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Anna E. Whitfield - One of the best experts on this subject based on the ideXlab platform.
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rna interference tools for the western flower thrips frankliniella occidentalis
Journal of Insect Physiology, 2015Co-Authors: Ismael E Badillovargas, Dorith Rotenberg, Brandi A Schneweis, Anna E. WhitfieldAbstract:The insect order Thysanoptera is exclusively comprised of small insects commonly known as thrips. The western flower thrips, Frankliniella occidentalis, is an economically important pest amongst thysanopterans due to extensive feeding damage and Tospovirus transmission to hundreds of plant species worldwide. Geographically-distinct populations of F. occidentalis have developed resistance against many types of traditional chemical insecticides, and as such, management of thrips and Tospoviruses are a persistent challenge in agriculture. Molecular methods for defining the role(s) of specific genes in thrips-Tospovirus interactions and for assessing their potential as gene targets in thrips management strategies is currently lacking. The goal of this work was to develop an RNA interference (RNAi) tool that enables functional genomic assays and to evaluate RNAi for its potential as a biologically-based approach for controlling F. occidentalis. Using a microinjection system, we delivered double-stranded RNA (dsRNA) directly to the hemocoel of female thrips to target the vacuolar ATP synthase subunit B (V-ATPase-B) gene of F. occidentalis. Gene expression analysis using real-time quantitative reverse transcriptase-PCR (qRT-PCR) revealed significant reductions of V-ATPase-B transcripts at 2 and 3 days post-injection (dpi) with dsRNA of V-ATPase-B compared to injection with dsRNA of GFP. Furthermore, the effect of knockdown of the V-ATPase-B gene in females at these two time points was mirrored by the decreased abundance of V-ATPase-B protein as determined by quantitative analysis of Western blots. Reduction in V-ATPase-B expression in thrips resulted in increased female mortality and reduced fertility, i.e., number of viable offspring produced. Survivorship decreased significantly by six dpi compared to the dsRNA-GFP control group, which continued decreasing significantly until the end of the bioassay. Surviving female thrips injected with dsRNA-V-ATPase-B produced significantly fewer offspring compared to those in the dsRNA-GFP control group. Our findings indicate that an RNAi-based strategy to study gene function in thrips is feasible, can result in quantifiable phenotypes, and provides a much-needed tool for investigating the molecular mechanisms of thrips-Tospovirus interactions. To our knowledge, this represents the first report of RNAi for any member of the insect order Thysanoptera and demonstrates the potential for translational research in the area of thrips pest control.
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Tospovirus-thrips interactions.
Annual Review of Phytopathology, 2005Co-Authors: Anna E. Whitfield, Diane E. Ullman, Thomas L. GermanAbstract:The complex and specific interplay between thrips, Tospoviruses, and their shared plant hosts leads to outbreaks of crop disease epidemics of economic and social importance. The precise details of the processes underpinning the vector-virus-host interaction and their coordinated evolution increase our understanding of the general principles underlying pathogen transmission by insects, which in turn can be exploited to develop sustainable strategies for controlling the spread of the virus through plant populations. In this review, we focus primarily on recent progress toward understanding the biological processes and molecular interactions involved in the acquisition and transmission of Tospoviruses by their thrips vectors.
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Thrips as vectors of Tospoviruses
Advances in Botanical Research, 2002Co-Authors: Diane E. Ullman, Ricardo Meideros, Leslie R. Campbell, Anna E. Whitfield, J. L. Sherwood, Thomas L. GermanAbstract:Publisher Summary This chapter reviews the thrips– Tospovirus pathosystem and the cellular and molecular determinants of thrips acquisition of Tospoviruses. Viruses in the genus Tospovirus (family Bunyaviridae) are transmitted by thrips and have become an ever increasing problem for the producers of agricultural and horticultural crops worldwide. The genus Tospovirus is the genus within the Bunyaviridae containing plant-infecting viruses. Tomato spotted wilt virus ( TSWV) is the type species of this genus. Thrips cause significant direct damage to plants, but it is their transmission of Tospoviruses that is most difficult to control and frequently causes the most severe damage to crops. At least ten species of thrips transmit Tospoviruses, all of which are in the Thysanopteran family Thripidae. Most thrips vector species deposit their eggs into plant tissue and the eggs hatch after 2–3 days, depending on temperature and plant host. For Tospoviruses to be transmitted by thrips, they must be acquired by the larvae. Thus, only immature thrips that acquire Tospoviruses or adults arising from such immatures are important to the transmission of the virus. This concept is extremely important in managing Tospoviruses, because only the plants that serve as hosts for both the insect and the virus are important in epidemics.
Renato O. Resende - One of the best experts on this subject based on the ideXlab platform.
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the functional analysis of distinct Tospovirus movement proteins nsm reveals different capabilities in tubule formation cell to cell and systemic virus movement among the Tospovirus species
Virus Research, 2017Co-Authors: Mikhail Oliveira Leastro, Vicente Pallás, Renato O. Resende, J A SancheznavarroAbstract:The lack of infectious Tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four Tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the ‘30K family’. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent Tospovirus species that have a distinct biological behavior.
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Resistance to Tospoviruses in Vegetable Crops: Epidemiological and Molecular Aspects.
Annual Review of Phytopathology, 2016Co-Authors: Massimo Turina, Richard Kormelink, Renato O. ResendeAbstract:During the past three decades, the economic impact of Tospoviruses has increased, causing high yield losses in a variety of crops and ornamentals. Owing to the difficulty in combating thrips vectors with insecticides, the best way to limit/prevent Tospovirus-induced diseases involves a management strategy that includes virus resistance. This review briefly presents current Tospovirus taxonomy, diversity, molecular biology, and cytopathology as an introduction to a more extensive description of the two main resistance genes employed against Tospoviruses: the Sw5 gene in tomato and the Tsw in pepper. Natural and experimental resistance-breaking (RB) isolates allowed the identification of the viral avirulence protein triggering each of the two resistance gene products; epidemiology of RB isolates is discussed to reinforce the need for allelic variants and the need to search for new/alternative resistance genes. Ongoing efforts for alternative resistance strategies are described not only for Tomato spotted wilt virus (TSWV) in pepper and tomato but also for other vegetable crops heavily impacted by Tospoviruses.
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The movement proteins (NSm) of distinct Tospoviruses peripherally associate with cellular membranes and interact with homologous and heterologous NSm and nucleocapsid proteins.
Virology, 2015Co-Authors: Mikhail Oliveira Leastro, Vicente Pallás, Renato O. Resende, Jesús A. Sánchez-navarroAbstract:Abstract Tospovirus is the only genus containing virus species which infect plants in the Bunyaviridae family. The aims of this study were to understand the in vivo membrane association of the movement protein (NSm) of the Tospovirus species Bean necrotic mosaic virus , Chrysanthemum stem necrosis virus , Tomato chlorotic spot virus and Tomato spotted wilt virus and the homologous and heterologous interactions among NSm and nucleocapsid protein (N). The results obtained by bimolecular fluorescence complementation (BiFC) assay and chemical treatments after membrane fractionation revealed that the four NSm proteins are associated with the biological membranes with the N- and C-termini oriented to the cytoplasm. BiFC analysis for protein–protein interactions showed: i) dimer formation for all NSm and N proteins; ii) interaction between NSm and the cognate N and iii) heterologous interactions between the NSm and N proteins. The implications of these interactions in the life cycle of Tospoviruses are discussed.
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Sequence diversity of NSM movement protein of Tospoviruses.
Archives of Virology, 2001Co-Authors: M. S. Silva, A. C. De Ávila, C. R. F. Martins, I. C. Bezerra, T. Nagata, Renato O. ResendeAbstract:In order to determine the diversity of the movement protein (NSM) among Tospoviruses, the NSM genes of five distinct Tospovirus species occurring in Brazil (Tomato chlorotic spot virus, Groundnut ring spot virus, Chrysanthemum stem necrosis virus, Zucchini lethal chlorosis virus and Iris yellow spot virus) were cloned, sequenced and compared with NSM sequences of other available Tospoviruses. The ‘D-motif’, a conserved region present in the majority of ‘30K superfamily’ virus movement proteins, is present in all NSM amino acid sequences available. In addition to the ‘D-motif’, a conserved phospholipase A2 motif was found. The NSM amino acid sequence comparisons among Tospovirus species revealed several conserved regions located in the internal part of the protein and diverse domains mainly located in the amino-terminus. Prediction of secondary structure showed similar patterns among all NSM proteins analyzed. Considering the geographical prevalence and phylogenetic analysis of N and NSM proteins, Tospoviruses were tentatively clustered in ‘American’ and ‘Eurasian’ groups. Both phylogenetic trees may reflect the natural evolution of Tospovirus species within distinct ecological niches. The sequence information obtained in this work would facilitate functional analysis of NSM during the Tospovirus infection process.
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rt pcr and dot blot hybridization methods for a universal detection of Tospoviruses
Fitopatologia Brasileira, 2001Co-Authors: Marcelo Eiras, Renato O. Resende, Alexandre A Missiaggia, Antonio Carlos De AvilaAbstract:Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the Tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all Tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all Tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.
H. R. Pappu - One of the best experts on this subject based on the ideXlab platform.
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Structure and genome organization of the large RNA of iris yellow spot virus (genus Tospovirus, family Bunyaviridae)
Archives of Virology, 2010Co-Authors: Sudeep Bag, K. L. Druffel, H. R. PappuAbstract:The structure and organization of the large (L) RNA of iris yellow spot virus (IYSV) was determined, and with this report, the complete genomic sequence of IYSV of the genus Tospovirus , family Bunyaviridae has been elucidated. The L RNA of IYSV was 8,880 nucleotides in length and contained a single open reading frame in the viral complementary (vc) strand. The primary translation product of 331.17 kDa shared many of the features of the viral RNA-dependent RNA polymerase (RdRp) coded by L RNAs of known Tospoviruses. The 5′ and 3′ termini of IYSV L RNA (vc) contain two untranslated regions of 33 and 226 nucleotides, respectively, and both termini have conserved terminal nucleotides, another common feature of Tospovirus genomic RNAs. Conserved motifs characteristic of RdRps of members of the family Bunyaviridae were present in the IYSV RdRp.
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nucleotide sequence and genome organization of the medium rna of iris yellow spot virus from the united states
Archives of Virology, 2009Co-Authors: Sudeep Bag, K. L. Druffel, T Salewsky, H. R. PappuAbstract:Iris yellow spot Tospovirus (IYSV) of the family Bunyaviridae causes a serious disease in onion in the USA and other parts of the world. Inspite of its economic importance, the complete genomic sequence of IYSV from the USA is not available. The genome structure and organization of the medium (M) RNA of a Washington (WA) isolate of IYSV were determined and compared to the corresponding region of two isolates previously described from Brazil and The Netherlands. Sequence analysis showed that the M-RNA was 4,817 nucleotides long and potentially coded for the movement protein (NSm) in the viral sense and the glycoprotein precursor (Gn and Gc) in the viral complementary sense. The predicted sizes of NSm and Gn/Gc precursor were 34.7 and 128.84 kDa, respectively. The two open reading frames are separated by a 380 nucleotide intergenic region. Phylogenetic analysis of the NSm and Gn/Gc genes from the WA isolate showed grouping that reflected their respective serogroups. The WA isolate formed a close cluster with the two previously reported IYSV isolates and the IYSV cluster was distinguishable from other Tospovirus species. This is the first report of complete genomic sequence of the M-RNA of IYSV from the US.
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response of early medium and late maturing peanut breeding lines to field epidemics of tomato spotted wilt
Peanut Science, 1999Co-Authors: A K Culbreath, H. R. Pappu, J W Todd, D W Gorbet, S L Brown, J Baldwin, Corley C Holbrook, F M ShokesAbstract:Abstract Epidemics of tomato spotted wilt, caused by tomato spotted wilt Tospovirus (TSWV), were monitored in field plots of runner-type peanut (Arachis hypogaea L.) cultivars Georgia Green and Geo...
Diane E. Ullman - One of the best experts on this subject based on the ideXlab platform.
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Tospovirus-thrips interactions.
Annual Review of Phytopathology, 2005Co-Authors: Anna E. Whitfield, Diane E. Ullman, Thomas L. GermanAbstract:The complex and specific interplay between thrips, Tospoviruses, and their shared plant hosts leads to outbreaks of crop disease epidemics of economic and social importance. The precise details of the processes underpinning the vector-virus-host interaction and their coordinated evolution increase our understanding of the general principles underlying pathogen transmission by insects, which in turn can be exploited to develop sustainable strategies for controlling the spread of the virus through plant populations. In this review, we focus primarily on recent progress toward understanding the biological processes and molecular interactions involved in the acquisition and transmission of Tospoviruses by their thrips vectors.
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Thrips as vectors of Tospoviruses
Advances in Botanical Research, 2002Co-Authors: Diane E. Ullman, Ricardo Meideros, Leslie R. Campbell, Anna E. Whitfield, J. L. Sherwood, Thomas L. GermanAbstract:Publisher Summary This chapter reviews the thrips– Tospovirus pathosystem and the cellular and molecular determinants of thrips acquisition of Tospoviruses. Viruses in the genus Tospovirus (family Bunyaviridae) are transmitted by thrips and have become an ever increasing problem for the producers of agricultural and horticultural crops worldwide. The genus Tospovirus is the genus within the Bunyaviridae containing plant-infecting viruses. Tomato spotted wilt virus ( TSWV) is the type species of this genus. Thrips cause significant direct damage to plants, but it is their transmission of Tospoviruses that is most difficult to control and frequently causes the most severe damage to crops. At least ten species of thrips transmit Tospoviruses, all of which are in the Thysanopteran family Thripidae. Most thrips vector species deposit their eggs into plant tissue and the eggs hatch after 2–3 days, depending on temperature and plant host. For Tospoviruses to be transmitted by thrips, they must be acquired by the larvae. Thus, only immature thrips that acquire Tospoviruses or adults arising from such immatures are important to the transmission of the virus. This concept is extremely important in managing Tospoviruses, because only the plants that serve as hosts for both the insect and the virus are important in epidemics.
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Thrips Transmission of Tospoviruses: Future Possibilities for Management
Thrips Biology and Management, 1995Co-Authors: Diane E. Ullman, Thomas L. German, John L. Sherwood, Daphne M. WestcotAbstract:Tomato spotted wilt Tospovirus (TSWV) is the type member of the Tospovirus genus in the family Bunyaviridae. The Tospoviruses are the only plant infecting members of this large family of animal viruses, many of which cause serious diseases of humans and domestic animals. The Tospoviruses are transmitted by at least eight species of thrips and cause serious epidemics in many food, fiber and ornamental crops around the world. The thrips vectors and the viruses making up the Tospoviruses have large, overlapping host ranges that make management of virus spread one of the greatest challenges facing agricultural and ornamental industries today. The relationship between TSWV and its vectors, among which the western flower thrips (WFT), is thought to be most important, has only recently begun to be investigated. Molecular and serological investigation of thrips cells is elucidating mechanisms governing virus entry to cells, the role of TSWV membrane glycoproteins in thrips acquisition, the processes of TSWV replication within thrips cells and virus movement from cell to cell. An understanding of these events will provide a foundation for management strategies to limit TSWV spread, such as production of transgenic plants that will block both thrips acquisition and inoculation, and new assays for detecting infective thrips populations.
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Tospovirus replication in insect vector cells immunocytochemical evidence that the nonstructural protein encoded by the s rna of tomato spotted wilt Tospovirus is present in thrips vector cells
Phytopathology, 1993Co-Authors: Diane E. UllmanAbstract:Tomato spotted wilt Tospovirus (TSWV) is an insect-transmitted virus that is the type member of the Tospovirus genus, which is the only genus in the family Bunyaviridae containing viruses that infect plants. Direct evidence that Tospoviruses replicate in their thrips vectors has been difficult to obtain because of limitations to definitively detect replicative intermediates of TSWV or to immunolabel vector tissues. A nonstructural protein is encoded by the small RNA of TSWV, and translation of the NSs protein occurs from a subgenomic RNA formed after transcription of viral sense RNA. This protein is designated as nonstructural because it is found only in TSWV-infected cells and has not been found in assembled virions []
Richard Kormelink - One of the best experts on this subject based on the ideXlab platform.
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Biochemical analysis of NSs from different Tospoviruses.
Virus Research, 2017Co-Authors: Marcio Hedil, Dryas De Ronde, Richard KormelinkAbstract:Tospoviruses suppress antiviral RNA interference by coding for an RNA silencing suppressor (NSs) protein. Previously, using NSs-containing crude plant and insect cell extracts, the affinity of NSs for double-stranded (ds)RNA molecules was demonstrated by electrophoretic mobility shifts assays (EMSAs). While NSs from tomato spotted wilt virus (TSWV) and groundnut ringspot virus (GRSV) were able to bind small and long dsRNA molecules, the one from tomato yellow ring virus (TYRV), a distinct Asian Tospovirus, only bound small dsRNA. Here, using bacterially expressed and purified NSs from GRSV and TYRV, it is shown that they are both able to bind to small and long dsRNA. Binding of siRNAs by NSs revealed two consecutive shifts, i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When NSs of TSWV resistance inducing (RI) and resistance breaking (RB) isolates were analyzed using extracts from infected plants only a major siRNA shift was observed. In contrast, plant extracts containing the respective transiently expressed NSs proteins showed only the lower shift with NSsRI but no shift with NSsRB. The observed affinity for RNA duplexes, as well as the two-stepwise shift pattern, is discussed in light of NSs as a suppressor of silencing and its importance for Tospovirus infection.
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Resistance to Tospoviruses in Vegetable Crops: Epidemiological and Molecular Aspects.
Annual Review of Phytopathology, 2016Co-Authors: Massimo Turina, Richard Kormelink, Renato O. ResendeAbstract:During the past three decades, the economic impact of Tospoviruses has increased, causing high yield losses in a variety of crops and ornamentals. Owing to the difficulty in combating thrips vectors with insecticides, the best way to limit/prevent Tospovirus-induced diseases involves a management strategy that includes virus resistance. This review briefly presents current Tospovirus taxonomy, diversity, molecular biology, and cytopathology as an introduction to a more extensive description of the two main resistance genes employed against Tospoviruses: the Sw5 gene in tomato and the Tsw in pepper. Natural and experimental resistance-breaking (RB) isolates allowed the identification of the viral avirulence protein triggering each of the two resistance gene products; epidemiology of RB isolates is discussed to reinforce the need for allelic variants and the need to search for new/alternative resistance genes. Ongoing efforts for alternative resistance strategies are described not only for Tomato spotted wilt virus (TSWV) in pepper and tomato but also for other vegetable crops heavily impacted by Tospoviruses.
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Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins
MDPI AG, 2016Co-Authors: Marcio Hedil, Richard KormelinkAbstract:The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how Tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like Tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far
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A distinct Tospovirus causing necrotic streak on Alstroemeria sp. in Colombia
Archives of Virology, 2010Co-Authors: Afshin Hassani-mehraban, Dick Peters, R. W. Goldbach, Marleen Botermans, J. Th. J. Verhoeven, Ellis Meekes, Janneke Saaijer, Richard KormelinkAbstract:A Tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests. Further analysis revealed a close serological relationship to tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV). A major part of the S-RNA segment, encompassing the nucleocapsid (N) protein gene, the 5′ untranslated region and a part of the intergenic region 3′ of the N gene, was cloned and sequenced. The deduced N protein sequence showed highest amino acid identity (82%) to that of TCSV, indicating that the virus represents a new Tospovirus species, for which the name Alstroemeria necrotic streak virus (ANSV) is coined. Phylogenetic analysis based on the N protein sequence revealed that this Alstroemeria-infecting Tospovirus clustered with Tospoviruses from the American continent. Frankliniella occidentalis was identified as potential vector species for ANSV.
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increase of tospoviral diversity in brazil with the identification of two new Tospovirus species one from chrysanthemum and one from zucchini
Phytopathology, 1999Co-Authors: I. C. Bezerra, Richard Kormelink, T. Nagata, R De O Resende, L Pozzer, A. C. De ÁvilaAbstract:ABSTRACT During a survey conducted in several different regions of Brazil, two unique Tospoviruses were isolated and characterized, one from chrysanthemum and the other from zucchini. The chrysanthemum virus displayed a broad host range, whereas the virus from zucchini was restricted mainly to the family Cucurbitaceae. Double-antibody sandwich-enzyme-linked immunosorbent assay and western immunoblot analyses demonstrated that both viruses were serologically distinct from all reported Tospovirus species including the recently proposed peanut yellow spot virus and iris yellow spot virus (IYSV) species. The nucleotide sequences of the nucleocapsid (N) genes of both viruses contain 780 nucleotides encoding for deduced proteins of 260 amino acids. The N proteins of these two viruses displayed amino acid sequence similarities with the previously described Tospovirus species ranging from 20 to 75%, but they were more closely related to each other (80%). Based on the biological and molecular features, these viruses are proposed as two new Tospovirus species, designated chrysanthemum stem necrosis virus (CSNV) and zucchini lethal chlorosis virus (ZLCV). With the identification of CSNV and ZLCV, in addition to tomato spotted wilt virus, groundnut ring spot virus, tomato chlorotic spot virus, and IYSV, Brazil harbors the broadest spectrum of Tospovirus species reported.