A Virus Capsid Component Mediates Virion Retention and Transmission by Its Insect Vector

A virus capsid component mediates virion retention and transmission by its insect vector

Angel Y. S. Chena, Gregory P. Walkerb, David Carterc, and James C. K. Nga,1

aDepartment of Plant Pathology and Microbiology and Center for Disease Vector Research; bDepartment of Entomology; and cCenter for Plant Cell Biology, University of California, Riverside, CA 92521

Edited* by George Bruening, University of California, Davis, CA, and approved August 25, 2011 (received for review June 14, 2011)

Numerous pathogens of humans, animals, and plants are trans- tor penetrates a phloem sieve element with its stylets and acquires mitted by specific arthropod vectors. However, understanding the virions by ingesting phloem sap. Acquisition success increases mechanisms governing these pathogen–vector interactions is with prolonged ingestion of phloem sap (from minutes to hours). hampered, in part, by the lack of easy-to-use analytical tools. We When they have been acquired, virions are retained in the vector investigated whitefly transmission of Lettuce infectious yellows for hours to days and are lost if the insect molts. Second, virions virus (LIYV) by using a unique immunofluorescent localization ap- do not circulate through the insect and invade the salivary glands, proach in which we fed virions or recombinant virus capsid com- in contrast to viruses that are transmitted in a circulative, per- ponents to whiteflies, followed by feeding them antibodies to the sistent manner; nor do they replicate in the vector (7). virions or capsid components, respectively. Fluorescent signals, in- During acquisition, virus-laden phloem sap enters the opening dicating the retention of virions, were localized in the anterior fore- at the tip of the maxillary stylets into a short lumen where the maxillary food and salivary canals merge. From this location, gut or cibarium of a whitefly vector biotype but not within those infected sap moves up the maxillary food canal into a region of a whitefly nonvector biotype. Retention of virions in these lo- fl referred to as the precibarium before entering the cibarium, cations strongly corresponded with the white y vector transmis- which functions as a sucking pump. Sap is then pumped out of sion of LIYV. When four recombinant LIYV capsid components the cibarium into the anterior foregut, the pharynx, followed by fl fi were individually fed to white y vectors, signi cantly more white- the esophagus. In a few early studies, transmission EM of viru- fl ies retained the recombinant minor coat protein (CPm). As dem- liferous leafhoppers and aphids have provided some evidence onstrated previously and in the present study, whitefly vectors that the foregut may be where virions of some semipersistently failed to transmit virions preincubated with anti-CPm antibodies transmitted viruses are retained, and from where they are even- but transmitted virions preincubated with antibodies recognizing tually released and inoculated into the plant (15–17). However, the major coat protein (CP). Correspondingly, the number of some uncertainties exist whether viruses retained in the foregut insects that specifically retained virions preincubated with anti- are related to transmission (15, 16). In addition, the viral deter- CPm antibodies were significantly reduced compared with those minants mediating virion retention in the foregut have not been that specifically retained virions preincubated with anti-CP anti- identified for any of these viruses. Cauliflower mosaic virus bodies. Notably, a transmission-defective CPm mutant was defi- (CaMV) transmission by aphids is noncirculative and generally cient in specific virion retention, whereas the CPm-restored virus regarded as semipersistent. A recent study localized CaMV vi- showed WT levels of specific virion retention and transmission. rion-like particles and a virus-encoded protein within the tip of These data provide strong evidence that transmission of LIYV is the maxillary stylets, rather than the foregut. Thus, there appears determined by a CPm-mediated virion retention mechanism in the to be significant variation in retention site among different anterior foregut or cibarium of whitefly vectors. semipersistently transmitted plant viruses (18). Interestingly, retention of plant virus virions in the stylets and foreguts of in- arthropod vector transmission | crinivirus | noncirculative transmission | sect vectors appears to be analogous to a feature occasionally semipersistent transmission | Bemisia tabaci exhibited by certain animal viruses. This feature, referred to as mechanical transmission (not to be confused with “mechanical leaf rub inoculation” coined by plant virologists), is thought to ransmission by arthropod (insect) vectors is essential to the occur when virions of viruses that do not replicate in arthropod Tinfection cycle of many viruses, including those that cause vectors are nonspecifically harbored in their mouthparts (and – diseases in humans, animals, and plants (1 3), and is mediated by even in the foreguts, as suggested in the case of retroviruses) and critical but poorly understood processes that vary across phases get transmitted from one vertebrate to another during blood of virus acquisition, retention, and inoculation. With few ex- meals taken by the vectors (19–21). For example, Chihota et al. ceptions, viral determinants and vector sites mediating trans- (22) showed that Lumpy skin disease virus can survive in mos- fi mission of vector-speci c viruses remain poorly investigated for quitoes without replication for at least 6 d and still remains MICROBIOLOGY numerous plant viruses, many of which cause serious diseases transmissible, and Smith et al. (23) demonstrated that Vesicular that constrain crop and fiber production worldwide (1, 4–6). stomatitis New Jersey virus can be mechanically transmitted Members of the genus Crinivirus (family Closteroviridae) are among domestic swine by biting flies after minutes of acquisition emerging viruses affecting many different crop plants (7). The and inoculation feeding. However, as will be inferred from our genomes of criniviruses are among the largest and most complex study, the interactions mediating virion retention associated with of the single-stranded positive-sense RNA viruses and, based on mechanical transmission of animal viruses may be more sophis- sequence information and biological data, are organized into two ticated than currently appreciated. separate components. RNA 1 encodes several functions including virus replication and synergism, whereas RNA 2 contains as many as 10 ORFs involved in cytopathology, virion assembly, and Author contributions: J.C.K.N. designed research; A.Y.S.C., G.P.W., D.C., and J.C.K.N. per- vector transmission (8–14). Criniviruses occur in low titer and formed research; A.Y.S.C., G.P.W., and J.C.K.N. analyzed data; and A.Y.S.C. and J.C.K.N. are restricted to the phloem of infected plants. They cannot be wrote the paper. transmitted by leaf-rub inoculation but are readily transmitted The authors declare no conflict of interest. in a noncirculative, semipersistent manner by specific whitefly *This Direct Submission article had a prearranged editor. vectors in the insect order Hemiptera. Molecular mechanisms 1To whom correspondence should be address E-mail: [email protected]. underlying this mode of transmission are poorly understood, but This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. several biological features are known or inferred. First, the vec- 1073/pnas.1109384108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1109384108 PNAS | October 4, 2011 | vol. 108 | no. 40 | 16777–16782 Downloaded by guest on September 27, 2021 Most of our knowledge on the whitefly transmission of criniviruses originates from studies of Lettuce infectious yellows virus (LIYV). Four LIYV RNA 2-encoded proteins—a heat shock protein 70 homologue, HSP70h; a 59-kDa protein, P59; the major coat protein, CP; and the minor coat protein, CPm—are components of the long flexuous rod shape virion (11). Purified virions of LIYV can be transmitted by the whitefly Bemisia tabaci biotype A via membrane feeding, a procedure in which insects acquire virions in artificial liquid diet trapped between a pair of stretched and closely spaced parafilm sheet. Thus, LIYV, unlike the potyviruses or CaMV, does not require a nonvirion virus protein “helper” component for acquisition and transmission by the insect vector (7, 11, 24). Recent studies have revealed that the CPm is essential for LIYV transmission by B. tabaci biotype A (10, 11), but much crucial information needed to understand the virus– vector interactions involved in LIYV transmission is still missing. Here, we used a unique membrane feeding and immunofluorescent localization system to provide unique insights on the mechanism of whitefly transmission of LIYV by demonstrating the correspondence between the specific retention of virions within the whitefly vector and successful transmission of LIYV. We also provide biochemical and molecular evidence that vector-specific retention and transmission of virions is mediated by the LIYV CPm. These results are significant not only from a plant virus–insect vector biology standpoint, but also from an evolutionary context given the parallels between noncirculative plant virus transmission and the mechanical transmission of animal viruses (20, 25). Fig. 1. Retention of LIYV virions in the anterior foregut or cibarium of whiteflies and transmissibility of LIYV by whiteflies B. tabaci biotypes A Results and B after sequential membrane feeding of the following solutions: (i) diet alone or diet containing virions, (ii) diet containing anti-LIYV IgG, and LIYV Virions Are Retained in Anterior Foregut or Cibarium of Whitefly fl (iii) diet containing a goat anti-rabbit IgG conjugated with Alexa Fluor 488. Vectors. To determine the retention site of LIYV in its white y The presence or absence of fluorescent signals in the dissected whitefly vector, we developed a membrane feeding and immunofluores- heads was analyzed by using fluorescence microscopy. (A) Widefield fluo- cent localization assay in which caged whiteflies were given rescence microscopy micrograph of the head of B. tabaci biotype A fed diet sequential access to basal artificial liquid diet containing (i) containing LIYV virions, with background transmitted light blocked. (B)The purified LIYV virions, (ii) anti-LIYV virion IgG, and (iii) goat image in A with background transmitted light unblocked. (C and D)The anti-rabbit IgG conjugated with Alexa Fluor 488. Afterward, the same as A and B, except that B. tabaci biotype A was fed diet without virions. heads were dissected from individual whiteflies and analyzed by (E) Confocal laser scanning microscopy micrograph of the head of a B. tabaci fluorescence microscopy. To first validate the assay, two groups biotype A fed diet containing LIYV virions (Movie S1). (F) Confocal laser of whitefly vectors, B. tabaci biotype A, were compared: in step 1 scanning microscopy micrograph of the head of a B. tabaci biotype B fed diet above, one group fed on diet containing WT LIYV virions containing LIYV virions. (G) Transmitted light view of F. The eye, cibarium (Cb), and stylets (S) are indicated. (Scale bars, 45 μm.) (H) The average per- whereas the other group fed on diet alone (i.e., no virions). fl Results from three independent experiments revealed the pres- centage of anterior foreguts or cibarium with uorescent signals between ence of bright green fluorescent signals distributed in the ante- virion-fed biotypes A and B and the corresponding percentage of LIYV transmissibility; the data were pooled from three independent experiments rior foregut or cibarium of virion-fed vectors, representative (Table S2). Error bars represent SE. images of which are shown in Fig. 1 A, B, and E and Movie S1. Two virion concentrations, 0.01 μg/μL and 0.1 μg/μL, were used, both of which were readily detectable in vitro under non- 1, and on a diet containing an anti-CMV antiserum in step 2. denaturing conditions by double antibody-sandwiched (DAS) fi μ μ No signal was observed in the cibarium, foregut, or maxillary ELISA (Table S1). In the rst two experiments, when 0.01 g/ L stylets (including the tips) of all 183 CMV-fed whiteflies we of virions were fed to the vectors, fluorescent signals were ob- examined (Fig. S1 A and B), even though the virion concen- served in the anterior foregut or cibarium of six of 10 and 17 of trations used in the feeding were readily detectable in vitro 65 individuals, respectively. Conversely, a much weaker signal was observed in the same regions of only one of 20 diet-fed (i.e., under nondenaturing conditions by triple antibody-sandwiched no virions) vectors in the first experiment (i.e., 19 of 20 showed (TAS) ELISA (Table S1). no signal), whereas, in the second experiment, none of 65 diet- fed vectors showed a signal in these regions. Representative Correspondence Between Virion Retention in Anterior Foregut or fl images of a diet-fed vector that displayed no fluorescent signal in Cibarium of White ies and LIYV Transmission. The above results led the specified regions are shown in Fig. 1 C and D. In the third us to hypothesize that LIYV virions retained in the anterior fl experiment, when the virion concentration was increased to foregut or cibarium are those that the white y vector transmits. 0.1 μg/μL, fluorescent signals were seen in the specified regions To determine if retention of virions in the foregut or cibarium of 18 of 48 vectors that fed on virion augmented diet, whereas corresponded to transmission success, we used the membrane none of 37 diet-fed vectors showed any signal. In all cases, no feeding and immunofluorescent localization technique in which signal was observed anywhere on the maxillary stylets of virion- whiteflies [with ∼100 vector (B. tabaci biotype A) or nonvector or diet-fed vectors (Fig. 1 A, B,andE), and only very rarely was (B. tabaci biotype B) whiteflies per cage] were fed a diet con- an extremely weak signal observed in the precibarium. To verify taining 0.4 μg/μL of LIYV virions or a diet without virions (Table that the signals in LIYV virion-fed vectors were not a result of S2). Then, half (∼50) of the whiteflies from each cage were nonspecific virion binding, we used a similar immunofluorescent transferred to a noninfected lettuce plant for overnight in- localization assay to analyze B. tabaci biotype A, except that oculation access feeding whereas the other half of the whiteflies they were fed on a diet containing 0.5 μg/μLor1μg/μLofthe from the same cage were subjected to the subsequent steps of the aphid transmitted Cucumber mosaic virus (CMV) virions in step membrane feeding and immunofluorescent localization assay as

16778 | www.pnas.org/cgi/doi/10.1073/pnas.1109384108 Chen et al. Downloaded by guest on September 27, 2021 described earlier. In three independent experiments comparing Recombinant LIYV CPm Is Localized in Anterior Foregut or Cibarium of vector whiteflies (biotype A) fed a diet with or without virions, Whitefly Vectors Following Acquisition. The above data indicate strong green fluorescent signals were seen in the anterior foregut that LIYV virions are retained specifically in the anterior foregut or cibarium of 16% to 63% (average, 39%) of virion-fed vectors or cibarium of the vector but not the nonvector whitefly; this (Fig. 1H), whereas faint signals were seen in the same regions of suggests that LIYV encodes determinants that function in the only 0% to 2% of diet-fed vectors (Table S2, experiments 1–3). specific recognition of retention sites within its whitefly vector. This was consistent with results of the LIYV virion acquisition Consequently, we conducted three experiments to determine experiments detailed earlier. The corresponding LIYV trans- which of the four capsid components are involved in retention in mission success by the other half of the virion-fed whiteflies that the vector B. tabaci biotype A. Each of the experiments had three were given an inoculation access to lettuce plants was 13 of 19 pairs of treatments (Fig. 2A): (i) unaugmented diet and diet test plants infected (68%; Fig. 1H). Similarly handled diet-fed augmented with 0.4 μg/μL of virions, each followed by diet whitefly vectors did not result in any LIYV transmission (Table augmented with anti-LIYV IgG; (ii) unaugmented diet and diet S2, experiments 1–3). The difference in transmission success was augmented with purified Escherichia coli expressed recombinant highly significant (P < 0.0001; Fisher exact test). LIYV CPm (rCPm; Fig. S2A), each followed by diet augmented In contrast, fluorescent signal in the anterior foregut or ciba- with anti-CPm IgG; and (iii) unaugmented diet and diet aug- rium was absent in almost all (99%) of the virion-fed nonvectors mented with one of the following purified E. coli expressed (biotype B; Fig. 1 F–H and Table S2, experiments 4–6). In a few recombinant capsid components—rCP, rHSP70h, or rP59 (Fig. cases, weak signals were seen in the same regions of only one or S2A)—each followed by diet augmented with the corresponding two nonvectors fed on virus augmented diet, and no signal was anticapsid protein IgG (anti-CP, anti-HSP70h, or anti-P59, re- observed in the same regions of all diet-fed nonvectors (Table spectively). The concentrations of the recombinant capsid com- S2, experiments 4–6). Correspondingly, for the half of the non- ponents used in membrane feeding are indicated in Tables S3– vector whiteflies that were transferred to lettuce plants after S5. Whiteflies in all treatments were then fed diet augmented feeding on virus-spiked diet or diet alone, no transmission was with goat anti-rabbit IgG conjugated with Alexa Fluor 488. observed (Fig. 1H and Table S2, experiments 4–6). Fluorescent signals were observed in the anterior foregut or MICROBIOLOGY

Fig. 2. Retention of recombinant LIYV capsid proteins in the anterior foregut or cibarium of whitefly vectors. Individual rLIYV capsid proteins, rCPm, rCP, rHSP70h, and rP59 produced from an E. coli expression system were purified, concentrated, and compared with LIYV virions for specific retention in B. tabaci biotype A. (A) Average percentage of whiteflies with their anterior foregut or cibarium labeled with fluorescent signals among virion-, rCPm-, rCP-, rHSP70h-, rP59-, and diet-fed whiteflies that subsequently fed on diet containing the specific antibodies indicated beneath the columns. Error bars represent SE; P values determined by Student t test from two or three independent experiments. (B) Left: Widefield fluorescence microscopy image of the dissected head of a whitefly that had fed on diet containing rCPm, followed sequentially by diet containing an anti-LIYV CPm IgG, and diet containing a goat anti-rabbit IgG conjugated with Alexa Fluor 488. Right: Images of heads dissected from whiteflies that had fed on diet containing rCP, rHSP70h, or rP59, followed sequentially by diet containing an anti-LIYV CP, anti-LIYV HSP70h, or anti-LIYV P59 IgG, respectively, and diet containing a goat anti-rabbit IgG conjugated with Alexa Fluor 488. Foregut regions with (Top) and without (Bottom) fluorescent signals are compared. Punctate fluorescent signals in the insets (Top)are enlarged. The positions of the eye and cibarium (Cb) are indicated. (Scale bars, 45 μm.) (C) Average percentage of anterior foregut or cibarium with fluorescent signals for B. tabaci biotype A that had fed on diet containing virions preincubated with anti-CP and anti-CPm IgGs. Error bars represent SE; P values determined by Student t test from two independent experiments.

Chen et al. PNAS | October 4, 2011 | vol. 108 | no. 40 | 16779 Downloaded by guest on September 27, 2021 cibarium of 40% to 47% of virion-fed vectors and 30% to 38% of on agro-pR6-5bM1 virions (Fig. 3 A, B, and E) but only 3% of rCPm-fed vectors (Fig. 2A and Tables S3–S5). The percentages vectors that had fed on agro-pR6-5b virions (Fig. 3 C–E and of virion-fed whiteflies and rCPm-fed whiteflies differed signifi- Table S6). Transmission using half of the vectors that had ac- cantly from their respective diet-fed controls in all three quired virions from the same membrane feeding cages as those experiments (P < 0.0001; Student t test; Fig. 2A). In contrast, that were subjected to immunofluorescent localization resulted only 4% of rCP-fed whiteflies, 10% of rHSP70h-fed whiteflies, in 11 infected among 15 test plants for vectors that had fed on and 10% of rP59-fed whiteflies showed positive signals, and none agro-pR6-5bM1 virions, and none infected among 19 test plants of these differed significantly from their respective diet-fed for vectors that had fed on agro-pR6-5b virions (Fig. 3E and controls (P > 0.05; Student t test; Fig. 2A and Tables S3–S5). The Table S6). The difference between the two transmission scores percentages of rCP-fed, rHSP70h-fed, and rP59-fed whiteflies was highly significant (P < 0.0001; Fisher exact test). with signals in their anterior foregut or cibarium were all significantly lower than in virion-fed and rCPm-fed whiteflies (P ≤ Discussion 0.0003; Student t test). Overall, although the average percen- Acquisition of a virus by an insect vector marks the beginning of tages of virion-fed and rCPm-fed whiteflies with signals in their an intimate relationship between the virus and the vector—a re- anterior foregut or cibarium were relatively high, they were lationship that necessitates the virus to equip itself with the statistically different from each other (P = 0.0228; Student t test; means to be retained at specific locations within the vector and data pooled over all three experiments; Fig. 2A). In most cases, eventually to be released and inoculated into a suitable plant the signals seen in rCPm-fed vectors occurred more in the form of multiple punctate spots as opposed to a larger, more continuous area of fluorescence as was typically observed in virion-fed vectors (compare Fig. 2B vs. Fig. 1 A and E and Movie S1). Similar punctate patterns were also seen in anterior foregut or cibarium of the few rCP-, rHSP70h-, rP59-, and diet-fed vectors that showed fluorescent signals (Fig. 2B). The above data are consistent with those of serological in- fectivity neutralization (SIN) experiments that implicated a role of the LIYV CPm in whitefly transmission (11). In that study, preincubation of virions with antibodies specific to the CPm before membrane feeding by B. tabaci biotype A successfully blocked the transmission of LIYV, whereas preincubation of virions with antibodies specific to the CP, HSP70h, and P59 did not. Here, we used SIN and immunofluorescent localization assays to determine the basis for CPm’s involvement in LIYV transmission. When SIN was performed by using anti-CP antibodies, 34% (118 of 350 observed) of the vectors showed fluorescent signals in their anterior foregut or cibarium (Fig. 2C). Correspondingly, consistent with the results presented in the work by Tian et al. (11), transmission was observed in two of six plants. In contrast, when SIN was performed by using anti-CPm antibodies, 18% (72 of 403 observed), i.e., a nearly twofold re- duction (P = 0.0024; Student t test) in percentage, of vectors had fluorescent signals in their anterior foregut or cibarium (Fig. 2C). Correspondingly, no transmission was observed in all six plants tested, which was also consistent with the previous study (11).

A Transmission-Defective Mutant Exhibits Altered Virion Retention in Foregut or Cibarium of Whitefly Vectors. As a stringent test of the hypothesis that the CPm plays an essential role for virion retention in the anterior foregut or cibarium of whitefly vectors and determines whitefly transmissibility of LIYV, another strategy was to compare WT and mutant LIYV virions for differences in virion retention and transmission. Previously, we characterized a transmission-defective mutant, which contains a single nucleotide de- letion in the CPm ORF that is predicted to result in a frameshift Fig. 3. Retention of virions of a CPm mutant LIYV (agro-pR6-5b) and a CPm- and premature termination of the protein (26). The cDNA se- restored LIYV (agro-pR6-5bM1) in the anterior foregut or cibarium of the quence corresponding to this mutant CPm ORF (p1-5b CPm) and whitefly vector and the corresponding virus transmissibility. Whiteflies (B. one in which an engineered mutation was rendered to restore the tabaci biotype A) were sequentially fed the following components: (i) diet CPm ORF (p1-5bM1 CPm) were each swapped into a WT LIYV containing virions, (ii) diet containing anti-LIYV virion IgG, and (iii) diet RNA 2 binary vector construct, agro-pR6, a construct that was containing a goat anti-rabbit IgG conjugated with Alexa Fluor 488. (A) engineered for the agrobacterium-mediated inoculation of plants. Widefield fluorescence microscopy micrograph of the head of B. tabaci Insertion of these cDNA sequences generated the agro-pR6-5b biotype A fed diet containing agro-pR6-5bM1 virions, with background and agro-pR6-5bM1 constructs, respectively (10). Consistent transmitted light blocked. (B) Image in A with background transmitted light unblocked. (C and D) Same as A and B except that B. tabaci biotype A was with our previous results, immunoblot analysis using antibodies fed diet containing agro-pR6-5b virions. The cibarium (Cb) is indicated. (Scale produced against LIYV virions showed that both virions con- μ B bars, 45 m.) (E) The average percentage (error bars represent SEs) of tained the approximately 28-kDa CP (Fig. S2 ) (10). Both viri- whiteflies with their anterior foregut or cibarium labeled with fluorescent ons also reacted positively with antibodies produced against the signals for agro-pR6-5bM1 (5bM1) virion- and agro-pR6-5b (5b) virion-fed LIYV CP in immunogold-labeling transmission EM, but only whiteflies and the corresponding LIYV transmissibility after half the virion- agro-pR6-5bM1 virions were recognized by antibodies to the fed whiteflies from each feeding cage (containing whiteflies that sub- LIYV CPm (Fig. S2C) (10). In immunofluorescent localization sequently fed on components ii and iii) were transferred to a noninfected and transmission experiments, fluorescent signals were observed lettuce plant. Values were determined from three or four independent in the anterior foregut or cibarium of 34% of vectors that had fed experiments.

16780 | www.pnas.org/cgi/doi/10.1073/pnas.1109384108 Chen et al. Downloaded by guest on September 27, 2021 host. Viral determinants and/or vector sites that participate in consistently detect LIYV by RT-PCR at the whole-insect level virion retention and transmission have been keenly studied for after they were given access to virions by membrane feeding (Fig. a number of noncirculatively transmitted viruses, including S3). Thus, the likeliest explanation why LIYV virions are not CMV, viruses in the genus Potyvirus, and CaMV (7, 27, 28). transmitted by B. tabaci biotype B is that they are not retained in Virions of these viruses are noncirculative and are retained or, in the foregut or cibarium of this insect. the case of CMV, thought to be retained on the stylets, but they Our results are unambiguous in determining that CPm plays differ in other regards. CMV and potyviruses are nonpersistently an essential role in mediating the binding of virions to the vec- transmitted, with transmission occurring in seconds to minutes tor’s anterior foregut or cibarium: (i) the rCPm but not the other after acquisition (7), whereas CaMV is considered to be semi- three recombinant capsid components specifically binds at these persistently transmitted, with transmission continuing over hours locations; and (ii) virions derived from the transmission-de- or days after acquisition (29). In contrast, all other semiper- fective mutant, agro-pR6-5b, which exhibits a truncated CPm sistently transmitted viruses whose retention sites have been genotype (10), are not retained in the anterior foregut or ciba- investigated previously are retained in the foreguts of their rium, whereas virions derived from the transmission-restored vectors—the aphid-transmitted Anthriscus yellows virus (AYV) agro-pR6-5bM1 are retained. Although the other three recom- and Parsnip yellow fleck virus (PYFV) (17), and the leafhopper- binant capsid components do not bind specifically to the whitefly transmitted Maize chlorotic dwarf virus (MCDV) (15, 16). Al- vector, we cannot rule out the possibility that, in the context of though the retention sites of these viruses have been known for the assembled virion, they may have some yet unknown functions more than 20 years, there has been little progress in dissecting associated with virus transmission. Because the CPm, CP, the transmission mechanisms. Viral molecular determinants of HSP70h, and P59 are interacting capsid components (11, 33, 34), foregut retention of virions have not been identified previously. it is intriguing that in the absence of a complete CPm, the agro- The limited knowledge is, in part, attributable to the lack of pR6-5b mutant is still capable of systemic plant infection and progress in development of reliable and user-friendly tools for in encapsidation, perhaps by one or more of the other three com- situ localization of virions within the vectors. Criniviruses, by ponents compensating for the truncated CPm (10). However, it virtue of their semipersistent transmission characteristics, have is clear from our data that virion retention and transmission by been postulated to be retained in the foreguts of their whitefly vectors are dependent on the presence of a complete CPm and vectors. However, as with AYV, PYFV, and MCDV, molecular cannot be substituted or compensated for. mechanisms underlying the retention of crinivirus virions had not The distinction among the fluorescent signals observed is been elucidated previously. This gap in knowledge can now be qualitative (i.e., they are meant to indicate the presence or ab- bridged with the membrane feeding and immunofluorescent lo- sence of virions or recombinant capsid proteins), not quantitative. calization assay, which has greatly facilitated routine large-scale Therefore, the signals could not be used to estimate the amount analysis of differently treated whiteflies. of virions or proteins present, nor could they be used to de- All the fluorescent signals seen in the whitefly vectors in the termine the retention affinity between the vector and the virion/ present study were observed within the anterior foregut or capsid protein. Qualitative differences in signals were distin- cibarium. In no instance were signals seen in the maxillary stylets guishable between the rCPm-fed vectors and the virion-fed vec- or the alimentary tract preceding the cibarium (only a very weak tors, with the former appearing more frequently as punctate spots signal was occasionally seen in the precibarium). These obser- and those of the latter occupying a more continuous and larger vations are clearly in agreement with the classical paradigm that area in the anterior foregut or cibarium. Several possibilities semipersistently transmitted viruses are retained within the fore- could account for the differences in distribution between whole gut regions of their insect vectors (7). However, they appeared to virion and expressed proteins. For example, CPms in intact viri- conflict with the results presented in the contemporary study by ons are specifically organized at one end of the filamentous LIYV Uzest et al. (18), who demonstrated that putative receptors of virion; in this configuration, the proteins may present multiple the semipersistently transmitted CaMV are located at the tip of copies of their vector binding surfaces. In contrast, the expressed the maxillary stylets of several aphid species. Although CaMV CPm by itself may be self-associating into more random olig- is considered to be a semipersistently transmitted virus, it has omers, causing the punctate appearance. Alternatively, an as- several characteristics that differ from other semipersistently sembled virion is a much bigger entity than a capsid component transmitted viruses. First, the optimum acquisition access period alone and clearly presents more opportunities for interaction with for the successful transmission of CaMV by aphid vectors varies recognizing antibodies than do retained recombinant capsid in a bi- or multiphasic manner, with transmission rate peaking at components. The latter explanation also could account for the several minutes and again at several hours of access feeding time slightly but statistically lower percentage of whiteflies showing (30). Second, unlike other semipersistently transmitted viruses, positive signals following rCPm feeding vs. virion feeding. CaMV is transmissible by leaf-rub inoculation and can invade Our findings support the ingestion–egestion hypothesis for both mesophyll and phloem cells, and is therefore able to be transmission of LIYV, which proposes that virions are acquired acquired relatively more quickly than typical semipersistently during ingestion, are retained at a binding site in the alimentary transmitted viruses that apparently must reach the phloem to canal, and then are dislodged and inoculated when the vector MICROBIOLOGY infect and be acquired (31). Moreover, aphid transmission of egests (i.e., regurgitates) into a previously uninfected plant (35). CaMV requires the participation of additional viral-encoded Virions that are retained at the tip of the maxillary stylets, such helper proteins (27), whereas the whitefly transmission of LIYV as the case for CaMV, in which the food and salivary canals are does not. Taken together, these characteristics suggest that dif- confluent, could potentially be dislodged and inoculated into ferent semipersistently transmitted viruses may not necessarily a plant by salivation or egestion. However, a retention site in the “behave” similarly within their respective insect vectors, and cibarium or foregut is not in the pathway of saliva secretion, and may even differ in vector retention sites. Thus, as reviewed by virions retained there could be dislodged and inoculated only Hogenhout et al. (32), knowledge of the biological transmission by egestion. Consequently, ingestion–egestion is the most likely features cannot be used to assume a specific vector retention site mechanism for transmission of noncirculative viruses that are for semipersistently transmitted viruses. retained in the cibarium or foregut. Our immunofluorescent localization and whitefly transmission In summary, our results provide unequivocal evidence that (i) data strongly indicate that LIYV virions transmitted by whitefly LIYV virions are retained within the anterior foregut or ciba- vectors are specifically those that are retained in their anterior rium of its whitefly vector, (ii) retention of virions at these sites foregut or cibarium. The absence of fluorescent signals in 99% of correlates with whitefly transmission of the virus, and (iii) individuals of the virion-fed nonvector B. tabaci biotype B, and binding of the virus to this retention site is mediated by the CPm. their inability to transmit LIYV, could not be a result of the lack These results offer insights into the transmission mechanism of of ingestion of virions from the artificial diet, as we were able to this and other criniviruses and virus–vector interactions in gen-

Chen et al. PNAS | October 4, 2011 | vol. 108 | no. 40 | 16781 Downloaded by guest on September 27, 2021 eral. These results also warrant a reexamination of the concept exception was that clearing was conducted for 1 h following the acquisition of mechanical transmission by arthropod vectors of animal feeding of the third solution. The construction of expression vectors for the viruses, which is believed to be mediated by a nonspecific in- E. coli expression of the recombinant capsid proteins, as well as their expres- teraction between the mouthparts of vectors and viruses they sion, postexpression processing, and concentration estimation are included transmit, especially for those viruses that remain viable for ex- in SI Materials and Methods. Antibodies (rabbit-raised polyclonal IgGs) cor- tended periods during their association with the vectors (22). responding to the respective capsid proteins were used at the following dilutions: anti-CPm IgG (1/250-fold of 2.5 mg/mL), anti-CP IgG (1/500-fold of Materials and Methods 1.2 mg/mL), anti-HSP70h IgG (1/250-fold of 1.9 mg/mL), and anti-P59 IgG (1/250-fold of 2 mg/mL). SIN assays using anti-CP and anti-CPm antisera were fl fl Virions at the retention sites of white y vectors were uorescently labeled by as previously described (11). To verify that fluorescent signals observed were the following procedure: adult whiteflies were placed in membrane feeding not caused by nonspecific binding to the insect by individual anticapsid cages (9, 11), with approximately 100 whiteflies per cage. The caged protein IgGs, diet-fed whiteflies were given access to basal artificial liquid whiteflies then were fed one of the following two components: (i) basal diet containing each of these IgGs. artificial liquid diet [15% sucrose and 1% BSA in 1× TE (10 mM Tris-HCl, 1 After the final membrane feeding, heads were removed from the whiteflies mM EDTA, pH 7.4)] or (ii) basal artificial liquid diet augmented with purified and examined under widefield fluorescence microscopy (bandpass filter, 450– LIYV virions (0.01 or 0.1 μg/μL) or CMV-Fny virions (1 or 0.5 μg/μL; the CMV- 490/long-pass filter, 520) with a Zeiss Axioskop microscope using a 40×/0.65 NA Fny virion augmented diet was used as a control for potential nonspecific objective, and images were taken using a Coolpix 995 digital camera (Nikon). virion retention). Afterward, the whiteflies were fed basal artificial liquid Confocal laser scanning microscopy of dissected whitefly heads was performed diet containing anti-LIYV polyclonal IgG raised in rabbit (1/833-fold dilution with an SP2 microscope (Leica Microsystems). Alexa Fluor 488 was imaged with of 2.3 mg/mL) or anti–CMV-Fny polyclonal antiserum raised in rabbit (diluted a20×/0.7 NA water objective using the 488-nm line of an Argon ion laser, with 1/800 fold), followed by basal artificial liquid diet containing a 1/200-fold a detection range set between 500 and 600 nm. Three-dimensional rendered dilution of goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Invi- confocal images were reconstructed with Imaris software (Bitplane). trogen). Whiteflies were given a 10- to 12-h acquisition access period for fl fl each of the aforementioned solutions. To remove unbound or non- Materials and methods used for white y and virus maintenance, white y fi specifically bound virions or antibodies present in the ingested solutions, the transmission, agroinoculation, virion preparation, virion quanti cation, fl food canals and foreguts of whiteflies were cleared or flushed out by ELISA, statistical analyses, and RT-PCR detection of LIYV in white ies are feeding whiteflies a basal artificial diet for several hours after the acquisi- included in SI Materials and Methods. tion feeding of the first and third solutions. To determine which of the LIYV capsid proteins (CP, CPm, HSP70h, or P59) ACKNOWLEDGMENTS. The authors thank Tom Smith for helpful discus- bind to the retention site with the whitefly vector, whiteflies were mem- sions; Katherine Hadikusumo, Chawin Mongkolsiriwattana, Eric Oduca, and Tongyan Tian for technical assistance; and Shou-wei Ding and A. L. N. brane-fed the same series of solutions as described earlier except that the first fl fi Rao for editorial comments. Funding was provided by startup funds from solution fed to the white ies was basal arti cial liquid diet augmented with the University of California, Riverside (UCR), College of Natural and fi each of the speci c recombinant capsid proteins rather than augmented with Agricultural Sciences and by a grantfromtheLosAlamosNational purified LIYV virions, and the second solution was basal artificial liquid diet Laboratory–UCR Collaborative Program in Pathogen-Induced Plant Infec- augmented with the corresponding antibodies to these proteins. Another tious Disease (to J.C.K.N.).

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