Complete genome sequence of datura leaf curl virus, a novel begomovirus infecting Datura innoxia in Sudan, related to begomoviruses causing tomato yellow leaf curl disease.

Several species in the genus Datura (family Solanaceae) are hosts for begomoviruses (family Geminiviridae), both in the New World and the Old World. Here, we report the genome characterization of two isolates of a novel Old World monopartite begomovirus infecting Datura innoxia in Sudan, which we propose to be named "datura leaf curl virus" (DaLCV). Pairwise sequence comparisons and phylogenetic analysis showed that DaLCV isolates are related to begomoviruses causing tomato yellow leaf curl disease, a global menace for tomato crops. Thus, the novel begomovirus could pose an additional threat to tomato cultivation in Africa.

Complete genome sequence and intracellular protein localization of Datura yellow vein nucleorhabdovirus.

A limited number of plant rhabdovirus genomes have been fully sequenced, making taxonomic classification, evolutionary analysis and molecular characterization of this virus group difficult. We have for the first time determined the complete genome sequence of 13,188 nucleotides of Datura yellow vein nucleorhabdovirus (DYVV). DYVV genome organization resembles that of its closest relative, Sonchus yellow net virus (SYNV), with six ORFs in antigenomic orientation, separated by highly conserved intergenic regions and flanked by complementary 3' leader and 5' trailer sequences. As is typical for nucleorhabdoviruses, all viral proteins, except the glycoprotein, which is targeted to the endoplasmic reticulum, are localized to the nucleus. Nucleocapsid (N) protein, matrix (M) protein and polymerase, as components of nuclear viroplasms during replication, have predicted strong canonical nuclear localization signals, and N and M proteins exclusively localize to the nucleus when transiently expressed as GFP fusions. As in all nucleorhabdoviruses studied so far, N and phosphoprotein P interact when co-expressed, significantly increasing P nuclear localization in the presence of N protein. This research adds to the list of complete genomes of plant-infecting rhabdoviruses, provides molecular tools for further characterization and supports classification of DYVV as a nucleorhabdovirus closely related to but with some distinct differences from SYNV.

Complementation between two tospoviruses facilitates the systemic movement of a plant virus silencing suppressor in an otherwise restrictive host.

BACKGROUND: New viruses pathogenic to plants continue to emerge due to mutation, recombination, or reassortment among genomic segments among individual viruses. Tospoviruses cause significant economic damage to a wide range of crops in many parts of the world. The genetic or molecular basis of the continued emergence of new tospoviruses and new hosts is not well understood though it is generally accepted that reassortment and/or genetic complementation among the three genomic segments of individual viruses could be contributing to this variability since plants infected with more than one tospovirus are not uncommon in nature. METHODOLOGY/PRINCIPAL FINDINGS: Two distinct and economically important tospoviruses, Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV), were investigated for inter-virus interactions at the molecular level in dually-infected plants. Datura (Datura stramonium) is a permissive host for TSWV, while it restricts the movement of IYSV to inoculated leaves. In plants infected with both viruses, however, TSWV facilitated the selective movement of the viral gene silencing suppressor (NSs) gene of IYSV to the younger, uninoculated leaves. The small RNA expression profiles of IYSV and TSWV in single- and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). No TSWV NSs-specific siRNAs were detected either in the inoculated or systemic leaves of dually-infected datura plants indicating a more efficient suppression of host silencing machinery in the presence of NSs from both viruses as compared to the presence of only TSWV NSs. CONCLUSION/SIGNIFICANCE: Our study identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the importance of virally-coded suppressors of gene silencing in virus infection of plants. This is the first experimental evidence of functional complementation between two distinct tospoviruses in the Bunyaviridae family.

Characterization of the necrosis determinant of the European genotype of pepino mosaic virus by site-specific mutagenesis of an infectious cDNA clone.

Mild and necrotic isolates have been described for the European (EU) genotype of pepino mosaic virus (PepMV), an important pathogen of tomato worldwide. In this study, we produced various infectious cDNA clones of an EU isolate with point mutations introduced by site-directed mutagenesis. Our results showed that the genetic determinant responsible for necrosis induction on tomato and Datura inoxia was amino acid 67 of TGBp3. This amino acid residue also acts as necrosis determinant in PepMV isolates belonging to the Chilean 2 genotype. This demonstrates that a single point mutation plays a role in necrosis induction by PepMV, irrespective of genotype.

Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan(®)MGB probes.

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan(®)MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidentalis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

Single mutation converts mild pathotype of the Pepino mosaic virus into necrotic one.

Pepino mosaic virus (PepMV) is a member of the Flexiviridae family, genus Potexvirus. PepMV isolates from the same genotype differ in the symptoms they induce in Datura inoxia and Solanum lycopersicum. Necrotic isolates cause necrosis on these plants whereas mild isolates are very often asymptomatic or induce mild mosaic. Sequence analysis of the genomes of mild (PepMV-P22) and necrotic (PepMV-P19) strains revealed that they differ by several nonsynonymous mutations. Eleven mutations are located in the RdRp coding region and one mutation is placed in the TGB3 coding region. To investigate whether these mutations affect the symptom induction, both PepMV-P22 and PepMV-P19 were mutagenized. As a result we found that the genetic determinant responsible for necrosis induction was amino acid 67 of TGB3. This showed that one point mutation is sufficient to alter the virus from mild to aggressive in tomato and D. inoxia.

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.

[TMV-infection localization and development of induced virus resistance in Nicotiana sanderae Hort., Datura stramonium L. and Datura metel]. / Lokalizatsiia VTM-infektsiï i rozvytok indukovanoï stiikosti u Nicotiana sanderae HORT., Datura stramonium L. ta Datura metel L.

The localization of virus infection and development of local and systemic induced resistance in plants Nicotiana sanderae Hort., Datura stramonium L. and D. metel L. infected by TMV have been studied. It was shown that acquired resistance to the second infection is induced in hypersensitive plants infected by TMV independent of localization mechanism. No definite correlation exists between these defense reactions.

[Effect of TMV-infection on protein and carbohydrate content in hypersensitive tobacco plants and their antiviral and hemagglutinating activity]. / Vplyv VTM-infektsiï na vmist bilkiv i vuhlevodiv u nadchutlyvykh roslynakh tiutiunu ta ïkhniu antyvirusnu i hemahliutynuval'nu aktyvnist'.

Changes in content of proteins and carbohydrates of sensitive and supersensitive tobacco and datura plants and their antiviral and hemagglutinating activity under tobacco mosaic virus infection were investigated. It was shown that the content of these substances was increased on early stages of virus infection in hypersensitive plants. Antiviral and hemagglutinating activity of the obtained substances was shown.

Detection of Tomato spotted wilt virus in its vector Frankliniella occidentalis by reverse transcription-polymerase chain reaction.

A method for rapid and reliable detection of Tomato spotted wilt virus (TSWV) (Tospovirus, Bunyaviridae) in its vector Frankliniella occidentalis (Thysanoptera Thripidae) would be a useful tool for studying the epidemiology of this virus. A RT-PCR method developed for this purpose is reported. The method was tested on thrips involved in laboratory transmission trials and on thrips collected in the field, whose capability to transmit TSWV was checked previously by leaf disk assays. The RT-PCR results were consistent with the results obtained by the leaf disk assays. Among thrips involved in laboratory experiments, 97% of the adults that transmitted TSWV were positive by RT-PCR; as did some non-transmitter adults reacted, whereas among field-collected thrips only the individuals able to transmit were positive by RT-PCR. In addition, healthy thrips were allowed to feed as adults on virus-infected leaves for 48 h, and then examined by RT-PCR immediately or after starving or feeding on virus-free plants for various times, to determine if virus ingested (but not transmissible) was also detectable. The virus was detectable immediately after the feed or within 12 and 24 h for individuals starved or fed on virus-free plants, respectively, but not after those periods. Thus, the method could detect rapidly and reliably the virus in vectors from the field, providing 24 h of starving to avoid positive RT-PCR results from thrips simply carrying the virus.