0 versus 4.4, P < 0.001; dsNcinOrco, 7.4 versus 7.0, P > 0.05), eliminated preference for RDV‐free plants (mean number of VG on RDV‐free versus RDV‐infected: dsGFP, 9.5 versus 5.1, P < 0.001; dsNcinOrco, 7.8 versus 7.0, P > 0.05), and impaired RDV transmission efficiency (mean transmission rates on MH63 versus MH63 + 2‐heptanol: dsGFP, 76% versus 35%, P < 0.001; dsNcinOrco, 51% versus 52%, P > 0.05).

CONCLUSION

Our results indicate that NcinOrco is essential for detecting a key host‐emitted repellent volatile, revealing how a plant virus may alter a core olfactory component to modify vector behavior and aid its transmission. Together, these findings contribute to the understanding of insect chemoreception under pathogenic stress and highlight a potential molecular interface in plant–virus–vector interactions. © 2026 Society of Chemical Industry.

RDV upregulates Orco gene expression to sensitize viruliferous vectors to 2‐heptanol, thereby mediating their foraging toward virus‐free plants and promoting viral transmission.

1. INTRODUCTION

Insects rely on a sensitive olfactory system to detect environmental volatile organic compounds (VOCs), which regulates essential behaviors such as host plant selection, mate choice and predator avoidance. ¹ , ² , ³ This olfactory process is mediated by a sophisticated molecular machinery involving odorant binding proteins (OBPs), chemosensory proteins (CSPs) and olfactory receptors (ORs). ⁴ Among these, ORs are particularly crucial for discriminating lipid‐soluble odorants. ⁵ , ⁶ The odorant receptor co‐receptor (Orco) plays a pivotal, conserved role by forming heteromeric complexes with ligand‐specific ORs. This interaction is essential for OR trafficking, complex stability and the efficiency of signal transduction, ultimately forming ligand‐gated cation channels. ⁷ , ⁸ , ⁹ , ¹⁰ Structurally distinct from vertebrate G‐protein coupled receptors, insect ORs and Orco feature a unique seven‐transmembrane topology with an intracellular N‐terminus. ¹¹ Although ligand‐binding ORs are highly divergent, Orco is exceptionally conserved across insect orders and is typically encoded by a single‐copy gene, making it a central hub in the insect olfactory circuitry. ¹² , ¹³ , ¹⁴ , ¹⁵

This conserved olfactory system is often exploited within the complex tripartite interactions among plant viruses, their insect vectors and host plants, which influences the epidemic and transmission of plant viruses. ¹⁶ , ¹⁷ Virus‐induced alterations in host plant physiology, particularly in VOC emission profiles, could directly manipulate vector behavior to promote viral acquisition or transmission—a phenomenon termed ‘host manipulation’. ¹⁸ , ¹⁹ , ²⁰ For instance, Cucumber mosaic virus infection increases ethylene emission from pepper plants to attract aphid vectors, ²¹ whereas Tomato spotted wilt virus elevates (Z)‐3‐hexenal release to lure thrips. ²² Likewise, Tomato yellow leaf curl virus (TYLCV) upregulates terpene synthase (TPS3 and TPS7) genes in tomato plants, enhancing myrcene emission and attracting whiteflies. ²³ Despite these documented examples of chemically mediated manipulation, the molecular mechanisms within the vector insect that underlie the perception of these virus‐induced volatiles remain poorly understood. Identifying the specific olfactory components, such as Orco and its partner ORs, that are modulated by viruses is critical for understanding the mechanistic basis of transmission.

Recent studies have shown that plant viruses could influence vector insects' perception of plant volatiles by directly regulating olfactory‐related genes, such as those encoding OBPs and ORs. Such regulation has been documented across several hemipteran vector species. For example, in planthoppers, Rice stripe virus alters the expression of multiple ORs in Laodelphax striatellus, with the Orco gene playing an important role in host‐seeking behavior. ²⁴ , ²⁵ Likewise, Southern rice black‐streaked dwarf virus regulates OR86 and OR127 in Sogatella furcifera through the Toll signaling pathway, thereby modulating vector host plants preference. ²⁶ In whiteflies, Cucurbit chlorotic yellows virus upregulates the expression of OBP5 in its whitefly vector, enhancing olfactory sensitivity to host plant volatiles and accelerating virus transmission, ²⁷ whereas TYLCV alters olfactory orientation by modulating the odorant receptor BtMEDOR6. ²³ Collectively, these findings highlight that key olfactory components—such as Orco and its partner ORs—play pivotal roles in virus–vector–host interactions, providing a foundation for understanding the mechanistic basis of viral transmission. However, despite these advances in planthoppers and whiteflies, the olfactory mechanisms specifically associated with virus regulation in rice leafhoppers, another crucial group of hemipteran vectors, remain largely unexplored.

Rice dwarf virus (RDV), a phytoreovirus transmitted in a persistent‐propagative manner mainly by green rice leafhoppers (Nephotettix cincticeps, GRLHs), is a major threat to rice production in Asia. ²⁸ , ²⁹ , ³⁰ , ³¹ , ³² Our previous study observed that RDV infection induces the emission of the volatile 2‐heptanol from rice plants. Intriguingly, this compound acts as a repellent specifically to viruliferous GRLHs (carrying RDV), redirecting them to feed on RDV‐free rice plants and thereby facilitating viral spread, while showing no effect on nonviruliferous GRLHs. ³³ , ³⁴ This presents a clear case of pathogen‐mediated behavioral manipulation via a plant volatile. This differential behavioral response implies that RDV infection may modulate the internal olfactory machinery of GRLHs. However, the specific molecular targets within the insect, particularly whether the Orco‐dependent olfactory pathway is exploited to mediate this critical behavioral shift towards 2‐heptanol, remain unknown.

In this study, we identified and characterized the Orco ortholog (NcinOrco) from GRLH transcriptomes. We analyzed its phylogenetic position, examined its expression patterns in viruliferous and nonviruliferous insects across developmental stages and tissues, and assessed the impact of RDV infection on its expression. Furthermore, using RNA interference (RNAi) coupled with behavioral assays, we functionally validated the role of NcinOrco in perceiving 2‐heptanol and its necessity for the subsequent virus transmission cycle. Our findings elucidate a molecular mechanism by which a plant virus exploits the vector's olfactory coreceptor to influence behavior, providing novel insights into plant–virus–vector interactions and offering a potential target for disrupting the transmission of insect‐borne viral diseases.

2. MATERIALS AND METHODS

2.1. Insects rearing

A nonviruliferous colony of GRLHs was originally obtained from rice paddy fields in Zhejiang University, Hangzhou, China, 2023, and these insects were reared in nylon cages (80‐mesh, 50 cm³) with susceptible rice seedlings (Taichung Native1, TN1) for three to four generations in a climate chamber at 27 ± 1 °C and 70 ± 5% relative humidity under a 14 h:10 h, light:dark photoperiod in Qingdao Agricultural University, Qingdao, China. ³⁵

The viruliferous colony of GRLHs was obtained as follows: nonviruliferous nymphs were fed on RDV‐infected rice plants, after the virus completed its latent period in the vector, these insects were transferred into glass tubes (containing RDV‐free rice seedlings) individually, with each tube assigned a unique reference number. Seedlings were replaced every 48 h, and those corresponding to each reference number were planted into a glasshouse with Kimura B nutrient solution under natural lighting. ³⁶ When typical symptoms of RDV infection (dwarfism and white chlorotic spots on the leaves), the RDV infection status of these rice plants was detected by real‐time (RT)‐PCR as described previously. ³⁷ The viruliferous insects were collected according to the detection, and maintained on RDV‐infected plants under the same conditions as above.

2.2. Rice plants

The rice cultivar used in this study was Minghui 63 (MH63). To obtain RDV‐infected rice plants, 10‐day‐old rice seedlings were exposed to viruliferous 4^th‐ to 5^th‐instar nymphs for 72 h, and nonviruliferous nymphs were used as controls. All of the rice seedlings were grown in a glasshouse under the same conditions as described above. At 40 days post‐transplantation, these RDV‐free and RDV‐infected rice plants were used in the relevant experiments.

2.3. Sequence alignment and phylogenetic analysis

The NcinOrco gene of GRLHs (OQ798996.1) was identified from the antennae and body transcriptome of nonviruliferous and viruliferous GRLHs. This protein was verified via a protein Blast search in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The transmembrane domain of the NcinOrco gene were separately predicted by TMHMM‐2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/). Multiple sequence alignment was conducted with other insects (Anopheles gambiae, Drosophila melanogaster, Tribolium castaneum, Bombyx mori, Acyrthosiphon pisum, Apis mellifera) by clustal w. ³⁸ A total of 87 amino acid (aa) sequences of Orco genes were selected to construct the phylogenetic tree by Mega 12.0 (based on the maximum‐likelihood method, the Jones–Taylor–Thornton model and 1000 bootstrap replications), ³⁹ and the final tree was optimized using an online tool, iTOL (https://itol.embl.de/). The specific Orco gene information can be found in Supporting Information, Table S1.

2.4. Tissue and temporal expression profiles analysis of the NcinOrco gene

The tissue and temporal expression profiles were determined by quantitative (q)RT‐PCR. For the tissue expression profiles, the head with or without antennae, thoraxes, abdomens, wings and legs were dissected from 20 of newly emerged nonviruliferous or viruliferous GRLHs were dissected under a stereoscope, and these tissues were quickly stored in liquid nitrogen (N₂) for RNA extraction. For the temporal expression profiles, a total of 20 individuals were collected per stage, including the 1^st‐ to 5^th‐instar nymphs, female and male adults. The total RNA of these samples was extracted using Trizol reagent following the procedure, and then transcribed into cDNA as described in a previous study. ⁴⁰ Each experiment was performed with three biological replicates. β‐actin was selected as a reference gene for normalizing the NcinOrco gene expression.

2.5. Effects of RDV infection on the NcinOrco gene in the head with antennae or in total GRLHs

The head tissues, including antennae, were dissected from both nonviruliferous and viruliferous GRLHs samples under a stereomicroscope, and mixed in groups of 20 heads per biological replicate. Immediately following dissection, the tissue samples or total insects were flash‐frozen in liquid N₂ to preserve RNA integrity for subsequent extraction. RNA extraction, reverse transcription and qRT‐PCR were performed following the identical protocol to that described above. Each experiment was performed with three biological replicates. The primers used for qRT‐PCR were designed on online tool (primer 3, https://bioinfo.ut.ee/primer3-0.4.0/) and listed in Table S2.

2.6. RNAi of NcinOrco gene

Based on the full‐length sequence of the NcinOrco gene, a specific NcinOrco dsRNA fragment (397 bp) was selected for synthesis. As a control, the green fluorescent protein (GFP) gene (GenBank accession no. U87973) was utilized to generate dsGFP. Both dsNcinOrco and dsGFP fragments were synthesized employing the T7 High Yield Transcription Kit (Vazyme, Nanjing, China) in strict accordance with the manufacturer's instructions. The reaction system was as follows: 8 μL dNTP (2 μL each of ATP, CTP, GTP and UTP), 2 μL of 10× reaction buffer, 1 μg PCR‐product template, 2 μL Enzyme Mix, and RNAase‐free water to make up the total volume to 20 μL. The above system was incubated at 37 °C for 12 h. Postincubation, the synthesized dsRNA was purified by a series of steps involving 3 m sodium acetate (pH 5.2) and phenol‐chloroform extraction (pH 4.7), followed by precipitation with isoamyl alcohol. The integrity and quality of the dsRNA were subsequently assessed by 1.2% agarose gel electrophoresis, whereas the concentration was quantified using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Following purification of dsRNA, microinjection was performed on 5^th‐instar nymphs of viruliferous GRLHs using the Smartouch Microinjection System (World Precision Instruments, Inc., Sarasota, FL, USA). The nymphs were injected with dsNcinOrco at a concentration of 5 μg μL⁻¹ (50 nL per insect), whereas the control group received dsGFP at the same concentration. After microinjection, these 5^th‐instar nymphs were transferred to RDV‐free MH63 rice seedlings. To evaluate gene silencing efficiency, head tissues were collected from both dsNcinOrco‐ and dsGFP‐injected newly emerged female adults at 48 h postinjection, with three biological replicates per group. Gene expression levels were subsequently quantified using qRT‐PCR.

2.7. Insect behavioral bioassay

Our previous study demonstrated that RDV infection triggers 2‐heptanol emission in rice plants, and that this volatile repels viruliferous GRLHs, causing them to feed on RDV‐free plants instead. ³³ Thus, we first examined the olfactory behavioral responses of viruliferous GRLHs following RNAi of the NcinOrco gene between RDV‐infected and RDV‐free rice plants. A two‐choice H‐shaped olfactometer was employed to evaluate olfactory behavior, as described previously. ³⁷ The apparatus consisted of two vertical plastic cylinders [height (H) = 30 cm, depth (D) = 5 cm], each connected to a smaller plastic cylinder (H = 8 cm, D = 6 cm) filled with Kimura B nutrient solution at the base. These two main cylinders were interconnected by a polypropylene (PP) tube (H = 20 cm, D = 2 cm) featuring a central hole for the placement of viruliferous GRLHs (Fig. S1). One RDV‐free and RDV‐infected rice plant were introduced into the olfactometer, and 15 dsGFP or dsNcinOrco‐treated viruliferous female GRLHs, starved for 4 h, were released into the central hole of the PP tube. The number of insects (dsGFP and dsNcinOrco) settling on each rice plant was recorded at 2, 4, 6, 8 and 24 h postinoculation (hpi). Five independent replications were conducted.

After separately validating the feeding preferences of dsGFP‐treated and dsNcinOrco‐treated of viruliferous GRLHs using RDV‐free and RDV‐infected rice plants, we referenced previous olfactory device to analyze the impact of the single standard compound 2‐heptanol (0.1 μg μL⁻¹; Sigma‐Aldrich, St Louis, MO, USA) on the olfactory behavior of dsGFP‐treated and dsNcinOrco‐treated of viruliferous female GRLHs. ⁴¹ The device comprised two polypropylene (PP) boxes (H = 8 cm, D = 6.5 cm). Each box lid was equipped with a 3‐cm‐diameter nylon hole for insect ventilation (Fig. S2). The two boxes were connected by a 20‐cm‐long, 2‐cm‐diameter PP tube, which also contained a central hole for inserting insects. In one PP box, a 0.15 g cotton ball was treated with 500 μL of a standard volatile and paraffin oil solution as the test group, whereas in the other PP box, a 0.15 g cotton ball received 500 μL pure paraffin oil as the control. Both boxes also contained water‐moistened cotton ball. Fifteen dsGFP‐treated and dsNcinOrco‐treated viruliferous GRLHs were placed into the central hole of this device to test the olfactory behavioral responses. The number of dsGFP‐treated and dsNcinOrco‐treated of viruliferous GRLHs settling on different PP box was recorded after being placed for 2, 4, 6, 8, 24 h. There were five biological replicates for test compound.

2.8. Effects of NcinOrco gene on the viral transmission

The device used for the effects of NcinOrco gene on RDV transmission assays was similar to that described in a previous study. ³⁴ The setup consisted of a perforated foam board floating on a large water tray filled with Kimura B nutrient solution. The foam board contained 10 columns in total: eight columns (four on the left and four on the right) each held six rice seedlings, totaling 48 plants, whereas the remaining two central columns were reserved for insect placement (Fig. S3). The rice combination was prepared as follows: MH63 plus synthetic 2‐heptanol in 10 μL lanolin paste (MH63 + 2‐hep + Lan) (left four columns) versus MH63 plus 10 μL pure lanolin paste (MH63 + Lan) (right four columns). First, a total of 48 dsGFP‐treated or dsNcinOrco‐ treated viruliferous GRLHs were placed into the two central columns of the foam board. The number of these viruliferous insects (dsGFP or dsNcinOrco) settling on the MH63 + Lan or MH63 + 2‐heptanol+Lan plants was recorded at 72 hpi, and then were detached from these rice plants. Following a further 25‐day period, the infection status of the plants was determined, and these data were used to calculate the RDV transmission rate.

2.9. Statistical analysis

The data analyses were conducted using the Spss 26.0 software. The relative quantification was calculated using the comparative 2^−ΔΔCt method. ⁴² The comparative analysis of Orco gene expression across various tissues and temporal stages was performed using one‐way ANOVA, with posthoc analysis conducted through Tukey's multiple range test at a significance level of P < 0.05. The data of the relative expression levels of NcinOrco in the head with antennae, as well as in female and male separately, from nonviruliferous and viruliferous GRLHs were analyzed using Student's t‐test. The data on the interference efficiency of the Orco gene also were analyzed using Student's t‐test. The odor selection preference data of dsGFP‐treated or dsNcinOrco‐treated viruliferous GRLHs between RDV‐free and RDV‐infected rice plant, or between 2‐heptanol and paraffin oil were analyzed by generalized linear mixed model (GLMM) with Poisson distribution. ³⁷ , ⁴³ The data were expressed as mean ± standard error (SEM).

3. RESULTS

3.1. Identification and phylogenetic analysis of NcinOrco

The olfactory receptor co‐receptor gene from N. cincticeps transcriptomes, NcinOrco, has an open reading frame encoding a 465‐aa protein predicted to contain seven transmembrane domains (TM‐I = 44–66 aa; TM‐II = 76–95 aa; TM‐III = 135–157 aa; TM‐IV = 206–228 aa; TM‐V = 337–359 aa; TM‐VI = 369–391 aa; TM‐VII = 439–461 aa), consistent with the conserved structure of insect Orco proteins [Fig. 1(A), (B)]. Multiple sequence alignment with Orco orthologs from diverse insect orders confirmed the high conservation of NcinOrco, particularly in its C‐terminal region [Fig. 1(A)]. Phylogenetic analysis placed NcinOrco within a well‐supported clade of hemipteran Orcos, where it showed the closest relationship to the ortholog from Macrosteles quadrilineatus (Fig. 2), corroborating its identity as the canonical Orco in GRLHs.

Figure 1.

Sequence analysis of NcinOrco. (A) Alignment of NcinOrco amino acid sequence with closely related species. The transmembrane domain is marked with a red bold line. Species abbreviations and GenBank accession nos are as follows: Agam, Anopheles gambiae (XP_312379); Apis, Acyrthosiphon pisum (XP_001951646); Amel, Apis mellifera (XP_006563267); Bmor, Bombyx mori (NP_001037060); Dmel, Drosophila melanogaster (NP_001097687); Tcas, Tribolium castaneum (EFA05687). (B) Transmembrane helical structures of NcinOrco amino acid sequences.

Figure 2.

Phylogenetic tree of Orco sequences from different insect species (in orders Hemiptera, Hymenoptera, Diptera, Coleoptera, Lepidoptera). The tree was constructed in Mega12.0 based on a clustal alignment, with branch lengths scaled to the percentage of sequence difference (see scale bar: 0.05%). Bootstrap values (from 1000 replicates) are shown at the nodes. The final phylogenetic trees were optimized and visualized by an online tool, iTOL (https://itol.embl.de/).

3.2. Expression profiles of NcinOrco gene

The qRT‐PCR analysis revealed that NcinOrco expression was developmentally regulated and tissue‐specific. In nonviruliferous GRLHs, transcript levels were highest in adults and 1^st‐instar nymphs compared to other nymphal stages [F = 34.093, df = 6, P < 0.001; Fig. 3(A)], and were predominantly localized to the head (which contains antennae) [F = 52.047, df = 5, P < 0.001; Fig. 3(C)]. Critically, a similar expression pattern was maintained in viruliferous GRLHs [Fig. 3(B): F = 129.908, df = 6, P < 0.001; Fig. 3(D): F = 56.126, df = 5, P < 0.001], indicating that RDV infection does not alter the spatial or temporal regulation of NcinOrco.

Figure 3.

Expression patterns of NcinOrco in nonviruliferous and viruliferous GRLHs. (A, B) Developmental stages and (C, D) various tissues of adults. Head + Ant, head with antennae; Head ‐ Ant, head without antennae. Different letters represent significant differences based on one‐way ANOVA followed by Tukey's multiple comparison test (P < 0.05, n = 3). Values are mean ± SEM.

3.3. RDV infection upregulates NcinOrco expression

Although RDV infection does not alter the relative expression patterns of NcinOrco, it significantly increased its transcript abundance. Compared to nonviruliferous controls, NcinOrco expression was significantly upregulated in the whole body of viruliferous female adults (t = 8.727, df = 4, P < 0.001) and male adults (t = 10.925, df = 4, P < 0.001) [Fig. 4(A)], more markedly, in the head (including antennae) of viruliferous GRLHs [Fig. 4(B); t = 9.571, df = 4, P < 0.001]. This finding indicates that RDV infection specifically enhances the expression of this key olfactory co‐receptor in its insect vector.

Figure 4.

Effects of RDV infection on the expression of NcinOrco gene in female and male adults of the GRLHs (A) or in the head (with antennae) of the GRLHs (B). Expression data of NcinOrco gene in the head (with antennae), female or male adults of nonviruliferous and viruliferous GRLHs were analyzed by Student's t‐test (***, P < 0.001; n = 3). Values are mean ± SEM.

3.4. NcinOrco is required for the repellent response to 2‐heptanol

In order to functionally characterize NcinOrco, we performed RNAi in viruliferous GRLHs. Injection of dsRNA targeting NcinOrco (dsNcinOrco) resulted in an approximately 74% reduction in transcript levels compared to the dsGFP control [Fig. 5(A); t = 5.850, df = 4, P = 0.004]. We then tested the odor preference of these insects. In a two‐choice assay, dsGFP‐treated viruliferous GRLHs exhibited a strong and persistent preference for RDV‐free over RDV‐infected plants. On average, 9.6 insects were found on RDV‐free compared to 5.1 on RDV‐infected plants. Statistical analysis revealed that this preference was significant at all time points measured [Fig. 5(B): 2 h, F _1,40 = 93.821, P < 0.001; 4 h, F _1,40 = 86.984, P < 0.001; 6 h, F _1,39 = 63.609, P < 0.001; 8 h, F _1,39 = 63.609, P < 0.001; 24 h, F _1,40 = 115.069, P < 0.001]. By contrast, this preference was completely abolished in dsNcinOrco‐treated insects, with an average of 7.8 insects on RDV‐free plants and 7.0 on RDV‐infected plants, showing no significant choice between the two plant types at any time point [Fig. 5(C): 2 h, F _1,40 = 2.440, P = 0.126; 4 h, F _1,40 = 3.766, P = 0.059; 6 h, F _1,40 = 1.353, P = 0.252; 8 h, F _1,40 = 3.766, P = 0.059; 24 h, F _1,40 = 0.609, P = 0.440].

Figure 5.

Functional analysis of NcinOrco in detection of rice plant volatiles. Relative expression levels of the NcinOrco gene in viruliferous GRLHs after 48 h of treatment with the dsNcinOrco (A). Plant selection preferences of dsGFP‐viruliferous (B) and dsNcinOrco‐viruliferous (C) GRLHs between MH63 and MH63‐RDV rice plants. The data on the interference efficiency of the NcinOrco gene was analyzed using Student's t‐test (**, P < 0.01; n = 3). All plant odor preference assays were performed with five replicates. Data of two treatments in the same column were analyzed by GLMM with Poisson distributions, and the sequential Bonferroni adjusted significance level is 0.05. Asterisks indicate significant differences between MH63 and MH63‐RDV rice plants (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Values are mean ± SEM (n = 5).

In order to directly link this behavioral change to the perception of the key volatile, we tested the response to synthetic 2‐heptanol. dsGFP‐treated controls were significantly repelled by 2‐heptanol versus a paraffin oil control throughout a 24‐h period, with an average of 9.0 insects choosing the paraffin oil control and 4.4 insects choosing 2‐heptanol [Fig. 6(A): 2 h, F _1,39 = 27.750, P < 0.001; 4 h, F _1,37 = 48.229, P < 0.001; 6 h, F _1,37 = 44.901, P < 0.001; 8 h, F _1,38 = 38.487, P < 0.001; 24 h, F _1,34 = 84.751, P < 0.001). In stark contrast, dsNcinOrco‐treated insects lost this repellent response, displaying no preference between 2‐heptanol and the control solvent, with an average of 7.0 insects on 2‐heptanol and 7.4 on the paraffin oil control [Fig. 6(B); 2 h, F _1,40 = 3.668, P = 0.063; 4 h, F _1,40 = 1.653, P = 0.206; 6 h, F _1,40 = 1.164, P = 0.287; 8 h, F _1,40 = 1.651, P = 0.206; 24 h, F _1,40 = 0.047, P = 0.830). These results demonstrate that NcinOrco is essential for viruliferous GRLHs to detect and behaviorally respond to the repellent volatile 2‐heptanol.

Figure 6.

Functional analysis of NcinOrco in detection of standard compound of 2‐heptanol. Olfactory response of dsGFP‐viruliferous (A) and dsNcinOrco‐viruliferous (B) GRLHs to 2‐heptanol. Data for the two treatments were analyzed by a GLMM with Poisson distributions, and the sequential Bonferroni adjusted significance level is 0.05. Asterisks indicate significant differences between 2‐heptanol and paraffin oil (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Values are mean ± SEM.

3.5. Silencing NcinOrco disrupts odor‐mediated behavior and impairs RDV transmission

Having established that NcinOrco mediates the repellent response to 2‐heptanol, we investigated the consequent impact on virus transmission. We first confirmed that the behavioral shift observed in olfactometers translated to actual feeding preference on rice plants. In a cage assay, dsGFP‐treated viruliferous GRLHs significantly preferred feeding on MH63 + Lan rice plants over MH63 + 2‐heptanol+Lan rice plants, with an average of 30 insects on MH63 + Lan rice plants and 12 on MH63 + 2‐heptanol+Lan rice plants [Fig. 7(A); F _1,3 = 232.692, P < 0.001]. This repellence was eliminated in dsNcinOrco‐treated insects, which showed no significant difference in distribution, with an average of 23 insects on MH63 + Lan rice plants and 18 on MH63 + 2‐heptanol+Lan rice plants [Fig. 7(B); F _1,3 = 0.182, P = 0.697].

Figure 7.

Functional analysis of NcinOrco in RDV transmission. The host plant odor preferences of dsGFP (A) or dsNcinOrco viruliferous (B) GRLHs between MH63 plus 10 μL pure lanolin paste (MH63 + Lan) and MH63 plus synthetic 2‐heptanol in 10 μL lanolin paste rice plants (MH63 + Lan + 2‐heptanol). RDV transmission rate by dsGFP (C) and dsNcinOrco (D) GRLHs viruliferous GRLHs between MH63 + Lan and MH63 + Lan + 2‐heptanol rice plants. All plant odor preference assays were performed with three replicates, each of 48 newly emerged female adults. Data of the host plant odor preferences of viruliferous GRLHs were analyzed by GLMM with Poisson distributions, and the sequential Bonferroni adjusted significance level is 0.05. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). RDV transmission rates by viruliferous GRLHs were analyzed by Student's t‐test. Values are mean ± SEM (n = 3).

Critically, this loss of odor‐guided behavior had a direct impact on transmission efficiency. dsGFP‐treated viruliferous insects transmitted RDV to MH63 + Lan rice plants at a significantly higher rate than to MH63 + 2‐heptanol+Lan rice plants with an average transmission rate of 76.2% on MH63 + Lan rice plants compared to 34.9% on MH63 + 2‐heptanol+Lan rice plants [Fig. 7(C); t = 9.827, df = 4, P < 0.001]. However, when NcinOrco was silenced, the transmission rate difference between the two plant treatments disappeared, with an average of 50.8% on MH63 + Lan plants and 52.4% on MH63 + 2‐heptanol+Lan [Fig. 7(D); t = 0.229, df = 4, P = 0.830]. This indicates that the ability to perceive 2‐heptanol via NcinOrco is exploited by the virus to effectively redirect them to RDV‐free plants, thereby facilitating its own spread.

4. DISCUSSION

Insects rely on sophisticated olfactory systems to identify and locate suitable hosts. The Orco protein plays a central role in insect physiology, influencing odorant perception, reproductive regulation and social behavior. ⁴ , ⁹ , ⁴⁴ Here, we identified and characterized the NcinOrco gene in the GRLHs (Fig. 1). The protein features seven transmembrane domains and shows sequence relatively high identity with Orco proteins from other insects, suggesting a high conservation of Orco during insect evolution. ⁴⁵ , ⁴⁶ , ⁴⁷ Notably, the C‐terminal domains exhibited the highest conservation, which is likely to be a result of their functional importance in mediating interactions between conventional ORs and Orco, as demonstrated in D. melanogaster. ⁹ , ¹¹

In this study, phylogenetic analysis revealed that the Orco proteins of Hemipteran insects form a distinct clade. Specifically, the NcinOrco gene was closely clustered with MquaOrco and HvirOrco (Fig. 2), indicating a high degree of homology among these three insects. Furthermore, the NcinOrco gene was more expressed in adults compared to nymphs (other than 1^st‐instar nymph), and showed higher expressions in head with antenna compared to other tissues (Fig. 3). Based on these findings and previous reports on the expression pattern and function of the Orco gene in other insects such as BodoOrco, RproOrco, MsepOrco, PbreOrco and AluOrco, ⁴⁸ , ⁴⁹ , ⁵⁰ , ⁵¹ , ⁵² we speculate that the NcinOrco gene may play a critical role in the perception of rice plant volatiles. Intriguingly, 1^st‐instar nymphs showed elevated NcinOrco expression compared to other nymphal stages, implying a potential role in early development by enhancing olfactory sensitivity to successfully locate hosts—a phenomenon warranting further study.

Our central finding was that RDV infection upregulated NcinOrco expression in viruliferous GRLHs (Fig. 4), implicating this gene in virus‐induced behavioral changes. To test this, we used RNAi to silence NcinOrco and observed a 74% reduction in transcript levels at 48 h postinjection [Fig. 5(A)]. Behavioral assays confirmed that dsNicnOrco‐treated viruliferous GRLHs lost their ability to discriminate between RDV‐infected (MH63‐RDV) and RDV‐free (MH63) rice plants, as well as between the volatile 2‐heptanol and clean air (Figs 5 and 6), indicating that NcinOrco is indeed essential for perceiving the RDV‐induced volatile 2‐heptanol. This finding is similar to the documented role of other Orco genes in Hemiptera, including LstrOrco, NluOrco, BtOrco and AluOrco, in host plant odor detection. ²⁵ , ⁴⁸ , ⁵³ , ⁵⁴ In a parallel case, Rice stripe virus infection in L. striatellus also was shown to trigger LstrOrco expression, which was crucial for host‐seeking behavior. ²⁵ Collectively, these observations suggest a conserved mechanism whereby viral infection influences insect vector behavior through Orco, yet whether this NcinOrco‐mediated perception promotes RDV transmission remains unclear.

The transmission of plant viruses is mediated by complex multilevel interactions among the viruses, their insect vectors and host plants. ¹⁶ Plant viruses can modify the composition and quantity of plant volatiles, thereby increasing the attractiveness of infected plants to nonviruliferous insect vectors or repelling viruliferous vectors toward virus‐free plants—a phenomenon that may promote viral spread and epidemic outbreaks. ¹⁹ , ²⁰ , ³⁷ , ⁵⁵ Our previous studies showed that a RDV‐induced volatile 2‐heptanol could facilitate viral transmission; ³⁴ however, the molecular mechanism by which 2‐heptanol facilitates the diffusion of RDV remains unclear. In this study, we first found that RDV increases the expression level of NcinOrco gene which influences the detection of 2‐heptanol by viruliferous GRLHs that promote viral transmission (Fig. 7). Although Orco serves as a conserved coreceptor, identifying its specific partner ORs involved in 2‐heptanol recognition—and elucidating the downstream signaling pathways—could provide more precise targets for disrupting the virus–vector chemosensory interaction. Such strategies aimed at blocking OR–Orco complexes may offer a more effective approach to prevent viruliferous insects from locating virus‐free hosts, ultimately curbing RDV transmission.

Beyond Orco and OR, other olfactory components such as OBPs and CSPs also are vital for volatile perception and are known targets of viral manipulation in other systems. ⁶ , ²³ , ²⁷ , ⁵⁶ , ⁵⁷ Future work should investigate whether RDV likewise modulates specific OBPs or CSPs in GRLHs, and if so, RNAi could be employed to assess the functional roles of these candidate proteins in the detection of 2‐heptanol, whereas fluorescence binding assays would serve to determine their direct binding affinities to this volatile compound. Furthermore, the RDV genome consists of 12 double‐stranded RNA segments (S1–S12), encoding seven structural (P1‐3, P5, P7‐9) and five nonstructural proteins (Pns4, Pns6, Pns10‐12). ⁵⁸ , ⁵⁹ , ⁶⁰ Thus, identifying which structural or nonstructural RDV protein(s) are responsible for the direct regulation of NcinOrco transcription will be crucial to fully elucidate the molecular dialog within this tripartite interaction.

In conclusion, this study suggests that NcinOrco may serve as a molecular target exploited by RDV to modulate vector behavior by enhancing the perception of a host plant volatile. By providing evidence that disruption of NcinOrco impairs virus transmission, our work contributes to the understanding of plant–virus–vector interactions and points to olfactory coreceptors as potential targets for developing behavior‐based interventions, such as RNAi or chemical inhibitors, aimed at interfering with the transmission of insect‐borne plant viruses.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

AUTHOR CONTRIBUTIONS

X.F.C. conceived and designed the experiment. X.F.C., Z.Q.S. and Z.F.L. performed research. X.F.C., Z.Q.S., W.X.T., W.M.Y., C.H.H. and L.Z.Z. analyzed the data and prepared the figures and tables. X.F.C. wrote the manuscript. L.Z.Z and Y.G.Y reviewed and revised the manuscript. All authors read and approved the paper.

Supporting information

Table S1. GenBank accession no. for 87 amino acid sequences of Orcos in a phylogenetic tree.

Table S2. Primer sequences used in this study.

Fig. S1. Schematic view of the two‐choice H‐shaped olfactometer for studying rice plant odor preference.

Fig. S2. Schematic representation of dual‐choice trap assays for GRLHs.

Fig. S3. Experimental design for RDV transmission rates by dsGFP‐ and dsNcinOrco‐treated viruliferous GRLHs, comparing MH63 plants with 10 μL lanolin paste containing 16.34 μg 2‐heptanol versus MH63 plants with 10 μL pure lanolin paste.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.