A plant virus manipulates the long-winged morph of insect vectors.

Wing dimorphism of insect vectors is a determining factor for viral long-distance dispersal and large-area epidemics. Although plant viruses affect the wing plasticity of insect vectors, the potential underlying molecular mechanisms have seldom been investigated. Here, we found that a planthopper-vectored rice virus, rice stripe virus (RSV), specifically induces a long-winged morph in male insects. The analysis of field populations demonstrated that the long-winged ratios of male insects are closely associated with RSV infection regardless of viral titers. A planthopper-specific and testis-highly expressed gene, Encounter, was fortuitously found to play a key role in the RSV-induced long-winged morph. Encounter resembles malate dehydrogenase in the sequence, but it does not have corresponding enzymatic activity. Encounter is upregulated to affect male wing dimorphism at early larval stages. Encounter is closely connected with the insulin/insulin-like growth factor signaling pathway as a downstream factor of Akt, of which the transcriptional level is activated in response to RSV infection, resulting in the elevated expression of Encounter. In addition, an RSV-derived small interfering RNA directly targets Encounter to enhance its expression. Our study reveals an unreported mechanism underlying the direct regulation by a plant virus of wing dimorphism in its insect vectors, providing the potential way for interrupting viral dispersal.

Key role of exportin 6 in exosome-mediated viral transmission from insect vectors to plants.

Exosomes play a key role in virus exocytosis and transmission. The exportin family is usually responsible for cargo nucleocytoplasmic trafficking, and they are frequently found in exosomes. However, the function of exportins sorted in exosomes remains unknown. Here, we successfully isolated "cup holder"-like exosomes from the saliva of ∼30,000 small brown planthoppers, which are vectors of rice stripe virus (RSV). RSV virions were packed in comparatively large exosomes. Four viral genomic RNAs at a certain ratio were identified in the saliva exosomes. The virions contained in the saliva exosomes were capable of replicating and causing disease in rice plants. Interference with each phase of the insect exosome system affected the transmission of RSV from the insect vectors to rice plants. Fragmented exportin 6 was coimmunoprecipitated with viral nucleocapsid protein in saliva and sorted to exosomes via interactions with the cargo sorting protein VPS37a. When the expression of exportin 6 was knocked down, the amounts of RSV secreted in saliva and rice plants were reduced by 60% and 74%, respectively. These results showed that exportin 6 acted as a vehicle for transporting RSV into exosomes to overcome the barrier of insect salivary glands for horizontal transmission. Exportin 6 would represent an ideal target that could be manipulated to control the outbreak of insect-borne viruses in the future.

Alternative Splicing Landscape of Small Brown Planthopper and Different Response of JNK2 Isoforms to Rice Stripe Virus Infection.

Alternative splicing (AS) is a frequent posttranscriptional regulatory event occurring in response to various endogenous and exogenous stimuli in most eukaryotic organisms. However, little is known about the effects of insect-transmitted viruses on AS events in insect vectors. The present study used third-generation sequencing technology and RNA sequencing (RNA-Seq) to evaluate the AS response in the small brown planthopper Laodelphax striatellus to rice stripe virus (RSV). The full-length transcriptome of L. striatellus was obtained using single-molecule real-time sequencing technology (SMRT). Posttranscriptional regulatory events, including AS, alternative polyadenylation, and fusion transcripts, were analyzed. A total of 28,175 nonredundant transcript isoforms included 24,950 transcripts assigned to 8,500 annotated genes of L. striatellus, and 5,000 of these genes (58.8%) had AS events. RNA-Seq of the gut samples of insects infected by RSV for 8 d identified 3,458 differentially expressed transcripts (DETs); 2,185 of these DETs were transcribed from 1,568 genes that had AS events, indicating that 31.4% of alternatively spliced genes responded to RSV infection of the gut. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, experienced exon skipping, resulting in three transcript isoforms. These three isoforms differentially responded to RSV infection during development and in various organs. Injection of double-stranded RNAs targeting all or two isoforms indicated that three or at least two JNK2 isoforms facilitated RSV accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors. IMPORTANCE Alternative splicing (AS) is a regulatory mechanism that occurs after gene transcription. AS events can enrich protein diversity to promote the reactions of the organisms to various endogenous and exogenous stimulations. It is not known how insect vectors exploit AS events to cope with transmitted viruses. The present study used third-generation sequencing technology to obtain the profile of AS events in the small brown planthopper Laodelphax striatellus, which is an efficient vector for rice stripe virus (RSV). The results indicated that 31.4% of alternatively spliced genes responded to RSV infection in the gut of planthoppers. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, produced three transcript isoforms by AS. These three isoforms showed different responses to RSV infection, and at least two isoforms facilitated viral accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors.

Flotillin 2 Facilitates the Infection of a Plant Virus in the Gut of Insect Vector.

Most plant viruses require insect vectors for transmission. One of the key steps for the transmission of persistent-circulative plant viruses is overcoming the gut barrier to enter epithelial cells. To date, little has been known about viral cofactors in gut epithelial cells of insect vectors. Here, we identified flotillin 2 as a plasma membrane protein that facilitates the infection of rice stripe virus (RSV) in its vector, the small brown planthopper. Flotillin 2 displayed a prominent plasma membrane location in midgut epithelial cells. The nucleocapsid protein of RSV and flotillin 2 colocalized on gut microvilli, and a nanomolar affinity existed between the two proteins. Knockout of flotillin 2 impeded the entry of virions into epithelial cells, resulting in a 57% reduction of RSV levels in planthoppers. The knockout of flotillin 2 decreased disease incidence in rice plants fed by viruliferous planthoppers from 40% to 11.7%. Furthermore, flotillin 2 mediated the infection of southern rice black-streaked dwarf virus in its vector, the white-backed planthopper. This work implies the potential of flotillin 2 as a target for controlling the transmission of rice stripe disease. IMPORTANCE Plant viral diseases are a major threat to world agriculture. The transmission of 80% of plant viruses requires vector insects, and 54% of vector-borne plant viruses are persistent-circulative viruses, which must overcome the barriers of gut cells with the help of proteins on the cell surface. Here, we identified flotillin 2 as a membrane protein that mediates the cell entry of rice stripe virus in its vector insect, small brown planthopper. Flotillin 2 displays a prominent cellular membrane location in midgut cells and can specifically bind to virions. The loss of flotillin 2 impedes the entry of virions into the midgut cells of vector insects and substantially suppresses viral transmission to rice. Therefore, flotillin 2 may be a promising target gene for manipulation in vector insects to control the transmission of rice stripe disease and perhaps that of other rice virus diseases in the future.

Coordination between terminal variation of the viral genome and insect microRNAs regulates rice stripe virus replication in insect vectors.

Maintenance of a balance between the levels of viral replication and selective pressure from the immune systems of insect vectors is one of the prerequisites for efficient transmission of insect-borne propagative phytoviruses. The mechanism regulating the adaptation of RNA viruses to insect vectors by genomic variation remains unknown. Our previous study demonstrated an extension of the 3'-untranslated terminal region (UTR) of two genomic segments of rice stripe virus (RSV). In the present study, a reverse genetic system for RSV in human cells and an insect vector, the small brown planthopper Laodelphax striatellus, was used to demonstrate that the 3'-terminal extensions suppressed viral replication in vector insects by inhibiting promoter activity due to structural interference with the panhandle structure formed by viral 3'- and 5'-UTRs. The extension sequence in the viral RNA1 segment was targeted by an endogenous insect microRNA, miR-263a, which decreased the inhibitory effect of the extension sequence on viral promoter activity. Surprisingly, the expression of miR-263a was negatively regulated by RSV infection. This elaborate coordination between terminal variation of the viral genome and endogenous insect microRNAs controls RSV replication in planthopper, thus reflecting a distinct strategy of adaptation of phytoviruses to insect vectors.

A Symbiotic Virus Facilitates Aphid Adaptation to Host Plants by Suppressing Jasmonic Acid Responses.

Symbiotic viruses exist in many insects; however, their functions in host insects are not well understood. In this study, we explored the role of acyrthosiphon pisum virus (APV) in the interaction of its host aphid Acyrthosiphon pisum with plants. APV is primarily located in aphid salivary glands and gut and propagated in the insect. APV is horizontally transmitted to host plants during aphid feeding, but the virus does not replicate in the host plant. When the pea host race of aphids colonized two low-fitness plants, Medicago truncatula and Vicia villosa, the virus titers in both the aphids and plants significantly increased. Furthermore, APV infection strongly promoted the survival rate of the pea host race on V. villosa. Transcriptomic analysis showed that only 0.85% of aphid genes responded to APV infection when aphids fed on V. villosa, with a fold change in transcript levels of no more than fourfold. The improved survival due to APV infection was apparently related to the inhibitory effect of the virus on levels of phytohormone jasmonic acid (JA) and JA-isoleucine. Our data suggest a benefit of the symbiotic virus to its aphid host and demonstrate a novel case of symbiotic virus-mediated three-species interaction.

A Plant Virus Ensures Viral Stability in the Hemolymph of Vector Insects through Suppressing Prophenoloxidase Activation.

Most plant viruses require vector insects for transmission. Viral stability in the hemolymph of vector insects is a prerequisite for successful transmission of persistent plant viruses. However, knowledge of whether the proteolytic activation of prophenoloxidase (PPO) affects the stability of persistent plant viruses remains elusive. Here, we explored the interplay between rice stripe virus (RSV) and the PPO cascade of the vector small brown planthopper. Phenoloxidase (PO) activity was suppressed by RSV by approximately 60%. When the PPO cascade was activated, we found distinct melanization around RSV particles and serious damage to viral stability in the hemolymph. Viral suppression of PO activity was derived from obstruction of proteolytic cleavage of PPOs by binding of the viral nonstructural protein NS3. These results indicate that RSV attenuates the PPO response to ensure viral stability in the hemolymph of vector insects. Our research provides enlightening cues for controlling the transmission of vector-borne viruses.IMPORTANCE Large ratios of vector-borne plant viruses circulate in the hemolymph of their vector insects before entering the salivary glands to be transmitted to plants. The stability of virions in the hemolymph is vital in this process. Activation of the proteolytic prophenoloxidase (PPO) to produce active phenoloxidase (PO) is one of the major innate immune pathways in insect hemolymph. How a plant virus copes with the PPO immune reaction in its vector insect remains unclear. Here, we report that the PPO affects the stability of rice stripe virus (RSV), a notorious rice virus, in the hemolymph of a vector insect, the small brown planthopper. RSV suppresses PPO activation using viral nonstructural protein. Once the level of PO activity is elevated, RSV is melanized and eliminated from the hemolymph. Our work gives valuable clues for developing novel strategies for controlling the transmission of vector-borne plant viruses.

Genome sequence of the small brown planthopper, Laodelphax striatellus.

Background: Laodelphax striatellus Fallén (Hemiptera: Delphacidae) is one of the most destructive rice pests. L. striatellus is different from 2 other rice planthoppers with a released genome sequence, Sogatella furcifera and Nilaparvata lugens, in many biological characteristics, such as host range, dispersal capacity, and vectoring plant viruses. Deciphering the genome of L. striatellus will further the understanding of the genetic basis of the biological differences among the 3 rice planthoppers. Findings: A total of 190 Gb of Illumina data and 32.4 Gb of Pacbio data were generated and used to assemble a high-quality L. striatellus genome sequence, which is 541 Mb in length and has a contig N50 of 118 Kb and a scaffold N50 of 1.08 Mb. Annotated repetitive elements account for 25.7% of the genome. A total of 17 736 protein-coding genes were annotated, capturing 97.6% and 98% of the BUSCO eukaryote and arthropoda genes, respectively. Compared with N. lugens and S. furcifera, L. striatellus has the smallest genome and the lowest gene number. Gene family expansion and transcriptomic analyses provided hints to the genomic basis of the differences in important traits such as host range, migratory habit, and plant virus transmission between L. striatellus and the other 2 planthoppers. Conclusions: We report a high-quality genome assembly of L. striatellus, which is an important genomic resource not only for the study of the biology of L. striatellus and its interactions with plant hosts and plant viruses, but also for comparison with other planthoppers.