Virology 479-480 (2015) 278–289

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Virology

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Review Insect vector-mediated transmission of plant viruses

Anna E. Whitfield a,n, Bryce W. Falk b, Dorith Rotenberg a a Department of Plant Pathology, Kansas State University, Manhattan, KS 66502, United States b Department of Plant Pathology, University of California, Davis, CA 95616, United States article info abstract

Article history: The majority of plant-infecting viruses are transmitted to their host plants by vectors. The interactions Received 24 January 2015 between viruses and vector vary in duration and specificity but some common themes in vector Returned to author for revisions transmission have emerged: 1) plant viruses encode structural proteins on the surface of the virion that 17 February 2015 are essential for transmission, and in some cases additional non-structural helper proteins that act to Accepted 6 March 2015 bridge the virion to the vector binding site; 2) viruses bind to specific sites in or on vectors and are Available online 29 March 2015 retained there until they are transmitted to their plant hosts; and 3) viral determinants of vector Keywords: transmission are promising candidates for translational research aimed at disrupting transmission or Capsid protein decreasing vector populations. In this review, we focus on well-characterized insect vector-transmitted Virus glycoprotein viruses in the following genera: Caulimovirus, Crinivirus, Luteovirus, Geminiviridae, Reovirus, Tospovirus, Aphid and Tenuivirus. New discoveries regarding these genera have increased our understanding of the basic Whitefly Thrips mechanisms of virus transmission by arthropods, which in turn have enabled the development of Planthopper innovative strategies for breaking the transmission cycle. Leafhopper & 2015 Elsevier Inc. All rights reserved. Hemipteran Virus–vector interactions

Contents

Introduction...... 278 Historical perspective of modes of virus transmission by insect vectors ...... 279 Noncirculative transmission ...... 280 Non-circulative, stylet-borne transmission ...... 280 Non-circulative, semipersistent transmission ...... 282 Introduction to the biology of persistent, circulative virus transmission ...... 282 Circulative, non-propagative transmission ...... 283 Circulative, propagative transmission ...... 284 Translational outcomes derived from basic virus–vector research ...... 286 Blocking transmission...... 286 Vector population suppression ...... 287 Summary...... 287 Acknowledgments...... 287 References...... 287

Introduction

There are more than 2000 virus species and those affecting plants include viruses of at least 21 families and 8 unassigned n Correspondence to: Department of Plant Pathology, 4024 Throckmorton Plant genera, many of which cause important diseases of various plants that Sciences Center, Kansas State University, Manhattan KS 66506, United States. fi Tel.: +1 785 532 3364; fax: +1 785 532 5692. humans grow for food and/or ber (Hull, 2014). In addition, many E-mail address: [email protected] (A.E. Whitfield). plant viruses have been found associated with non-cultivated plants, http://dx.doi.org/10.1016/j.virol.2015.03.026 0042-6822/& 2015 Elsevier Inc. All rights reserved. A.E. Whitfield et al. / Virology 479-480 (2015) 278–289 279

Table 1 Viruses and their associated vectors and transmission strategies.

Text in blue indicates plant viruses that are non-circulative in their respective insect vectors, they do not enter the body as part of the transmission process. Text in red indicates viruses that do enter the body as part of the transmission process and either circulate (indicated by superscript 1) or circulate and replicate (indicatedbysuperscript2)withinthe insect vector body. CP¼capsid protein or major capsid protein; HC-Pro¼helper component proteinase, P2¼non-virion helper component protein, P3¼protein anchored in the n CaMV virion CPm¼minor capsid protein, CP-RT¼capsid protein readthrough domain, GN¼glycoprotein N, P2 ¼outer capsid protein encoded by RDV segment 2,G ¼glycoprotein (indicated by superscript 3). and new plant viruses are being discovered every day. Virus (Ammar et al., 2009; Hogenhout et al., 2008; Ng and Falk, 2006). diseases make up 47% of the new emerging diseases affecting However, recent studies suggest additional complexities for plant plants (Anderson et al., 2004). Thus, plant infecting viruses are very virus:vector relationships (Blanc et al., 2014; Gutierrez et al., 2013). successful. As virus hosts, plants differ from animals and even Here we discuss the current state of this knowledge, but also recent bacteria in several ways, but one that is critical in terms of virus exciting translational applications of fundamental knowledge of biology is that plants are sessile. In order to survive, plant-infecting virus:insect vector interactions that has opened up new doors for viruses must have an efficient means to move from one plant host plant virus and insect vector control. to another. To do so, the great majority of plant viruses utilize specific vectors to ensure their ability to move from one plant to another, and to ensure their survival, plant viruses encode for Historical perspective of modes of virus transmission by insect specific proteins that facilitate this process (Table 1)(Kritzman vectors et al., 2002; Moreno et al., 2012; Moritz et al., 2004). Although there are different types of plant-associated organisms including fungi, The biology of plant virus transmission has been studied for more nematodes and various types of invertebrates that serve as vectors than 100 years (Ando, 1910; Gutierrez et al., 2013; Takami, 1901). Since for different plant viruses, the majority of plant viruses utilize then many studies have examined the specificity of insect vector- specific plant feeding insects as their primary vector(s), and here mediated plant virus transmission and clearly demonstrated that we focus on insect-transmitted plant viruses. Proteins encoded by there are specific molecular determinants required (e.g. see Pirone, different plant viruses have been identified to specifically interact 1964; Rochow, 1970; Storey, 1933). These studies led to discoveries of with their respective insect vectors and facilitate virus transmission, virus proteins that in part, determined vector-specificinteractions and there are many excellent papers and reviews on these subjects (Table 1). Because plant virologists realized that vector transmission of 280 A.E. Whitfield et al. / Virology 479-480 (2015) 278–289

Table 2 potyviruses and CaMV require intact virions plus additional non- Genera within the family Potyviridae have diverse vectors but within a genus, the structural virus protein(s). Govier and Kassanis (1974a, 1974b) used related viruses are transmitted by a similar vector. simple, but elegant experiments to show that more than virions were

Genus Type of vector needed for aphid transmission of Potato virus Y (PVY), and they coined the term “helper component” as the “virus-induced” factor that Potyvirus Aphid aphids must acquire simultaneously with, or prior to acquiring virions Ipomovirus Whitefly of PVY. Since these seminal discoveries the potyvirus-encoded helper Macluravirus Aphid component, or HCPro, has been very well studied (Valli et al., 2014) Rymovirus Eriophyid mite Tritimovirus Eriophyid mite and its role in potyvirus transmission by aphids is well known (Blanc Poacevirus Eriophyid mite et al., 1998, 2014; Ng and Falk, 2006; Peng et al., 1998). Thus, it is now Brambyvirus Not confirmed understood that two strategies are recognized for viruses that are Bymovirus Plasmodiophorid (root infecting unicellular parasite) transmitted by insects (not just aphids) in a noncirculative manner. These are the capsid strategy and the helper strategy (reviewed in Blanc et al., 2014; Ng and Falk, 2006). However, what is still in its plant viruses is specific, and well before we were able to analyze virus infancy is our ability to identify the insect vector receptors that are genomes so readily by contemporary sequencing and bioinformatics involved in interacting with “transmission proteins” to allow for analysis, vector transmission properties were used as critical criteria successful virus transmission. But some recent studies using power- for plant virus taxonomy. Today's sequence data-based taxonomy ful, contemporary imaging technologies have in some cases give largely supports these earlier findings. Within a given plant virus much greater understanding of at least where the hypothetical genus, all species utilize the same type of vector and show the same receptors are located, and in some cases dynamic interactions that transmission relationship; for example, all members of the genus are necessary for vector-mediated virus acquisition and transmission Potyvirus are transmitted by various aphid vectors in a non-circulative to occur (Blanc et al., 2014). (non-persistent) manner. However, virus species of other genera even within the same virus family may have other types of vectors. Other Non-circulative, stylet-borne transmission examples from the Potyviridae are the Ipomoviruses and Tritimo- viruses that are transmitted by whiteflies and eriophyid mites, CaMV is a para-retrovirus (family Caulimoviridae) and virions respectively. Table 2 shows as an example the vectors for viruses have isometric T¼7 capsids of approximately 52 nm in diameter, within the different genera of the family Potyviridae. composed primarily of the P4 (gag or capsid) protein (Fig. 1). An While some plant viruses may be transmitted by several elaborate and intricate tritrophic interaction determines aphid different vector species (e.g. aphids and the non-persistent trans- transmission of CaMV. CaMV can be acquired by aphids probing, mitted potyviruses and Cucumber mosaic virus (CMV)), other transient puncturing of the epidermal, mesophyll, parenchyma viruses are highly specific, being transmitted perhaps by a single cells of infected leaf tissues, but also by when aphids feed on species of insect vector, as shown by the circulative-propagative phloem tissues, i.e. long periods of sustained ingestion from the transmitted rhabdoviruses. Early research into the biology of insect phloem (Palacios et al., 2002). As alluded to above, CaMV uses a transmission of plant virus:vector interactions gave rise to terms helper strategy to achieve aphid transmission (Lung and Pirone, describing transmission relationships based on acquisition and 1974), but unlike for aphid transmission of potyviruses, the aphid inoculation thresholds, as well as retention of the virus by its transmission of CaMV is more complex. CaMV aphid transmission vector(s). Thus, four basic types of insect vector:plant virus requires interactions between three CaMV-encoded proteins, one transmission relationships were described: non-persistent; semi- of which, P2, also interacts with the aphid stylet. Another CaMV- persistent; persistent:circulative and persistent:propagative (see encoded protein, P3, is anchored within the virion capsid shell, and Ng and Falk, 2006). More recent terminology emphasizes how CaMV-encoded P2 is the non-virion helper component protein plant viruses interact with their insect vectors (Blanc et al., 2014). which binds by its N0-terminus to the aphid stylet, but its Some bind to specific insect vector cuticular locations without C0-terminus also binds to the N0-terminal region of P3 (Blanc entering cells (noncirculative) and others enter the insect gut and et al., 2014; Hoh et al., 2010; Plisson et al., 2005). circulate or replicate within the insect vector body (circulative; see CaMV replicates in the plant cell cytoplasm, and large (up to Fig. 1 and Table 1). 4.5 μM in diameter), electron dense distinct viroplasms composed mostly of the CaMV-encoded P6 protein and progeny virions are visible by light and electron microscopy as inclusion bodies in the Noncirculative transmission cytoplasm (Drucker et al., 2002; Shalla et al., 1980). But CaMV- infected plant cells also contain another type of cytoplasmic inclu- Noncirculative plant viruses are retained in the stylet or foregut. sion body, the electron-lucent IB (elIB; Espinoza et al., 1991). The Early studies with potyviruses and Cauliflower mosaic virus (CaMV) primary component of these elIBs is the CaMV-encoded P2 protein first demonstrated that aphid transmission of these viruses results (Drucker et al., 2002; Espinoza et al., 1991; Khelifa et al., 2007). not from mere contamination of virions on aphid stylets, but from Because the elIBs contain essentially all of the P2 within the cell and specificinteractions.Pirone (1964) showed that by using high because P2 is essential for binding CaMV to virions and aphid stylets, concentrations of two noncirculative aphid-transmitted viruses, the elIBs are referred to recently as transmission bodies (TBs; Bak Cucumber mosaic virus (CMV) and Alfalfa mosaic virus (AMV), that et al., 2013; Martiniere et al., 2013). Recent, work by this group aphids could acquire these viruses from solutions held between two demonstrated that CaMV acquisition by aphids results from dynamic parafilm membranes and subsequently transmit these viruses to plant cell and virus responses to aphid activity. plants. However, similarly high concentrations of infectious Tobacco Biological data clearly demonstrated that the three CaMV- mosaic virus (TMV) and the potyvirus, Turnip mosaic virus (TuMV) encoded proteins, P2, P3 and P4, are required for CaMV acquisition could not. CMV, AMV and TuMV were known to be aphid-transmitted by aphids, but detailed microscopic examination of CaMV-infected in nature, so what explained the non-transmissibility of TuMV in plant cells shows that these proteins are not co-localized (Bak these types of feeding experiments? We now know that viruses et al., 2013; Drucker et al., 2002; Espinoza et al., 1991; Khelifa et al., such as CMV contain virus-encoded aphid transmission determinants 2007). However, when aphids explore cells via probing activity as part of the virion capsid protein (Perry et al., 1998, 1994), but they produce minute wounds that quickly heal. Upon probing, A.E. Whitfield et al. / Virology 479-480 (2015) 278–289 281

Fig. 1. Virus localization sites in insect vectors. Non-circulative viruses are retained in the insect stylet (A) or foregut (B). Non-propagative circulative (yellow circles) viruses are generally phloem limited and penetrate the insect body via the midgut or hindgut. Circulative viruses use a hemolymph route to reach the salivary glands. In contrast, circulative propagative viruses (red ovals) enter the insect at the anterior region of the midgut and/or filter chamber region. Propagative viruses may use a hemolymph route and others such as the Rhabdoviruses also use a neurotropic route to reach the salivary glands. Propagative viruses replicate in the midgut cells and other insect tissues. Some propagative viruses are phloem limited while others are widely distributed in plant tissues. The salivary glands are the final destination for circulative transmission, and viruses reach the salivary glands via the hemolymph or other routes such as the nervous tissue (neurotropic route) or through connective tissues. Reoviruses use tubules to move cell to cell in the midgut and another uses the tubular structure to traverse the basal lamina (C). Insets: Magnification of an insect stylet showing the proposed site of virion attachment at the tip of the stylet in the common duct region (A). Numbers designate the different strategies for virion binding and retention in the stylet: capsid strategy, direct binding of capsid protein to the stylet (1), helper component strategies for caulimoviruses, two virus proteins serve as a “bridge” between the virion and the stylet (2) and potyviruses, one virus protein (HC-Pro) binds to the aphid stylet and to the virus (3). Inset B: Magnification of the foregut retention site and proposed capsid binding strategy used by Criniviruses. The minor capsid protein (CPm) is the viral attachment protein. Inset C: The steps in the reovirus infection cycle and spread to adjacent cells modeled on Rice dwarf virus. Rice dwarf virus enters cells using the endocytic pathway and after virion release from the vesicle the replication cycle begins. Progeny virions move cell-to-cell via tubule structures composed of virus nonstructural protein. This enables virions to move directly from one cell to another without an extracellular phase. Modified from Ng and Falk (2006), Ammar et al. (2009), Blanc et al. (2014), Miyazaki et al. (2013) and Whitfield and Rotenberg (2015).

CaMV-induced TBs “sense” cell wounding and almost immediately naming this activity as “virus perceptive behavior” (Martiniere respond by dissociating and redistributing P2 onto cellular micro- et al., 2013), and additional work shows that CaMV virions from tubules (Bak et al., 2013; Martiniere et al., 2013). The TB dissocia- viroplasms also relocalize, probably to be able to interact with P2 tion and P2 relocalization occurs in cells only very near to aphid and be transmitted by aphid vectors (Bak et al., 2013). probing, and can be induced by some chemical treatments or cell Uzestetal.,(2010,2007)used creative and high resolution wounding (Martiniere et al., 2013). Furthermore, the relocalization approaches to identify the aphid stylet binding sites for the CaMV- of P2 is temporary and reversible for when chemical inducers are encoded P2. They created P2:GFP and P2 mutant:GFP fusion proteins removed, or after aphids were removed from leaves, P2 redistrib- by recombinant baculovirus expression in Sf9cells(Uzest et al., 2007). uted back into TBs (Martiniere et al., 2013). The authors interpreted They then performed aphid stylet in vitro binding assays with these their data suggesting that TBs “sense” aphid activity and reorganize proteins and used epifluorescence microscopy to carefully examine as an active response to increase the probability of CaMV transmis- aphid stylets for GFP fluorescence. Several important findings sion by aphid vectors (Martiniere et al., 2013). They proposed emerged from these experiments. First, GFP fluorescence was seen 282 A.E. Whitfield et al. / Virology 479-480 (2015) 278–289 only when stylets were tested with P2:GFP fusion proteins, not when antibodies to the CP cover most of the LIYV virion, CP-treated LIYV they were tested with GFP only. Second, the P2:GFP bound to vector virions were still very efficiently transmitted by B. tabaci. These aphid stylets but not the stylet of non-vector aphids. Third, a mutant results strongly supported the hypothesis that the CPm is a LIYV- P2:GFP with a Q to Y substitution at amino acid 6 in P2 (P2Rev5:GFP) encoded protein involved in the transmission of LIYV by B. tabaci. did not bind to vector aphid stylets. This mutation had previously Subsequent mutations in the CPm which did not affect the ability been shown to disable the biological activity of P2, rendering it unable of LIYV to form virions or to systemically infect N. benthamiana to support CaMV aphid transmission (Moreno et al., 2005). Most plants, did abolish the ability of the mutant LIYVs to be transmitted important, the fluorescence for P2:GFP was not randomly distributed, by B. tabaci (Stewart et al., 2010). or scattered along the stylets, but was localized to a unique and tiny Chen et al. (2011a) combined biology and molecular biology, region at the aphid stylet tip (Uzest et al., 2007). Furthermore, the coupled with contemporary imaging technologies in attempts to ability of stylets to bind the P2:GFP was abolished by proteinase K, but identify LIYV virion binding sites in vector whiteflies. They fed not by trypsin, pronase E, n-hexane, chloroform:methanol or sodium vector and non-vector whiteflies (B. tabaci A and B biotypes, metaperiodate (Uzest et al., 2007). The interpretation from these data respectively) sequentially on artificial diets containing virions or was that there is a specific stylet region containing specificnon- LIYV virion capsid proteins produced by expression in E. coli, glycosylated proteinaceous receptors. These receptors are most likely followed by solutions containing different antibodies (including embedded in chitin within the salivary canal extremity stylet. More fluorescent-labeled antibodies) to bind to LIYV virions or capsid recent ultrastructural examination showed that this region is con- proteins in whiteflies. Then whiteflies were examined by both wide served among aphids (Uzest et al., 2010). These authors referred to field fluorescence microscopy and confocal laser scanning micro- this unique anatomical feature to be called the “acrostyle” (Uzest et al., scopy to visualize where the fluorescent antibodies could be found. 2010), and suggested that due to its uniqueness, this is an ideal place They showed that fluorescence was localized only in a precise for receptors for “CaMV and perhaps those of other noncirculative region of the foregut, the anterior foregut region (cibarium) of the viruses” (Uzest et al., 2007). A biotype B. tabaci vector whitefly. Essentially no binding, as assessed by fluorescence, was seen when non-vector B biotype B. Non-circulative, semipersistent transmission tabaci fed on the same preparations. This strongly suggested that LIYV virions bound to this specificlocationinvectorwhiteflies (Fig. 1B). While non-circulative, nonpersistent transmission of plant Of the LIYV capsid proteins evaluated (CP, CPm, HSP70h, P59), only viruses is only so far found among viruses transmitted by aphid CPm was found to bind within the anterior foregut and again, only in vectors, several aphid, whitefly and leafhopper-transmitted viruses vector Biotype A B. tabaci. They also used a recombinant LIYV CPm show a non-circulative, semipersistent transmission relationship mutant, which was not transmissible by A biotype B. tabaci,and (Ng and Falk, 2006). Viruses showing this type of transmission showed that it also did not bind, but when the mutation was restored relationship are retained by viruliferous vectors for longer time to give wildtype CPm, they observed LIYV transmission and specific periods than are viruses transmitted in a nonpersistent manner binding in the anterior foregut. (Ng and Falk, 2006), and they are lost by viruliferous vectors during Like the work with CaMV, the work by Chen et al., (2011a) molting. The latter property supports the hypothesis that these demonstrated the specificity of binding only in vector species, and viruses are not internalized within insect vector guts, but are likely only at a precise location, for LIYV this is within the stylet/foreguts. retained in chitin-lined areas that are lost during vector insect Furthermore, unlike CaMV which is inoculated to plants by aphid molting. Attempts to localize several viruses transmitted in a non- probing, LIYV is transmitted to, and acquired from plants by circulative semipersistent manner suggested that they were not B. tabaci feeding, i.e. sustained periods of ingestion from plant borne at the tips of the vector stylets, but definitive localization vascular tissues. Because CaMV virions are located in the vector and correlation with the vectors' ability to transmit viruses with aphid acrostyle, inoculation to plants likely occurs when aphids these properties has been mostly lacking (Blanc et al., 2014). Recent salivate during probing, the saliva flows through the acrostyle findings with the Bemisia tabaci-transmitted Lettuce infectious region. However because LIYV is in the foregut, the work of Chen yellows virus (LIYV, genus Crinivirus, family Closteroviridae)have et al. (2011a) suggests that release of LIYV from the anterior foregut identified not only the LIYV-encoded protein determining its during inoculation to plants most likely can occur during egestion transmission by B. tabaci, but also where in the whitefly the LIYV or regurgitation by the viruliferous whitefly, and not merely by virions are retained (Chen et al., 2011a; Stewart et al., 2010; Tian salivation. The authors noted that due to virion binding in the et al., 1999), thereby giving some suggestion as to the type of vector anterior foregut, which is physically separated from the maxillary behavior involved in transmitting LIYV back to plants. stylet and salivary duct, LIYV virions thus cannot be released Tian et al. (1999) showed that the filamentous LIYV virions were during salivation (2011a), but that salivation could serve to release structurally complex, composed of at least four LIYV-encoded viruses like CaMV which are in the acrostyle at the stylet tip (Uzest proteins, and that the purified virions could be acquired in vitro, et al., 2010), a location where the salivary and food canals are and subsequently transmitted to plants by B. tabaci. This work confluent. demonstrated for the first time that LIYV has a capsid and not helper strategy for its whitefly vector transmission. The two major virion proteins are the CPm and CP (minor capsid protein and Introduction to the biology of persistent, circulative virus major capsid protein, respectively). The CPm covered only about transmission 10% of the virion from one end, thus showing that virions were morphologically polar (Tian et al., 1999, see Fig. 2). As purified Circulative viruses, by definition, enter the insect body and virions were transmissible by vector whiteflies, they performed disseminate to various tissue systems prior to their transmission to experiments in attempts to identify which of them are likely to be plant hosts. Circulative viruses include both those that disseminate vector transmission determinants. They first incubated purified but do not replicate in the body of the insect (non-propagative) and LIYV virions with antibodies specific to each of the four virion those that replicate (propagative) in different tissues. The precise proteins separately, and then after centrifugation fed the super- route of dissemination from point of entry (Gutierrez et al., 2013)to natant to vector whiteflies and tested their ability to transmit LIYV the salivary glands has been well-described, with some variation, for to plants. Only antibodies to the LIYV CPm neutralized the ability of different types of circulative viruses (Fig. 1). Virus dissemination is a B. tabaci to acquire and transmit LIYV to plants. Even though major defining feature of vector competency, and as such, has been a A.E. Whitfield et al. / Virology 479-480 (2015) 278–289 283

Fig. 2. Virion structures and viral attachment proteins. TEM and immunogold labeling analysis of partially purified LIYV virions (A and B). The virion in (A) was labeled using antiserum to the LIYV capsid protein (CP). Virions in (B) and were labeled using antiserum to the LIYV CP minor (CPm), the determinant of insect transmissibility. Bars represent 224 nm. Arrows in (B) indicate LIYV virion termini labeled using CPm antiserum. Arrow in (A) indicates a virion terminal region unlabeled with LIYV CP antiserum. Panels A and B were reproduced and modified from Tian et al. (1999). Structural model of the CaMV virion (C). The cryo-EM reconstruction shows the capsid protein (P4) in yellow and the P3 ectodomain (gray) decorating the surface of the virion. The helper component protein (P2) is not a structural protein but binds to the aphid stylet and P3- decorated virion to enable transmission. (D) Enlarged view of P3 (gray) in the cryo-EM difference map surrounded by three adjacent capsid hexamers (yellow). The ectodomains of 3 antiparallel dimeric coiled-coil P3 molecules are displayed as ribbons. Panels C and D reproduced and modified from Hoh et al. (2010) with permission from American Society for Microbiology. Drawing of an icosahedral luteovirid showing the coat protein (CP) in beige and the read through domain (RTD) in blue (E). The RTP is produced via translational readthrough of a leaky stop codon at the end of the CP gene. The RTD is predicted to be highly disordered and the CP:RTP stoichiometry in luteovirids is not known (Chavez and Cilia et al. 2012). The CP and N-terminus of the RTD are essential for aphid transmission (modified from DeBlasio et al., in press).

Diagram of TSWV virion (F). A double-layered membrane of host origin (blue) is shown with the viral-encoded proteins GN and GC (green) projecting from the surface in monomeric and dimeric configurations. The genomic RNA is presented as ribonucleoprotein (RNP) complex created by its association with many copies of N protein (peach). A few copies of the virion-associated RNA-dependent RNA polymerase (RdRp or L) are shown (purple) in association with the RNPs. Panel F is reproduced from Whitfield et al. (2005a). primary focus of vector–virus research. Examination of various associated interactions between virus and vector are well- char- interactions between vectors and circulative viruses has revealed acterized for luteovirus transmission by aphid vectors (Gray et al., commonalities and unique aspects pertaining to pathways to the 2014). Luteovirids are acquired when the aphid feeds on the salivary glands, viral determinants of transmission, and in some phloem tissues of infected plants. These viruses are relatively systems, vector components that respond to or interact directly with small, simple icosahedral virions that enter the insect body viral proteins. through the alimentary canal. The majority of species of luteovirids cross the hindgut, however, in a few species, the point of entry is the midgut (Garret et al., 1996; Gildow, 1993; Reinbold et al., 2003). Circulative, non-propagative transmission The virions interact with molecules on the surface of the gut epithelial cells, enter in a receptor-mediated endocytic fashion and The luteoviruses are transmitted by aphid vectors in a non- traverse the epithelial cell layer without uncoating (Gildow, 1993). propagative, circulative manner. The dissemination pathway and Subsequently, virions are delivered to the space between the basal 284 A.E. Whitfield et al. / Virology 479-480 (2015) 278–289 plasmalemma and basal lamina via the exocytic pathway. This role in virus transmission (Gray et al. 2014). A recent study of endo- and exocytic virion transport process is termed transcytosis. Wheat dwarf virus (WDV, genus Mastrevirus) movement in the The mechanism for virus movement through the basal lamina is leafhopper vector Psammotettix alienus, reported that the entire not well understood. Virions reach the hemocoel, where bacterial process from virus acquisition to transmission occurred in 5 min, endosymbionts are hypothesized to enhance vector transmission following the same dissemination route as other geminiviruses but efficiency (Gray et al., 2014). Once the virions enter the hemocoel deviating remarkably from the 8-h latent period required for TYLCV they must circulate to and penetrate the accessory salivary glands in whiteflies and the 6–12-h latent period for MSV in the leafhopper (ASG) to be inoculated. For the majority of luteovirids character- Cicadulina mbila (Ghanim et al., 2001; Storey, 1928; Wang et al., 2014). ized, the gut is not a major barrier to virus entry and it is common For members of the Geminiviridae, several lines of evidence for viruses to enter the hemocoel of non-vector aphids (Gray and implicate the viral CP as the sole determinant of insect transmissi- Gildow, 2003). The basal lamina and basal plasmalemma of the bility. The CP comprises the twinned icosahedral particles that ASG are both significant barriers to transmission for various identify geminiviruses. In elegant recombinant chimera virus experi- luteovirid-aphid species combinations and the major determinants ments, exchanging CP ORFs between Beet curly top virus (BCTV, of vector competence (Gray et al., 2014; Gray and Gildow, 2003; leafhopper vector) and African cassava mosaic virus (ACMV, whitefly Peiffer et al., 1997). As these viruses do not replicate in the vector, vector) resulted in vector ‘switching’,defining role of the CP in vector higher virus accumulation in plants and/or longer feeding periods specificity (Briddon et al., 1990). Other experiments provide direct increase the amount of virus harbored by the aphid and the evidence that the CP serves as the viral attachment protein (VAP) to efficiency of transmission (Gray et al., 1991). insect vector guts, a first step in acquisition. A recombinant CP of The luteovirus virion is an icosahedral, T¼3 structure that is TYLCV fed to whiteflies bound to the midgut, and in competition composed primarily of the capsid protein (CP). The minor compo- assays with wildtype virus reduced the amount of virus in whiteflies nent of the virion is the CP-readthrough protein (CP-RTP), gener- (Wang et al., 2014). Likewise, feeding leafhopper (P. alienus)vectors ated by translational readthrough of the CP stop codon resulting in recombinant WDV CP localized the recombinant CP to the midgut, i.e., a C-terminal extension of the CP (Fig. 2). Numerous studies provide site of entry, and sequentially feeding leafhoppers antibodies raised evidence that the CP and CP-RTP are the major determinants of against wildtype WDV CP and virus reduced the proportion of insects insect acquisition and transmission of luteoviruses, and no other harboring the virus in various tissues along the route of dissemination luteovirid proteins have been implicated in these coordinated and virus accumulation in the vector (Wang et al., 2014). Collectively, processes (Brault et al., 2000; Gray et al., 2014; Liu et al., 2009; these findings support the hypothesis that although geminiviruses Peter et al., 2008). Virions made of the CP alone can transcytose are transmitted by diverse vector species, they use a similar route of through the gut, indicating that the CP is sufficient to deliver dissemination in the vector and the viral CP is the viral determinant the virus to the hemocoel. The RTP contains a highly conserved of this process. N-terminal region and a variable C-terminal region and these domains have distinct functional roles. The N-terminal half of the RTP is important for association with the ASG and the Circulative, propagative transmission C-terminal region is dispensable for transmission but plays a role in accumulation and tissue tropism in plants (Brault et al., 2000; The family Reoviridae is a large virus family with 15 genera that Bruyere et al., 1997; Peter et al., 2009). The full length RTP is infect humans, animals, plants, insects, and fungi (Attoui et al., detectable in plant tissues, but in purified virion preparations, the 2012). Members of the Reoviridae have genomes composed of protein is significantly smaller due to proteolytic processing of multiple (9–12) segments of linear double-stranded RNA (dsRNA) the C-terminal region (Brault et al., 1995; Wang et al., 1995). The that are encased in a non-enveloped particle (Attoui et al., 2012). CP-RTP also has a proline hinge domain that is important for Reoviruses have icosahedral symmetry with a diameter of approxi- incorporation of CP-RTP into virions. The N-terminus of the RTP is mately 60–85 nm made up of one or more structural layers of required for aphid transmission and is thought to mediate inter- capsid protein(s). There are three plant-infecting genera of the actions with the salivary glands (Peter et al., 2008; Brault et al., Reoviridae: Fijivirus, Phytoreovirus, and Oryzavirus that are trans- 1995, 2000). Mutations in CP-RTP have been shown abrogate mitted in a persistent-propagative manner by planthopper (Hemi- transmission but have no apparent effect on persistence in the ptera: Delphacidae) or leafhopper (Hemiptera: Cicadellidae) vector as long as the mutant CP-RTP is incorporated into virions vectors. Despite the similarities in vector transmission of reo- (Peter et al., 2008). viruses, there are some variations in reovirus tissue tropism and Much like the family Potyviridae, the virus genera in the family dissemination routes in their vectors that have been recently Geminiviridae are transmitted by vectors in a virus genus-specific documented. Vector transmission of plant-infecting reoviruses is manner. For example, whiteflies transmit viruses in the genus well characterized due to several tools and features of these Begomovirus, e.g., Tomato yellow leaf curl virus (TYLCV), while viruses: 1) vector cell monolayers (VCM) are available for studying leafhopper vectors transmit viruses in the genus Mastrevirus, e.g., virus infection at the cellular level with a synchronous infection; 2) Maize streak virus (MSV). Like luteovirids, the route of the Bego- the viral genome is segmented which enables viral gene function movirus begins with the insect feeding on phloem sap of infected to be studied by RNA interference (RNAi), despite the lack of an plants and virions are ingested and travel through the alimentary infectious clone system and 3) the leafhopper and planthopper canal (reviewed in Gray et al., 2014). The virions traverse the gut at vectors are amenable to RNAi for reducing transcript abundance of the midgut region and the majority accumulate in the filter potential host proteins that are part of the virus infection cycle. chamber region (Cicero and Brown, 2011; Ghanim et al., 2001). In Application of these technologies to the study of reovirus–vector most cases, begomoviruses do not replicate in their vectors and interactions has enabled significant advancements in understand- virions move through the gut via a transcytotic pathway much like ing initial virus entry into cells, movement between cells, mechan- the luteovirids. Virions are released from gut cells, travel through isms of dissemination and viral protein function. the insect hemocoel, and reach the primary salivary glands, moving The Phytoreovirus, Rice dwarf virus (RDV) and its vector, Nepho- through different salivary gland physical barriers for transmission tettix cincticeps, is the best characterized virus–vector system to occur (Cicero and Brown, 2011; Ghanim et al., 2001). Like within this family. The generalized route of dissemination for luteovirids, it is hypothesized that bacterial endosymbionts resid- plant-infecting Reoviruses starts with virus entry into the ing in bacteriocytes, specialized cells in the hemocoel, may play a alimentary canal and entering cells of the epithelial cells of filter A.E. Whitfield et al. / Virology 479-480 (2015) 278–289 285 chamber region and then the anterior midgut (Chen et al., 2011b). 2005a). The type member, Tomato spotted wilt virus (TSWV), is Virus traverses the basal lamina to infect the muscle cells, and transmitted efficiently by western flower thrips, Frankliniella occi- travels to the salivary glands via a hemolymph route. The study of dentalis, and the virus vector interactions for this virus and vector RDV entry and movement in VCMs enabled the discovery of a species combination has been well characterized. The acquisition unique tubule-mediated mechanism of virus delivery to surround- and transmission of tospoviruses is an insect-stage specific process. ing cells (Fig. 1). Initial entry into VCMs and midgut tissues is Larval thrips acquire TSWV and virus enters the anterior midgut mediated by the viral structural protein P2, a minor component of epithelial cells. The virus spreads to surrounding gut cells and the outer capsid of RDV (Omura et al., 1998). The virion binds to traverses the basal lamina to infect the circular and longitudinal cells and is taken up by clathrin-mediated endocytosis (Wei et al., muscle cells. In contrast to reoviruses, tospoviruses have not been 2007). P2 is also a fusion protein that facilitates virion release from observed in tubule-like structures in insect vectors despite the the endocytic vesicle and upon release from the vesicle the virus ability for these viruses to induce tubule formation in insect tissue replication cycle begins (Zhou et al., 2007). After assembly of culture cells (Storms et al., 1995). Virions have not been observed in progeny virions, newly generated RDV virions can associate with the hemocoel of thrips and it is thought that virus moves from the tubule structures or accumulate in multi-vesicular bodies in the midgut to the salivary glands through connective structures (i.e., vector cells (Wei et al., 2009). RDV-induced tubule structures were tubular salivary glands, ligaments) and/or directly between tissues first observed in VCMs and are composed primarily of a nonstruc- during insect stages when the midgut and salivary tissues are in tural protein, Pns10 (Wei et al., 2006, 2008). TEM examination of close contact (Kritzman et al., 2002; Montero Astúa, 2012; Moritz tubules revealed that RDV virions are encased within the structure et al., 2004). The timing of virus movement from the initial site of in a narrow row. The Pns10 tubules are associated with actin-based entry, the midgut, to the site for virus replication and delivery to filopodia, protrude from the surface of cells, and are capable of plants, the salivary glands is the latent period and at 24 C is 109 h penetrating neighboring cells (Wei et al., 2006). Experiments (Wijkamp and Peters, 1993). Virus can reach the salivary glands demonstrated that RDV exploits these tubules to move into during the second larval stage, but the primary transmitters are adjacent cells without release outside of the cell (Wei et al., adults because they are winged and are more mobile. Once the 2006). Pharmacological experiments with VCMs led to the hypoth- insect is infected, the virus persists for the duration of the lifespan. esis that the endomembrane system and myosin motors associated The pupal stages do not feed on plants so they neither acquire nor with actin filaments are required for tubule-mediated transport of transmit virus. Adult insects can feed on infected plants and RDV to adjacent cells (Wei et al., 2008). A direct interaction between sustain midgut infections, but they do not transmit the virus cytoplasmic actin of the vector, N. cincticeps,butnotactinofan (Assis Filho et al., 2004). It is thought that a developmental specific inefficient vector indicates that the ability to interact via tubules barrier or other changes in the vector prevents virus from reaching through specific interactions with Pns10 and actin is a determinant the salivary glands of adult thrips. The development of transcrip- of vector specificity (Chen et al., 2015). In vivo studies with N. cincticeps, tome and proteome resources for thrips are now available for F. provide evidence that the tubule movement strategy facilitates RDV occidentalis, and research aimed at identifying and characterizing movement in the microvilli of the gut epithelial cells and in the muscle vector molecules that interact with and respond to TSWV is cells encircling the gut (Chen et al., 2012). RNAi knockdown of Pns10 in beginning to describe the vector–virus interactome (Badillo- vector feeding experiments inhibited formation of tubules, prevented Vargas et al., 2012; Rotenberg and Whitfield, 2010). intercellular spread, and reduced leafhopper transmission efficiency of The major tospovirus determinants of thrips transmission are the virus (Chen et al., 2012). These data conclusively show that the the viral glycoproteins that project from the surface of the virion Pns10 tubules facilitate the intercellular spread of RDV in the leafhop- (Fig. 2). Several research groups observed that serial mechanical per vector. inoculation of TSWV to plants led to development of virus In contrast to RDV tubule-mediated transport within microvilli of populations with decreased thrips transmissibility (Nagata et al., midgut epithelial cells and visceral muscles, analysis of the Fijivirus, 2000; Resende Rde et al., 1991; Sin et al., 2005). Analysis of Southern rice black-streaked dwarf virus (SRBSDV) in the vector transmission-deficient virus populations revealed changes in the Sogatella furcifera, revealed that tubules are involved in virus escape medium genome segment, the segment that encodes the glyco- through the basal lamina. SRBSDV-induced tubules are comprised of proteins. To specifically map the genome segment that encodes the the P7-1 protein (Liu et al., 2011). RNAi experiments demonstrated determinants of transmissibility, a virus genome reassortment that P7-1 and the associated tubules are required for virus spread but study was conducted between thrips transmissible and non- knockdown did not affect virus replication (Jia et al., 2014). A primary transmissible isolates (Sin et al., 2005). The ability to be trans- difference between SRBSDV and RDV tubules is that the SRBSDV mitted by thrips was always associated with virus isolates that tubules crossed the basal lamina and appear to provide a route for contained an M segment from the thrips-transmissible isolate. rapid movement of the virus from gut epithelial cells into visceral Further analysis of single lesion isolates derived from a serially- muscle cells. The basal lamina is a major barrier to virus escape from mechanically passaged virus further mapped the transmission to gut cells. Correspondingly, the latent period for SRBSDV is shorter the glycoprotein ORF. Using a different approach to study the role than the RDV latent period, 6 to 9 days for SRBSDV in contrast with of viral proteins in transmission, the GN protein was expressed in 2 to 3 weeks for RDV (Honda et al., 2007; Pu et al., 2012). The insect cells using a recombinant baculovirus and feeding experi- discovery of tubule structures in insect vectors and the role in virus ments with the protein demonstrated that GN could bind to thrips movement along the actin cytoskeleton provides new insight into midguts and block entry into midguts (Whitfield et al., 2004). The virus movement in vectors and highlights similarities of the virus GN protein was capable of binding to midguts in the absence of lifecycle in insect and plant hosts. Additionally, these findings for other viral proteins indicating that it is a VAP or an important reoviruses emphasize the importance of understanding interactions component of the viral attachment complex. The GC protein of for each virus–vector combination within a virus family. other Bunyaviruses has been shown to be involved in fusion

Tospoviruses are members of the family Bunyaviridae and like with host membranes and the TSWV GC protein has similar all viruses in this family, they have enveloped virions that encap- characteristics supporting the hypothesis that it also plays a similar sidate three-ssRNA genome segments that have helical symmetry role in entry of virus into thrips (Garry and Garry, 2004; Whitfield, and are covered in nucleocapsid proten (N). All tospoviruses are Ullman, German, 2005b). transmitted in a circulative, persistent-propagative manner by The Tenuiviruses are non-enveloped viruses that replicate in their thrips, small insects in the order Thysanoptera (Whitfield et al., planthopper and leafhopper vectors. The dissemination route of these 286 A.E. Whitfield et al. / Virology 479-480 (2015) 278–289 viruses in their vectors have been described in detail, and initial species display differences in reproductive tissue tropisms that are studies found that tenuiviruses infect organs including the digestive associated with the ability of a species to be transovarially and respiratory tracts, Malpighian tubules, leg muscles, fat bodies, transmitted. For RSV, the mechanism of transovarial transmission brain, salivary glands, and reproductive tracts of both sexes (Nault and was documented using a yeast two hybrid assay to identify virus- Ammar, 1989; Zheng et al., 2014). The generalized dissemination route host protein interactions and functional validation of the interac- for Tenuiviruses begins with entry into and replication in the midgut tion using RNA interference (RNAi) methods. The RSV major epithelial cells, traversing the basal lamina, and infection of the nucleocapsid protein, pc3, interacted with vitellogenin, the major visceral muscles surrounding the gut. The virus then occurs in the yolk protein precursor of egg-laying animals, in yeast two-hybrid salivary glands, and specifically, Rice stripe virus (RSV) has been assays (Huo et al., 2014). Functional analysis using RNAi to knock- observed in the reproductive organs of both sexes of the small brown down vitellogenin transcripts resulted in a significant reduction in planthopper (Wu et al., 2014). However, there are some key differ- virus in the ovariole and demonstrated the importance of the ences between Tenuivirus species with regards to tissue tropism. For protein in transovarial transmission. These findings support the example, Rice grassy stunt virus (RGSV) in the small brown planthop- hypothesis that RSV directly binds to vitellogenin and appropriates per, Nilaparvata lugens, spreads intercellularly in the midgut epithe- the vitellogenin transport route to enter L. striatellus oocytes. lium and then traverses the basal lamina and infects the principal and accessory salivary glands (Zheng et al., 2014). This virus is not found in neural tissues or ovarioles (Zheng et al., 2014). In contrast, RSV in Translational outcomes derived from basic virus–vector Laodelphax striatellus is found in the ovarioles, spreads between research epithelial cells and is found in the principal salivary glands and absent in the accessory salivary glands (Deng et al., 2013; Wu et al., 2014). Exploiting the binding and functional properties of viral deter- The Tenuivirus genome is composed of four to six negative minants of virus acquisition is an exciting and real possibility for strand RNA segments coated in nucleocapsid protein, and the the development of new interdictive strategies that mitigate virions are ribonucleoprotein structures with helical symmetry. pathogen dispersal and disease. For the majority of plant virus– Based on sequence comparisons, the tenuiviruses are most closely vector interactions described in this review, viral components related to viruses in the family Bunyaviridae; however the lack of directly involved in virus acquisition are well defined. The identi- an envelope and the greater number of genome segments pre- fication and functional analysis of VAPs in vector–virus relations cludes taxonomic placement of this genus into the Bunyaviridae. have enabled innovative strategies for virus transmission disrup- Interestingly, the Tenuiviruses encode membrane glycoproteins tion and vector pest control. Recombinant VAPs can be used to like those of the Tospoviruses but enveloped virus particles have block binding of native virus to vector molecules that coordinate not been found in plants or insect vectors. In a protein localization virus entry into gut tissues, culminating into reduced acquisition study with plants, tenuivirus glycoproteins of RSV were documen- and viral loads, and subsequently prevention of transmission. ted to be processed into two proteins, designated Pc2-N and Pc2-C Another use is engineering VAPs to deliver insecticidal chemistries based on their location at the N- and C-termini of the polyprotein, to the insect hemolymph by way of the natural dissemination route and localized in the plant cell in a similar manner as the TSWV from point of entry, the gut epithelium. The bottom line is that glycoproteins (Yao et al., 2014). The N-terminal protein was these strategies rely on molecular interactions between VAPs and targeted to the Golgi and the C-terminal protein to the ER. When points of entry. Here, we discuss new frontiers in transmission expressed together, the N-terminal protein and C-terminal protein disruption strategies facilitated by Tospovirus and Luteovirus VAPs. co-localize to the Golgi. In insect cells, both proteins were found in the ER (Zhao et al., 2012). Blocking transmission Notably, there are currently no described roles for these glycoproteins in plants and insects and it appears that Tenuiviruses TSWV GN is a one of two viral transmembrane-bound structural are efficiently acquired and disseminated in insect vectors without proteins decorating the envelope of the virion and it plays an essential the envelope that is required for Tospoviruses. In their study, Yao role in the attachment of the virus to thrips midguts. A soluble et al. (2014) hypothesized that the RSV glycoproteins may function recombinant form of GN (GN-S) expressed from a baculovirus-SF21 as helper components that assist in virus acquisition by vectors cell-culture system has been the workhorse for TSWV acquisition and much like the helper components of non-circulative viruses. This transmission disruption studies. Demonstration of the capacity of hypothesis is yet to be tested but could be an explanation for the purified GN-S to specifically bind larval thrips guts, block TSWV retention of the genes that encode these glycoproteins in the acquisition and to reduce virus accumulation (Whitfield et al., 2004) tenuivirus genome. Understanding the role of the glycoproteins and subsequent transmission (Whitfield et al., 2008)whenapplied in the Tenuivirus infection cycle will be an interesting pursuit for exogenously to western flower thrips (WFT), led to the development these viruses because it is highly unlikely that these viruses have of a proof-of-concept tomato transgenics-based strategy to determine retained a “vestigial” gene. One emerging theme for the propaga- if ingestion of plant-expressed GN-S could inhibit TSWV acquisition tive plant viruses that lack envelopes is that they induce the and transmission by WFT (Montero-Astúa et al., 2014). TSWV accu- formation of tubules in the insect vector. The tubule dissemination mulation (titer) in young larval thrips exposed to TSWV-infected strategy may be a unique mechanism of dissemination in vectors transgenic plants for a 24-h AAP was significantly reduced compared for propagative viruses that lack membranes. to those exposed to infected non-transgenic plants. Interestingly, non- As with other circulative plant viruses, non-structural proteins transgenic and transgenic plants supported similar titers, making it play essential roles in Tenuivirus dissemination in the insect unlikely that reduced larval acquisition/titer resulted from exposure to vector body and transovarial transmission. In the case of RSV in source plants harboring low levels of virus. It appears that the GN-S- L. striatellus, ribonucleoprotein (RNP) interactions with NS4, a non- transgenic plants interfered with the infection of larval thrips by structural protein, facilitates tissue tropism in the insect vector TSWV. The reduction in titer persisted through the adult stage and (Wu et al., 2014). In the insect gut, RNPs co-localized with fibrillary transmission efficiency (number of transmitting adults) was signifi- inclusions of NS4 and knockdown of NS4 by RNAi slowed virus cantly reduced, indicating that the initial virus inoculum dose is spread and reduced transmission efficiency (Wu et al., 2014). important for vector competence. These studies show that blocking The ability to disseminate to and infect reproductive tissues is a or even significantly reducing the amount of virus acquired and prerequisite for transovarial transmission of viruses. Tenuivirus accumulated in the body can be an effective transmission reduction A.E. Whitfield et al. / Virology 479-480 (2015) 278–289 287 strategy for tospoviruses (Montero-Astúa et al., 2014; Rotenberg et al., insect and plant hosts, and in at least one case, the tubule has been 2009; Whitfield and Rotenberg, in press). documented to be a new strategy for escape through midgut barriers in the insect. Another new discovery for vector transmission of plant Vector population suppression viruses was the use of the vitellogenin uptake pathway for virus invasion of insect eggs, and this pathway for transovarial transmis- PEMV, a member of the genus Enamovirus within the family sion of viruses is parallel to transovarial transmission of other insect- Luteoviridae, is transmitted by Acyrthosiphon pisum, the pea aphid. associated microbes. While the virus components of the vector PEMV CP is the primary constituent of the luteovirus virion and a interaction are well-defined in most economically important sys- critical determinant of acquisition and transmission by the aphid tems, the identification of vector molecules that interact and respond vector. In the absence of other PEMV proteins, CP has the capacity to virus are just beginning to be characterized and we expect that the to bind to and enter the aphid hindgut to travel to the hemocoel via use of new research technologies will enable the functional analysis transcytosis (Liu et al., 2009). In addition, entry of luteovirids into of these vector components in the virus transmission process. For the aphid hemocoel is not vector specific, implying that the PEMV now, the commonalities in the role of viral structural proteins and CP can target aphids that are not vectors. Taking advantage of these initial sites of virus entry or retention indicate that these are characteristics of CP movement along the natural dissemination candidate targets for disrupting virus transmission by a wide range route to the hemocoel and the proline hinge region of the CP-RTP, a of vectors. The successful use of viral CP to deliver toxins to vectors feature determined to be critical for protein solubility and efficient and use of viral proteins to prevent transmission provide hope that transport of fused foreign sequences to the hemocoel, a PEMV CP the basic knowledge of virus binding and entry to vectors can (plus proline hinge) – neurotoxin fusion protein was engineered to provide a platform for development of a new control strategies for determine if the CP could serve as a vehicle to transport neurotoxic viruses and their vectors, a situation where current control options chemistries to the aphid hemocoel (Bonning et al., 2014). The are often limited and ineffective. neurotoxin of choice was a spider-derived insect-specific toxin that, in its native form, is not orally harmful to insects; it is toxic if delivered artificially to the hemocoel (Pal et al., 2013). Feeding CP- Acknowledgments toxin fusion protein to aphids via a membrane apparatus, delivered the toxin to the hemocoel and efficaciously resulted in high We thank Aurulie Bak for helpful comments and Michelle Cilia mortality of PEMV-vector (A. pisum and Myzus persicae) and non- for the Luteovirid drawing. This work was supported by the USDA vector (Rhopalosiphum padi and Aphis glycines) aphids belonging to NIFA/AFRI Grant numbers 2012-68004-20166 (DR and AEW) and two different tribes (Aphidini and Macrosiphini). This finding 2007-35319-18326 (AEW) and by the National Science Foundation supports the idea that the gut is not the major barrier for CAREER Grant IOS-0953786 (AEW); and in part by the University of California, and a USDA NIFA grant to BWF. 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