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1.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502559

RESUMO

Stable transgenic rice line (named KRSV-1) with strong resistance against rice stripe virus was generated using the gene sequence of disease-specific protein by RNA interference. Comprehensive safety assessment of transgenic plants has turned into a significant field of genetic modification food safety. In this study, a safety assessment of KRSV-1 was carried out in a stepwise approach. The molecular analysis exhibited that KRSV-1 harbored one copy number of transgene, which was integrated into the intergenic non-coding region of chromosome 2 associated with inter-chromosomal translocations of 1.6-kb segments of chromosome 8. Then, transcriptomics and proteomics analyses were carried out to detect the unintended effects as a result of the integration of the transgene. Although 650 dramatically differentially expressed genes (DDEGs) and 357 differentially expressed proteins were detected between KRSV-1 and wild-type (WT) by transcriptomics and proteomics analyses, no harmful members in the form of toxic proteins and allergens were observed. Encouragingly, the nutritional compositions of seeds from KRSV-1 were comparable with WT seeds. The results of this entire study of molecular analysis, transcriptome and proteome profile of KRSV-1 revealed that no detrimental changes in the form of toxic proteins and allergens were detected in the transgenic rice line due to the integration of the transgene.


Assuntos
Genoma de Planta/genética , Oryza/genética , Doenças das Plantas/genética , Tenuivirus/genética , Biologia Computacional , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Oryza/crescimento & desenvolvimento , Oryza/virologia , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas/genética , Proteoma/genética , Tenuivirus/patogenicidade , Transcriptoma/genética
2.
Virol J ; 16(1): 89, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277670

RESUMO

A tenuivirus, referred to here as JKI 29327, was isolated from a black medic (Medicago lupulina) plant collected in Austria. The virus was mechanically transmitted to Nicotiana benthamiana, M. lupulina, M. sativa, Pisum sativum and Vicia faba. The complete genome was determined by high throughput sequencing. The genome of JKI 29327 consists of eight RNA segments closely related to those of melon chlorotic spot virus (MeCSV) isolate E11-018 from France. Since segments RNA 7 and 8 of JKI 29327 are shorter, its genome is slightly smaller (by 247 nts) than that of E11-018. Pairwise comparisons between the predicted virus proteins of JKI 29327 and their homologues in E11-018 showed aa identities ranging from 80.6 to 97.2%. Plants infected with E11-081 gave intermediate DAS-ELISA reactions with polyclonal antibodies to JKI 29327. Since JKI 29327 and E11-018 appear to be closely related both serologically and genetically, we propose to regard JKI 29327 as the black medic strain of MeCSV. To our knowledge, JKI 29327 represents the second tenuivirus identified from a dicotyledonous plant. Serological and molecular diagnostic methods were developed for future detection.


Assuntos
Cucurbitaceae/virologia , Doenças das Plantas/virologia , Tenuivirus/genética , Tenuivirus/isolamento & purificação , Áustria , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Ervilhas/virologia , Filogenia , RNA Viral/genética , Tabaco/virologia , Vicia faba/virologia , Proteínas Virais/genética
3.
Virology ; 533: 137-144, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31247402

RESUMO

Angiotensin-converting enzyme (ACE) plays diverse roles in the animal kingdom. However, whether ACE plays an immune function against viral infection in vector insects is unclear. In this study, an ACE gene (LsACE) from the small brown planthopper was found to respond to Rice stripe virus (RSV) infection. The enzymatic activities of LsACE were characterized at different pH and temperature. Twenty planthopper proteins were found to interact with LsACE. RSV infection significantly upregulated LsACE expression in the testicle and fat body. When the expression of LsACE in viruliferous planthoppers was inhibited, the RNA level of the RSV SP gene was upregulated 2-fold in planthoppers, and all RSV genes showed higher RNA levels in the rice plants consumed by these planthoppers, leading to a higher viral infection rate and disease rating index. These results indicate that LsACE plays a role in the immune response against RSV transmission by planthoppers.


Assuntos
Hemípteros/imunologia , Hemípteros/virologia , Proteínas de Insetos/imunologia , Insetos Vetores/imunologia , Insetos Vetores/virologia , Peptidil Dipeptidase A/imunologia , Tenuivirus/fisiologia , Sequência de Aminoácidos , Animais , Hemípteros/genética , Hemípteros/fisiologia , Proteínas de Insetos/genética , Insetos Vetores/genética , Insetos Vetores/fisiologia , Oryza/virologia , Peptidil Dipeptidase A/genética , Filogenia , Doenças das Plantas/virologia , Tenuivirus/classificação , Tenuivirus/genética , Tenuivirus/isolamento & purificação
4.
J Gen Virol ; 100(5): 877-888, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30990404

RESUMO

Persistent propagative plant viruses are usually transmitted between a vector insect and a host plant. To adapt to the two different organisms, viruses may show distinct genomic replication or gene expression patterns. To verify this hypothesis, we applied an aboslute real-time quantitative PCR method to measure and compare the replication levels of four genomic RNA segments and the expression levels of seven genes of rice stripe virus (RSV) according to the infection time in the small brown planthopper and rice plant, respectively. In the vector insect, RNA3 began replicating later than the other segments, and RNA2 remained nearly constant during the infection process. RNA1 was the dominant segment, and a difference of over 300-fold appeared among the four segments. In rice plants, the size of the four segments increased with infection time, but decreased to a low level in the late infection period. The ratios of the four segments varied by no more than 15-fold. In planthoppers, three expression patterns were observed for the seven viral genes during viral infection, while in rice plants, the expression patterns of the seven viral genes were similar. These results reflect distinct genomic replication and gene expression patterns in a persistent propagative plant virus in adapting to vector insects and host plants.


Assuntos
Regulação Viral da Expressão Gênica , Hemípteros/virologia , Insetos Vetores/virologia , Oryza/virologia , Tenuivirus/crescimento & desenvolvimento , Tenuivirus/genética , Replicação Viral , Animais , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real
5.
Pest Manag Sci ; 75(7): 1979-1985, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30609247

RESUMO

BACKGROUND: Persistent plant viruses transfer from insect gut to the hemolymph, and finally to the salivary glands before inoculation into the plant hosts with saliva during insect feeding. Virus accumulation in saliva is an important indicator for the transmission ability of an insect vector. In order to evaluate the transmission ability of the small brown planthopper to rice stripe virus (RSV), we successfully measured accumulation of RSV in the saliva of planthoppers via the absolute real-time quantitative polymerase chain reaction method by quantifying the copy numbers of viral genes. RESULTS: After feeding on an artificial diet for 24 h, the copy numbers of viral genes of capsid protein (CP) and disease-specific protein (SP) can be detected in the saliva collected from as few as ten viruliferous planthoppers and ten non-viruliferous planthoppers after infected with RSV for 7 days. When the expression of planthopper G protein pathway suppressor 2 or c-Jun N-terminal kinase was knocked down, the copy numbers of CP and SP in the saliva varied accordingly. CONCLUSION: Our study provided an accurate and convenient detection system to evaluate the transmission efficiency of RSV by small brown planthoppers, and this method may also be suitable for other persistent plant viruses. © 2019 Society of Chemical Industry.


Assuntos
Hemípteros/virologia , Saliva/virologia , Tenuivirus/genética , Animais , Proteínas do Capsídeo/genética , Regulação Viral da Expressão Gênica , Insetos Vetores/virologia , Ninfa/virologia , Doenças das Plantas/virologia , Reação em Cadeia da Polimerase em Tempo Real , Carga Viral
6.
Arch Virol ; 164(1): 297-301, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30302581

RESUMO

A mechanically transmissible virus tentatively named "melon chlorotic spot virus" (MeCSV) was isolated in southeastern France from a melon plant showing chlorotic spots and yellowing of the older leaves. Its complete sequence was obtained by Illumina and Sanger sequencing. The genome comprises eight RNAs for a total size of 20,079 nt and is distantly related to Ramu stunt virus and maize yellow stunt virus, two tentative tenuiviruses. MeCSV differs from other tenuiviruses by its number of genomic fragments, by being readily mechanically transmissible, and by infecting only dicotyledonous hosts. MeCSV should thus be considered a member of a tentative new species related to tenuiviruses.


Assuntos
Magnoliopsida/virologia , Doenças das Plantas/virologia , Tenuivirus/genética , Tenuivirus/isolamento & purificação , Petunia/virologia , Filogenia , Folhas de Planta/virologia
7.
Virology ; 527: 122-131, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30500711

RESUMO

Persistent plant viruses circulate between host plants and vector insects, possibly leading to the genetic divergence in viral populations. We analyzed the single nucleotide polymorphisms (SNPs) of Rice stripe virus (RSV) when it incubated in the small brown planthopper and rice. Two SNPs, which lead to nonsynonymous substitutions in the disease-specific protein (SP) of RSV, produced three genotypes, i.e., GG, AA and GA. The GG type mainly existed in the early infection period of RSV in the planthoppers and was gradually substituted by the other two genotypes during viral transmission. The two SNPs did not affect the interactions of SP with rice PsbP or with RSV coat protein. The GG genotype of SP induced stronger immune responses than those of the other two genotypes in the pattern recognition molecule and immune-responsive effector pathways. These findings demonstrated the population variations of RSV during the circulation between the vector insect and host plant.


Assuntos
Hemípteros/imunologia , Insetos Vetores/imunologia , Oryza/virologia , Doenças das Plantas/virologia , Tenuivirus/genética , Animais , Regulação da Expressão Gênica/imunologia , Genótipo , Hemípteros/genética , Hemípteros/virologia , Proteínas de Insetos/genética , Insetos Vetores/genética , Insetos Vetores/virologia , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Tenuivirus/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
BMC Plant Biol ; 18(1): 219, 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30286719

RESUMO

BACKGROUND: Most plant viruses depend on vector insects for transmission. Upon viral infection, virus-derived small interfering RNAs (vsiRNAs) can target both viral and host transcripts. Rice stripe virus (RSV) is a persistent-propagative virus transmitted by the small brown planthopper (Laodelphax striatellus, Fallen) and can cause a severe disease on rice. RESULTS: To investigate how vsiRNAs regulate gene expressions in the host plant and the insect vector, we analyzed the expression profiles of small RNAs (sRNAs) and mRNAs in RSV-infected rice and RSV-infected planthopper. We obtained 88,247 vsiRNAs in rice that were predominantly derived from the terminal regions of the RSV RNA segments, and 351,655 vsiRNAs in planthopper that displayed relatively even distributions on RSV RNA segments. 38,112 and 80,698 unique vsiRNAs were found only in rice and planthopper, respectively, while 14,006 unique vsiRNAs were found in both of them. Compared to mock-inoculated rice, 273 genes were significantly down-regulated genes (DRGs) in RSV-infected rice, among which 192 (70.3%) were potential targets of vsiRNAs based on sequence complementarity. Gene ontology (GO) analysis revealed that these 192 DRGs were enriched in genes involved in kinase activity, carbohydrate binding and protein binding. Similarly, 265 DRGs were identified in RSV-infected planthoppers, among which 126 (47.5%) were potential targets of vsiRNAs. These planthopper target genes were enriched in genes that are involved in structural constituent of cuticle, serine-type endopeptidase activity, and oxidoreductase activity. CONCLUSIONS: Taken together, our results reveal that infection by the same virus can generate distinct vsiRNAs in different hosts to potentially regulate different biological processes, thus reflecting distinct virus-host interactions.


Assuntos
Interações Hospedeiro-Patógeno/genética , Insetos Vetores/virologia , Oryza/virologia , RNA Interferente Pequeno/genética , Tenuivirus/genética , Animais , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Hemípteros/genética , Hemípteros/virologia , Proteínas de Insetos/genética , Insetos Vetores/genética , Oryza/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Viral , Tenuivirus/patogenicidade
9.
Virology ; 524: 32-44, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30142571

RESUMO

High-throughput deep sequencing and variant detection showed that variations of Rice stripe virus (RSV) populations obtained from small brown planthopper-transmitted rice plants and sap-inoculated N. benthamiana plants were single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels). The SNPs were more uniform across RSV genome, but InDels occurred mainly in the intergenic regions (IRs) and in the 5' or 3' noncoding regions. There were no clear patterns of InDels, although the inserted sequences were all from virus itself. Six, one, and one non-synonymous substitutions were respectively observed in the RdRP ORF, IR and the movement protein ORF. These non-synonymous substitutions were found to be stable, resulting in new consensus sequences in the NBL11 RSV population. Furthermore, the numbers of SNPs and InDels in RSV genome from N. benthamiana plants were much higher than that from O. sativa plants. These differences are likely caused by selection pressures generated by different host plants.


Assuntos
Genoma Viral/genética , Hemípteros/virologia , Oryza/virologia , Doenças das Plantas/virologia , Polimorfismo de Nucleotídeo Único/genética , Tenuivirus/genética , Animais , DNA Intergênico/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Mutação INDEL , Análise de Sequência de RNA , Tenuivirus/isolamento & purificação , Tabaco/virologia
10.
New Phytol ; 219(3): 1085-1096, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29882354

RESUMO

A large number of plant RNA viruses circulate between plants and insects. For RNA viruses, host alternations may impose a differential selective pressure on viral populations and induce variations in viral genomes. Here, we report the variations in the 3'-terminal regions of the multiple-segment RNA virus Rice stripe virus (RSV) that were discovered through de novo assembly of the genome using RNA sequencing data from infected host plants and vector insects. The newly assembled RSV genome contained 16- and 15-nt extensions at the 3'-termini of two genome segments compared with the published reference RSV genome. Our study demonstrated that these extensional sequences were consistently observed in two RSV isolates belonging to distinct genetic subtypes in RSV-infected rice, wheat and tobacco. Moreover, the de novo assembled genome of Southern rice black-streaked dwarf virus also contained 3'-terminal extensions in five RNA segments compared with the reference genome. Time course experiments confirmed that the 3'-terminal extensions of RSV were enriched in the vector insects, were gradually eliminated in the host plant and potentially affected viral replication. These findings indicate that variations in the 3'-termini of viral genomes may be different adaptive strategies for plant RNA viruses in insects and plants.


Assuntos
Variação Genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Insetos Vetores/virologia , Oryza/virologia , Tenuivirus/genética , Animais , Sequência de Bases , Nucleotídeos/genética , Doenças das Plantas/virologia , Reoviridae/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Tenuivirus/isolamento & purificação , Tenuivirus/ultraestrutura , Replicação Viral/genética
11.
Arch Virol ; 163(5): 1317-1323, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29392491

RESUMO

The nonstructural protein pc6 encoded by rice grassy stunt virus (RGSV) plays a significant role in viral cell-to-cell movement, presumably by transport through plasmodesmata (PD). We confirmed the association of pc6 with PD, and also elucidated the mechanisms of protein targeting to PD. Several inhibitor treatments showed conclusively that pc6 is targeted to PD via the ER-to-Golgi secretory system and actin filaments. In addition, VIII-1 myosin was also found to be involved in pc6 PD targeting. Deletion mutants demonstrated that C-terminal amino acid residues 209-229 (transmembrane domain 2; TM2) are essential for pc6 to move through PD.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas do Movimento Viral em Plantas/metabolismo , Plasmodesmos/virologia , Tenuivirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Doenças das Plantas/virologia , Transporte Proteico , Via Secretória , Deleção de Sequência , Tenuivirus/química , Tenuivirus/genética , Tabaco/virologia , Proteínas não Estruturais Virais/genética
12.
Virus Res ; 247: 15-20, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29374519

RESUMO

Rice stripe virus (RSV) transmitted by the vector, small brown planthopper (SBPH), can cause a severe rice disease. The nucleocapsid (N) protein is the major component of RSV ribonucleoprotein particles (RNPs), and it plays important roles in viral persistent-propagative transmission by SBPH. To gain further insights into the vector components enabling RSV transmission, a GAL4-based yeast two-hybrid system was utilized to find unknown vector factors that interact with the N protein. Thirteen different proteins were identified as factors that interact with the N protein. The interaction between 60S ribosomal protein L18 (RPL18) and the N protein was further studied. Although the expression of RPL18 was not altered in insects during RSV infection, RPL18 was validated to bind directly to RSV RNPs and interact with RSV N protein. Knockdown of RPL18 dramatically reduced viral RNA and protein levels, especially viral protein expression, indicating a requirement for RPL18 in RSV translation and replication. Our results provide evidence that RPL18 is a critical factor required for RSV accumulation in SBPH, which suggests that the vector factor RPL18 may be as a potential target to develop for controlling the transmission of rice virus.


Assuntos
Regulação Viral da Expressão Gênica , Hemípteros/genética , Insetos Vetores/genética , Proteínas do Nucleocapsídeo/genética , RNA Viral/genética , Proteínas Ribossômicas/genética , Tenuivirus/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Hemípteros/metabolismo , Hemípteros/virologia , Insetos Vetores/metabolismo , Insetos Vetores/virologia , Proteínas do Nucleocapsídeo/metabolismo , Oryza/virologia , Doenças das Plantas/virologia , Ligação Proteica , RNA Viral/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tenuivirus/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Replicação Viral
13.
J Virol ; 92(1)2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29046442

RESUMO

Most segmented negative-sense RNA viruses employ a process termed cap snatching, during which they snatch capped RNA leaders from host cellular mRNAs and use the snatched leaders as primers for transcription, leading to the synthesis of viral mRNAs with 5' heterogeneous sequences (HSs). With traditional methods, only a few HSs can be determined, and identification of their donors is difficult. Here, the mRNA 5' ends of Rice stripe tenuivirus (RSV) and Rice grassy stunt tenuivirus (RGSV) and those of their host rice were determined by high-throughput sequencing. Millions of tenuiviral HSs were obtained, and a large number of them mapped to the 5' ends of corresponding host cellular mRNAs. Repeats of the dinucleotide AC, which are complementary to the U1G2 of the tenuiviral template 3'-U1G2U3G4UUUCG, were found to be prevalent at the 3' termini of tenuiviral HSs. Most of these ACs did not match host cellular mRNAs, supporting the idea that tenuiviruses use the prime-and-realign mechanism during cap snatching. We previously reported a greater tendency of RSV than RGSV to use the prime-and-realign mechanism in transcription with leaders cap snatched from a coinfecting reovirus. Besides confirming this observation in natural tenuiviral infections, the data here additionally reveal that RSV has a greater tendency to use this mechanism in transcribing genomic than in transcribing antigenomic templates. The data also suggest that tenuiviruses cap snatch host cellular mRNAs from translation- and photosynthesis-related genes, and capped RNA leaders snatched by tenuiviruses base pair with U1/U3 or G2/G4 of viral templates. These results provide unprecedented insights into the cap-snatching process of tenuiviruses.IMPORTANCE Many segmented negative-sense RNA viruses (segmented NSVs) are medically or agriculturally important pathogens. The cap-snatching process is a promising target for the development of antiviral strategies against this group of viruses. However, many details of this process remain poorly characterized. Tenuiviruses constitute a genus of agriculturally important segmented NSVs, several members of which are major viral pathogens of rice. Here, we for the first time adopted a high-throughput sequencing strategy to determine the 5' heterogeneous sequences (HSs) of tenuiviruses and mapped them to host cellular mRNAs. Besides providing deep insights into the cap snatching of tenuiviruses, the data obtained provide clear evidence to support several previously proposed models regarding cap snatching. Curiously and importantly, the data here reveal that not only different tenuiviruses but also the same tenuivirus synthesizing different mRNAs use the prime-and-realign mechanism with different tendencies during their cap snatching.


Assuntos
Genoma Viral , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Tenuivirus/genética , Transcrição Genética , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Oryza/virologia , RNA Mensageiro/genética , RNA Viral , Tenuivirus/metabolismo
14.
PLoS Pathog ; 13(10): e1006662, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28977024

RESUMO

MicroRNAs (miRNAs) are small regulatory RNAs processed from primary miRNA transcripts, and plant miRNAs play important roles in plant growth, development, and response to infection by microbes. Microbial infections broadly alter miRNA biogenesis, but the underlying mechanisms remain poorly understood. In this study, we report that the Rice stripe virus (RSV)-encoded nonstructural protein 3 (NS3) interacts with OsDRB1, an indispensable component of the rice (Oryza sativa) miRNA-processing complex. Moreover, the NS3-OsDRB1 interaction occurs at the sites required for OsDRB1 self-interaction, which is essential for miRNA biogenesis. Further analysis revealed that NS3 acts as a scaffold between OsDRB1 and pri-miRNAs to regulate their association and aids in vivo processing of pri-miRNAs. Genetic evidence in Arabidopsis showed that NS3 can partially substitute for the function of double-stranded RNA binding domain (dsRBD) of AtDRB1/AtHYL1 during miRNA biogenesis. As a result, NS3 induces the accumulation of several miRNAs, most of which target pivotal genes associated with development or pathogen resistance. In contrast, a mutant version of NS3 (mNS3), which still associated with OsDRB1 but has defects in pri-miRNA binding, reduces accumulation of these miRNAs. Transgenic rice lines expressing NS3 exhibited significantly higher susceptibility to RSV infection compared with non-transgenic wild-type plants, whereas the transgenic lines expressing mNS3 showed a less-sensitive response. Our findings revealed a previously unknown mechanism in which a viral protein hijacks OsDRB1, a key component of the processing complex, for miRNA biogenesis and enhances viral infection and pathogenesis in rice.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/genética , Oryza/virologia , Proteínas de Ligação a RNA/metabolismo , Tenuivirus/genética , Proteínas Virais/metabolismo , Oryza/genética , Interferência de RNA/fisiologia , Proteínas de Ligação a RNA/genética
15.
Virus Genes ; 53(6): 898-905, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28589385

RESUMO

The amount of Rice stripe virus (RSV) maintained through transovarial transmission was analyzed during the development and reproduction of its vector, Laodelphax striatellus. Reverse transcription quantitative PCR analysis was used to quantify RNA expressed from the RSV coat protein (CP) gene as an estimate of RSV content in nymphs and adults of L. striatellus at various developmental stages. The 18S ribosome RNA gene of L. striatellus was chosen as the reference for calculating RSV CP expression using the comparative Ct method. Based on the CP transcript levels, the amount of RSV did not differ significantly throughout the nymphal stage or between adult females of different ages; however, RSV content tended to increase slightly as males became older. The average RSV content in males was 1.30-2.49 times that in females. The amount of RSV in L. striatellus adults was compared between generations. The RSV content of female adults did not differ significantly between the parent and progeny populations three of three different females. L. striatellus grown to adults on a susceptible cultivar and five RSV-resistant cultivars were compared to analyze whether the amount of RSV varied among cultivars. Although the amount of RSV in L. striatellus adults differed significantly among the six rice cultivars evaluated, the difference seemed independent of whether resistance genes were present. In addition, the percentage of viruliferous insects was similar among cultivars.


Assuntos
Hemípteros/virologia , Insetos Vetores/virologia , Oryza/virologia , Tenuivirus/genética , Animais , Feminino , Insetos/virologia , Masculino , RNA Ribossômico 18S/genética , Proteínas Virais/genética
16.
Virol J ; 14(1): 90, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28468626

RESUMO

BACKGROUND: The small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA isolation when processing a large number of samples. RESULTS: We established a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 103 fold. Compared to the traditional RT-PCR with RNA isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA isolation. CONCLUSIONS: A simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA isolation step. It offers a convenient alternative to the traditional RT-PCR method.


Assuntos
Hemípteros/virologia , Insetos Vetores/virologia , Vírus de RNA/isolamento & purificação , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , DNA Complementar/isolamento & purificação , Dicistroviridae/genética , Dicistroviridae/isolamento & purificação , Immunoblotting/métodos , Insetos/virologia , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , RNA Viral/análise , Reoviridae/genética , Reoviridae/isolamento & purificação , Sensibilidade e Especificidade , Tenuivirus/genética , Tenuivirus/isolamento & purificação
17.
Virus Res ; 235: 14-23, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28392445

RESUMO

Rice stripe virus (RSV) is an important pathogen of rice. The RSV genome consists of four single-stranded RNA segments that encode seven viral proteins. A previous report found that NS3 is a viral suppressor of RNA silencing and self interacts. Using a model that predicts protein structure, we identified amino acid residues or motifs, including four α-helix motifs, required for NS3 self-interaction. We then used yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays to study the interactions between full-length NS3 and its truncated and alanine substitution mutants. Y2H and BiFC results showed that the N-terminal region of NS3 is essential for self-interaction. All α-helix deletion mutants and substitution mutants lost the ability to self-interact. To identify the relationship between NS3 self-interaction and silencing suppressor activity, we used a GFP silencing system in Nicotiana benthamiana with Agrobacterium-mediated transient overexpression of each mutated NS3 protein. All of the deletion and the α-helix substitution mutants that had lost the ability to self-interact also lost their silencing suppressor ability. The substitution of amino acids with alanine at positions 70-75, 76-83, and 173-177, however, resulted in mutants that were able to self-interact but were unable to function as silencing suppressors. These results suggest that RSV requires NS3 self-interaction to suppress RNA silencing and to thereby counter host defenses.


Assuntos
Interações Hospedeiro-Patógeno , Mapas de Interação de Proteínas , Tenuivirus/fisiologia , Tabaco/imunologia , Tabaco/virologia , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Análise Mutacional de DNA , Fluorometria , Modelos Moleculares , Conformação Proteica , Tenuivirus/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Fatores de Virulência/química , Fatores de Virulência/genética
18.
Virology ; 506: 73-83, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28359901

RESUMO

Rice stripe tenuivirus (RSV) is a filamentous, negative-strand RNA virus causing severe diseases on rice in Asian countries. The viral particle is composed predominantly of a nucleocapsid protein (NP) and genomic RNA. However, the molecular details of how the RSV NP interacts with genomic RNA during particle assembly remain largely unknown. Here, we modeled the NP-RNA complex and show that polar amino acids within a predicted groove of NP are critical for RNA binding and protecting the RNA from RNase digestion. RSV NP formed pentamers, hexamers, heptamers, and octamers. By modeling the higher-order structures, we found that oligomer formation was driven by the N-terminal amino arm of the NP. Deletion of this arm abolished oligomerization; the N-terminally truncated NP was less able to interact with RNA and protect RNA than was the wild type. These findings afford valuable new insights into molecular mechanism of RSV NPs interacting with genomic RNA.


Assuntos
Nucleocapsídeo/metabolismo , Oryza/virologia , Doenças das Plantas/virologia , RNA Viral/metabolismo , Tenuivirus/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Nucleocapsídeo/química , Nucleocapsídeo/genética , Ligação Proteica , RNA Viral/química , RNA Viral/genética , Alinhamento de Sequência , Tenuivirus/química , Tenuivirus/genética
19.
Mol Phylogenet Evol ; 109: 343-350, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28189616

RESUMO

Rice stripe virus (RSV) is an insect-borne tenuivirus of economical significance. It is endemic to the rice-growing regions of East Asia and exhibits more genetic diversity in Yunnan Province of China. To gain more insights into the molecular epidemiology and evolution of RSV, recombination analyses were conducted and potential events were detected in each of the four RNA segments of RSV. Bayesian coalescent method was then applied to the time-stamped coding sequences of the CP gene. The nucleotide substitution rate and the divergence time were estimated. Age calculations suggested that the first diversification event of the RSV isolates analyzed might take place in the early 20th century, and RSV has existed in Yunnan long before notice. Surveys of codon usage variation showed that the RSV genes had influences other than mutational bias. In codon choice, RSV conformed to neither vector small brown planthopper nor host rice, although the former exerted a more dominant influence on shaping codon usage pattern of RSV. In addition, CpG dinucleotide deficiency was observed in RSV.


Assuntos
Evolução Molecular , Tenuivirus/classificação , Teorema de Bayes , China , Códon , Variação Genética , Oryza/virologia , Filogenia , Tenuivirus/genética
20.
Virol J ; 13(1): 202, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27912765

RESUMO

BACKGROUND: Virus infection induces and suppresses host gene expression on a global level. Rice stripe virus (RSV) is the type species of the genus Tenuivirus and infects rice and Arabidopsis plants. Microarray-based and next generation sequencing-based transcriptomic approaches have been used to study rice-RSV interactions. However, our knowledge of the response of Arabidopsis plants to RSV infection is limited, and it requires further investigation to determine the similarities (or differences) in virus-host interactions between monocot and dicot hosts infected with RSV. METHODS: We characterized transcriptome changes in Arabidopsis thaliana infected with rice stripe virus (RSV) with RNA-seq based digital gene expression (DGE) analysis. The transcriptomes of RSV-infected samples were compared to those of mock-treated samples at 14 and 21 days post-infection (dpi) during different stages of symptom development. RESULTS: We identified 624 differentially expressed genes (DEGs) in Arabidopsis influenced by RSV at 14 dpi and 21 dpi, among which at 14 dpi, 255 transcripts were induced, and 38 were repressed; at 21 dpi, 146 were induced, and 237 were repressed. Functional annotation indicated that these DEGs were related to multiple biological functions, including defense response, secondary metabolism, protein amino acid phosphorylation and response to abiotic stress. CONCLUSIONS: Importantly, the transcription of genes related to host defense systems was activated by RSV infection at an early stage of symptom development (14 dpi), whereas over the infection period (21 dpi), the host defense response systems were suppressed. A total of 52 genes were continuously differentially expressed between the two time points, indicating that the majority of DEGs were transient and unique to a particular time point during symptom development. The DEGs, particularly the defense response genes, identified in this study are candidates suitable for further functional analysis during the RSV-Arabidopsis interaction.


Assuntos
Arabidopsis/virologia , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno , Doenças das Plantas/virologia , Análise de Sequência de RNA/métodos , Tenuivirus/crescimento & desenvolvimento , Tenuivirus/genética , Fatores de Tempo
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