ann revual tospovirusses

26 Jul 2005 11:53 AR AR250-PY43-19.tex XMLPublishSM(2004/02/24) P1: KUV10.1146/annurev.phyto.43.040204.140017

Annu. Rev. Phytopathol. 2005. 43:459–89doi: 10.1146/annurev.phyto.43.040204.140017

Copyright c© 2005 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on May 19, 2005

TOSPOVIRUS-THRIPS INTERACTIONS

Anna E. Whitfield,1 Diane E. Ullman,2 andThomas L. German1

1Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706;email: [email protected], [email protected] of Entomology, University of California, Davis, California 95616;email: [email protected]

Key Words Bunyaviridae, insect vector, membrane glycoproteins, Thysanoptera,Tomato spotted wilt virus

■ Abstract The complex and specific interplay between thrips, tospoviruses, andtheir shared plant hosts leads to outbreaks of crop disease epidemics of economicand social importance. The precise details of the processes underpinning the vector-virus-host interaction and their coordinated evolution increase our understanding ofthe general principles underlying pathogen transmission by insects, which in turncan be exploited to develop sustainable strategies for controlling the spread of thevirus through plant populations. In this review, we focus primarily on recent progresstoward understanding the biological processes and molecular interactions involved inthe acquisition and transmission of Tospoviruses by their thrips vectors.

INTRODUCTION

Insects in the order Thysanoptera (commonly known as thrips) include many im-portant direct crop pests and at least 10 species that transmit viruses in the genusTospovirus, the only plant-infecting genus in the family Bunyaviridae. Thripstransmission of tospoviruses impacts a diverse number of food, fiber, and orna-mental crops encompassing hundreds of plant species (14), resulting in crop diseaseepidemics of worldwide economic and social significance. The biology that in-fluences the coordinated evolution of thrips, tospoviruses, and their plant hostshas provided an opportunity for scientists from many disciplines to address issuesof applied and basic significance since the early decades of the 1900s. Manythrips that serve as tospovirus vectors have extensive plant host ranges. Bothtospoviruses and thrips vector species thrive in diverse climates, which explainstheir worldwide importance.

The interest in the Thysanoptera, their role as tospovirus vectors, and the biol-ogy of tospoviruses is global. Growers and pest control advisors have recognizedthat advances in our ability to manage thrips as direct pests and as vectors re-quires a broad and integrated research approach at the interface of entomology,

0066-4286/05/0908-0459$20.00 459

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26 Jul 2005 11:53 AR AR250-PY43-19.tex XMLPublishSM(2004/02/24) P1: KUV

460 WHITFIELD � ULLMAN � GERMAN

ecology, systematics, and virology. To expand our knowledge of the fundamen-tal principles underlying virus-vector relationships, scientists are beginning todissect the specific series of events governing the complex interaction betweenthrips, tospoviruses, and their plant hosts. Recent progress toward understandingthe biological processes and molecular interactions that lead to virus acquisition,movement within the vector, and transmission to plant hosts are covered in thisreview.

TOSPOVIRUSES

The disease known as spotted wilt was first described in Australia in 1915 (12), andthrips were implicated as vectors of the disease-causing agent several years later(107). In 1930, Samuel et al. (121) demonstrated that a virus, which they namedTomato spotted wilt virus (TSWV), was the causative agent of the disease. Morethan 50 years later, Francki and colleagues (88) noted the similarity between TSWVand viruses in the family Bunyaviridae: a large group of membrane-bound, mostlyarthropod-transmitted, animal-infecting viruses with tripartite negative-strandedRNA genomes. They suggested that it was the first plant-infecting member of amonotypic group of plant viruses (36). During the following two decades, dataon the molecular biology of TSWV (reviewed in 1, 41, 44, 93, 123, 124) and theclosely related Impatiens necrotic spot virus (INSV) (25, 73) confirmed (27) thevalidity of the taxonomic status of these and related viruses.

The family Bunyaviridae includes animal-infecting viruses of the genera Or-thobunyavirus, Hantavirus, Nairovirus, and Phlebovirus and the Tospovirus genus,which consists of the plant-pathogenic, thrips-transmitted members of the fam-ily. TSWV is the type member of the genus that currently includes 14 species(Table 1) separated primarily on the basis of the serological properties and aminoacid sequence identity of the viral structural proteins (23, 24). Typical of the fam-ily Bunyaviridae, TSWV has a genome consisting of three negative or ambisensessRNAs designated S (2.9 kb), M (4.8 kb), and L (8.9 kb). The RNAs have apanhandle conformation created by base pairing of about 60 complementary nu-cleotides at the 3′ and 5′ ends of each strand (28). The core of the virion containsribonucleoproteins (RNPs) composed of the ssRNA components encapsidated bythe nucleoprotein (N) and a few copies of the viral RNA-dependent RNA poly-merase (RdRp or L protein). The 80–120-nm pleiomorphic virus particles areformed by enclosure of the RNPs in a host-derived membrane studded with sur-face projections composed of two viral glycoproteins, GN and GC (Figure 1).

Although the details of the genomic organization and expression strategy (asshown in Figure 2) of TSWV are well characterized, gene product function inTSWV is just beginning to emerge. The ambisense 2.9-kb S RNA encodes a52.4-kDa nonstructural protein (NSs, not present in the mature virus particle) inthe viral (v) sense and the 29-kDa N protein in the viral complementary (vc) sense(29). Both proteins are expressed by translation of subgenomic RNA species thatpossibly terminate at a long, stable intergenic hairpin structure (67). The tospovirus

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TOSPOVIRUS-THRIPS INTERACTIONS 461

TABLE 1 Recognized Tospovirus species and their documented vectors

Tospovirus species Thrips vectors

Capsicum chlorosis virus (83) ?

Chrysanthemum stem necrosis virus (113) Frankliniella occidentalis (95)F. schultzei (94)

Groundnut bud necrosis virus (111) F. schultzei (6)Thrips palmi (70)Scirtothrips dorsalis (6)

Groundnut ringspot virus (23) F. occidentalis (156)F. schultzei (156)

Impatiens necrotic spot virus (70) F. occidentalis (22)

Iris yellow spot virus (20) T. tabaci (40)

Melon yellow spot virus (58) T. palmi (57)

Peanut chlorotic fanspot virus (17) S. dorsalis (17)

Peanut yellow spot virus (110) S. dorsalis (109)

Tomato chlorotic spot virus (23) F. intonsa (156)F. occidentalis (156)F. schultzei (156)

Tomato spotted wilt virus (12) F. bispinosa (151)F. fusca (120)F. intonsa (156)F. occidentalis (37)F. schultzei (121)T. setosus (65)T. tabaci (107)

Watermelon bud necrosis virus (53) T. palmi (92, 126)

Watermelon silver mottle virus (160) T. palmi (161)

Zucchini lethal chlorotic virus (113) F. zucchini (100)

NSs protein has been shown to function in suppression of RNA silencing during theplant-infection phase of the virus life cycle. (13, 132). The NSs genes of the relatedphleboviruses and orthobunyaviruses can influence the virulence of these viruses intheir animal hosts (11, 150), which has led scientists to speculate about the possiblerole of the tospovirus counterpart in the thrips infection cycle. The nucleocapsidprotein (N) contributes to the viral replication cycle in a structural and, perhaps,regulatory manner by participating in the complex interactions among the RNPcomponents leading to the initiation of viral RNA transcription and replication.Consistent with its role in fulfilling this putative function, the N protein has beenshown to form dimers in the absence of RNA (56, 136) and to cooperatively bindssRNA but not dsRNA (114). Interestingly, mutated forms of N protein serve aspotent dominant-negative inhibitors of virus replication (118).

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Figure 2 Genomic organization and expression strategy of Tomato spotted wilt virus.vRNA represents virion sense RNA, the predominate form of RNA in the virion. vcRNArepresents viral complementary sense RNA. Open boxes in the RNAs indicate openreading frames expressible from either v or vc RNA. Stippled boxes at the 5′ terminiof mRNAs represent nontemplated cap structures. The flexuous lines represent virus-encoded proteins.

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The M RNA encodes a 33.6-kDa nonstructural protein (NSm) in the v senseand a 127.4-kDa precursor to the two viral membrane glycoproteins, GN andGC, in the vc sense. Using an expression strategy similar to that of the S RNA, theNSm open reading frame (ORF) is expressed from a subgenomic RNA transcribedfrom the vcRNA, and GN and GC are translated as a polyprotein from a singleORF on a subgenomic mRNA transcribed from the vRNA (67). The resultingGN/GC polyprotein is cleaved in the absence of other viral proteins to produce theindividual membrane components (2). The role of GN and GC in virus assemblyand transmission by thrips is treated in detail in the following sections. The roleof NSm in cell-to-cell movement is supported by its early expression profile, itsassociation with (68) and ability to alter the size exclusion limit of plasmodesmata(129), and the observation that it forms tubules, a feature of other viral movementproteins (30, 50, 128).

The L RNA is of entirely negative polarity, with one ORF located on the vcstrand corresponding to a primary translation product of 2875 amino acids, witha predicted molecular mass of 331 kDa. This protein contains a cluster of nucleicacid polymerase motifs (26) including the highly conserved serine-aspartic acid-aspartic acid (SDD) element found in the RdRp of all segmented negative-strandRNA viruses (134). Sequence and functional analyses of the L protein support therole of a multifunctional viral RdRp that, by analogy to other negative-strand RNAviruses, has NTPase, polymerase, nuclease, helicase, and polymerization activities.In several negative-strand RNA virus families, these discrete functions are carriedout by individual proteins produced from unique mRNAs; however, the singlemultifunctional protein organization of TSWV L RNA is characteristic of virusesin the Orthobunyavirus, Hantavirus, and Phlebovirus genera of the Bunyaviridae(134). Functional analysis has shown that an RdRp activity is associated withdetergent-disrupted TSWV virion preparations (3, 148) and that the L protein isthe source of this activity (15).

A brief overview of the replication cycle provides insight into the complex inter-play transpiring between virus and host during the infection process. By analogy tothe animal-infecting members of the Bunyaviridae, it is reasonable to hypothesizethat tospoviruses infect insect cells by binding to a host cell receptor through themediation of a viral surface glycoprotein (Figure 3). A subsequent fusion event ofthe viral and host membranes, possibly initiated by low pH (155), releases the ge-nomic RNA segments in association with multiple copies of N protein (RNPs) intothe cytoplasm. The RNP serves as a template for transcription of viral mRNAs,catalyzed by the virion-associated viral RdRp. Typical of segmented negative-strand RNA viruses, TSWV mRNAs are not polyadenylated and have eukaryoticcap structures and nontemplated heterogenous sequences of host origin at their5′ends. These are generated during a process, termed cap-snatching, during whichthe viral polymerase cleaves some 10–20 nucleotides along with the cap struc-ture from a host mRNA and incorporates them into the 5′ terminus of the newlysynthesized viral mRNA (32, 33, 69, 149). These messages then associate withthe host cell translational apparatus to produce the viral protein complement and

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Figure 3 Transmission electron micrographs showing the likely steps in virus fusionand entry to the larval midgut of Frankliniella occidentalis. Protein A-gold was usedas the tag in all micrographs. (A) A thin section of a larval thrips midgut epithelialcell. Virions ingested from an infected plant are labeled with polyclonal antibodyto the Tomato spotted wilt virus (TSWV) membrane glycoproteins. Inset shows alabeled virion fusing with the apical membrane. [Reprinted with permission from theAmerican Phytopathological Society from (145, figure 1).] (B) Thin section of larvalthrips midgut epithelial cell immunostained with antibodies against TSWV membraneglycoproteins and 15-nm Protein A-gold. The membrane just above a dense massis labeled, suggesting that TSWV membrane glycoproteins are bound to the apicalmembrane. (C) Thin section adjacent to that shown in B immunostained with antibodiesagainst TSWV N protein and 15-nm Protein A-gold. Labeling of the mass suggeststhat TSWV ribonucleoproteins or virions have entered the cell. ap, apical membrane;c, cytoplasm; mv, microvilli; lu, lumen; v, virus; unmarked arrowhead, dense mass ofribnucleoprotein; unmarked arrow, gold particle on apical membrane.

additional copies of the RdRp that generate full-length antigenomic RNAs. Theseantigenomic RNAs, in turn, serve as templates for de novo synthesis of manygenomic RNA copies. The panhandle structure formed by the complementary 3′

and 5′ ends of each viral genomic segment most likely serve as promoters forreplication. Host proteins are then probably involved in the process of templateselection, stabilization of the polymerase complex, and catalytic events resultingin extension of the RNA strands (4, 31, 84).

Tospovirus replication has been described in the context of a plant infection;however, immunocytochemical and microscopic identification of TSWV NSs pro-tein in cells of Frankliniella occidentalis established that viral replication alsooccurs in the insect vector (141, 158). Therefore, in contrast to viruses that havenonpersistent or circulative modes of transmission, adaptation and selection eventsthat influence the rate of virus replication, movement, assembly, and counterde-fense strategies in the insect have an increased influence on transmission to planthosts. For example, the rate of virus replication in the midgut and the extent of

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virus migration from the midgut to the visceral muscle cells and the salivary glandsare crucial factors in the determination of vector competence (98). Consequently,fully understanding the transmission process requires a detailed consideration ofthe multiple points of interaction between virus and vector that govern entrance,replication, movement within, and exit from the vector. Taken together, these stepscomprise the events culminating in the infection of a susceptible plant host.

COEVOLUTION OF THRIPS AND TOSPOVIRUSES

Although tospoviruses can be mechanically transmitted under experimental con-ditions, tospovirus dispersal and survival in nature depends on passage to plants bythrips vectors. The dispersal and survival of TSWV depends upon the coexistenceof virus and vector populations under conditions in which their genetic and physio-logical make-up form a compatible vector-virus interaction. The environment andthe plant-host interactions with the virus and the insect also influence every phaseof the infection cycle. Thus, coevolution between thrips and tospoviruses mustgreatly influence the observed variability between virus isolates, epidemics, andeven the emergence of new tospoviruses (Figure 4). Tremendous genetic variabilityin tospovirus populations is provided by the high error rate inherent in RNA repli-cation by the viral RdRp (21) and the reassortment of genomic segments betweendifferent virus isolates in planta (108). Little is known about the thrips genome,but all of the vector species are characterized by striking morphological diversity,which suggests genetically variable populations (92). Tospovirus and thrips vectorpopulations certainly meet the basic requirements for rapid coevolution.

An example of likely coevolution between tospviruses and their vectors is pro-vided by the altered status of Thrips tabaci as a vector of TSWV. Forty years agothis species transmitted all known isolates of TSWV worldwide (119), but nowappears to be incapable of transmitting modern TSWV isolates (82, 156). Newvector-virus relationships have also arisen, possibly as a result of coevolutionaryevents. For example, Thrips palmi can now transmit several newly emergenttospoviruses in cucurbits (57, 92, 126), and F. bispinosa emerged as a vectorof TSWV (151), while Thrips tabaci emerged as a vector of Iris yellow spot virus(IYSV) (40).

The precise mechanisms underlying the formation of these new vector-virusrelationships are not well understood, but certainly plant host-vector relationshipsand thrips reproductive strategies play roles as well (16). Many plant hosts sup-port mixed virus infection and multiple thrips species, which provide an arena forgenetic exchange. Reassortment of RNA segments from resistance-breaking orthrips-transmissible isolates could endow nontransmissible isolates with resistance-breaking characteristics and new transmission qualities (108). This evidencesupports the notion that reassortment occurring in mixed infections could con-tribute to the appearance of isolates with a variety of new characteristics,including the emergence of new vector species or reemergence of a vector likeT. tabaci.

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THRIPS LIFE CYCLE, REPRODUCTIVE STRATEGIES,AND FEEDING BEHAVIOR

Thrips vector species are well equipped for their role as vectors of tospoviruses. Thethrips life cycle and its relevance to the tospovirus transmission cycle is illustratedin Figure 5. Adult females lay eggs on plants and after eclosion, there are twowingless larval stages that feed on plant leaves and flowering parts. The ensuingpupal stages are nonfeeding and depending on the thrips species, this stage occursin the soil or on plants. Winged adults that strongly resemble the larvae emergeand tend to disperse widely.

Most of the vector species have high fecundity and short reproductive cycles.They are haplodiploid: Females are diploid and males are haploid. Thrips have tworeproductive strategies: Arrhenotokous thrips reproduce sexually and thelotokusthrips reproduce asexually (16, 92). Knowledge of Thysanopteran reproductivestrategies can help us understand the genetic basis of transmission efficiency andvariability in tospovirus epidemics, e.g., thelotokus, and not arrhentokus, T. tabacipopulations are efficient vectors of certain tospoviruses (16).

The thrips species that transmit TSWV are polyphagous, piercing-sucking in-sects that feed on a variety of cell types (92, 138). Many thrips species feed onpollen and flower structures, as well as on vegetative portions of plants (75, 138).To feed, the insect punctures the leaf epidermis and ingests the cytoplasm frommesophyll cells (138), either collapsing single cells or destroying several cells.Intact plant organelles and virions were observed in thrips guts with the use of EM(132, 139). Evidence from electronic monitoring suggests that the WFT has at leasttwo modes of feeding. They make many probes of short duration during whichthey apparently salivate into and empty the contents of single or small groups ofplant cells probably just under the epidermal surface (63). Less often, probes ofmuch longer duration are made that consist of a short period of salivation followedby what appears to be a long period of ingestion (51). This type of feeding tends tobe very destructive to leaves, with the damage extending through all or several leafcell layers (51, 63, 138). Sakimura (119) suggested that thrips were more likely totransmit virus during brief shallow probes; however, more research in this area isneeded to define the impact of feeding behaviors on virus transmission to plants.

TOSPOVIRUS-THRIPS INTERACTIONS

Virus Dissemination and Replication in Vector Thrips

To understand the specific events that lead to transmission of tospoviruses in nature,it is critical to identify the anatomical features of the insect that mediate viralmovement and replication in the insect. Dissemination of the virus through theinsect illustrates its ability to bind and travel across host membranes. Here, wedescribe the tissue systems and six membranes encountered by virions along theirpath from the alimentary canal to the salivary glands (Figure 6). [For a more

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Figure 6 Thrips internal organs and their putative role in virus passage to the salivaryglands. This line drawing depicts the putative path of tospoviruses through the thrips andillustrates the membrane barriers the virus must pass before successful inoculation of aplant can occur. Tospoviruses enter the midgut lumen and move across the apical mem-brane of the brush border [1]. Tospoviruses replicate in the midgut and by an unknownmechanism cross the basement membrane [2] into the visceral muscle cells wherereplication continues as indicated by the presence of viroplasm [inset, 3]. The virusesmust then exit these cells across their basal membrane [inset, 4] and enter the salivarygland [5]. Virions exit the salivary gland across the apical membrane [6] and flow withthe salivary secretions into the plant during thrips feeding. Hg, hindgut; Mc, mouth-cone; Mg, midgut; E, esophagus; EF, efferent salivary duct; L, lumen; PSg, primarysalivary gland; s, cross sections of muscle; TSg, tubular salivary gland; VP, viroplasm;DM, dense mass.

detailed description of thrips internal anatomy, see (138).] Upon ingestion of viralparticles, virions travel through the foregut into the midgut, the primary site ofTSWV-binding and entry into insect cells (8, 97, 139). A brush border of microvilliextends into the midgut lumen and forms the first membrane barrier encounteredby the virus (see magnified inset in Figure 6, denoted by the number 1). Virusparticles move across the microvilli to the basal surface of the columnar epithelialcells of the midgut, which is formed by the basement membrane, the next membraneencountered by the virus. The virus exits the midgut epithelia (see magnified areain Figure 6, denoted by the number 2) by crossing the basal membrane that is

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encircled by alternating series of longitudinal and circular muscle cells (90, 137,141). TSWV has been observed in these muscle cells, and entry and exit fromthese muscle cells presumably consitute the third and fourth membrane barriersthat virions must cross on their path to the salivary glands (7, 8, 97, 98, 101).

The primary salivary glands are thought to play a critical role in virus acquisi-tion and transmission. Little is known about the basal membrane of these glands;however, tospoviruses entering the salivary gland must traverse this membrane(Figure 6, see magnified inset, the number 5). The lumen of each lobe is lined withmicrovilli (Figure 6, see magnified inset, the number 6), and this represents thelast membrane the virus must cross in order for transmission to occur. Once insidethe salivary gland lumen, virions can move with saliva into a canal that leads to anefferent salivary canal, a common salivary reservoir, and then a duct that ultimatelyallows virus-laden saliva to exit the combined salivary-food canal.

As previously outlined, viral infection in larval thrips begins in the midgut afterwhich the virus replicates, spreads throughout the midgut, and subsequently infectsthe muscle cells surrounding the midgut and the primary salivary glands (7, 97, 98,135, 139, 141, 144, 145, 158). Presence of nonstructural proteins (NSs) and viralinclusions in the insect support the contention that tospoviruses replicate in theirthrips vectors (141, 158) and the presence of viral inclusions in midgut epithelialcells, muscles surrounding the alimentary canal, and the primary salivary glands ofthrips indicated that virus replicates in these tissues (141, 144, 145, 158). TSWVglycoproteins were immunolocalized to membranes thought to be part of the Golgicomplex in thrips cells (145). Additionally, TSWV proteins were associated withmembrane-bound structures in midgut epithelial cells that appeared to be fusingwith basal membrane (145), and Nagata et al. (98) observed virions budding fromthe basal side of infected midgut cells. Viral proteins were also associated withvesicles that were similar in appearance to clathrin vesicles; furthermore, thesevesicles were often observed near vacuoles that also contained viral proteins (145).It is likely that virions and/or viral proteins associated with secretory vesicles couldfunction in virus cell-to-cell movement via the exocytic pathway.

Models for Virus Movement in Thrips

Although contemporary electron microscopic evidence clearly shows virus infec-tion of several thrips organs and tissues, little is known about the route virus takesin the insect to eventually reach the salivary glands. Three hypothetical modelshave been proposed to attempt to explain the mechanisms underlying tospovirusmovement from the thrips midgut to the salivary glands:

1. By analogy to other persistently transmitted viruses, virus could enter the in-sect gut, traverse the midgut and muscle cell barriers, and enter the hemocoel.The virus would then circulate and may infect other organs, but ultimatelyinfect the salivary glands, allowing for successful transmission to plants. Todate, there is no direct evidence to support this hypothesis.

2. Examination of thrips anatomy showed that two structures connect themidgut to the primary salivary glands: tubular salivary glands (138) and thin

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ligament-like structures (97). On the basis of immunolabeling of TSWV inthrips, several authors propose that the ligament-like structures may serve asa conduit for virus transport by connecting the midgut to the salivary glands(8, 97, 98). Nagata et al. (97, 98) found that infection of the ligament-likestructure preceded salivary gland infection. Early infection sites in the sali-vary gland occurred at the point of contact with the ligament-like structure,which led the authors to propose that the ligament-like tissue connects themidgut to the salivary glands, thereby facilitating cell-to-cell spread of virusfrom the midgut to the salivary gland. Transmission electron microscopicevidence will be very useful in confirming the presence of virus in theseligament-like structures. Although the tubular salivary glands would seemto be convenient structures to support virus movement, there is no reportedevidence of virus infection in these structures (98; D.E. Ullman & D.M.Westcot, unpublished data).

3. Based on thrips ontogeny, Moritz et al. (91) demonstrated that the proxim-ity of organs changes during thrips development. They proposed that virusmovement from midgut tissues to the primary salivary glands occurs whenthere is direct contact between membranes of the visceral muscle cells and theprimary salivary glands during larval development. These authors showedthat the primary salivary glands, midgut, and visceral muscles of the firstlarval stage of F. occidentalis are compressed into one area of the thoraxwhere they lie in direct contact with one another through the early secondinstar stage. It is proposed that virus moves from the midgut and musclesto the salivary gland when these tissues lie in direct proximity to one an-other in the larval insect (91). As the insect grows, the brain and primarysalivary glands move forward, while the first midgut loop moves back intothe metathorax, resulting in spatial separation of these organs and prevent-ing virus movement between these tissues. This hypothesis is compellingbecause it is consistent with the evidence that only adults that feed as larvalstages can transmit virus.

Virus movement in the thrips vector is an exciting area of investigation. Weexpect the near future to hold explanations for potential barriers to virus survival inthe hemolymph and the mechanisms of virus passage from the midgut and visceralmuscles to the primary salivary glands. Innovative techniques such as laser-capturemicrodissection, immunohistochemistry, and real-time quantitative RT-PCR willprovide the needed tools to unravel the mysteries remaining in understanding theroute tospoviruses take while navigating the insect vector.

Thrips Stage-Dependent Acquisition of Tospovirus

Tospoviruses are transmitted in a persistent propagative fashion and are transsta-dially passed in their insect vector. Figure 5 illustrates the essential elements ofthe tospovirus transmission cycle. The thrips-tospovirus relationship is unique be-cause adult thrips can only transmit TSWV if acquisition occurs in the larval stages(77). Larval acquisition of the virus is an essential determinant of adult vector

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competency, and furthermore, acquisition rates decrease as larval thrips develop(147). For example, when first instar larvae were allowed to acquire TSWV, 47%of the concomitant adults transmitted virus. In contrast, 12% of adults from co-horts acquiring virus as second instars were able to inoculate plants (97, 98,147). Many plant species can be infected by polyphagous thrips adults as theydisperse and sample potential hosts; however, plant species that do not supportthrips development are dead ends for the virus and do not contribute to epidemicdevelopment.

Adult thrips that feed on infected plants do not become viruliferous even if theyare allowed lengthy feeding on tospovirus-infected plants (119, 138, 146, 147).EM observations by Ullman et al. (139) showed that virions were present in midgutepithelial cells of adult F. occidentalis shortly after the acquisition access period(AAP). The work of Ullman et al. (139) and Nagata et al. (97) with TSWV-MT2and BR01, respectively, suggest that persistent infection of adult midgut cells isa rare occurrence. Recent work with different isolates of TSWV shows that viruscan infect midgut cells (101) and, in some cases, spreads to muscle cells in insectsfed as adults (7). Ohnishi et al. (101) found that TSWV (originally isolated frompepper in Japan) infected adult Thrips setosus midguts, and by three days post-AAP, the infection spread through the insect midgut. By five days post-AAP, theinfection had diminished; furthermore, they found that virus was unable to spreadbeyond the basal lamina of adult thrips midguts. These results suggest that the basallamina serves as a potential barrier to virus movement out of the insect midgut.Other researchers, working with a TSWV isolate recently isolated from peanut inGeorgia, found that TSWV infected adult midguts (7). Adult F. occidentalis andF. fusca sustained midgut infections and the virus also spread to the surroundingmuscle tissue. Virus was not found in ligament-like tissue or salivary glands, andthrips given AAP as adults were unable to transmit. Importantly, all these studiesagree that adult thrips, whether they support midgut infection or not, are unableto transmit TSWV and virus infection does not spread to the salivary gland unlessacquisition initially occurs during the larval stages of life. These experiments wereall performed with different isolates of TSWV, and these isolates varied in theirability to initiate successful infection of adult midguts, escape the midgut, andinfect muscles. Virus genotype, thrips genotype, and environmental conditionslikely play a critical role in these differential interactions.

Vector specificity between thrips species and virus isolates does occur (99,147). Working with four tospovirus species and four thrips species, Nagata et al.(99) found that viruses persisted and perhaps replicated in insects that were unableto transmit virus. Thrips fell into three categories: transmitters with detectablelevels of virus, nontransmitters with detectable virus, and nontransmitters with-out detectable virus. For example, Thrips palmi contained detectable amounts ofChrysanthemum stem necrosis virus (CNSV) from larval stages to adulthood, butthey were unable to transmit this virus. These data provide evidence that virus en-ters and replicates in the insect, and one possible explanation for their inability totransmit is that virus was unable to infect the salivary glands. In other virus-vectorcombinations, virus did not persist and was not transmitted; Thrips tabaci did not

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contain detectable levels of Groundnut ringspot virus and Tomato chlorotic spotvirus and was unable to transmit either virus. Two possible explanations for theseobservations are that virus replication was not supported in these insects or viruswas unable to enter midgut cells to initiate an infection. The barriers contributingto vector specificity may well vary with vector species and virus isolate, as hasbeen observed in other virus-vector interactions, particularly the persistently trans-mitted Luteoviruses (47). Further work examining these incompatible interactionswill help us understand the differences between tospovirus vectors and nonvectors.

Is TSWV a Pathogen of Thrips?

Tospoviruses have an intimate association with their insect vectors. The effect ofvirus infection on the insect has been the subject of much research, and recentfindings are strengthening our understanding of this intricate relationship. Onedifficulty in comparing investigations of the impact of tospovirus infection onthrips fitness lies in the genetic variability of the thrips populations and the geneticdiversity of tospovirus species, isolates, and populations. Two studies indicatedthat TSWV and INSV, respectively, decreased thrips fitness (22, 115). In both ofthese studies, thrips spent either their entire lives or a significant portion of theirlarval development on infected plants. The pathogenicity of the viruses to thripscould not be fully established because the nutritional status of infected plants mayhave caused the decrease in fitness. Using the BR01 isolate of TSWV, Wijkampet al. (157) assessed the effect of infection on thrips developmental time, repro-duction rate, and survival. Larvae were given a short AAP, reared to adulthood onnoninfected D. stramonium, and then moved to Petunia x hybrida ‘Blue Magic’to assess their infection status. They found no significant differences between vir-uliferous, nonviruliferous, and control thrips (157). A serious concern in assessingthis investigation is that Petunia is not a host for thrips. Thus, the uniformly poorfitness observed may have occurred because a nonhost plant was being used for theinvestigation. Maris et al. (81) confirmed that TSWV-BR01 caused no differencein mortality among thrips reared on leaf disks of noninfected and infected plants.In addition, these authors found that development from egg to adult required 1 to2 days less on TSWV-infected leaf disks. Medeiros et al. (85) found that TSWV-BR01 infection of F. occidentalis appears to induce several genes that are charac-teristically initiated as part of the insect defense response to pathogens, providingevidence that thrips mount an immune response to TSWV, or at least to isolateBR01. In contrast to the findings of Marais et al. (81) and Wijkamp et al. (157),experiments with another TSWV isolate yielded different results. Newly emergedthrips were given a brief AAP, reared to adulthood on bean, and transmission as-sessed on datura leaf disks. Thrips that transmitted virus consistently until deathhad shorter life spans and higher virus titers than nontransmitters. The latter groupdid not differ significantly in their longevity from insects fed on noninfected plants.(N.K.K. Kumar, A.E. Whitfield, T.L. German & D.E. Ullman, unpublished data).

Recent work provides clarification of these results and further insight into thisaspect of thrips-tospovirus relationships. Stumpf & Kennedy (130) examined the

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effect of TSWV isolate (CFL and RG2), temperature, and plant host on the insectvector. They found that TSWV-infected F. fusca reared on infected foliage tooklonger to develop into adults and were smaller than noninfected thrips also rearedon infected foliage, indicative of a direct effect of TSWV on thrips. Noninfectedthrips reared on noninfected leaves took longer to develop than noninfected thripson infected leaves, indicating that plant infection status also affected thrips. Nonin-fected females reared on virus-free host foliage were intermediate in developmenttime. The TSWV isolate also had an effect on the insects. CFL-infected femalestook 0.6 days longer to reach the adult stage than females infected with RG2and there was no effect on male developmental time. When taken together, it ap-pears that TSWV isolates may differ in pathogenicity to thrips, as well as causediverse nutritional changes in plants. In addition, thrips may vary in their abilityto adapt to nutritional changes in their plant hosts and may have different defensesagainst the virus isolates. These new research findings are beginning to explainsome of the complexity of the virus-vector relationship; discrepancies betweenresults achieved with different TSWV isolates and tospovirus species; and shedlight on the variability observed in the field epidemiology of the tospoviruses.

VIRUS ENTRY

Virus Entry Mechanisms

Virus entry into a host cell entails a complex series of events that culminate with thefusion of viral and host membranes. Viruses bind to the molecules on the host cell,and virion entry commonly occurs by one of two mechanisms, pH-independententry or pH-dependent entry. Virus entry via the pH-independent pathway beginswith virus attachment and is followed by direct fusion of viral membrane with hostplasma membrane. During pH-dependent entry, virus attachment is followed byformation of a cellular compartment (e.g., endosome). Virions are engulfed by thisvesicle, and a change in compartmental pH or fusion with an acidic compartment(e.g., lysosome) alters the conformation of the viral fusion protein. The subsequentconformational change in the viral fusion protein exposes the fusion peptide orfusion loop (89, 127). The fusion peptide inserts into the target membrane andleads to the fusion of virion and host membranes and the subsequent release of thevirion contents into the cytoplasm (reviewed in 125).

Evidence indicates that some members of the Bunyaviridae enter cells by pH-dependent receptor-mediated endocytosis (49, 55, 103). Researchers working withHantaan virus (HTN) showed that agents that disrupt clathrin-dependent endocy-tosis inhibited infection of cells; however, agents that disrupt caveolae-dependentendocytosis did not inhibit infection of cells (55). These results were supportedwith immunolabeling experiments that showed HTN localizing to endosomes andlater to lysosomes (55). Further support for pH-dependent entry comes fromstudies of the Orthobunyaviruses. The envelope glycoprotein GC of La Crossevirus (LAC) and California encephalitis (CE) undergoes a conformational change

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after exposure to acidic pH rendering it susceptible to protease cleavage, con-sistent with endocytic entry (46, 49, 103). Furthermore, conformation-specificGC antibodies did not recognize GC after virions were incubated at acidic pH(103). CE- and LAC-infected cells form syncytia (multinucleate cells) when theextracellular pH is lowered, which indicates that virus entry is stimulated by lowpH (46, 49). These data provide strong evidence that bunyaviruses enter cells ina pH-dependent process. Although the TSWV internalization pathway has notbeen studied in such detail, by analogy to other members of the family Bun-yaviridae, it is likely that TSWV enters thrips cells in a pH-dependent mannerand that the two membrane glycoproteins are involved in the entry process (154,155).

TSWV Glycoproteins

Sequence analysis of the TSWV glycoprotein gene has provided insight into theirbiology and function (Figure 7). The glycoproteins are encoded as a polyprotein

Figure 7 A schematic of the Tomato spotted wilt virus (TSWV) glycoprotein openreading frame. The top box represents the polyprotein, with light gray and dark grayboxes representing the low and medium areas of hydrophobicity, respectively. Putativesignal peptidase cleavage sites (S1 and S2), N- and O-linked glycosylation sites andtransmembrane domains are marked and were predicted as previously described (154).The dark horizontal lines designated GN and GC represent the predicted mature proteins,and numbers specify amino acid positions of the proposed N and C termini of theproteins. Figure not drawn to scale.

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that is cleaved to generate the two mature glycoproteins. Analysis of the TSWV(isolate MT2) polyprotein amino acid sequence revealed two hydrophobic do-mains that likely serve as signal peptides. The N-terminal hydrophobic domainfrom residues 9 to 31 may serve as a signal sequence for GN and cleavage is pre-dicted to occur at amino acid 35 (TDA-KV). The hydrophobic domain from aminoacid 438–455 may be a signal peptide for GC and the predicted cleavage occursat residue 464 (SMA-QT). Similar, but not identical, hydrophobic regions andcleavage sites were reported for another isolate of TSWV (61). If the hydrophobicregion from residue 438 to 455 serves as a signal peptide, then cleavage by a signalpeptidase could be the proteolytic process that separates the two glycoproteins, asobserved for HTN (78). Research on the expression and processing of the TSWVGPs indicates that the polyprotein is cleaved when expressed in heterologous bac-ulovirus and Semliki forest virus expression systems (2, 61, 153), indicating thatother TSWV proteins do not cleave the glycoprotein precursor. Protein sequencingwould define the exact ends of the glycoproteins and would confirm if the glyco-protein precursor is cleaved by a signal peptidase. Both GN and GC are anchoredin the viral membrane by hydrophobic regions. The hydrophobic regions spanningamino acid residues 309 to 339 and 346 to 369 are likely membrane anchors forGN. This is a particularly long hydrophobic region, and a similar region is seen inother members of the Bunyaviridae (78). Residues 1062 to 1085 are predicted tofunction as the GC membrane anchor. Based on sequence analysis, we can predictthat the glycoproteins have cytoplasmic tails of approximately 69 and 50 aminoacids for GN and GC, respectively. It is expected that these cytoplasmic tails in-teract with ribonucleoprotein complexes and play a vital role in virion packagingand perhaps entry. There are nine possible N-glycosylation sites on the GP ORF;however, it is expected that not all are used due to proximity to transmembrane re-gions and protein orientation. Biochemical analysis of the glycoproteins confirmsthat they are glycosylated, but site usage and glycan role in pathogenesis has yet tobe described (2, 99, 153, 154). Processing and glycosylation of the glycoproteinsin infected thrips has not been examined.

In addition to playing a role in virus binding and entry, the GPs likely playa role in virion assembly. Tospoviruses, like other bunyaviruses, bud from theGolgi (60, 64, 74). In infected plants, assembled virions appear to be retrogradetransported to the ER (60). Based on evidence from mammalian expression ofthe GPs, GN and GC efficiently travel from the ER to the Golgi as heterodimerswhen expressed together (61). However, GC expressed alone is retained in theER, and GN expressed alone localizes to the Golgi. When GC is coexpressed withGN, it is transported to and retained in the Golgi (61). These data suggest that GN

contains the Golgi targeting signal and GC localizes to the Golgi via an interactionwith GN. The processing pathway observed for TSWV GPs is consistent with thepathway described for other viruses in the Bunyaviridae (18, 61, 105). Althoughthe TSWV GN Golgi localization signal has yet to be mapped, by analogy withother GN proteins, it resides in the transmembrane domain and/or cytoplasmic tail(42).

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Virus Entry-TSWV Glycoproteins

One of the most exciting areas of thrips-tospovirus research today is the investi-gation of TSWV entry into thrips midgut cells. The best-supported hypothesis ofvirus acquisition involves the presence of a thrips midgut receptor that binds theGPs. In line with this hypothesis, the subsequent fusion of the virion membraneand a cellular membrane would allow the contents of the virion to enter the thripscell cytoplasm where it replicates. Previous findings support the hypothesis thatthe TSWV GPs are determinants of thrips acquisition and probably serve as viralattachment and fusion proteins. The two TSWV GPs decorate the surface of thevirion (Figure 1) and therefore are probably the first viral components that interactwith molecules in the thrips midgut. Isolates of TSWV that are serially, mechan-ically passed to plants generate mutations and deletions in the GP ORF and theresulting viruses are not compromised in their ability to infect plants; however,they are no longer thrips transmissible (96, 112). This loss of thrips transmissibilityindicates that the GPs play important roles in virus infection of thrips and that theGPs are necessary for thrips acquisition.

For other members of the Bunyaviridae, the GPs are important in virus entry(49, 80, 104, 131). Antibodies to GC (66, 104) and/or GN (76, 122) neutralize virusinfection. Another study revealed that when CE-infected cells were treated withGC monoclonal antibody (MAb), virus fusion was inhibited without compromis-ing virus attachment (49). Reassortment studies with hantaviruses and orthobun-yaviruses have shown that virulence maps to the M RNA segment and that theL segment is partly related to virulence (34, 48, 54). Mutations in both the GN andGC protein have been identified that attenuate disease. Researchers studying HTN,the prototype member of the genus Hantavirus, mapped a virulence determinantto the GN protein transmembrane domain (34). Mutations at the carboxyl terminusof HTN GN affect virulence, and antibodies that neutralize infection recognize thissame region (34, 76). Likewise, a mutation in the transmembrane region of HTNGC reduced virulence in mice (52). A variant isolate of LAC was identified that wasrestricted in its ability to infect the mosquito vector, and the change occurred in theGC glycoprotein (131). These data indicate that both glycoproteins are importantin virulence of bunyaviruses. Studies with purified LAC GN and GC indicate thatpretreatment of a mosquito cell line with GN and GC inhibited LAC infection. Incontrast, pretreatment with GC alone inhibited infection of vertebrate cell lines,suggesting that the GPs have unique roles in different hosts (80). Based on thesedata, GPs are involved in virus attachment and entry into host cells whether it is avertebrate or invertebrate host. By analogy to other members of the Bunyaviridae,the GP/thrips receptor hypothesis seems reasonable and is consistent with the roleof GPs in vector acquisition of bunyaviruses by other arthropod vectors.

Direct evidence from immunolabeling experiments with the anti-idiotypic an-tibodies (antibodies that mimic the epitope recognized by GN and GC monoclonalantibodies) to TSWV GN and GC suggests that both proteins play a role in virusacquisition by thrips. When GN and GC anti-idiotypic antibodies were incubated

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with dissected thrips, they specifically labeled the midgut, the expected locationof a cellular receptor (9). The GC anti-idiotype labeled the internal epithelial plas-malemma and GN anti-idiotype labeled the basal membrane of the midgut andweakly labeled the internal membranes (9). When examined by TEM, GC anti-idiotype labeled the apical plasmalemma of the microvilli lining the midgut cellsand receptosome-like structures in the cytoplasm of those cells (D.E. Ullman & S.Kumm, unpublished data). Additionally, anti-idiotypic antibodies to GN and GC

also recognized a 50-kDa thrips protein, receptor candidate, in the gel overlay assay(9, 86). These data provide support for the GP-receptor hypothesis; however, anti-idiotypes mimic one epitope and may not reflect binding by a glycoprotein and/orhetero- or homo-oligomeric protein complexes that may exist on the virion surface.

GN INVOLVEMENT IN VIRUS BINDING Several pieces of direct and indirect evi-dence suggest that the GN protein is involved in virus binding and/or entry. GN

contains an arginine-glycine-aspartic acid (RGD) motif near the N terminus, whichis characteristic of cell adhesion molecules (67), and could serve as a receptor-binding region. The RGD motif on GN is intriguing because this motif is knownto interact with β-integrins on cell surfaces (106, 133). Several viruses have beenshown to bind β-integrin receptors via RGD motifs in the context of their viralattachment proteins (5, 35, 116). Moreover, hantaviruses use integrins as receptors(38, 39). Research with LAC provides insight into possible TSWV GN partici-pation in virus entry. Enzymatic removal of GC, but not GN, from LAC virionsresulted in an increased ability to bind mosquito midguts (79, 80). However, thetreated virions exhibited reduced binding to cultured mosquito and mammaliancells (79, 80). This finding highlights the importance of GN in virus binding tovectors and suggests that LAC GN may mediate attachment to insect midguts. Insupport of the GN/vector interaction, sequence analysis of isolates of LAC withdifferent passage histories revealed that the GN coding sequence is more stablethan the GC coding sequence (10). These results provide evidence that GN playsan important role in TSWV binding and/or entry into insect guts.

To directly determine the role(s) of GN in binding to thrips guts, we expressedand purified a soluble, recombinant form of GN (GN-S). Because GN is an inte-gral membrane protein, we expressed the ectodomain of GN from a recombinantbaculovirus in insect cells (SF21), thus creating a protein that was soluble in the ab-sence of detergents (117). Soluble, recombinant proteins are essential in functionalstudies with living organisms and cells in which membrane integrity is imperativefor determination of glycoprotein function. By expressing GN individually, weexamined its role in virus binding and entry in the absence of other viral proteins(154). Thrips were fed purified protein and then cleared by feeding on a sucrosesolution so only proteins that were retained in the midgut were detected. We fo-cused our study on the thrips midgut because it is the site of virus entry (139). Asstated earlier, the thrips midgut consists of a single layer of epithelial cells thatis surrounded by longitudinal and circular muscle cells (90). Midgut muscle andepithelial cells have distinct labeling patterns when stained with Texas Red phal-

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loidin. We found that GN-S could bind the midgut epithelial cells of larval thripswithout assistance from other TSWV proteins (Figure 8). The specificity of theGN-S/thrips interaction was supported by the failure of another structural TSWVprotein, N, to bind thrips midgut epithelial cells. Furthermore, we demonstratedthat the viral attachment protein, glycoprotein B, from another enveloped virus,Human cytomegalovirus (HCMV), did not bind to thrips guts (154).

Because GN-S bound larval thrips guts in a specific manner, we assayed itfor the ability to inhibit TSWV acquisition (154). Larval thrips were fed equalamounts of purified virus, virus and GN-S, or virus and HCMV gB. Acquisitionwas measured by detection of N protein in dissected midguts. We found that GN-Sinhibited TSWV acquisition by larval thrips (Figure 9), whereas HCMV gB didnot, again indicating a specific interaction between GN and thrips. Our findingsthat GN-S binds larval thrips guts and inhibits TSWV acquisition provide evidencethat GN plays a role in virus binding and/or entry into the vector midgut, but donot preclude a role for GC.

GC: A POSSIBLE FUSION PROTEIN? Accumulating evidence indicates that theTSWV GC protein may serve as a fusion protein mediating entry into the in-sect vector cells (155). First, sequence comparison of orthobunyavirus GC, whichwas shown to be involved in fusion (46, 49, 103), and TSWV GC suggests that theprotein may serve a similar role for both genera in virus entry (19, 67). Tospovirusand orthobunyavirus GC proteins share a highly conserved domain in the core ofthe GC coding region (19, 67). In TSWV-MT2, this conserved region encompassesamino acid 674 to 727. This is notable because fusion peptides can be highly con-served within virus families (152). Second, GC contains hydrophobic domains thatcould serve as a fusion peptide or loop following the pH-dependent conformationalchange (refer to Figure 7). The orthobunyavirus-tospovirus conserved region over-laps with the area of low hydrophobicity from amino acid 465 to 783 and is close tothe smaller more hydrophobic region from residue 752 to 772. Third, we demon-strated that GC is cleaved at low pH, suggesting it undergoes a conformationalchange consistent with it playing a role in pH-dependent endocytosis (155). ApH-dependent cleavage of GC was observed by Western blot analysis. At neutraland alkaline pH, the GC protein molecular mass was approximately 85 kDa, but atacidic pH we observed an 85-kDa protein and a 72-kDa protein that reacted withthe GC MAb. In contrast to GC, we observed no change in the mobility of the GN

protein at acid, neutral, or high pH. These data are consistent with results obtainedwith LAC and CE (46, 49) and provide evidence that the GN protein does notundergo a pH-dependent conformational change, or if it does, it remains proteaseresistant (155).

The Search for a TSWV Receptor in Thrips

Thus far, researchers studying tospoviruses have been unable to identify a virusreceptor in thrips. This is not surprising because no virus receptor has been identi-fied for an arthropod vector of any animal- or plant-infecting virus. Experimental

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approaches used to identify a thrips receptor include gel overlay assays of ho-mogenized thrips and a thrips cDNA expression library (9, 86, 87). The work ofBandla et al. (9) supports the hypothesis that GN and/or GC interact with a receptormolecule in thrips. A 50-kDa protein in thrips (F. occidentalis) was identified asa candidate TSWV receptor by gel overlay analysis (9). A consistent differencein band intensity was observed between larval and adult thrips, a result that iscompatible with known TSWV-thrips biology (i.e., efficiency of virus acquisitionby larvae is reduced as the vector ages). Anti-idiotypic antibodies to GN and GC,as well as purified, recombinant forms of GN and GC recognized the 50-kDa thripsprotein in the gel overlay assay (9, 153). The putative 50-kDa thrips receptor wasimmunoprecipitated with anti-idiotypic antibodies and anti–GN/GC TSWV conju-gate (86). Kikkert et al. (59) identified a 94-kDa protein in thrips that binds virusin the gel overlay assay, but this protein was not present in the midgut of larvalthrips and may be involved in virus specificity in other insect tissues. The 50-kDaprotein has not been purified in a large enough quantity to sequence, largely dueto the small size of the insect. More research is needed to determine the specificinvolvement of the 50- and 94-kDa proteins in virus infection.

A larval F. occidentalis cDNA library was constructed, expressed via phagedisplay, and screened for TSWV-binding proteins using a modified gel-overlayassay (87). Several interesting proteins were identified using this assay, but afterexpression and further analysis, the most promising candidate did not bind virus(S.F. Hanson & T.L. German, unpublished data). The search for a receptor iscomplicated by the likelyhood that the receptor exists on the surface of the thripsgut. Cell surface proteins are often quite complex; for example, the receptor mayrequire chaperones to attain the proper three-dimensional structure necessary forreceptor-ligand interactions to occur, or it may be glycosylated and these glycansmay play a role in receptor-ligand interactions or stability of the receptor in themilieu of the insect midgut.

Because attempts to identify a tospovirus receptor have been unfruitful, welook to other members of the Bunyaviridae for enlightenment on the topic of virusreceptors. The only genus within the family Bunyaviridae for which a receptor hasbeen identified is the genus Hantavirus. Gavrilovskaya et al. (39) demonstratedthat antibodies to β3 integrins and β1 integrins respectively inhibit pathogenicand nonpathogenic hantavirus infection of cells. Interestingly, the hantavirus GPsdo not contain an RGD motif, and RGD peptides have no effect on hantavirusinfection. They concluded that pathogenic and nonpathogenic hantaviruses usedifferent cellular receptors for virus entry, and they interact with integrins in anRGD-independent manner (38, 39). Although integrins have been identified ashantavirus receptors, their role, whether it be binding and/or entry, has not beendefined, and the role of GN and GC in viral attachment and/or fusion has not beendefined for the Hantavirus genus.

Based on the two genera that have been studied in any detail (Orthobunyavirusand Hantavirus), it appears that closely related species can use the same recep-tor but members belonging to the same genus that are not closely related use a

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different receptor (39, 104). When LAC GC was used in blocking experiments,the soluble protein was capable of blocking infection of closely related viruses(California serogroup), but GC was unable to block infection by viruses fromdifferent serogroups within the same genus (104). These results indicate that Cali-fornia serogroup bunyaviruses may share a common receptor, but other orthobun-yaviruses use different receptors (104). Whether or not this finding applies to allmembers of the Bunyaviridae is unresolved and may make finding receptors and/orcontrol strategies difficult. In addition to the β-integrins that have been implicatedin hantavirus entry, a 30-kDa protein was identified in Vero-E6 cells that bindsHantaan virus (62). Polyclonal antibodies generated to this protein reduce virusinfection by 70% (62). The molecular mass of this protein is much smaller thanthat of β integrins, approximately 100-kDa, suggesting that these viruses may bindmore than one receptor and/or that these proteins are involved in different steps ofthe virus entry process.

Interestingly, integrins, the only identified receptor for a member of the familyBunyaviridae, are a family of cell-adhesion receptors found in many eukaryoticorganisms. Integrins have not been identified in thrips, but are present in otherwell-studied insects such as Drosophila melanogaster and Anopheles gambiae.A novel βV integrin was identified in D. melanogaster and found to be midguttissue specific (159). Since then, other integrins have been identified and have beenfound to play an important role in control of epithelial cell morphogenesis (102),and these receptors may play a role in arthropod cellular immune response (71). Bycomparison to D. melanogaster, integrins may play a role in thrips development. Ifa developmentally regulated integrin is implicated in thrips acquisition of TSWV,it may explain the decrease in vectorial capacity as thrips mature.

CONCLUDING REMARKS

We are optimistic that in the next decade significant progress will be made inunderstanding the complex biological processes involved in the transmission ofexisting and emerging tospoviruses and their thrips vectors. The negative impactof diseases caused by tospoviruses on a global scale will sustain internationallycollaborative research programs, and the utilization of new technologies will openthe doors to unique avenues of investigation. The field of genomics and proteomicswill provide a mechanism to identify genetic determinants of vector competence.Comparative analysis of virus isolates and thrips species will lead to the discoveryof virulence factors and identify important barriers to transmission. Recombi-nant forms of viral proteins will continue to be useful for delving deeper intomechanisms of tospovirus-thrips interactions and may become tools in the de-velopment of procedures to control both viral disease and thrips as pests. Thetospovirus-thrips relationship provides a rare opportunity to directly compare andcontrast pathogenicity in plant and animal hosts. Recent developments in our un-derstanding of RNA silencing as a defense mechanism against viruses (and the

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corresponding counterdefense deployed by viruses) in their plant hosts will be ex-tended to replication of viruses in their vectors. Clarifying the roles of viral genessuch as NSm, known to be involved in cell-to-cell movement in plants but with noapparent corresponding function in the insect phase of replication, will increaseour understanding of animal virus replication mechanisms.

There are over 300, mainly arthropod-transmitted, viruses in the family Bun-yaviridae alone. In addition to the devastating agricultural problems caused bythrips and Tospoviruses, animal viruses in this family also cause acute debilitat-ing diseases (Oropouche virus), encephalitis (La Crosse virus), acute respiratorydisease (Sin Nombre virus) or hemorrhagic fevers (Rift Valley fever virus). It isreasonable to predict that research on the virus-vector relationship of Bunyaviruseswill lead to both an improved understanding of basic biological concepts and thedevelopment of effective measures to lessen their impact on a wide range of humanactivities.

ACKNOWLEDGMENTS

We thank Eileen Rendahl for graphic design assistance with figures and DorithRotenberg for critical reading of the chapter. This work was supported by UnitedStates Department of Agriculture grant 99-35303-8271 (T.L.G. and D.E.U.) andby Hatch funds (WISO4316) (T.L.G.).

The Annual Review of Phytopathology is online athttp://phyto.annualreviews.org

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TOSPOVIRUS-THRIPS INTERACTIONS C-1

Figure 1 Diagram of TSWV virion. A double-layered membrane of host origin(blue) is shown with the viral-encoded proteins GN and GC (green) projecting fromthe surface in monomeric and dimeric configurations. The genomic RNA is present-ed as noncovalently closed circles in the form of a ribonucleoprotein (RNP) complexcreated by its association with many copies of N protein (peach). A few copies of thevirion-associated RNA-dependent RNA polymerase (RdRp or L) are shown (purple)in association with the RNPs.

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C-2 WHITFIELD ■ ULLMAN ■ GERMAN

Figure 4 Conditions driving rapid coevolution between thrips vectors and tospo-viruses include the genetic diversity that occurs in thrips populations, the potentialfor them to carry and encounter mixtures of viral genotypes in plants, and the inher-ent genetic diversity that arises in RNA virus populations. The reassortment of theviral genome allows for virus populations with new characteristics to arise, includ-ing new vector relationships and the ability to overcome host resistance. Graphicdesign by Eileen Rendahl. Peanut photo courtesy of John Sherwood.


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Figure 5 Graphic representation of the thrips life cycle and the tospovirus trans-mission cycle. Thrips eggs are oviposited into plant tissue and within a few days thefirst instar larvae emerge. Virus acquisition occurs solely during the larval stagesafter which the virus is passed transstadially to the adult. The pupal stages are non-feeding and do not move, although they do maintain virus infection. In nature,Frankliniella occidentalis pupates in the soil. Many other vector species, e.g., Thripstabaci, pupate in the foliage. Adults emerge and have a tendency to disperse widely.Only adult thrips (male and female) that acquired the virus during their larval stagescan transmit tospoviruses. Graphic design by Eileen Rendahl. Thrips and kalanchoephotographs produced by Jack Kelley Clark.


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C-4 WHITFIELD ■ ULLMAN ■ GERMAN

Figure 8 Soluble GN (GN-S) binds larval F. occidentalis guts in an in vivo bindingassay. Larval thrips were fed GN-S and after feeding, thrips guts were cleared fortwo hours on a 5% sucrose solution. Thrips were then dissected, fixed in 4%paraformaldehyde, and permeabilized. The guts were immunolabeled with 6xHisMAb conjugated to Alexafluor 488 (green). Actin was stained with Texas Red phal-loidin (red ) and the visceral muscles and midgut epithelial cells are labeled. Stainingwas visualized by confocal microscopy. (A) Thrips fed BSA, (B) thrips fed GN-Sshowing that labeling is associated with midgut epithelial cell layers. The scale baris 50 �m. [Reprinted from (154, figure 7) with permission from ASM Press.]


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Figure 9 In vivo thrips feeding experiment showing that soluble GN (GN-S) inhibitsacquisition of TSWV. Thrips were given two-hour acquisition access periods (AAP)on (A) TSWV alone or (B) TSWV and GN-S. All treatments contained the same con-centration of virus. Thrips were then allowed to feed on sucrose solution for twohours to clear their guts after the AAP. Acquisition was measured by immunolabel-ing with TSWV nucleocapsid polyclonal antibody and secondary antibody conjugat-ed to alexafluor 647 (blue). Actin was stained with Texas Red phalloidin (red ) .Thrips guts were imaged with a confocal microscope. Insects fed the combination ofGN-S and TSWV had reduced amounts of virus in their guts when compared toinsects fed TSWV alone. The scale bar is 50 �m.


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P1: KUV

July 14, 2005 11:17 Annual Reviews AR250-FM

Annual Review of PhytopathologyVolume 43, 2005

CONTENTS

FRONTISPIECE, Robert K. Webster xii

BEING AT THE RIGHT PLACE, AT THE RIGHT TIME, FOR THE RIGHT

REASONS—PLANT PATHOLOGY, Robert K. Webster 1

FRONTISPIECE, Kenneth Frank Baker

KENNETH FRANK BAKER—PIONEER LEADER IN PLANT PATHOLOGY,R. James Cook 25

REPLICATION OF ALFAMO- AND ILARVIRUSES: ROLE OF THE COAT PROTEIN,John F. Bol 39

RESISTANCE OF COTTON TOWARDS XANTHOMONAS CAMPESTRIS pv.MALVACEARUM, E. Delannoy, B.R. Lyon, P. Marmey, A. Jalloul, J.F. Daniel,J.L. Montillet, M. Essenberg, and M. Nicole 63

PLANT DISEASE: A THREAT TO GLOBAL FOOD SECURITY, Richard N. Strangeand Peter R. Scott 83

VIROIDS AND VIROID-HOST INTERACTIONS, Ricardo Flores,Carmen Hernandez, A. Emilio Martınez de Alba, Jose-Antonio Daros,and Francesco Di Serio 117

PRINCIPLES OF PLANT HEALTH MANAGEMENT FOR ORNAMENTAL PLANTS,Margery L. Daughtrey and D. Michael Benson 141

THE BIOLOGY OF PHYTOPHTHORA INFESTANS AT ITS CENTER OF ORIGIN,Niklaus J. Grunwald and Wilbert G. Flier 171

PLANT PATHOLOGY AND RNAi: A BRIEF HISTORY, John A. Lindboand William G. Doughtery 191

CONTRASTING MECHANISMS OF DEFENSE AGAINST BIOTROPHIC AND

NECROTROPHIC PATHOGENS, Jane Glazebrook 205

LIPIDS, LIPASES, AND LIPID-MODIFYING ENZYMES IN PLANT DISEASE

RESISTANCE, Jyoti Shah 229

PATHOGEN TESTING AND CERTIFICATION OF VITIS AND PRUNUS SPECIES,Adib Rowhani, Jerry K. Uyemoto, Deborah A. Golino,and Giovanni P. Martelli 261

MECHANISMS OF FUNGAL SPECIATION, Linda M. Kohn 279

vii

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P1: KUV

July 14, 2005 11:17 Annual Reviews AR250-FM

viii CONTENTS

PHYTOPHTHORA RAMORUM: INTEGRATIVE RESEARCH AND MANAGEMENT

OF AN EMERGING PATHOGEN IN CALIFORNIA AND OREGON FORESTS,David M. Rizzo, Matteo Garbelotto, and Everett M. Hansen 309

COMMERCIALIZATION AND IMPLEMENTATION OF BIOCONTROL, D.R. Fravel 337

EXPLOITING CHINKS IN THE PLANT’S ARMOR: EVOLUTION AND EMERGENCE

OF GEMINIVIRUSES, Maria R. Rojas, Charles Hagen, William J. Lucas,and Robert L. Gilbertson 361

MOLECULAR INTERACTIONS BETWEEN TOMATO AND THE LEAF MOLD

PATHOGEN CLADOSPORIUM FULVUM, Susana Rivasand Colwyn M. Thomas 395

REGULATION OF SECONDARY METABOLISM IN FILAMENTOUS FUNGI,Jae-Hyuk Yu and Nancy Keller 437

TOSPOVIRUS-THRIPS INTERACTIONS, Anna E. Whitfield, Diane E. Ullman,and Thomas L. German 459

HEMIPTERANS AS PLANT PATHOGENS, Isgouhi Kaloshianand Linda L. Walling 491

RNA SILENCING IN PRODUCTIVE VIRUS INFECTIONS, Robin MacDiarmid 523

SIGNAL CROSSTALK AND INDUCED RESISTANCE: STRADDLING THE LINE

BETWEEN COST AND BENEFIT, Richard M. Bostock 545

GENETICS OF PLANT VIRUS RESISTANCE, Byoung-Cheorl Kang, Inhwa Yeam,and Molly M. Jahn 581

BIOLOGY OF PLANT RHABDOVIRUSES, Andrew O. Jackson, Ralf G. Dietzgen,Michael M. Goodin, Jennifer N. Bragg, and Min Deng 623

INDEX

Subject Index 661

ERRATA

An online log of corrections to Annual Review of Phytopathology chaptersmay be found at http://phyto.annualreviews.org/

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ann revual tospovirusses

Documents

thrips vectors

thrips vector species

tospovirus vectors

plant populations

hundreds of plant species

virusvector relationships

plantinfecting genus

insect vector