The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population.

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

The Sw-5b NLR Immune Receptor Induces Early Transcriptional Changes in Response to Thrips and Mechanical Modes of Inoculation of Tomato spotted wilt orthotospovirus.

The NLR (nucleotide-binding leucine-rich repeat) class immune receptor Sw-5b confers resistance to Tomato spotted wilt orthotospovirus (TSWV). Although Sw-5b is known to activate immunity upon recognition of the TSWV movement protein NSm, we know very little about the downstream events that lead to resistance. Here, we investigated the Sw-5b-mediated early transcriptomic changes that occur in response to mechanical and thrips-mediated inoculation of TSWV, using near-isogenic tomato lines CNPH-LAM 147 (Sw5b+/+) and Santa Clara (Sw-5b-/-). We observed earlier Sw-5b-mediated transcriptional changes in response to thrips-mediated inoculation compared with that in response to mechanical inoculation of TSWV. With thrips-mediated inoculation, differentially expressed genes (DEGs) were observed at 12, 24, and 72 h postinoculation (hpi). Whereas with mechanical inoculation, DEGs were observed only at 72 hpi. Although some DEGs were shared between the two methods of inoculation, many DEGs were specific to either thrips-mediated or mechanical inoculation of TSWV. In response to thrips-mediated inoculation, an NLR immune receptor, cysteine-rich receptor-like kinase, G-type lectin S-receptor-like kinases, the ethylene response factor 1, and the calmodulin-binding protein 60 were induced. Fatty acid desaturase 2-9, cell death genes, DCL2b, RIPK/PBL14-like, ERF017, and WRKY75 were differentially expressed in response to mechanical inoculation. Our findings reveal Sw-5b responses specific to the method of TSWV inoculation. Although TSWV is transmitted in nature primarily by the thrips, Sw-5b responses to thrips inoculation have not been previously studied. Therefore, the DEGs we have identified in response to thrips-mediated inoculation provide a new foundation for understanding the mechanistic roles of these genes in the Sw-5b-mediated resistance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome-enabled insights into the biology of thrips as crop pests.

BACKGROUND: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. RESULTS: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. CONCLUSIONS: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

Sequence analysis of the medium and small RNAs of impatiens necrotic spot virus reveals segment reassortment but not recombination.

The complete sequence of the medium (M) and small (S) RNA genome segments were determined for twelve isolates of impatiens necrotic spot virus from eight plant species. The M- and S-RNAs of these isolates shared 97-99% and 93-98% nucleotide sequence identity, respectively, with the corresponding full-length sequences available in public databases. Phylogenetic analysis based on the M- or S-RNA sequences showed incongruence in the phylogenetic position of some isolates, suggesting intraspecies segment reassortment. The lack of phylogenetic discordance in individual and concatenated sequences of individual genes encoded by M- or S-RNAs suggests that segment reassortment rather than recombination is driving evolution of these INSV isolates.

Topical paromomycin with or without gentamicin for cutaneous leishmaniasis.

BACKGROUND: There is a need for a simple and efficacious treatment for cutaneous leishmaniasis with an acceptable side-effect profile. METHODS: We conducted a randomized, vehicle-controlled phase 3 trial of topical treatments containing 15% paromomycin, with and without 0.5% gentamicin, for cutaneous leishmaniasis caused by Leishmania major in Tunisia. We randomly assigned 375 patients with one to five ulcerative lesions from cutaneous leishmaniasis to receive a cream containing 15% paromomycin-0.5% gentamicin (called WR 279,396), 15% paromomycin alone, or vehicle control (with the same base as the other two creams but containing neither paromomycin nor gentamicin). Each lesion was treated once daily for 20 days. The primary end point was the cure of the index lesion. Cure was defined as at least 50% reduction in the size of the index lesion by 42 days, complete reepithelialization by 98 days, and absence of relapse by the end of the trial (168 days). Any withdrawal from the trial was considered a treatment failure. RESULTS: The rate of cure of the index lesion was 81% (95% confidence interval [CI], 73 to 87) for paromomycin-gentamicin, 82% (95% CI, 74 to 87) for paromomycin alone, and 58% (95% CI, 50 to 67) for vehicle control (P<0.001 for each treatment group vs. the vehicle-control group). Cure of the index lesion was accompanied by cure of all other lesions except in five patients, one in each of the paromomycin groups and three in the vehicle-control group. Mild-to-moderate application-site reactions were more frequent in the paromomycin groups than in the vehicle-control group. CONCLUSIONS: This trial provides evidence of the efficacy of paromomycin-gentamicin and paromomycin alone for ulcerative L. major disease. (Funded by the Department of the Army; ClinicalTrials.gov number, NCT00606580.).

Infection with a plant virus modifies vector feeding behavior.

Vector infection by some animal-infecting parasites results in altered feeding that enhances transmission. Modification of vector behavior is of broad adaptive significance, as parasite fitness relies on passage to a new host, and vector feeding is nearly always essential for transmission. Although several plant viruses infect their insect vectors, we have shown that vector infection by a plant virus alters feeding behavior. Here we show that infection with Tomato spotted wilt virus (TSWV), type member of the only plant-infecting genus in the Bunyaviridae, alters the feeding behavior of its thrips vector, Frankliniella occidentalis (Pergande). Male thrips infected with TSWV fed more than uninfected males, with the frequency of all feeding behaviors increasing by up to threefold, thus increasing the probability of virus inoculation. Importantly, infected males made almost three times more noningestion probes (probes in which they salivate, but leave cells largely undamaged) compared with uninfected males. A functional cell is requisite for TSWV infection and cell-to-cell movement; thus, this behavior is most likely to establish virus infection. Some animal-infecting members of the Bunyaviridae (La Crosse virus and Rift Valley fever virus) also cause increased biting rates in infected vectors. Concomitantly, these data support the hypothesis that capacity to modify vector feeding behavior is a conserved trait among plant- and animal-infecting members of the Bunyaviridae that evolved as a mechanism to enhance virus transmission. Our results underscore the evolutionary importance of vector behavioral modification to diverse parasites with host ranges spanning both plant and animal kingdoms.

Dehydration and tomato spotted wilt virus infection combine to alter feeding and survival parameters for the western flower thrips, Frankliniella occidentalis.

Dehydration and tomato spotted wilt virus (TSWV) infection substantially impact the feeding of western flower thrips, Frankliniella occidentalis. Until now, the dynamics between these biotic and abiotic stresses have not been examined for thrips. Here, we report water balance characteristics and changes in other biological parameters during infection with TSWV for the western flower thrips. There were no apparent differences in water balance parameters during TSWV infection of male or female thrips. Our results show that, although water balance characteristics of western flower thrips are minimally impacted by TSWV infection, the increase in feeding and activity when dehydration and TSWV are combined suggests that virus transmission could be increased under periods of drought. Importantly, survival and progeny generation were impaired during TSWV infection and dehydration bouts. The negative impact on survival and reproduction suggests that the interactions between TSWV infection and dehydration will likely reduce thrips populations. The opposite effects of dehydration on feeding/activity and survival/reproduction for virus infected thrips suggest the impact of vectorial capacity will likely be minor for TSWV transmission. As water stress significantly impacts insect-plant-virus dynamics, these studies highlight that all interactions and effects need to be measured to understand thrips-TSWV interactions in their role as viral vector to plants.

Acylsugar-mediated resistance as part of a multilayered defense against thrips, orthotospoviruses, and beyond.

Resistant varieties are critical tools for crop production, and single-resistance genes providing strong protection against pests or pathogens are deployed in agriculture. Durability of these traits is threatened by emergence of resistance-breaking pests and pathogens. This review focuses on acylsugar-mediated resistance in tomato. Wild tomatoes have type-IV trichomes that exude chemically complex mixtures of acylsugars altering behavior and suppressing multiple pest species, and with thrips and whiteflies (WF), suppressing virus transmission, for example, Tomato spotted wilt orthotospovirus and Tomato yellow leaf curl virus, respectively. Marker-assisted selection and bioassays led to development of advanced cultivated tomato breeding lines rich in acylsugar variations, allowing acylsugar-mediated resistance to be combined with other resistance traits providing a layered defense system that reduces pest populations and virus disease prevalence. This strategy also holds promise for enhancing durability of virus resistance genes by reducing the intensity of selection for resistance-breaking variants.

Sex-biased proteomic response to tomato spotted wilt virus infection of the salivary glands of Frankliniella occidentalis, the western flower thrips.

Successful transmission of tomato spotted wilt virus (TSWV) by Frankliniella occidentalis requires robust infection of the salivary glands (SGs) and virus delivery to plants during salivation. Feeding behavior and transmission efficiency are sexually-dimorphic traits of this thrips vector species. Proteins secreted from male and female SG tissues, and the effect of TSWV infection on the thrips SG proteome are unknown. To begin to discern thrips factors that facilitate virus infection of SGs and transmission by F. occidentalis, we used gel- and label-free quantitative and qualitative proteomics to address two hypotheses: (i) TSWV infection modifies the composition and/or abundance of SG-expressed proteins in adults; and (ii) TSWV has a differential effect on the male and female SG proteome and secreted saliva. Our study revealed a sex-biased SG proteome for F. occidentalis, and TSWV infection modulated the SG proteome in a sex-dependent manner as evident by the number, differential abundance, identities and generalized roles of the proteins. Male SGs exhibited a larger proteomic response to the virus than female SGs. Intracellular processes modulated by TSWV in males indicated perturbation of SG cytoskeletal networks and cell-cell interactions, i.e., basement membrane (BM) and extracellular matrix (ECM) proteins, and subcellular processes consistent with a metabolic slow-down under infection. Several differentially-abundant proteins in infected male SGs play critical roles in viral life cycles of other host-virus pathosystems. In females, TSWV modulated processes consistent with tissue integrity and active translational and transcriptional regulation. A core set of proteins known for their roles in plant cell-wall degradation and protein metabolism were identified in saliva of both sexes, regardless of virus infection status. Saliva proteins secreted by TSWV-infected adults indicated energy generation, consumption and protein turnover, with an enrichment of cytoskeletal/BM/ECM proteins and tricarboxylic acid cycle proteins in male and female saliva, respectively. The nonstructural TSWV protein NSs - a multifunctional viral effector protein reported to target plant defenses against TSWV and thrips - was identified in female saliva. This study represents the first description of the SG proteome and secretome of a thysanopteran and provides many candidate proteins to further unravel the complex interplay between the virus, insect vector, and plant host.

Development of an IPM Strategy for Thrips and Tomato spotted wilt virus in Processing Tomatoes in the Central Valley of California.

Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus; genus Orthotospovirus; family Tospoviridae) is a thrips-transmitted virus that can cause substantial economic losses to many crops, including tomato (Solanum lycopersicum). Since 2005, TSWV emerged as an economically important virus of processing tomatoes in the Central Valley of California, in part due to increased populations of the primary thrips vector, western flower thrips (WFT; Frankliniella occidentalis). To develop an understanding of the epidemiology of TSWV in this region, population densities of WFT and incidence of TSWV were monitored in California's processing tomato transplant-producing greenhouses and associated open fields from 2007 to 2013. Thrips were monitored with yellow sticky cards and in tomato flowers, whereas TSWV incidence was assessed with indicator plants and field surveys for virus symptoms. All thrips identified from processing tomato fields were WFT, and females were three-fold more abundant on sticky cards than males. Symptoms of TSWV infection were observed in all monitored processing tomato fields. Incidences of TSWV ranged from 1 to 20%, with highest incidence found in late-planted fields. There was no single primary inoculum source, and inoculum sources for thrips/TSWV varied depending on the production region. These results allowed us to develop a model for TSWV infection of processing tomatoes in the Central Valley of California. The model predicts that low levels of primary TSWV inoculum are amplified in early-planted tomatoes and other susceptible crops leading to highest levels of infection in later-planted fields, especially those with high thrips populations. Based upon these findings, an integrated pest management (IPM) strategy for TSWV in processing tomatoes in California was devised. This IPM strategy focuses on strategic field placement (identification of high-risk situations), planting TSWV- and thrips-free transplants, planting resistant varieties, monitoring for TSWV symptoms and thrips, roguing infected plants, thrips management targeting early generations, extensive sanitation after harvest, and strategic cropping to avoid overlap with winter bridge crops.

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.

Distribution and ecology of Frankliniella occidentalis (Thysanoptera: Thripidae) bacterial symbionts.

Bacterial populations in Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) collected in diverse California environments consisted of two bacterial symbionts: BFo-1 and BFo-2 (B = bacteria, Fo = Frankliniella occidentalis, numbers reflect different types). Dual infections of BFo-1 and BFo-2 were found in 50% of the thrips, 18% had neither bacterium, and 24 and 8% were infected solely with BFo-1 and BFo-2, respectively. No other bacteria consistently infected F. occidentalis. Dual infections occurred more often in male thrips and in thrips of both sexes from southern mountain and valley sites. As average collection year or month minimum temperature decreased, infections of BFo-1, alone or in dual infections, increased significantly. As yearly precipitation increased, infection with BFo-1 alone also increased. F. occidentalis color morphology did not affect bacterial infection. BFo-1 created weak biofilms at 25 and 32 degrees C; BFo-2 made strong biofilms at 25 degrees C and no biofilms at 32 degrees C. When the bacteria were grown in culture together, weak biofilms formed at both temperatures studied, although there was no way to determine what each bacterium contributed to the biofilm. BFo-1 and BFo-2 grew at similar rates at 25 and 30 degrees C. Our data show BFo-1 and BFo-2 occur in natural populations of F. occidentalis and support the hypothesis BFo have a symbiotic relationship with F. occidentalis. Regional differences in bacterial prevalence suggest bacterial infection is associated with environmental conditions, and altitude, temperature, and precipitation may be important factors.

A thrips vector of tomato spotted wilt virus responds to tomato acylsugar chemical diversity with reduced oviposition and virus inoculation.

There is increasing evidence that acylsugars deter insect pests and plant virus vectors, including the western flower thrips (WFT), Frankliniella occidentalis (Pergande), vector of tomato spotted wilt virus (TSWV). Acylsugars are sugar-polyesters composed of saturated, un-saturated, and variously branched short and long chain fatty acids (FAs) esterified to a glucose (acylglucose) or sucrose (acylsucrose) moiety. We sought to understand how acylsucrose amount and composition of associated FA profiles interacted to mediate resistance to WFT oviposition and TSWV inoculation on tomato leaves. Towards this goal, we examined WFT oviposition and TSWV inoculation behavior on tomato lines bred to exude varying amounts of acylsucrose in association with diverse FA profiles. Our data show that as acylsucrose amounts increased, WFT egg-laying (oviposition) decreased and TSWV inoculation was suppressed. Western flower thrips also responded to FA profiles that included iC4, iC11, nC12 and nC10 FA. These findings support improving acylsugar-mediated resistance against WFT by breeding tomatoes exuding greater amounts of acylsucrose associated with specific FA profiles. We show that increasing acylsucrose amount output by type IV trichomes and selecting for particular FA profiles through advanced breeding profoundly affects WFT behavior in ways that benefit management of WFT as direct pests and as TSWV vectors.

Characterization of bacterial symbionts in Frankliniella occidentalis (Pergande), Western flower thrips.

Many insects have associations with bacteria, although it is often difficult to determine the intricacies of the relationships. In one such case, facultative bacteria have been discovered in a major crop pest and virus vector, the Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Several bacterial isolates have been studied in Netherlands greenhouse thrips populations, with molecular data indicating that these bacteria were similar to Escherichia coli, although biochemical properties suggested these microbes might actually be most similar to plant pathogenic bacteria in the genus Erwinia. We focused on the bacterial flora of the Hawaiian Islands thrips population where these gut bacteria were first reported in 1989. We also analyzed a German population and a 1965 California population preserved in ethanol. Culture and culture-independent techniques revealed a consistent microflora that was similar to the Netherlands isolates studied. The similarity among thrips microbes from multiple populations and environments suggested these bacteria and their hosts share a widespread association. Molecular phylogeny based on the 16S rRNA gene and biochemical analysis of thrips bacteria suggested two distinctive groups of microbes are present in thrips. Phylogenetic analysis also revealed support for one thrips bacterial group having a shared ancestry with Erwinia, whereas the second group of thrips bacteria fell out with E. coli, but without support. Although species-specific relationships were indeterminable due to the conservative nature of 16S, there is strong indication that thrips symbionts belong to two different genera and originated from environmental microbes.

Acylsugar amount and fatty acid profile differentially suppress oviposition by western flower thrips, Frankliniella occidentalis, on tomato and interspecific hybrid flowers.

Tomatoes (Solanum lycopersicum L.) have been bred to exude higher amounts or different types of the specialized plant metabolites, acylsugars, from type IV trichomes. Acylsugars are known to deter several herbivorous insect pests, including the western flower thrips (WFT), Frankliniella occidentalis (Pergande); however, all previous studies investigated the effect of acylsugars on leaves, or acylsugar extracts obtained from leaves. In spite of the WFT predilection for flowers, there is a gap in knowledge about flower defenses against thrips damage. This is especially important in light of their capacity to acquire and inoculate viruses in the genus Orthotospovirus, such as Tomato spotted wilt orthotospovirus (TSWV), in flowers. Therefore, we turned our attention to assessing thrips oviposition differences on flowers of 14 entries, including 8 interspecific hybrids, 5 tomato lines bred for specific acylsugar-related characteristics (type IV trichome densities, acylsugar amount, sugar moiety and fatty acid profile), and a fresh market tomato hybrid, Mt. Spring, which only produces trace amounts of acylsugars. Our results show that the density of the acylsugar droplet bearing type IV trichomes is greatest on sepals, relative to other flower structures, and accordingly, WFT avoids oviposition on sepals in favor of trichome-sparse petals. In concordance with past studies, acylsugar amount was the most important acylsugar-related characteristic suppressing WFT oviposition. Certain acylsugar fatty acids, specifically i-C5, i-C9 and i-C11, were also significantly associated with changes in WFT oviposition. These results support continued breeding efforts to increase acylsugar amounts and explore modifications of fatty acid profile and their roles in deterring thrips oviposition. The finding that acylsugar production occurs and reduces thrips oviposition in tomato flowers will be important in efforts to use acylsugar-mediated resistance to reduce incidence of orthotospoviruses such as TSWV in tomato by deterring virus transmission and development of thrips vector populations in the crop.

Tospovirus-thrips interactions.

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

Heterologous expression of the Mi-1.2 gene from tomato confers resistance against nematodes but not aphids in eggplant.

The Mi-1.2 gene in tomato (Solanum lycopersicum) is a member of the nucleotide-binding leucine-rich repeat (NBLRR) class of plant resistance genes, and confers resistance against root-knot nematodes (Meloidogyne spp.), the potato aphid (Macrosiphum euphorbiae), and the sweet potato whitefly (Bemisia tabaci). Mi-1.2 mediates a rapid local defensive response at the site of infection, although the signaling and defensive pathways required for resistance are largely unknown. In this study, eggplant (S. melongena) was transformed with Mi-1.2 to determine whether this gene can function in a genetic background other than tomato. Eggplants that carried Mi-1.2 displayed resistance to the root-knot nematode Meloidogyne javanica but were fully susceptible to the potato aphid, whereas a susceptible tomato line transformed with the same transgene was resistant to nematodes and aphids. This study shows that Mi-1.2 can confer nematode resistance in another Solanaceous species. It also indicates that the requirements for Mi-mediated aphid and nematode resistance differ. Potentially, aphid resistance requires additional genes that are not conserved between tomato and eggplant.

Chimeric cDNA Sequences from Citrus tristeza virus Confer RNA Silencing-Mediated Resistance in Transgenic Nicotiana benthamiana Plants.

ABSTRACT RNA silencing has been shown to be an important mechanism for conferring resistance in transgenic, virus-resistant plants. We used this approach to evaluate resistance in Nicotiana benthamiana plants transformed with chimeric coding and noncoding sequences from Citrus tristeza virus (CTV). Several independent transgenic plant lines were generated, using two constructs (pCTV1 and pCTV2) designed to produce self-complementary transcripts. The pCTV1 contained cDNA sequences from the CTV capsid protein (CP), p20, and 3' untranslated region (UTR); and pCTV2 contained CP, p23, and 3' UTR sequences. Heterologous recombinant Potato virus X (PVX) containing either homologous or heterologous CTV sequences was used to challenge plants and resistance was evaluated phenotypically and validated with reverse-transcriptase polymerase chain reaction and northern hybridization analysis. Transgenic plants (T1 generation) for each construct showed resistance to recombinant PVX constructs used for challenge experiments when PVX contained p20 or UTR (for CTV1 plants), or p23 or UTR (for CTV2 plants). However, no resistance was seen when plants were challenged with PVX containing the CTV CP. T2 generation plants also showed resistance even when challenged with PVX containing the cognate CTV sequences obtained from heterologous CTV isolates. The presence of transgene-specific small interfering RNAs in the resistant CTV1 and CTV2 plants indicated that resistance was mediated by post-transcriptional gene silencing.