Arboviruses antagonize insect Toll antiviral immune signaling to facilitate the coexistence of viruses with their vectors.

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.

A nonstructural protein encoded by a rice reovirus induces an incomplete autophagy to promote viral spread in insect vectors.

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.

Scavenging H2O2 of plant host by saliva catalase of leafhopper vector benefits viral transmission.

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.

A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions.

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.

The Detection of Anthrax Biomarker DPA by Ratiometric Fluorescence Probe of Carbon Quantum Dots and Europium Hybrid Material Based on Poly(ionic)- Liquid.

Bacillus anthracis has gained international attention as a deadly bacterium and a potentially deadly biological warfare agent. Dipicolinic acid (DPA) is the main component of the protective layer of anthracis spores, and is also an anthrax biomarker. Therefore, it is of great significance to explore an efficient and sensitive DPA detection method. Herein, a novel ratio hybrid probe (CQDs-PIL-Eu3+) was prepared by a simple one-step hydrothermal method using carbon quantum dots (CQDs) as an internal reference fluorescence and a covalent bond between CQDs and Eu3+ by using a polyionic liquid (PIL) as a bridge molecule. The ratiometric fluorescence probe was found to have the characteristics of sensitive fluorescence visual sensing in detecting DPA. The structure and the sensing properties of CQDs-PIL-Eu3+ were investigated in detail. In particular, the fluorescence intensity ratio of Eu3+ to CQDs (I616/I440) was linear with the concentration of DPA in the range of 0-50 µM, so the detection limit of the probe was as low as 32 nm, which was far lower than the DPA dose released by the number of anthrax spores in human body (60 µM) and, thus, can achieve sensitive detection. Therefore, the ratiometric fluorescence probe in this work has the characteristics of strong anti-interference, visual sensing, and high sensitivity, which provides a very promising scheme for the realization of anthrax biomarker DPA detection.

Berberine alleviates intestinal barrier dysfunction in glucolipid metabolism disorder hamsters by modulating gut microbiota and gut-microbiota-related tryptophan metabolites.

BACKGROUND: Barberry plants can be considered as useful additives and functional compounds in various industries, especially in the food industry. Berberine (BBR), the most important functional compound in the barberry roots, has recently been used to treat obesity, diabetes, and atherosclerosis. Gut microbiota and the intestinal barrier play an important role in the development of glucolipid metabolism disorders (GLMDs). However, the association of gut microbiota metabolism disorder and the intestinal barrier dysfunction effect of BBR in GLMDs remains elusive. RESULTS: The results showed that administration of BBR could increase the number of colonic glands and goblet cell mucus secretion, improve the intestinal barrier function, and reduce the serum glycolipid level in GLMD hamsters. Interestingly, BBR was metabolized into 12 metabolites by gut microbiota, and the main metabolic pathways were oxidation, demethylation, and hydrogenation. In addition, BBR significantly improved the species diversity and uniformity of gut microbiota and promoted the proliferation of beneficial microbiota. Furthermore, the levels of tryptophan metabolites, such as indole, indole-3-acetamide, indole-3-acetaldehyde, indole-3-pyruvic acid, and indole-3-acetic acid were significantly altered by BBR. Both the intestinal tight junction proteins and intestinal immune factors were altered by BBR. CONCLUSION: BBR could alleviate intestinal barrier dysfunction of GLMDs by modulating gut microbiota and gut-microbiota-related tryptophan metabolites, which may be one of the pharmacological mechanisms for the treatment of GLMDs. © 2022 Society of Chemical Industry.

Fibrillar structures induced by a plant reovirus target mitochondria to activate typical apoptotic response and promote viral infection in insect vectors.

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.

Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector.

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.

Filamentous Structures Induced by a Phytoreovirus Mediate Viral Release from Salivary Glands in Its Insect Vector.

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.

DNA methylation affects metastasis of renal cancer and is associated with TGF-ß/RUNX3 inhibition.

BACKGROUND: Renal cell carcinoma accounts for 2-3% of all cancers and metastasis increased the malignancy of renal cancer. However, the role of methylation in metastasis of renal cancer is poorly understood. METHODS: We performed targeted gene array to compare the differential expressions of methylation regulated genes in metastatic and primary renal cancer tissues. Quantitative methylation specific PCR was performed to examine the CpG methylation levels of Runt related transcription factor 3 (RUNX3) and transforming growth factor (TGF)-ß. Western blot was performed to detect the expression of target genes. Murine xenograft renal cancer model was established to assay gene expression, methylation level, tumor growth and animal survival in vivo. RESULTS: RUNX3 and TGF-ß levels were decreased in metastatic renal cancer tissues as a result of their CpG methylation. Metastatic xenograft model displayed decreased expression levels of RUNX3 and TGF-ß and higher CpG methylation levels, bigger tumor size and shorter survival time, all which were restored by treatment with a methylation inhibitor. CONCLUSIONS: Hypermethylation in CpG islands promotes metastasis of renal cancer and is associated with TGF-ß and RUNX3 inhibition.

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

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.

The speed of tubule formation of two fijiviruses corresponds with their dissemination efficiency in their insect vectors.

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.

Development of continuous cell culture of brown planthopper to trace the early infection process of oryzaviruses in insect vector cells.

UNLABELLED: Rice ragged stunt virus (RRSV), an oryzavirus in the family Reoviridae, is transmitted by the brown planthopper, Nilaparvata lugens, in a persistent-propagative manner. Here, we established a continuous cell line of brown planthopper to investigate the mechanism underlying the formation of the viroplasm, the putative site for viral replication and assembly, during infection of RRSV in its insect vector cells. Within 24 h of viral infection of cultured cells, the viroplasm had formed and contained the viral nonstructural proteins Pns6 and Pns10, known to be constituents of viroplasm. Core capsid protein P3, core particles, and newly synthesized viral RNAs were accumulated inside the viroplasm, while outer capsid protein P8 and virions were accumulated at the periphery of the viroplasm, confirming that the viroplasm induced by RRSV infection was the site for viral replication and assembly. Pns10 formed viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm matrix was largely composed of Pns10. Pns6 was recruited in the viroplasm by direct interaction with Pns10. Core capsid protein P3 was recruited to the viroplasm through specific association with Pns6. Knockdown of Pns6 and Pns10 expression using RNA interference inhibited viroplasm formation, virion assembly, viral protein expression, and viral double-stranded RNA synthesis. Thus, the present study shows that both Pns6 and Pns10 of RRSV play important roles in the early stages of viral life cycle in its insect vector cells, by recruiting or retaining components necessary for viral replication and assembly. IMPORTANCE: The brown planthopper, a commonly distributed pest of rice in Asia, is the host of numerous insect endosymbionts, and the major vector of two rice viruses (RRSV and rice grassy stunt virus). For the first time, we successfully established the continuous cell line of brown planthopper. The unique uniformity of brown planthopper cells in the monolayer can support a consistent, synchronous infection by endosymbionts or viral pathogens, improving our understanding of molecular insect-microbe interactions.

Virus-induced tubule: a vehicle for rapid spread of virions through basal lamina from midgut epithelium in the insect vector.

UNLABELLED: The plant reoviruses, plant rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent propagative manner. These viruses induce the formation of viral inclusions to facilitate viral propagation in insect vectors. The intestines of insect vectors are formed by epithelial cells that lie on the noncellular basal lamina surrounded by visceral muscle tissue. Here, we demonstrate that a recently identified plant reovirus, southern rice black-streaked dwarf virus (SRBSDV), exploits virus-containing tubules composed of virus-encoded nonstructural protein P7-1 to directly cross the basal lamina from the initially infected epithelium toward visceral muscle tissues in the intestine of its vector, the white-backed planthopper (Sogatella furcifera). Furthermore, such tubules spread along visceral muscle tissues through a direct interaction of P7-1 and actin. The destruction of tubule assembly by RNA interference with synthesized double-stranded RNA targeting the P7-1 gene inhibited viral spread in the insect vector in vitro and in vivo. All these results show for the first time that a virus employs virus-induced tubule as a vehicle for viral spread from the initially infected midgut epithelium through the basal lamina, facilitating the rapid dissemination of virus from the intestine of the insect vector. IMPORTANCE: Numerous plant viruses are transmitted in a persistent manner by sap-sucking insects, including thrips, aphids, planthoppers, and leafhoppers. These viruses, ingested by the insects, establish their primary infection in the intestinal epithelium of the insect vector. Subsequently, the invading virus manages to transverse the basal lamina, a noncellular layer lining the intestine, a barrier that may theoretically hinder viral spread. The mechanism by which plant viruses cross the basal lamina is unknown. Here, we report that a plant virus has evolved to exploit virus-induced tubules to pass through the basal lamina from the initially infected midgut epithelium of the insect vector, thus revealing the previously undescribed pathway adapted by the virus for rapid dissemination of virions from the intestine of the insect vector.

New model for the genesis and maturation of viroplasms induced by fijiviruses in insect vector cells.

Plant reoviruses are thought to replicate and assemble within cytoplasmic, nonmembranous structures called viroplasms. Here, we established continuous cell cultures of the white-backed planthopper (Sogatella furcifera Horváth) to investigate the mechanisms for the genesis and maturation of the viroplasm induced by Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus in the family Reoviridae, during infection of its insect vector. Electron and confocal microscopy revealed that the viroplasm consisted of a granular region, where viral RNAs and nonstructural proteins P6 and P9-1 accumulated, and a filamentous region, where viral RNAs, progeny cores, viral particles, as well as nonstructural proteins P5 and P6 accumulated. Our results suggested that the filamentous viroplasm matrix was the site for the assembly of progeny virions. Because viral RNAs were produced by assembled core particles within the filamentous viroplasm matrix, we propose that these viral RNAs might be transported to the granular viroplasm matrix. P5 formed filamentous inclusions and P9-1 formed granular inclusions in the absence of viral infection, suggesting that the filamentous and granular viroplasm matrices were formed primarily by P5 and P9-1, respectively. P6 was apparently recruited in the whole viroplasm matrix by direct interaction with P9-1 and P5. Thus, the present results suggested that P5, P6, and P9-1 are collectively required for the genesis and maturation of the filamentous and granular viroplasm matrix induced by SRBSDV infection. Based on these results, we propose a new model to explain the genesis and maturation of the viroplasms induced by fijiviruses in insect vector cells.

[Influence of different processing methods and mature stages on 3,29-dibenzoyl rarounitriol of Trichosanthes kirilowii seeds].

OBJECTIVE: To study the different mature stages and the best processing methods on the quality of Trichosanthes kirilowii seeds. METHODS: The content of 3,29-dibenzoyl rarounitriol in Trichosanthes kirilowii seeds was determined by HPLC. The sample of different mature stages such as immature, near mature and fully mature and processed by different methods were studied. RESULTS: Fully mature Trichosanthes kirilowii seeds were better than the immatured, and the best processing method was dried under 60degrees C, the content of 3,29-dibenzoyl rarounitriol reached up to 131.63microlg/mL. CONCLUSION: Different processing methods and different mature stages had a significant influence on the quality of Trichosanthes kirilowii seeds.

Leafhopper salivary carboxylesterase suppresses JA-Ile synthesis to facilitate initial arbovirus transmission in rice phloem.

Plant jasmonoyl-L-isoleucine (JA-Ile) is a major defense signal against insect feeding, but whether or how insect salivary effectors suppress JA-Ile synthesis and thus facilitate viral transmission in the plant phloem remains elusive. Insect carboxylesterases (CarEs) are the third major family of detoxification enzymes. Here, we identify a new leafhopper CarE, CarE10, that is specifically expressed in salivary glands and is secreted into the rice phloem as a saliva component. Leafhopper CarE10 directly binds to rice jasmonate resistant 1 (JAR1) and promotes its degradation by the proteasome system. Moreover, the direct association of CarE10 with JAR1 clearly impairs JAR1 enzyme activity for conversion of JA to JA-Ile in an in vitro JA-Ile synthesis system. A devastating rice reovirus activates and promotes the co-secretion of virions and CarE10 via virus-induced vesicles into the saliva-storing salivary cavities of the leafhopper vector and ultimately into the rice phloem to establish initial infection. Furthermore, a virus-mediated increase in CarE10 secretion or overexpression of CarE10 in transgenic rice plants causes reduced levels of JAR1 and thus suppresses JA-Ile synthesis, promoting host attractiveness to insect vectors and facilitating initial viral transmission. Our findings provide insight into how the insect salivary protein CarE10 suppresses host JA-Ile synthesis to promote initial virus transmission in the rice phloem.

An insect cell line derived from the small brown planthopper supports replication of rice stripe virus, a tenuivirus.

A cell line from the small brown planthopper (SBPH; Laodelphax striatellus) was established to study replication of rice stripe virus (RSV), a tenuivirus. The SBPH cell line, which had been subcultured through 30 passages, formed monolayers of epithelial-like cells. Inoculation of cultured vector cells with RSV resulted in a persistent infection. During viral infection in the SBPH cell line, the viral non-structural protein NS3 co-localized with the filamentous ribonucleoprotein particles of RSV, as revealed by electron and confocal microscopy. The knockdown of NS3 expression due to RNA interference induced by synthesized double-stranded RNAs from the NS3 gene significantly inhibited viral infection in the SBPH cell line. These results demonstrated that NS3 of RSV might be involved in viral replication or assembly. The persistent infection of the SBPH cell line by RSV will enable a better understanding of the complex relationship between RSV and its insect vector.

Development of an insect vector cell culture and RNA interference system to investigate the functional role of fijivirus replication protein.

An in vitro culture system of primary cells from white-backed planthopper, an insect vector of Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, was established to study replication of the virus. Viroplasms, putative sites of viral replication, contained the nonstructural viral protein P9-1, viral RNA, outer-capsid proteins, and viral particles in virus-infected cultured insect vector cells, as revealed by transmission electron and confocal microscopy. Formation of viroplasm-like structures in non-host insect cells upon expression of P9-1 suggested that the matrix of viroplasms observed in virus-infected cells was composed basically of P9-1. In cultured insect vector cells, knockdown of P9-1 expression due to RNA interference (RNAi) induced by synthesized double-stranded RNA (dsRNA) from the P9-1 gene strongly inhibited viroplasm formation and viral infection. RNAi induced by ingestion of dsRNA strongly abolished viroplasm formation, preventing efficient viral spread in the body of intact vector insects. All these results demonstrated that P9-1 was essential for viroplasm formation and viral replication. This system, combining insect vector cell culture and RNA interference, can further advance our understanding of the biological activities of fijivirus replication proteins.

A plant nonenveloped double-stranded RNA virus activates and co-opts BNIP3-mediated mitophagy to promote persistent infection in its insect vector.

Mitophagy that selectively eliminates damaged mitochondria is an essential mitochondrial quality control mechanism. Recently, mitophagy has been shown to be induced in host cells infected by a few animal viruses. Here, we report that southern rice black-streaked dwarf virus (SRBSDV), a plant nonenveloped double-stranded RNA virus, can also trigger mitophagy in its planthopper vector to prevent mitochondria-dependent apoptosis and promote persistent viral propagation. We find that the fibrillar structures constructed by the nonstructural protein P7-1 of SRBSDV directly target mitochondria via interaction with the mitophagy receptor BNIP3 (BCL2 interacting protein 3), and these mitochondria are then sequestered within autophagosomes to form mitophagosomes. Moreover, SRBSDV infection or P7-1 expression alone can promote BNIP3 dimerization on the mitochondria, and induce autophagy via the P7-1-ATG8 interaction. Furthermore, SRBSDV infection stimulates the phosphorylation of AMP-activated protein kinase (AMPK), resulting in BNIP3 phosphorylation via the AMPKα-BNIP3 interaction. Together, P7-1 induces BNIP3-mediated mitophagy by promoting the formation of phosphorylated BNIP3 dimers on the mitochondria. Silencing of ATG8, BNIP3, or AMPKα significantly reduces virus-induced mitophagy and viral propagation in insect vectors. These data suggest that in planthopper, SRBSDV-induced mitophagosomes are modified to accommodate virions and facilitate persistent viral propagation. In summary, our results demonstrate a previously unappreciated role of a viral protein in the induction of BNIP3-mediated mitophagy by bridging autophagosomes and mitochondria and reveal the functional importance of virus-induced mitophagy in maintaining persistent viral infection in insect vectors.Abbreviations: AMPK: AMP-activated protein kinase; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; CASP3: caspase 3; dsRNA: double strand RNA; ER: endoplasmic reticulum; FITC: fluorescein isothiocyanate; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GFP: green fluorescent protein; GST: glutathione S-transferase; padp: post-first access to diseased plants; Phos-tag: Phosphate-binding tag; PINK1: PTEN induced kinase 1; Sf9: Spodoptera frugiperda; SQSTM1: sequestosome 1; SRBSDV: southern rice black-streaked dwarf virus; STK11/LKB1: serine/threonine kinase 11; TOMM20: translocase of outer mitochondrial membrane 20; RBSDV: rice black-streaked dwarf virus; TUNEL: terminal deoxynucleotidyl dUTP nick end labeling; ULK1: unc-51 like autophagy activating kinase 1; VDAC1: voltage dependent anion channel 1.