Thomas L. German

Thrips as vectors of tospoviruses

Publisher Summary This chapter reviews the thrips– tospovirus pathosystem and the cellular and molecular determinants of thrips acquisition of tospoviruses. Viruses in the genus Tospovirus (family Bunyaviridae) are transmitted by thrips and have become an ever increasing problem for the producers of agricultural and horticultural crops worldwide. The genus Tospovirus is the genus within the Bunyaviridae containing plant-infecting viruses. Tomato spotted wilt virus ( TSWV) is the type species of this genus. Thrips cause significant direct damage to plants, but it is their transmission of tospoviruses that is most difficult to control and frequently causes the most severe damage to crops. At least ten species of thrips transmit tospoviruses, all of which are in the Thysanopteran family Thripidae. Most thrips vector species deposit their eggs into plant tissue and the eggs hatch after 2–3 days, depending on temperature and plant host. For tospoviruses to be transmitted by thrips, they must be acquired by the larvae. Thus, only immature thrips that acquire tospoviruses or adults arising from such immatures are important to the transmission of the virus. This concept is extremely important in managing tospoviruses, because only the plants that serve as hosts for both the insect and the virus are important in epidemics.

Variation in Tomato spotted wilt virus titer in Frankliniella occidentalis and its association with frequency of transmission.

Tomato spotted wilt virus (TSWV) is transmitted in a persistent propagative manner by Frankliniella occidentalis, the western flower thrips. While it is well established that vector competence depends on TSWV acquisition by young larvae and virus replication within the insect, the biological factors associated with frequency of transmission have not been well characterized. We hypothesized that the number of transmission events by a single adult thrips is determined, in part, by the amount of virus harbored (titer) by the insect. Transmission time-course experiments were conducted using a leaf disk assay to determine the efficiency and frequency of TSWV transmission following 2-day inoculation access periods (IAPs). Virus titer in individual adult thrips was determined by real-time quantitative reverse transcriptase-PCR (qRT-PCR) at the end of the experiments. On average, 59% of adults transmitted the virus during the first IAP (2 to 3 days post adult-eclosion). Male thrips were more efficient at transmitting TSWV multiple times compared with female thrips of the same cohort. However, females harbored two to three times more copies of TSWV-N RNA per insect, indicating that factors other than absolute virus titer in the insect contribute to a successful transmission event. Examination of virus titer in individual insects at the end of the third IAP (7 days post adult-eclosion) revealed significant and consistent positive associations between frequency of transmission and virus titer. Our data support the hypothesis that a viruliferous thrips is more likely to transmit multiple times if it harbors a high titer of virus. This quantitative relationship provides new insights into the biological parameters that may influence the spread of TSWV by thrips.

Immunoprecipitation of a 50-kDa protein: a candidate receptor component for tomato spotted wilt tospovirus (Bunyaviridae) in its main vector, Frankliniella occidentalis

A 50-kDa protein that binds to viral particles in solid-phase assays and that is recognized by anti-idiotypic antibodies made against anti-viral glycoproteins G1/G2 (anti-Ids) has been proposed as a receptor candidate for tomato spotted wilt tospovirus (TSWV) in its main thrips vector, Frankliniella occidentalis Pergande (Bandla et al., 1998. Phytopathology 88, 98-104). Here we show the immunoprecipitation of the 50-kDa protein by anti-Ids and by an anti-G1/G2-TSWV conjugate - a new immunoprecipitation method. In addition, we show that anti-Ids made against anti-G1 (anti-IdG1) block virus replication in an insect tissue replication assay. The results indicate that (a) the TSWV-50-kDa protein interaction occurs in solution, as it must do in vivo; (b) G1 is a viral attachment protein; and (c) the 50-kDa protein is a candidate host factor essential for TSWV entry. These results provide additional support for the role of the 50-kDa thrips protein as a viral receptor. Additionally these experiments provide the basis for testing saturable binding and represent an important step toward the first cloning and identification of a cellular receptor for a plant virus.

Expression and Characterization of a Soluble Form of Tomato Spotted Wilt Virus Glycoprotein GN

ABSTRACT Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, GN and GC, are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play important roles in the entry of TSWV into the insect midgut, the first site of infection. To directly test the hypothesis that GN plays a role in TSWV acquisition by thrips, we expressed and purified a soluble, recombinant form of the GN protein (GN-S). The expression of GN-S allowed us to examine the function of GN in the absence of other viral proteins. We detected specific binding to thrips midguts when purified GN-S was fed to thrips in an in vivo binding assay. The TSWV nucleocapsid protein and human cytomegalovirus glycoprotein B did not bind to thrips midguts, indicating that the GN-S-thrips midgut interaction is specific. TSWV acquisition inhibition assays revealed that thrips that were concomitantly fed purified TSWV and GN-S had reduced amounts of virus in their midguts compared to thrips that were fed TSWV only. Our findings that GN-S binds to larval thrips guts and decreases TSWV acquisition provide evidence that GN may serve as a viral ligand that mediates the attachment of TSWV to receptors displayed on the epithelial cells of the thrips midgut.

A soluble form of the Tomato spotted wilt virus (TSWV) glycoprotein G(N) (G(N)-S) inhibits transmission of TSWV by Frankliniella occidentalis.

Tomato spotted wilt virus (TSWV) is an economically important virus that is transmitted in a persistent propagative manner by its thrips vector, Frankliniella occidentalis. Previously, we found that a soluble form of the envelope glycoprotein G(N) (G(N)-S) specifically bound thrips midguts and reduced the amount of detectable virus inside midgut tissues. The aim of this research was to (i) determine if G(N)-S alters TSWV transmission by thrips and, if so, (ii) determine the duration of this effect. In one study, insects were given an acquisition access period (AAP) with G(N)-S mixed with purified virus and individual insects were assayed for transmission. We found that G(N)-S reduced the percent of transmitting adults by eightfold. In a second study, thrips were given an AAP on G(N)-S protein and then placed on TSWV-infected plant material. Individual insects were assayed for transmission over three time intervals of 2 to 3, 4 to 5, and 6 to 7 days post-adult eclosion. We observed a significant reduction in virus transmission that persisted to the same degree throughout the time course. Real-time reverse transcription polymerase chain reaction analysis of virus titer in individual insects revealed that the proportion of thrips infected with virus was reduced threefold when insects were preexposed to the G(N)-S protein as compared to no exposure to protein, and nontransmitters were not infected with virus. These results demonstrate that thrips transmission of a tospovirus can be reduced by exogenous viral glycoprotein.

In situ localization of pea enation mosaic virus double-stranded ribonucleic acid.

Abstract Precursor ([ 3 H]uridine) incorporation studies in the presence or absence of actinomycin D (AMD) showed that AMD-insensitive nucleic acid synthesis occurred only in the nuclei in pea enation mosaic virus (PEMV)-infected pea plant tissues. Ferritin-labeled antibody studies showed that ds-RNA was present only in nuclei. In vitro hybridization of the nucleic acid from various infected or healthy cell fractions with [ 125 I]PEMV-ss-RNA indicated that PEMV-ds-RNA was primarily associated with nuclei-enriched fractions from infected cells. An in situ hybridization technique, which utilized autoradiography to detect the subcellular location of material which hybridized with [ 125 I]PEMV-ss-RNA, was developed. This method confirmed that PEMV-ds-RNA was localized in the nuclei of PEMV-infected tissues.

Mealybug Wilt of Pineapple

Mealybug wilt of pineapple (MBW), first described in Hawaii in the early 1900’s (46), is now reported in most areas of the world where pineapple is grown (6, 7, 13/14, 22). The circumstances surrounding MBW epidemics are complex involving multi-trophic interactions between mealybugs (MB), ants, mealybug predators and parasites, pineapple plants and other plant species (58). Symptom expression is variable and apparently linked to environmental conditions as well as variations in mealybug populations. Several generations of entomologists and plant pathologists have been challenged by the study of MBW and the etiology is yet to be fully explained. A number of hypotheses involving mealybug salivary toxins, “latent transmissible factors” or viruses have been proposed to explain the cause of the disease but none of these have been substantiated. Recently, a pineapple closterovirus (PCV) was described that appears to be associated with the disease (35, 36, 38, 39) although its role in the mealybug wilt syndrome, if any, is not fully understood. In this chapter we will present the historical background concerning the biology and epidemiology of MBW, data showing the association of PCV with pineapple plants and mealybugs and describe an apparent influence of PCV on pineapple growth.

Purification and properties of the replicative forms and replicative intermediates of pea enation mosaic virus

Abstract Pea enation mosiac virus consists of two nucleoprotein components with sedimentation coefficients of 95 and 115 S. Three single-stranded RNAs were obtained from purified preparations of virus. Replicative forms and replicative intermediates have been isolated from diseased tissue. These molecules were characterized with respect to buoyant density, melting properties, approximate molecular weight and length distribution. Ribonucleic acid isolated from purified virus was labeled in vitro with 125 I and the product was used in molecular hybridization experiments. It was shown that replicative form RNA hybridizes to virus RNA with greater specificity than to controls.

Interaction Between Tomato spotted wilt virus N Protein Monomers Involves Nonelectrostatic Forces Governed by Multiple Distinct Regions in the Primary Structure.

ABSTRACT The ambisense RNA genome of Tomato spotted wilt virus (TSWV) isby interaction with numerous copies of the virus encoded nucleocapsid (N) protein to form a subvirion structure called a ribonucleo-protein (RNP). RNPs are central to the viral replication cycle because they, and not free viral RNA, serve as templates for viral gene expression and genome replication. N protein monomers bind to viral RNA molecules in a cooperative manner. We have examined regions of the N protein that are involved in the N-N interactions that likely contribute to the cooperative binding of N to viral RNA. We created random and alanine scanning mutants of N and then screened the mutants for defects in N-N interaction using reverse and forward yeast two-hybrid assays. Our experiments identified residues in three distinct regions of the primary structure of the protein, residues 42 to 56, 132 to 152, and in the C-terminal 26 amino acids, that contribute to N-N dimerization or multimerization.interactions between N monomers mediated by the residues we identified are of a nonelectrostatic nature.

Disruption of vector transmission by a plant-expressed viral glycoprotein.

Vector-borne viruses are a threat to human, animal, and plant health worldwide, requiring the development of novel strategies for their control. Tomato spotted wilt virus (TSWV) is one of the 10 most economically significant plant viruses and, together with other tospoviruses, is a threat to global food security. TSWV is transmitted by thrips, including the western flower thrips, Frankliniella occidentalis. Previously, we demonstrated that the TSWV glycoprotein GN binds to thrips vector midguts. We report here the development of transgenic plants that interfere with TSWV acquisition and transmission by the insect vector. Tomato plants expressing GN-S protein supported virus accumulation and symptom expression comparable with nontransgenic plants. However, virus titers in larval insects exposed to the infected transgenic plants were three-log lower than insects exposed to infected nontransgenic control plants. The negative effect of the GN-S transgenics on insect virus titers persisted to adulthood, as shown by four-log lower virus titers in adults and an average reduction of 87% in transmission efficiencies. These results demonstrate that an initial reduction in virus infection of the insect can result in a significant decrease in virus titer and transmission over the lifespan of the vector, supportive of a dose-dependent relationship in the virus-vector interaction. These findings demonstrate that plant expression of a viral protein can be an effective way to block virus transmission by insect vectors.