RESUMO
Leaf yellowing is a well-known phenotype that attracts phloem-feeding insects. However, it remains unclear how insect-vectored plant pathogens induce host leaf yellowing to facilitate their own transmission by insect vectors. Here, we report that an effector protein secreted by rice orange leaf phytoplasma (ROLP) inhibits chlorophyll biosynthesis and induces leaf yellowing to attract leafhopper vectors, thereby presumably promoting pathogen transmission. This effector, designated secreted ROLP protein 1 (SRP1), first secreted into rice phloem by ROLP, was subsequently translocated to chloroplasts by interacting with the chloroplastic glutamine synthetase (GS2). The direct interaction between SRP1 and GS2 disrupts the decamer formation of the GS2 holoenzyme, attenuating its enzymatic activity, thereby suppressing the synthesis of chlorophyll precursors glutamate and glutamine. Transgenic expression of SRP1 in rice plants decreased GS2 activity and chlorophyll precursor accumulation, finally inducing leaf yellowing. This process is correlated with the previous evidence that the knockout of GS2 expression in rice plants causes a similar yellow chlorosis phenotype. Consistently, these yellowing leaves attracted higher numbers of leafhopper vectors, caused the vectors to probe more frequently, and presumably facilitate more efficient phytoplasma transmission. Together, these results uncover the mechanism used by phytoplasmas to manipulate the leaf color of infected plants for the purpose of enhancing attractiveness to insect vectors.
Assuntos
Cloroplastos , Glutamato-Amônia Ligase , Hemípteros , Insetos Vetores , Oryza , Phytoplasma , Folhas de Planta , Animais , Hemípteros/microbiologia , Glutamato-Amônia Ligase/metabolismo , Glutamato-Amônia Ligase/genética , Phytoplasma/fisiologia , Folhas de Planta/microbiologia , Folhas de Planta/metabolismo , Oryza/microbiologia , Oryza/genética , Insetos Vetores/microbiologia , Cloroplastos/metabolismo , Doenças das Plantas/microbiologia , Clorofila/metabolismo , Plantas Geneticamente Modificadas , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genéticaRESUMO
Many plant arboviruses are persistently transmitted by piercing-sucking insect vectors. However, it remains largely unknown how conserved insect Toll immune response exerts antiviral activity and how plant viruses antagonize it to facilitate persistent viral transmission. Here, we discover that southern rice black-streaked dwarf virus (SRBSDV), a devastating planthopper-transmitted rice reovirus, activates the upstream Toll receptors expression but suppresses the downstream MyD88-Dorsal-defensin cascade, resulting in the attenuation of insect Toll immune response. Toll pathway-induced the small antibacterial peptide defensin directly interacts with viral major outer capsid protein P10 and thus binds to viral particles, finally blocking effective viral infection in planthopper vector. Furthermore, viral tubular protein P7-1 directly interacts with and promotes RING E3 ubiquitin ligase-mediated ubiquitinated degradation of Toll pathway adaptor protein MyD88 through the 26 proteasome pathway, finally suppressing antiviral defensin production. This virus-mediated attenuation of Toll antiviral immune response to express antiviral defensin ensures persistent virus infection without causing evident fitness costs for the insects. E3 ubiquitin ligase also is directly involved in the assembly of virus-induced tubules constructed by P7-1 to facilitate viral spread in planthopper vector, thereby acting as a pro-viral factor. Together, we uncover a previously unknown mechanism used by plant arboviruses to suppress Toll immune response through the ubiquitinated degradation of the conserved adaptor protein MyD88, thereby facilitating the coexistence of arboviruses with their vectors in nature.
Assuntos
Arbovírus , Insetos Vetores , Transdução de Sinais , Receptores Toll-Like , Animais , Arbovírus/imunologia , Receptores Toll-Like/metabolismo , Insetos Vetores/virologia , Insetos Vetores/imunologia , Doenças das Plantas/virologia , Doenças das Plantas/imunologia , Reoviridae/fisiologia , Reoviridae/imunologia , Hemípteros/virologia , Hemípteros/imunologia , Oryza/virologia , Oryza/imunologia , Proteínas de Insetos/metabolismo , Imunidade InataRESUMO
Viral diseases seriously threaten rice production. Plasmodesmata (PD)-associated proteins are deemed to play a key role in viral infection in host plants. However, few PD-associated proteins have been discovered in rice to afford viral infection. Here, inspired by the infection mechanism in insect vectors, we identified a member of the Flotillin family taking part in the cell-to-cell transport of rice stripe virus (RSV) in rice. Flotillin1 interacted with RSV nucleocapsid protein (NP) and was localized on PD. In flotillin1 knockout mutant rice, which displayed normal growth, RSV intercellular movement was retarded, leading to significantly decreased disease incidence. The PD pore sizes of the mutant rice were smaller than those of the wild type due to more callose deposits, which was closely related to the upregulation of two callose synthase genes. RSV infection stimulated flotillin1 expression and enlarged the PD aperture via RSV NP. In addition, flotillin1 knockout decreased disease incidences of southern rice black-streaked dwarf virus (SRBSDV) and rice dwarf virus (RDV) in rice. Overall, our study reveals a new PD-associated protein facilitating virus cell-to-cell trafficking and presents the potential of flotillin1 as a target to produce broad-spectrum antiviral rice resources in the future.
Assuntos
Hemípteros , Proteínas de Membrana , Oryza , Viroses , Animais , Plasmodesmos/metabolismo , Proteínas Virais/metabolismo , Oryza/metabolismo , Doenças das Plantas , Hemípteros/metabolismoRESUMO
Viruses can hijack autophagosomes as the nonlytic release vehicles in cultured host cells. However, how autophagosome-mediated viral spread occurs in infected host tissues or organs in vivo remains poorly understood. Here, we report that an important rice reovirus, rice gall dwarf virus (RGDV) hijacks autophagosomes to traverse multiple insect membrane barriers in the midgut and salivary gland of leafhopper vector to enhance viral spread. Such virus-containing double-membraned autophagosomes are prevented from degradation, resulting in increased viral propagation. Mechanistically, viral nonstructural protein Pns11 induces autophagy and embeds itself in the autophagosome membranes. The autophagy-related protein 5 (ATG5)-ATG12 conjugation is essential for initial autophagosome membrane biogenesis. RGDV Pns11 specifically interacts with ATG5, both in vitro and in vivo. Silencing of ATG5 or Pns11 expression suppresses ATG8 lipidation, autophagosome formation, and efficient viral propagation. Thus, Pns11 could directly recruit ATG5-ATG12 conjugation to induce the formation of autophagosomes, facilitating viral spread within the insect bodies. Furthermore, Pns11 potentially blocks autophagosome degradation by directly targeting and mediating the reduced expression of N-glycosylated Lamp1 on lysosomal membranes. Taken together, these results highlight how RGDV remodels autophagosomes to benefit viral propagation in its insect vector.
Assuntos
Orthoreovirus , Oryza , Reoviridae , Animais , Autofagossomos/metabolismo , Autofagia , Insetos Vetores , Insetos/metabolismo , Oryza/metabolismo , Reoviridae/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Catalase (CAT) is the main reactive oxygen species (ROS)-scavenging enzyme in plants and insects. However, it remains elusive whether and how insect saliva CAT suppresses ROS-mediated plant defense, thereby promoting initial virus transmission by insect vectors. Here, we investigated how leafhopper Recilia dorsalis catalase (RdCAT) was secreted from insect salivary glands into rice phloem, and how it was perceived by rice chaperone NO CATALASE ACTIVITY1 (OsNCA1) to scavenge excessive H2O2 during insect-to-plant virus transmission. We found that the interaction of OsNCA1 with RdCAT activated its enzymatic activity to decompose H2O2 in rice plants during leafhopper feeding. However, initial insect feeding did not significantly change rice CATs transcripts. Knockout of OsNCA1 in transgenic lines decreased leafhopper feeding-activated CAT activity and caused higher H2O2 accumulation. A devastating rice reovirus activated RdCAT expression and promoted the cosecretion of virions and RdCAT into leafhopper salivary cavities and ultimately into the phloem. Virus-mediated increase of RdCAT secretion suppressed excessive H2O2, thereby promoting host attractiveness to insect vectors and initial virus transmission. Our findings provide insights into how insect saliva CAT is secreted and perceived by plant chaperones to suppress the early H2O2 burst during insect feeding, thereby facilitating viral transmission.
Assuntos
Catalase , Hemípteros , Peróxido de Hidrogênio , Insetos Vetores , Oryza , Saliva , Animais , Peróxido de Hidrogênio/metabolismo , Hemípteros/virologia , Hemípteros/fisiologia , Saliva/virologia , Saliva/enzimologia , Catalase/metabolismo , Catalase/genética , Insetos Vetores/virologia , Oryza/virologia , Oryza/genética , Oryza/enzimologia , Reoviridae/fisiologia , Doenças das Plantas/virologia , Floema/virologiaRESUMO
BACKGROUND: The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation. METHODS: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. RESULTS: First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. CONCLUSION: These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.
Assuntos
Animais Geneticamente Modificados , Galactosiltransferases , Edição de Genes , Transplante de Rim , Transplante Heterólogo , Animais , Transplante Heterólogo/métodos , Transplante de Rim/métodos , Suínos , Edição de Genes/métodos , Galactosiltransferases/genética , Sistemas CRISPR-Cas , Macaca mulatta , Técnicas de Transferência Nuclear , Xenoenxertos , Humanos , Sobrevivência de Enxerto/imunologia , Rejeição de Enxerto/imunologia , Apirase , Antígenos CDRESUMO
Plant viruses are a group of intracellular pathogens that persistently threaten global food security. Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years, including basic research and technologies for preventing and controlling plant viral diseases. Here, we review these milestones and advances, including the identification of new crop-infecting viruses, dissection of pathogenic mechanisms of multiple viruses, examination of multilayered interactions among viruses, their host plants, and virus-transmitting arthropod vectors, and in-depth interrogation of plant-encoded resistance and susceptibility determinants. Notably, various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants. We also recommend future plant virology studies in China.
Assuntos
Patologia Vegetal , Vírus de Plantas , Doenças das Plantas/genética , Plantas/genética , Plantas/metabolismo , ChinaRESUMO
In the field, many insect-borne crop viral diseases are more suitable for maintenance and spread in hot-temperature areas, but the mechanism remains poorly understood. The epidemic of a planthopper (Sogatella furcifera)-transmitted rice reovirus (southern rice black-streaked dwarf virus, SRBSDV) is geographically restricted to southern China and northern Vietnam with year-round hot temperatures. Here, we reported that two factors of endoplasmic reticulum-associated degradation (ERAD) machinery, the heat shock protein DnaJB11 and ER membrane protein BAP31, were activated by viral infection to mediate the adaptation of S. furcifera to high temperatures. Infection and transmission efficiencies of SRBSDV by S. furcifera increased with the elevated temperatures. We observed that high temperature (35°C) was beneficial for the assembly of virus-containing tubular structures formed by nonstructural protein P7-1 of SRBSDV, which facilitates efficient viral transmission by S. furcifera. Both DnaJB11 and BAP31 competed to directly bind to the tubule protein P7-1 of SRBSDV; however, DnaJB11 promoted whereas BAP31 inhibited P7-1 tubule assembly at the ER membrane. Furthermore, the binding affinity of DnaJB11 with P7-1 was stronger than that of BAP31 with P7-1. We also revealed that BAP31 negatively regulated DnaJB11 expression through their direct interaction. High temperatures could significantly upregulate DnaJB11 expression but inhibit BAP31 expression, thereby strongly facilitating the assembly of abundant P7-1 tubules. Taken together, we showed that a new temperature-dependent protein quality control pathway in the ERAD machinery has evolved for strong activation of DnaJB11 for benefiting P7-1 tubules assembly to support efficient transmission of SRBSDV in high temperatures. We thus deduced that ERAD machinery has been hitchhiked by insect-borne crop viruses to enhance their transmission in tropical climates.
Assuntos
Temperatura Alta/efeitos adversos , Insetos Vetores/virologia , Doenças das Plantas/virologia , Reoviridae/imunologia , Animais , Degradação Associada com o Retículo Endoplasmático/imunologia , Insetos Vetores/imunologia , Orthoreovirus/patogenicidadeRESUMO
Spinareoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (9-12 linear segments) dsRNA genomes of 23-29 kbp. Spinareovirids have a broad host range, infecting animals, fungi and plants. Some have important pathogenic potential for humans (e.g. Colorado tick fever virus), livestock (e.g. avian orthoreoviruses), fish (e.g. aquareoviruses) and plants (e.g. rice ragged stunt virus and rice black streaked dwarf virus). This is a summary of the ICTV Report on the family Spinareoviridae, which is available at ictv.global/report/spinareoviridae.
Assuntos
Fungos , RNA de Cadeia Dupla , Animais , Humanos , Plantas , Especificidade de Hospedeiro , FilogeniaRESUMO
A novel virus named Aconitum amalgavirus 1 (AcoAV-1) was identified in Chinese aconite (Aconitum carmichaelii) plants. The complete genome of AcoAV-1 is 3,370 nucleotides long, containing two partially overlapping open reading frames encoding a putative coat protein and a RNA-dependent RNA polymerase, respectively. Its fusion protein shares 34.9%-50.7% amino acid sequence identity with other amalgaviruses. Phylogenetic analysis showed that this virus formed a clade with blueberry latent virus and four other related viruses, suggesting that it belongs to the genus Amalgavirus in the family Amalgaviridae.
Assuntos
Aconitum , Vírus de RNA , Aconitum/genética , Genoma Viral , Nucleotídeos , Fases de Leitura Aberta , Filogenia , Vírus de RNA/genética , RNA Viral/genética , RNA Polimerase Dependente de RNARESUMO
Numerous plant viruses that cause significant agricultural problems are persistently transmitted by insect vectors. We wanted to see if apoptosis was involved in viral infection process in the vector. We found that a plant reovirus (rice gall dwarf virus, RGDV) induced typical apoptotic response during viral replication in the leafhopper vector and cultured vector cells, as demonstrated by mitochondrial degeneration and membrane potential decrease. Fibrillar structures formed by nonstructural protein Pns11 of RGDV targeted the outer membrane of mitochondria, likely by interaction with an apoptosis-related mitochondrial protein in virus-infected leafhopper cells or nonvector insect cells. Such association of virus-induced fibrillar structures with mitochondria clearly led to mitochondrial degeneration and membrane potential decrease, suggesting that RGDV Pns11 was the inducer of apoptotic response in insect vectors. A caspase inhibitor treatment and knockdown of caspase gene expression using RNA interference each reduced apoptosis and viral accumulation, while the knockdown of gene expression for the inhibitor of apoptosis protein improved apoptosis and viral accumulation. Thus, RGDV exploited caspase-dependent apoptotic response to promote viral infection in insect vectors. For the first time, we directly confirmed that a nonstructural protein encoded by a persistent plant virus can induce the typical apoptotic response to benefit viral transmission by insect vectors.
Assuntos
Apoptose/fisiologia , Hemípteros/virologia , Reoviridae/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Colágenos Fibrilares/metabolismo , Insetos Vetores/virologia , Insetos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/virologia , Vírus de Plantas/metabolismo , Reoviridae/genética , Reoviridae/patogenicidade , Reoviridae/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Plant viruses typically cause severe pathogenicity in plants, even resulting in the death of plants. Many pathogenic plant viruses are transmitted in a persistent manner via insect vectors. Interestingly, unlike in the plant hosts, persistent viruses are either nonpathogenic or show limited pathogenicity in their insect vectors, while taking advantage of the cellular machinery of insect vectors for completing their life cycles. This review discusses why persistent plant viruses are nonpathogenic or have limited pathogenicity to their insect vectors while being pathogenic to plants hosts. Current advances in cell biology of virus-insect vector interactions are summarized, including virus-induced inclusion bodies, changes of insect cellular ultrastructure, and immune response of insects to the viruses, especially autophagy and apoptosis. The corresponding findings of virus-plant interactions are compared. An integrated view of the balance strategy achieved by the interaction between viral attack and the immune response of insect is presented. Finally, we outline progress gaps between virus-insect and virus-plant interactions, thus highlighting the contributions of cultured cells to the cell biology of virus-insect interactions. Furthermore, future prospects of studying the cell biology of virus-vector interactions are presented.
Assuntos
Interações Hospedeiro-Patógeno , Insetos Vetores , Vírus de Plantas , Plantas , Animais , Insetos Vetores/virologia , Células Vegetais/virologia , Doenças das Plantas/virologia , Vírus de Plantas/fisiologia , Plantas/virologiaRESUMO
Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.
Assuntos
Autofagia/fisiologia , Hemípteros/virologia , Insetos Vetores/virologia , Vírus de Plantas/metabolismo , Reoviridae/fisiologia , Animais , Linhagem Celular , Insetos/virologia , Vírion/metabolismo , Replicação Viral/genéticaRESUMO
Numerous viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. These viruses circulate in the vector body, enter the salivary gland, and then are released into the apical plasmalemma-lined cavities, where saliva is stored. The cavity plasmalemma of vector salivary glands thus represents the last membrane barrier for viral transmission. Here, we report a novel mechanism used by a persistent virus to overcome this essential barrier. We observed that the infection by rice gall dwarf virus (RGDV), a species of the genus Phytoreovirus in the family Reoviridae, induced the formation of virus-associated filaments constructed by viral nonstructural protein Pns11 within the salivary glands of its leafhopper vector, Recilia dorsalis Such filaments attached to actin-based apical plasmalemma and induced an exocytosis-like process for viral release into vector salivary gland cavities, through a direct interaction of Pns11 of RGDV and actin of R. dorsalis Failure of virus-induced filaments assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene inhibited the dissemination of RGDV into salivary cavities, preventing viral transmission by R. dorsalis For the first time, we show that a virus can exploit virus-induced inclusion as a vehicle to pass through the apical plasmalemma into vector salivary gland cavities, thus overcoming the last membrane barrier for viral transmission by insect vectors.IMPORTANCE Understanding how persistent viruses overcome multiple tissue and membrane barriers within the insect vectors until final transmission is the key for viral disease control. The apical plasmalemma of the cavities where saliva is stored in the salivary glands is the last barrier for viral transmission by insect vectors; however, the mechanism is still poorly understood. Here we show that a virus has evolved to exploit virus-induced filaments to perform an exocytosis-like process that enables viral passage through the apical plasmalemma into salivary cavities. This mechanism could be extensively exploited by other persistent viruses to overcome salivary gland release barriers in insect vectors, opening new perspectives for viral control.
Assuntos
Hemípteros/virologia , Reoviridae/fisiologia , Proteínas não Estruturais Virais/metabolismo , Liberação de Vírus , Actinas/metabolismo , Animais , Exocitose , Insetos Vetores/virologia , Microscopia de Fluorescência , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Reoviridae/ultraestrutura , Glândulas Salivares/ultraestrutura , Glândulas Salivares/virologia , Células Sf9 , Montagem de Vírus , Replicação ViralRESUMO
BACKGROUND: In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear. METHODS: In present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm. RESULTS: Here we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment. CONCLUSIONS: In summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.
Assuntos
Hemípteros/citologia , Hemípteros/virologia , Insetos Vetores/citologia , Insetos Vetores/virologia , Oryza/virologia , Vírus de Plantas/fisiologia , Rhabdoviridae/metabolismo , Animais , Células Cultivadas , China , Técnicas de Silenciamento de Genes , Hemípteros/ultraestrutura , Insetos Vetores/ultraestrutura , Técnicas do Sistema de Duplo-Híbrido , Proteínas Estruturais Virais/metabolismo , Replicação ViralRESUMO
UNLABELLED: Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/µg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE: Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/µg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector.
Assuntos
Hemípteros/virologia , RNA Interferente Pequeno/metabolismo , Reoviridae/crescimento & desenvolvimento , Reoviridae/imunologia , Animais , Células Cultivadas , Epitélio/imunologia , Epitélio/virologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/virologiaRESUMO
Numerous viruses can be transmitted by their corresponding vector insects; however, the molecular mechanisms enabling virus transmission by vector insects have been poorly understood, especially the identity of vector components interacting with the virus. Here, we used the yeast two-hybrid system to study proteomic interactions of a plant virus (Rice stripe virus, RSV, genus Tenuivirus) with its vector insect, small brown planthopper (Laodelphax striatellus). Sixty-six proteins of L. striatellus that interacted with the nucleocapsid protein (pc3) of RSV were identified. A virus-insect interaction network, constructed for pc3 and 29 protein homologs of Drosophila melanogaster, suggested that nine proteins might directly interact with pc3. Of the 66 proteins, five (atlasin, a novel cuticular protein, jagunal, NAC domain protein, and vitellogenin) were most likely to be involved in viral movement, replication, and transovarial transmission. This work also provides evidence that the novel cuticular protein, CPR1, from L. striatellus is essential for RSV transmission by its vector insect. CPR1 binds the nucleocapsid protein (pc3) of RSV both in vivo and in vitro and colocalizes with RSV in the hemocytes of L. striatellus. Knockdown of CPR1 transcription using RNA interference resulted in a decrease in the concentration of RSV in the hemolymph, salivary glands and in viral transmission efficiency. These data suggest that CPR1 binds RSV in the insect and stabilizes the viral concentration in the hemolymph, perhaps to protect the virus or to help move the virus to the salivary tissues. Our studies provide direct experimental evidence that viruses can use existing vector proteins to aid their survival in the hemolymph. Identifying these putative vector molecules should lead to a better understanding of the interactions between viruses and vector insects.
Assuntos
Hemípteros/metabolismo , Proteínas de Insetos/metabolismo , Insetos Vetores/virologia , Tegumento Comum/fisiologia , Proteômica/métodos , Tenuivirus/metabolismo , Animais , Linhagem Celular , Biologia Computacional , Feminino , Técnicas de Silenciamento de Genes , Imunoprecipitação , Ligação Proteica , Mapas de Interação de Proteínas , Transporte Proteico , Interferência de RNA , Reprodutibilidade dos Testes , Frações Subcelulares/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/metabolismoRESUMO
Most plant viruses are transmitted by hemipteroid insects. Some viruses can be transmitted from female parent to offspring usually through eggs, but the mechanism of this transovarial transmission remains unclear. Rice stripe virus (RSV), a Tenuivirus, transmitted mainly by the small brown planthopper (Laodelphax striatellus), is also spread to the offspring through the eggs. Here, we used the RSV-planthopper system as a model to investigate the mechanism of transovarial transmission and demonstrated the central role of vitellogenin (Vg) of L. striatellus in the process of virus transmission into the eggs. Our data showed Vg can bind to pc3 in vivo and in vitro and colocalize in the germarium. RSV filamentous ribonucleoprotein particles (RNPs) only accumulated in the terminal filaments and pedicel areas prior to Vg expression and was not present in the germarium until Vg was expressed, where RSV RNPs and Vg had colocalized. Observations by immunoelectron microscopy (IEM) also indicated that these two proteins colocalized in nurse cells. Knockdown of Vg expression due to RNA interference resulted in inhibition of the invasion of ovarioles by RSV. Together, the data obtained indicated that RSV RNPs may enter the nurse cell of the germarium via endocytosis through binding with Vg. Finally, the virus enters the oocytes through nutritive cords, using the same route as for Vg transport. Our results show that the Vg of L. striatellus played a critical role in transovarial transmission of RSV and shows how viruses can use existing transovarial transportation systems in insect vectors for their own purposes.
Assuntos
Flores/virologia , Insetos Vetores/metabolismo , Oryza/virologia , Tenuivirus/patogenicidade , Vitelogeninas/metabolismo , Animais , Imunofluorescência , Hemípteros/virologia , Imunoprecipitação , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase em Tempo Real , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) are two closely related fijiviruses transmitted by the small brown planthopper (SBPH) and white-backed planthopper (WBPH), respectively. SRBSDV has a latent period 4 days shorter than that of RBSDV, implying a more efficient spread in insect vector. Currently, the mechanisms underlying this higher efficiency are poorly understood. However, our recent studies have implicated a role of virus induced tubular structures in the dissemination of fijiiruses within their insect vectors. METHODS: Immunofluorescence labeling was performed to visualize and compare the dynamics of P7-1 tubule formation of the RBSDV and SRBSDV in their own vector insects and nonhost Spodoptera frugiperda (Sf9) cells. RESULTS: Tubule formation of SRBSDV P7-1 was faster than that of RBSDV P7-1. For RBSDV, P7-1 formed tubules were observed at 3-days post-first access to diseased plants (padp) in SBPH. For SRBSDV, these structures were detected as early as 1 day padp in WBPH. Importantly, similar phenomena were observed when P7-1 proteins from the two viruses were expressed alone in Sf9 cells. CONCLUSIONS: Our research revealed a relationship between the speed of P7-1 tubule formation and virus dissemination efficiency and also supports a role of such tubular structures in the spread of reoviruses within insect vectors.
Assuntos
Interações Hospedeiro-Patógeno , Insetos Vetores/virologia , Substâncias Macromoleculares/metabolismo , Reoviridae/fisiologia , Animais , Células Cultivadas , Reoviridae/crescimento & desenvolvimento , Fatores de TempoRESUMO
Rice gall dwarf virus (RGDV), a plant reovirus that threatens rice production in Southeast Asia and Southern China, is transmitted by the leafhopper vector Recilia dorsalis in a persistent-propagative manner. To assess the direct effects of RGDV on R. dorsalis, we established an infected leafhopper population from eggs laid by viruliferous females using the water-soaked filter paper culture method. Life history parameters indicated that the virus was harmful to its vector in terms of all biotic indices, including reduced survival rate, emergence rate, fecundity, and longevity of adults, compared with a nonviruliferous control population. Those findings were supported by systematic monitoring of viruliferous rates of R. dorsalis in different overwintering generations. To better elucidate the adverse effects of RGDV on its vector, we measured fecundity at the molecular level using quantitative reverse-transcription polymerase chain reaction and Western blot assays, which revealed differential expression of vitellogenin (Vg) in viruliferous versus nonviruliferous adult females. We infer that RGDV reduced levels of Vg transcript and protein product, resulting in the lower fecundity of its vector. Overall, this study demonstrates how RGDV exerts an adverse effect on R. dorsalis, which hinders the expansion of viruliferous populations of the insect.