Alice K. Inoue-Nagata

Tissue tropism related to vector competence of Frankliniella occidentalis for tomato spotted wilt tospovirus

The development of tomato spotted wilt tospovirus (TSWV) infection in the midgut and salivary glands of transmitting and non-transmitting thrips, Frankliniella occidentalis, was studied to elucidate tissue tropism and the virus pathway within the body of this vector. Immunohistological techniques used in this study showed that the midgut, foregut and salivary glands were the only organs in which virus accumulated. The first signals of infection, observed as randomly distributed fluorescent granular spots, were found in the epithelial cells of the midgut, mainly restricted to the anterior region. The virus subsequently spread to the circular and longitudinal midgut muscle tissues, a process which occurred late in the larval stage. In the adult stage, the infection occurred in the visceral muscle tissues, covering the whole midgut and foregut, and was abolished in the midgut epithelium. The infection of the salivary glands was first observed 72 h post-acquisition, and simultaneously in the ligaments connecting the midgut with these glands. The salivary glands of transmitting individuals appeared heavily or completely infected, while no or only a low level of infection was found in the glands of non-transmitting individuals. Moreover, the development of an age-dependent midgut barrier against virus infection was observed in second instar larvae and adults. The results show that the establishment of TSWV infection in the various tissues and the potential of transmission seems to be regulated by different barriers and processes related to the metamorphosis of thrips.

Impeded Thrips Transmission of Defective Tomato spotted wilt virus Isolates

Two defective RNA-containing isolates (Pe-1 and 16-2) and an envelope-deficient (env ) isolate of Tomato spotted wilt virus (TSWV) were tested for their transmissibility by Frankliniella occidentalis. The Pe-1 isolate contained a truncated L RNA segment that barely interfered with symptom expression and replication of the wild-type (wt) L RNA segment. This isolate was transmitted with an efficiency of 51%, a value comparable to that found for wt TSWV (54%). Isolate 16-2, which contained a genuine defective interfering L RNA as concluded from its ability to suppress wt L RNA synthesis and attenuation of symptom expression, was not transmitted at all. The midguts of all larvae that ingested Pe-1 became infected, whereas limited midgut infections were found in 24% of the larvae that ingested 16-2. This difference in infection could be explained by the presence of a low number of infectious units in the inoculum ingested from plants as demonstrated in infection experiments and verified by northern blot analysis. The env isolate failed to infect the midgut after ingestion and could not be transmitted by any thrips stage. This isolate also cannot infect primary thrips cell cultures. Taken together, these results suggest that the envelope of TSWV contains the determinants required for binding and subsequent infection of thrips cells.

Molecular and biological characterization of Tomato chlorotic mottle virus suggests that recombination underlies the evolution and diversity of Brazilian tomato begomoviruses

ABSTRACT Tomato chlorotic mottle virus (ToCMoV) is an emerging begomovirus species widely distributed throughout tomato-growing regions of Brazil. ToCMoV appears to have expanded its geographic range recently, invading tomato-growing areas that were free of begomovirus infection before 2004. We have determined the first complete genome sequence of an infectious ToCMoV genome (isolate BA-Se1), which is the first begomovirus species isolated in the northeast of Brazil. When introduced by particle bombardment into tomato, the cloned ToCMoV-[BA-Se1] DNA-A and DNA-B components caused typical chlorotic mottle symptoms. The cloned virus was whitefly-transmissible and, although it was infectious in hosts such as Nicotiana benthamiana, pepper, tobacco, and Nicandra physaloides, it was unable to infect Arabidopsis thaliana, bean, N. glutinosa, and Datura metel. Sequence and biological analyses indicate that ToCMoV-[BA-Se1] is a typical New World begomovirus sp. requiring both DNA-A and DNA-B components to establish systemic infections. Although evidence of multiple recombination events was detected within the ToCMoV-[BA-Se1] DNA-A, they apparently occurred relatively long ago, implying that recombination probably has not contributed to the recent emergence of this species.

A Study of Weeds as Potential Inoculum Sources for a Tomato-Infecting Begomovirus in Central Brazil

Tomato severe rugose virus (ToSRV) is the most important begomovirus species in Brazilian tomato production. Many weeds are associated with tomato, and some are hosts of begomoviruses. Only one species of weed, Nicandra physaloides, has been found to be infected with ToSRV. In this study, four weed species were investigated for their capacity to be infected by ToSRV and serve as a potential source of inoculum for tomato. Begomoviruses from naturally infected Crotalaria spp., Euphorbia heterophylla, N. physaloides, and Sida spp. were successfully transferred to tomato plants by biolistic inoculation. ToSRV was the major virus transferred to tomato. In contrast, other begomoviruses were transferred to weeds, such as Sida micrantha mosaic virus and Euphorbia yellow mosaic virus. Furthermore, a new strain of Sida micrantha mosaic virus is reported. We also confirmed that Crotalaria spp., E. heterophylla, and Sida spp. are infected with ToSRV but at low viral titers and in mixed infections with weed-infecting begomoviruses. Thus, it was demonstrated that weeds are potential sources of ToSRV for tomato in central Brazil.

Effects of Temperature and Host on the Generation of Tomato Spotted Wilt Virus Defective Interfering RNAs

ABSTRACT The generation of defective interfering (DI) RNA molecules of tomato spotted wilt tospovirus (TSWV) was studied by serially passaging in-ocula from plant to plant under different controlled conditions. DI RNAs were generated at higher rates in plants at 16 degrees C than in plants incubated at higher temperatures. Another factor promoting the TSWV DI RNA generation was the use of high virus concentrations in the inocula. The solanaceous species Capsicum annuum, Datura stramonium, Lycopersicon esculentum, Nicotiana benthamiana, and N. rustica supported the generation of DI RNAs, whereas the virus recovered from the inoculated composite species, Emilia sonchifolia, remained free of any DI RNA under all conditions tested. This study resulted in a strategy to maintain DI RNA-free TSWV isolates, as well as in an efficient way to produce a large population of different DI RNA species. A single DI RNA species usually became dominant in an isolate after a few rounds of serial inoculations. The possible mechanisms involved in TSWV DI RNA generation under different inoculation circumstances are discussed.

First Report of Natural Occurrence of Zucchini Lethal Chlorosis Tospovirus on Cucumber and Chrysanthemum Stem Necrosis Tospovirus on Tomato in Brazil

During a field survey in 1994, five cucumber (Cucumis sativus) cv. Hokushin plants showing symptom of yellowing, mottling, and vein banding on the leaves were collected from a commercial field of the Federal District. By electron microscopy, quasi-spherical particles with double membrane, typical tospovirus-like particles were found in the infected leaf material. All samples strongly reacted with antibody of zucchini lethal chlorosis tospovirus (ZLCV), but not with antibodies of other to-spoviruses reported in Brazil (1): tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), groundnut ringspot virus (GRSV), chrysanthemum stem necrosis virus (CSNV), or iris yellow spot virusonion isolate (IYSV-BR). The virus was identified as ZLCV, which was first isolated in 1994 from zucchini (Cucurbita pepo) in São Paulo State, Brazil. Tomato (Lycopersicon esculentum) plants showing stem necrosis and necrotic spots and rings on the leaves were collected in Viçosa, Minas Gerais State. By electron microscopy, molecular studies, and enzyme-linked immunosorbent assay with antibodies of the six tospoviruses occurring in Brazil, the virus was identified as CSNV. This virus was first reported in 1995 on a Chrysanthemum sp. in São Paulo State and recently reported in the Netherlands from Dendranthema indicum. This is the first report of the natural occurrence of ZLCV and CSNV on cucumber and tomato, respectively. Reference: (1) A. C. de Ávila et al. 1998. Pages 32-34 in: Int. Symp. on Tospoviruses and Thrips in Floral and Vegetable Crops, 4th.

Development of a locus-specific, co-dominant SCAR marker for assisted-selection of the Sw-5 (Tospovirus resistance) gene cluster in a wide range of tomato accessions

The best levels of broad-spectrum Tospovirus resistance reported in tomatoes thus far are conferred by the Sw-5 locus. This locus contains at least five paralogues (denoted Sw-5a through Sw-5e), of which Sw-5b represents the actual resistance gene. Here we evaluated a panel of seven PCR primer pairs matching different sequences within a genomic region spanning the Sw-5a and Sw-5b gene cluster. Primer efficiency evaluation was done employing tomato isolines with and without the Sw-5 locus. One primer pair produced a single and co-dominant polymorphism between susceptible and resistant isolines. Sequence analysis of these amplicons indicated that they were specific for the Sw-5 locus and their differences were due to insertions/deletions. The polymorphic SCAR amplicon encompass a conserved sequence of the promoter region of the functional Sw-5b gene, being located in the position −31 from its open reading frame. This primer pair was also evaluated in field assays and with a collection of accessions known to be either susceptible or resistant to tospoviruses. An almost complete correlation was found between resistance under greenhouse/field conditions and the presence of the marker. Therefore, this primer pair is a very useful tool in marker-assisted selection systems in a large range of tomato accessions.

Sustained NIK-mediated antiviral signalling confers broad-spectrum tolerance to begomoviruses in cultivated plants.

Begomovirus-associated epidemics currently threaten tomato production worldwide due to the emergence of highly pathogenic virus species and the proliferation of a whitefly B biotype vector that is adapted to tomato. To generate an efficient defence against begomovirus, we modulated the activity of the immune defence receptor nuclear shuttle protein (NSP)-interacting kinase (NIK) in tomato plants; NIK is a virulence target of the begomovirus NSP during infection. Mutation of T474 within the kinase activation loop promoted the constitutive activation of NIK-mediated defences, resulting in the down-regulation of translation-related genes and the suppression of global translation. Consistent with these findings, transgenic lines harbouring an activating mutation (T474D) were tolerant to the tomato-infecting begomoviruses ToYSV and ToSRV. This phenotype was associated with reduced loading of coat protein viral mRNA in actively translating polysomes, lower infection efficiency and reduced accumulation of viral DNA in systemic leaves. Our results also add some relevant insights into the mechanism underlying the NIK-mediated defence. We observed that the mock-inoculated T474D-overexpressing lines showed a constitutively infected wild-type transcriptome, indicating that the activation of the NIK-mediated signalling pathway triggers a typical response to begomovirus infection. In addition, the gain-of-function mutant T474D could sustain an activated NIK-mediated antiviral response in the absence of the virus, further confirming that phosphorylation of Thr-474 is the crucial event that leads to the activation of the kinase.

Molecular and biological characterization of a new Brazilian begomovirus, euphorbia yellow mosaic virus (EuYMV), infecting Euphorbia heterophylla plants

To date, no begomovirus has been fully characterized from Euphorbia heterophylla, a widely distributed weed, in Brazil. Here, we show the occurrence of a new begomovirus on E. heterophylla plants showing bright yellow mosaic. The bipartite viral genome was cloned from 10 samples, and all clones are almost identical to each other (95.6-98.8% nucleotide sequence identity). The DNA-A sequences shared a maximum nucleotide sequence identity of 87.3% with euphorbia mosaic Peru virus (EuMPV) and thus were classified as belonging to a novel begomovirus species, tentatively named Euphorbia yellow mosaic virus (EuYMV). The EuYMV DNA-B sequences share a maximum nucleotide sequence identity of 56.2% with a euphorbia mosaic virus (EuMV) isolate from Mexico. Phylogenetic analysis demonstrated that this new virus belongs to a different lineage than EuMV isolates from Central America.

High incidence of Tomato chlorosis virus alone and in mixed infection with begomoviruses in two tomato fields in the Federal District and Goiás state, Brazil

Tomato chlorosis virus (ToCV), a species in the Crinivirus genus, was first reported in tomatoes in Brazil (state of Sao Paulo) in 2008. This was followed by reports in several other Brazilian states. Tomato plants with chlorotic spots and leaf roll symptoms are frequently observed in tomato fields with high whitefly populations in Central Brazil. These plants could be infected with a begomovirus, a crinivirus, or with both viruses. A survey of two selected tomato fields in the Federal District and Goias State was conducted in 2012 and 2013 specifically to determine the occurrence of begomoviruses and criniviruses. A total of 150 samples were collected and subjected to RT-PCR for ToCV detection and PCR for begomovirus detection. About 48% of the tested plants were infected with both viruses, 32% were infected with ToCV alone and 20% were only infected with the begomovirus ToSRV. The increasing incidence of ToCV associated with high whitefly populations in the field highlights the need for studying this virus disease to clarify its impact on tomato crops and minimize its potential damage.