Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Paving the Way to Tospovirus Infection: Multilined Interplays with Plant Innate Immunity
    Zhu, Min ; Grinsven, Irene Louise Van; Kormelink, Richard ; Tao, Xiaorong - \ 2019
    Annual Review of Phytopathology 57 (2019). - ISSN 0066-4286 - p. 41 - 62.
    Antiviral RNAi - Effector/avirulence determinant - NLR - R gene - tospovirus - Viral RNA silencing suppressor

    Tospoviruses are among the most important plant pathogens and cause serious crop losses worldwide. Tospoviruses have evolved to smartly utilize the host cellular machinery to accomplish their life cycle. Plants mount two layers of defense to combat their invasion. The first one involves the activation of an antiviral RNA interference (RNAi) defense response. However, tospoviruses encode an RNA silencing suppressor that enables them to counteract antiviral RNAi. To further combat viral invasion, plants also employ intracellular innate immune receptors (e.g., Sw-5b and Tsw) to recognize different viral effectors (e.g., NSm and NSs). This leads to the triggering of a much more robust defense against tospoviruses called effector-Triggered immunity (ETI). Tospoviruses have further evolved their effectors and can break Sw-5b-/Tsw-mediated resistance. The arms race between tospoviruses and both layers of innate immunity drives the coevolution of host defense and viral genes involved in counter defense. In this review, a state-of-The-Art overview is presented on the tospoviral life cycle and the multilined interplays between tospoviruses and the distinct layers of defense.

    Tomato Chlorotic Spot Virus (TCSV) Putatively Incorporated a Genomic Segment of Groundnut Ringspot Virus (GRSV) Upon a Reassortment Event
    Silva, João Marcos Fagundes ; Oliveira, Athos Silva de; Almeida, Mariana Martins Severo de; Kormelink, Richard ; Nagata, Tatsuya ; Resende, Renato Oliveira - \ 2019
    Viruses 11 (2019)2. - ISSN 1999-4915
    groundnut ringspot virus - reassortment - tomato chlorotic spot virus - tospovirus - virus evolution

    Tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV) share several genetic and biological traits. Both of them belong to the genus Tospovirus (family Peribunyaviridae), which is composed by viruses with tripartite RNA genome that infect plants and are transmitted by thrips (order Thysanoptera). Previous studies have suggested several reassortment events between these two viruses, and some speculated that they may share one of their genomic segments. To better understand the intimate evolutionary history of these two viruses, we sequenced the genomes of the first TCSV and GRSV isolates ever reported. Our analyses show that TCSV and GRSV isolates indeed share one of their genomic segments, suggesting that one of those viruses may have emerged upon a reassortment event. Based on a series of phylogenetic and nucleotide diversity analyses, we conclude that the parental genotype of the M segment of TCSV was either eliminated due to a reassortment with GRSV or it still remains to be identified.

    Tospovirus : induction and suppression of RNA silencing
    Hedil, Marcio - \ 2016
    Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Richard Kormelink. - Wageningen : Wageningen University - ISBN 9789462577848 - 137
    tospovirus - rna - plants - immunity - gene silencing - biochemical pathways - rna interference - viral proteins - plant viruses - tospovirus - rna - planten - immuniteit - uitschakelen van genexpressie - biochemische omzettingen - rna-interferentie - viruseiwitten - plantenvirussen

    While infecting their hosts, viruses must deal with host immunity. In plants the antiviral RNA silencing pathway is an important part of plant innate immunity. Tospoviruses are segmented negative-stranded RNA viruses of plants. To counteract the antiviral RNA silencing response in plants, tospoviruses have evolved a silencing suppressor function via its NSs protein. This viral protein has previously been shown to bind dsRNA that likely arises from secondary RNA folding structures in viral RNAs. The aim of the present research was to further investigate the interaction between tospoviruses and the plant antiviral RNA silencing response, including the target sequences in the viral RNA and the further role of the NSs protein as part of the tospovirus counterdefence strategy.

    In order to identify the target and inducer for RNA silencing against tospoviruses, small RNAs purified from plants infected with three tospoviral species, tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), were probed against the viral RNA segments of these three different tospoviruses (Chapter 3). Virus-derived siRNAs (vsiRNAs) were found to be derived from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) from where hardly any vsiRNAs were found. Similar profiles were observed with the closely related GRSV as well as the distantly related TYRV. Dicer cleavage assays using Drosophila melanogaster embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3′-UTR did not show more rapid/strong silencing, and profiling of the corresponding siRNAs generated outside the context of a viral infection still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP region/structure is a weak inducer of RNA silencing and plays a minor role in the amplification of a strong antiviral RNA silencing response.

    Next, a biochemical analysis was performed using E. coli-expressed and purified NSs from GRSV and TYRV. The binding of both purified NSs proteins to small and long dsRNA indicated that this is likely a generic feature of all tospoviral NSs proteins (Chapter 4). Binding of siRNAs to NSs furthermore revealed two shifts on polyacrylamide gels i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When the NSs protein of TSWV resistant breaker (RB) isolates (of Tsw-gene based resistance), which lack RSS activity when transiently expressed, were analyzed using extracts from infected plants still a major (second) shift of siRNAs was observed, similar to the case with extracts containing TSWV resistant inducer (RI) isolates. In contrast, plant extracts containing transiently expressed NSs proteins alone (no infection) showed only the smaller, first shift for NSsRI but no shift for NSsRB.

    The ability of NSs to suppress systemic silencing is demonstrated for the NSs proteins of TSWV, GRSV and TYRV, and their relative strengths to suppress local and systemic silencing were compared (Chapter 5). A system was developed to quantify suppression via GFP silencing constructs, allowing comparison of relative RNA silencing suppressor strength. In this case NSs proteins of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Moreover, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream of the biogenesis of siRNAs and their sequestration.

    In summary, evidence is presented showing that sequences from all three genomic segments from tospovirus are targeted by the plant RNA silencing machinery. The predicted hairpin sequence in the IGR is poorly targeted. Biochemical experiments with purified NSs proteins further support the view that binding to small and long dsRNA is a characteristic common to all tospovirus NSs proteins. Furthermore, tospovirus NSs proteins suppress systemic silencing and there are indications that local and systemic silencing suppression can be uncoupled in NSs. Collectively, these results add to our current understanding of the tospovirus-plant interaction involving antiviral RNA silencing and the viral counter-defence (NSs protein). Lastly, the results of the research presented in this thesis are discussed in light of the current knowledge on RNA silencing and to present some future perspectives and questions that remain open and/or resulted from this thesis (Chapter 6).

    The Sw-5 gene cluster : analysis of tomato resistance against tospoviruses
    Silva de Oliveira, A. - \ 2015
    Wageningen University. Promotor(en): Monique van Oers; R. de Oliveira Resende, co-promotor(en): Richard Kormelink. - Wageningen : Wageningen University - ISBN 9789462575769 - 158
    solanum lycopersicum - tomaten - ziekteresistentie - plantenvirussen - tospovirus - genen - tomatenbronsvlekkenvirus - plantenveredeling - resistentieveredeling - solanum lycopersicum - tomatoes - disease resistance - plant viruses - tospovirus - genes - tomato spotted wilt virus - plant breeding - resistance breeding
    The complete nucleotide sequence of chrysanthemum stem necrosis virus
    Dullemans, A.M. ; Verhoeven, J.Th.J. ; Kormelink, R.J.M. ; Vlugt, R.A.A. van der - \ 2015
    Archives of Virology 160 (2015)2. - ISSN 0304-8608 - p. 605 - 608.
    1st report - tospovirus - identification - diversity - zucchini
    The complete genome sequence of chrysanthemum stem necrosis virus (CSNV) was determined using Roche 454 next-generation sequencing. CSNV is a tentative member of the genus Tospovirus within the family Bunyaviridae, whose members are arthropod-borne. This is the first report of the entire RNA genome sequence of a CSNV isolate. The large RNA of CSNV is 8955 nucleotides (nt) in size and contains a single open reading frame of 8625 nt in the antisense arrangement, coding for the putative RNA-dependent RNA polymerase (L protein) of 2874 aa with a predicted Mr of 331 kDa. Two untranslated regions of 397 and 33 nt are present at the 5' and 3' termini, respectively. The medium (M) and small (S) RNAs are 4830 and 2947 nt in size, respectively, and show 99 % identity to the corresponding genomic segments of previously partially characterized CSNV genomes. Protein sequences for the precursor of the Gn/Gc proteins, N and NSs, are identical in length in all of the analysed CSNV isolates
    The Tomato spotted wilt virus cell-to-cell movement protein (NSM) triggers a hypersensitive response in Sw-5 containing resistant tomato lines and Nicotiana benthamiana transformed with the functional Sw-5b resistance gene copy.
    Hallwass, M. ; Silva de Oliveira, A. ; Dianese, E.C. ; Lohuis, D. ; Boiteux, L.S. ; Inoue-Nagata, A.K. ; Resende, R.O. de; Kormelink, R.J.M. - \ 2014
    Molecular Plant Pathology 15 (2014)9. - ISSN 1464-6722 - p. 871 - 880.
    mosaic-virus - lycopersicon-esculentum - nonstructural protein - capsicum-chinense - coat protein - plant-cells - rna segment - tswv - tospovirus - tobacco
    Although the Sw-5 gene cluster has been cloned, and Sw-5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum SR1 plants transformed with a copy of the Sw-5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N.benthamiana transformed with Sw-5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non-structural TSWV proteins tested, the TSWV cell-to-cell movement protein (NSM) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non-replicative pEAQ-HT expression vector system. HR was induced in Sw-5b-transgenic N.benthamiana as well as in resistant near-isogenic tomato lines after agroinfiltration with a functional cell-to-cell movement protein (NSM) from a resistance-inducing (RI) TSWV strain (BR-01), but not with NSM from a Sw-5 resistance-breaking (RB) strain (GRAU). This is the first biological demonstration that Sw-5-mediated resistance is triggered by the TSWV NSM cell-to-cell movement protein.
    Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus
    Ronde, D. de; Butterbach, P.B.E. ; Lohuis, H. ; Hedil, M. ; Lent, J.W.M. van; Kormelink, R.J.M. - \ 2013
    Molecular Plant Pathology 14 (2013)4. - ISSN 1464-6722 - p. 405 - 415.
    mediated plant transformation - capsicum-chinense - cell-death - disease-resistance - lycopersicon-esculentum - viral suppressors - sw-5 gene - potato - tospovirus - agrobacterium
    As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSsRI protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the NRI/RB protein, or when NSsRB was expressed. Although NSsRI was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSsRB had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene
    Molecular Diagnosis of Iris Yellow Spot Virus (IYSV) on Onion in Iran
    Beikzadeh, N. ; Jafarpour, B. ; Rouhani, H. ; Peters, D. ; Hassani-Mehraban, A. - \ 2012
    Journal of Agricultural Science and Technology (JAST) 14 (2012)5. - ISSN 1680-7073 - p. 1149 - 1158.
    genetic diversity - tospovirus - population - disease
    Viral symptoms indicative of Iris yellow spot virus (IYSV) were observed on onion in several fields near Chenaran in Khorasan Razavi Province. Mechanical inoculation of herbaceous hosts with onion sap extracts from symptomatic plants showed similar symptoms to those described for IYSV. The mechanically transmitted virus reacted only with antisera specific to IYSV in DAS-ELISA but not with antisera specific to seven other tospoviruses. In RT-PCR, a DNA fragment approximately 822 bp in size was amplified from infected Nicotiana benthamiana by using primers specific to the nucleocapsid (N) gene of IYSV. After cloning and sequencing, the deduced N protein sequence of two isolates (GenBank accession no. HQ148173 and HQ148174) showed 98% amino acid identity with a Sri Lankan isolate, 96% with a Dutch isolate and 92% with a Brazilian isolate. To our knowledge, this is the first molecular characterization of IYSV in Iran
    Infection of Alstroemeria Plants with Tomato yellow ring virus in Iran
    Beikzadeh, N. ; Bayat, H. ; Jafarpour, B. ; Rohani, H. ; Peters, D. ; Hassani-Mehraban, A. - \ 2012
    Journal of Phytopathology 160 (2012)1. - ISSN 0931-1785 - p. 45 - 47.
    Alstroemeria cv. Ovation plants with virus-like necrotic spots and streaks on leaves and petals were observed in greenhouses in Khorasan Razavi (Mashhad) and Markazi (Mahallat) provinces, Iran. Samples with virus-like symptoms reacted positively in enzyme-linked immunosorbent assay with a polyclonal antibody raised against Tomato yellow ring virus (TYRV) nucleocapsid (N) protein. TYRV-specific primers were used in a reverse transcription-polymerase chain reaction to amplify the N gene. The deduced amino acid sequences of the obtained amplicon revealed 99% identity to the N protein of an isolate of TYRV isolated from tomato (TYRV-t)
    Identification of Iris yellow spot virus on Leek (Allium porrum) in Sri Lanka
    Widana Gamage, S.M.K. ; Hassani-Mehraban, A. ; Peters, D. - \ 2010
    Plant Disease 94 (2010)8. - ISSN 0191-2917 - p. 1070 - 1070.
    Virus-host interactions of tomato yellow ring virus, a new tospovirus from Iran
    Hassani-Mehraban, A. - \ 2008
    Wageningen University. Promotor(en): R.W. Goldbach, co-promotor(en): Richard Kormelink. - [S.l.] : S.n. - ISBN 9789085049395 - 130
    tospovirus - bunyaviridae - identificatie - waardplanten - gastheer parasiet relaties - iran - plant-microbe interacties - tospovirus - bunyaviridae - identification - host plants - host parasite relationships - iran - plant-microbe interactions
    During the past decades, an increasing number of new tospovirus species occurring in various agricultural and horticultural crops have been reported. The emergence of new tospoviruses may be attributed to intensified international trading, to increasing problems to control their thrips vectors, but certainly also by better recognition based on new diagnostic tools.
    The works presented in the thesis first focused on comprehensive characterisation and identification of a tospovirus species occurring in different crops in Iran, and next on transgenic approaches to control this virus.
    In Chapter 2, five presumed Iranian tospovirus isolates from tomato, chrysanthemum, gazania, soybean and potato, collected in 2002, were analysed. All isolates induced necrotic local lesions on Petunia hybrida, indicative for tospoviruses. None of the available antisera against known tospoviruses reacted with the isolates, suggesting that if these were tospoviruses, they should belong to a novel species. As a next step the viral nucleoprotein (N) gene of the tomato isolate was cloned and sequenced and this information demonstrated that it represented a new tospovirus species for which the name Tomato yellow ring virus (TYRV) was proposed. Subsequently the N gene sequences of the chrysanthemum and gazania isolates were also obtained and showed these isolates to represent TYRV as well. Back-inoculation of the tomato isolate induced resembling chlorotic and necrotic spots on leaves and yellow rings on the tomato fruits. The complete S RNA sequence of this isolate revealed the generic topology of a tospoviral S RNA, containing both NSS (suppressor of silencing) and N (nucleocapsid) genes separated by a long non-coding intergenic region including a predicted hairpin structure. Multiple sequence alignment of the N protein of TYRV with those of established tospovirus species revealed the closest relationship (74% identity) to Iris yellow spot virus (IYSV).
    In chapter 3, the isolates from soybean and potato were analysed. Although in ELISA assays these isolates scored positive with antiserum raised against the TYRV-tomato isolate, they failed to amplify in RT-PCR when using primers derived from the latter. The N gene sequences of these isolates indeed revealed a sequence divergence of 8% compared to that of TYRV-tomato, indicating these two belonged to different strains of TYRV (denoted TYRV-s, while the strain occurring in tomato was named TYRV-t). Additional differences between the two strains were found in their respective S RNA non-coding intergenic regions. Differences in their host range and symptom expression underscored the decision to treat them as separate strains. A preliminary cross-protection study indicated that TYRV-t and TYRV-s mutually exclude each other, indicating that the strains represent stable, isolated lineages which do not easily converge despite their geographical overlap.
    In chapter 4, extended inverted repeat transgenic cassettes for broad tospoviral resistance were constructed and tested. These transgene cassettes contained partial N gene sequences from 5 different tomato-infecting tospoviruses, including TYRV-t from Iran, in such arrangement that transgenic expression would deliver a ds hairpin RNA. Using Nicotiana benthamiana as a model plant, transgenic lines harboring an inverted repeat construct interspaced with a sense-oriented intron were obtained with high frequencies of resistance up to 100% for all 5 tospoviruses in F2. By analysing the siRNA content of the transgenic plants it could be verified that the transgenic resistance was based on RNA silencing (or shortly RNAi). Whilst these transgenics were fully resistant to TYRV-t (whose N gene sequence was used in the transgene cassette), they were fully susceptible to TYRV-s, demonstrating again how narrow transgenic resistance based on RNAi is. Surprisingly upon co-inoculation with TYRV-s, TYRV-t also could overcome the transgenic resistance. Mass spectrometric analysis of viral ribonucleocapsid protein (RNP) purified from a mixed-infected transgenic line revealed that the N protein of both strains were present and hence hetero-encapsidation as possible mechanism to rescue TYRV-t from these plants could be excluded. Experiments involving the expression of the viral suppressor protein (NSs) from TYRV-s using a PVX vector, indicated that rescue of TYRV-t by TYRV-s was based on the expression of the TYRV-s RNAi suppressor.
    Since most of the operational transgenic tospovirus resistance approaches are based on RNAi, involving the production of transgenic viral siRNAs, in chapter 5 the production and involvement of viral siRNA molecules during a natural tospovirus infection process was investigated. Special attention was given to the intergenic hairpin region of the S RNA segment. Total small RNA was isolated from TYRV-t infected N. benthamiana and mapped to the S RNA segment. These studies demonstrated the occurrence of a hot spot for siRNA induction within the S RNA, but surprisingly this hotspot was not mapped in the IGR hairpin but at the start of NSS ORF where a much shorter hairpin structure was predicted. Surprisingly, fewest siRNAs mapped to the intergenic region which is predicted to fold into a long hairpin, despite additional experiments involving DICER cleavage by Drosophila melanogaster embryo extract and YFP (yellow fluorescent protein)-hairpin constructs indicating this region to be a potential inducer and target for RNAi.
    In chapter 6, the observations done in the experimental chapters are discussed in a broader context and a model for tospovirus-induced RNA silencing presented.

    The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants
    Lacorte, C.C. ; Ribeiro, S.G. ; Lohuis, H. ; Goldbach, R.W. ; Prins, M.W. - \ 2007
    Protein Expression and Purification 55 (2007)1. - ISSN 1046-5928 - p. 17 - 22.
    transgenic plants - fluorescence microscopy - nucleocapsid protein - beta-glucuronidase - mosaic-virus - expression - system - gene - tospovirus - resistance
    Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a potentially useful protein fusion partner. To evaluate this potential, the N protein was fused to the Aequorea victoria green fluorescent protein (GFP), either at the amino or carboxy terminus, and expressed in plants from binary vectors in Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens and evaluated after 4 days, revealing an intense GFP fluorescence under UV light. Microscopic analysis revealed that upon expression of the GFP:N fusion a small number of large aggregates were formed, whereas N:GFP expression led to a large number of smaller aggregates scattered throughout the cytoplasm. A simple purification method was tested, based on centrifugation and filtration, yielding a gross extract that contained large amounts of N:GFP aggregates, as confirmed by GFP fluorescence and Western blot analysis. These results show that the homopolymerization properties of the N protein can be used as a fast and simple way to purify large amounts of proteins from plants.
    The role of weeds in the spread of Tomato spotted wilt virus by thrips tabaci (Thysanoptera: Thripidae) in tobacco crops
    Chatzivassiliou, E.K. ; Peters, D. ; Katis, N.I. - \ 2007
    Journal of Phytopathology 155 (2007)11-12. - ISSN 0931-1785 - p. 699 - 705.
    western flower thrips - frankliniella-occidentalis thysanoptera - insect vector - host-range - transmission - tospovirus - epidemiology - populations - disease - hawaii
    Oviposition of Thrips tabaci, larval development and their potential to acquire Tomato spotted wilt virus (TSWV) from infected Amaranthus retroflexus, Datura stramonium, Lactuca serriola, Solanum nigrum and Sonchus oleraceus plants and the ability of the adults to transmit this virus to these weeds and tobacco (Nicotiana tabacum cv. Basmas) were studied. When a T. tabaci female was given an oviposition period of 4¿days, an average of 21 larvae were produced on leaves of D. stramonium, 17.5 on S. nigrum, 16.3 on L. serriola and 14.3 on S. oleraceus. Significantly higher numbers of larvae were found on tobacco (29.5), and lower numbers on A. retroflexus (1.9). In a choice test, when females were placed onto leaves of these weed species and tobacco in a Petri dish, D. stramonium was preferred over tobacco. Equal numbers of larvae emerged on leaves of tobacco, of L. serriola and of S. nigrum. Oviposition was lower on A. retroflexus and S. oleraceus in this test. After the transfer of newborn 24¿h old larvae to leaf discs of tobacco or to one of the weeds 87, 84, 82, 71, 64 and 17% became pupa on tobacco, D. stramonium, L. serriola, S. nigrum, A. retroflexus or S. oleraceus respectively. After acquisition of virus by newborn-24¿h old larvae from L. serriola, D. stramonium, S. nigrum and A. retroflexus 69.5, 51.4, 32.6 and 22% of the adults became transmitters. No transmission could be recorded on S. oleraceus due to a high larval mortality. Males appeared to be more efficient transmitters than females. Tobacco was more susceptible to TSWV than petunia and the weed species, while among weeds, S. nigrum was the most and A. retroflexus the least susceptible species.
    Tomato spotted wilt virus particle assembly : studying the role of the structural proteins in vivo
    Snippe, M. - \ 2006
    Wageningen University. Promotor(en): R.W. Goldbach, co-promotor(en): Richard Kormelink. - [S.l. ] : S.n. - ISBN 9789085043263 - 128
    solanum lycopersicum - tomaten - tomatenbronsvlekkenvirus - tospovirus - viruseiwitten - virale regulatoire eiwitten - glycoproteïnen - fluorescentiemicroscopie - genexpressieanalyse - solanum lycopersicum - tomatoes - tomato spotted wilt virus - tospovirus - viral proteins - viral regulatory proteins - glycoproteins - fluorescence microscopy - genomics
    Members of the Bunyaviridae have spherical, enveloped virus particles that acquire their lipid membrane at the Golgi complex. For the animal-infecting bunyaviruses, virus assembly involves budding of ribonucleoprotein particles (RNPs) into vacuolised lumen of the Golgi complex, after which the enveloped particles are secreted. The maturation of tomato spotted wilt virus (TSWV), a bunyavirus infecting plants, is different in that virions acquire their membrane by wrapping of a Golgi stack around RNPs after which the enveloped particles eventually accumulate in large vesicles in the plant cell. TSWV also multiplies in its insect vector thrips, and here particles are secreted from salivary gland cells into the gland ducts. The latter seems a logic requirement to allow virus passage to healthy host plants.

    To further study the process of TSWV particle assembly, the interactions between the structural N, Gn and Gc proteins in mature virus particles, as well as their intracellular behaviour invivo havebeen the main target of this Ph. D. thesis.

    After an introductory chapter on bunyavirus particle assembly (chapter 1), the protein composition of purified TSWV RNPs and enveloped particles was studied in chapter 2.In enveloped virus preparations, the three major structural proteins, i.e. the nucleocapsid protein (N) and the two envelope glycoproteins Gn andGc, were detected in monomeric as well as oligomeric forms. GlycoproteinGcbut not Gn was observed tightly bound to RNPs, suggesting Gc is involved in RNP envelopment. Analysis of cytoplasmic RNPs and mature virus particles for other viral proteins revealed, surprisingly, the presence of the so-called nonstructural protein NSs. Whereas mature virus particles contained only traces of NSs, RNP preparations clearly contained larger amounts of this protein, which could be related to an earlier reported difference in transcriptional/replicational activity between both.

    To study the process of virus assembly in more detail, fluorescence microscopy methods were employed for the in vivo detection of protein interactions, rendering information concerning the intracellular localisation simultaneously (chapter 3). For this a system was set up in mammalian cells and as a first protein to be studied the cytosolic N protein was selected. This protein was already known to form homo-oligomeric structures in vitro. Using fusions of N with either yellow or cyan fluorescent protein (YFP and CFP, respectively), pairs were created to function as a donor (CFP) and acceptor (YFP) fluorophore for fluorescence resonance energy transfer (FRET). Using acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to measure FRET, N was observed to form homodimers and -multimers throughout the cytoplasm before eventually accumulating in a non-Golgi, perinuclear region ona microtubuli- and actin-dependent manner.

    In a similar way, potential in vivo interactions between N and the viral glycoproteins were investigated (chapter 4). While no interaction between N and Gn was observed, these studies demonstrated interaction between N andGc, in

    agreementwith the earlier observation (chapter 2) that some Gc remains tightly bound to purified RNPs. The interactions between N andGclocalised to the non-Golgi perinuclear area, similar to transiently expressed N. These data provided further support for the idea that interactions between N andGcare involved in envelopment of the viral RNPs. While studying the possible formation of heterodimers of Gn andGc, it appeared that with the constructs used, FRET could not be applied for this purpose, as fluorescence from the two fluorophore fusion proteins was never observed in the same cell. This could be due to the fact that interaction between the two glycoproteins interfered with proper folding of (one of) the fluorophores, resulting in greatly reduced and possibly undetectable fluorescence.

    Another intriguing question-relevant to a broader cell biological field as well-concerns the signal responsible for Golgi localisation of the two glycoproteins during infection. Previous work had shown that Gn carries a Golgi retention signal and is able to rescueGcfrom the ER to the Golgi, suggesting a heterodimerisation of Gn and Gc. For a number of other bunyaviruses, the Golgi retention signal had been mapped to the C-terminal transmembrane domain (TMD) and / or cytoplasmic tail of Gn. Using C-terminal deletion mutants of TSWV Gn and chimeric Gc and vesicular stomatitis virus glycoprotein (VSV-G) constructs (chapter 5), it was shown that both the TMD and the first 30 amino acids of the 60 residues-sized cytoplasmic tail of Gn are necessary for Golgi localisation. The lumenal domain was shown not to be required for Golgi localisation, nor was its presence required for rescuing ofGc. By deletion mapping the 20 most C-terminal residues of the cytoplasmic tail were shown to be crucial for interaction withGc.

    Large cells containing multiple nuclei were frequently observed whenGcwas expressed. This phenomenon was further investigated in chapter 6, and was shown probably to result from a cell fusion activity of this glycoprotein. Cell fusion is not likely to occur during the plant infection cycle, but may play a role in the infection cycle or the process of virus entry within the thrips vector. The fusion was not pH-dependent and not observed with Gn.

    Chapter 7 discusses the major findings of this Ph. D. research in a broader perspective, and presents a model for TSWV particle assembly in which all observations have been accomodated.
    Herbivore arthropods benefit from vectoring plant viruses
    Belliure, B. ; Janssen, A. ; Maris, P.C. ; Peters, D. ; Sabelis, M.W. - \ 2005
    Ecology Letters 8 (2005)1. - ISSN 1461-023X - p. 70 - 79.
    spotted-wilt-virus - western flower thrips - frankliniella-occidentalis - jasmonic acid - insect vector - tomato leaves - dwarf virus - transmission - tospovirus - aphid
    Plants infected with pathogens often attract the pathogens' vectors, but it is not clear if this is advantageous to the vectors. We therefore quantified the direct and indirect (through the host plant) effects of a pathogen on its vector. A positive direct effect of the plant-pathogenic Tomato spotted wilt virus on its thrips vector (Frankliniella occidentalis) was found, but the main effect was indirect; juvenile survival and developmental rate of thrips was lower on pepper plants that were damaged by virus-free thrips than on unattacked plants, but such negative effects were absent on plants that were damaged and inoculated by infected thrips or were mechanically inoculated with the virus. Hence, potential vectors benefit from attacking plants with virus because virus-infected plants are of higher quality for the vector's offspring. We propose that plant pathogens in general have evolved mechanisms to overcome plant defences against their vectors, thus promoting pathogen spread
    Tomato spotted wilt virus infection improves host suitability for its vector Frankliniella occidentalis
    Maris, P.C. ; Joosten, N.N. ; Goldbach, R.W. ; Peters, D. - \ 2004
    Phytopathology 94 (2004)7. - ISSN 0031-949X - p. 706 - 711.
    western flower thrips - insect vector - tospovirus - transmission - thysanoptera - aphididae - homoptera - pepper
    The effect of Tomato spotted wilt virus (TSWV) infection on plant attractiveness for the western flower thrips (Frankliniella occidentalis) was studied. Significantly more thrips were recovered on infected than were recovered on noninfected pepper (Capsicum annuum) plants in different preference tests. In addition, more offspring were produced on the virus-infected pepper plants, and this effect also was found for TSWV-infected Datura stramonium. Thrips behavior was minimally influenced by TSWV-infection of host plants with only a slight preference for feeding on infected plants. Offspring development was positively affected since larvae hatched earlier from eggs and subsequently pupated faster on TSWV-infected plants. These results show a mutualistic relationship between F. occidentalis and TSWV.
    Restricted spread of tomato spotted wilt virus
    Maris, P.C. ; Joosten, N.N. ; Goldbach, R.W. ; Peters, D. - \ 2003
    Phytopathology 93 (2003)10. - ISSN 0031-949X - p. 1223 - 1227.
    western flower thrips - frankliniella-occidentalis thysanoptera - insecticide resistance - in-field - tospovirus - capsicum - transmission - weeds - chrysanthemum - strains
    Spread of Tomato spotted wilt virus (TSWV) and population development of its vector Frankliniella occidentalis were studied on the pepper accessions CPRO-1 and Pikante Reuzen, which are resistant and susceptible to thrips, respectively. Viruliferous thrips were released on plants of each accession (nonchoice tests) or on plants in a 1:1 mixture of both accessions (choice tests) in small cages containing 8 or 16 plants. Significantly fewer CPRO-1 plants became infected in the primary infection phase in both tests. fit the nonchoice test, virus infection of the resistant plants did not increase after the initial infection, but all plants eventually became infected when mixtures of both cultivars were challenged in the secondary infection phase. Secondary spread of TSWV from an infected resistant or susceptible source plant was significantly slower to resistant plants than to susceptible plants, independent of source plant phenotype. The restricted introduction and spread of TSWV in the thrips-resistant cultivar was confirmed in a large-scale greenhouse experiment. The restricted and delayed TSWV spread to plants of the resistant accession in both the cage and the greenhouse experiment was explained by impeded thrips population development. The results obtained indicate that thrips resistance may provide a significant protection to TSWV infection, even when the crop is fully susceptible to the virus.
    Thrips resistance in pepper and its consequences for the acquisition and inoculation of Tomato spotted wilt virus by the Western Flower Thrips
    Maris, P.C. ; Joosten, N.N. ; Peters, D. ; Goldbach, R.W. - \ 2003
    Phytopathology 93 (2003)1. - ISSN 0031-949X - p. 96 - 101.
    frankliniella-occidentalis thysanoptera - insecticide resistance - mosaic-virus - transmission - tospovirus - capsicum - vector - craccivora - pathogen - strains
    Different levels of thrips resistance were found in seven Capsicum accessions. Based on the level of feeding damage, host preference, and host suitability for reproduction, a thrips susceptible and a resistant accession were selected to study their performance as Tomato spotted wilt virus (TSWV) sources and targets during thrips-mediated virus transmission. Vector resistance did not affect the virus acquisition efficiency in a broad range of acquisition access periods. Inoculation efficiency was also not affected in short inoculation periods, but was significantly lower on plants of the thrips resistant accession during longer inoculation access periods. Under the experimental conditions used, the results obtained show that transmission of TSWV is little affected by vector resistance. However, due to a lower reproduction rate on resistant plants and a lower preference of thrips for these plants, beneficial effects of vector resistance might be expected under field conditions.
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