Tomato Yellow Ring Virus Research Articles

DETECTION OF EIGHT DIFFERENT TOSPOVIRUS SPECIES BY A MONOCLONAL ANTIBODY AGAINST THE COMMON EPITOPE OF NSS PROTEIN

Rabbit antisera against the nucleocapsid protein (NP) have been commonly used for detection of tospoviruses and classification into serogroups or serotypes. Mouse monoclonal antibodies (MAbs) with high specificity to the NPs have also been widely used to identify tospovirus species. Recently, a serogroup-specific MAb against the NSs protein of Watermelon silver mottle virus (WSMoV) was produced by our laboratory to react with five members of WSMoV serogroup, i.e., WSMoV, Capsicum chlorosis virus (CaCV), Calla lily chlorotic spot virus (CCSV), Peanut bud necrosis virus (PBNV) and Watermelon bud necrosis virus (WBNV). The epitope recognized by the NSs MAb was determined and the comparison with the reported sequences of tospoviral NSs proteins revealed that the epitope is highly conserved at the N-terminal region of NSs proteins among members of WSMoV and Iris yellow spot virus (IYSV) serogroups, and Melon yellow spot virus (MYSV) serotype. When the NSs MAb was further used to react with the crude antigens of MYSV serotype, IYSV and Tomato yellow ring virus (TYRV) of IYSV serogroup, strong serological reactions, both in ELISA and western blotting, were observed. Thus, our results indicated that the NSs MAb is a useful and convenient tool for detection of the eight tospovirus species. It is also suggested that these eight Asian-type tospoviruses, i.e., WSMoV, CaCV, CCSV, PBNV, WBNV, MYSV, IYSV and TYRV, may share a common evolutionary ancestor.

Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

RNAi-Mediated Transgenic Tospovirus Resistance Broken by IntraspeciesSilencing Suppressor Protein Complementation

Extension of an inverted repeat transgene cassette, containing partial nucleoprotein (N) gene sequences from four different tomato-infecting Tospovirus spp. with a partial N gene sequence from the tomato strain of Tomato yellow ring virus (TYRV-t), renders transgenic Nicotiana benthamiana plants additionally resistant to this strain but not to the soybean strain of this Tospovirus sp. (TYRV-s), both strains exhibiting 14.4% nucleotide sequence divergence in their N genes. Surprisingly, coinoculation of the TYRV-t-resistant transgenic lines with both TYRV-t and TYRV-s resulted in rescue of the former. Mass-spectrometric analysis of the viral ribonucleocapsids accumulating in the transgenic plants showed the presence of the N proteins of both strains excluding hetero-encapsidation as rescue mechanism and indicating suppression of TYRV-t N gene transcript breakdown by RNA interference. Prior (Potato virus X [PVX]-vector-mediated) expression of the TYRV-s silencing suppressor (NS(s)) gene also allowed TYRV-t to break the resistance. This phenomenon was also observed when the homologous (TYRV-t) NS(s) gene was provided from a PVX replicon, demonstrating that TYRV can break RNA-mediated host resistance upon a priori expression of its NS(s) protein. Remarkably, mixed inoculation of TYRV-t with other Tospovirus spp. or nonrelated viruses did not result in resistance breaking, indicating that the rescuing activity of NS(s)-though based on suppressing RNA silencing-is species-dependent.

A member of a new Tospovirus species isolated in Italy from wild buckwheat (Polygonum convolvulus)

Electron microscopy of extracts from diseased Polygonum convolvulus plants from Piedmont (Italy) revealed particles with the morphological features of a tospovirus. Sequencing of the full-length small (S) and medium (M) genome segments indicated that the virus is a member of a new Tospovirus species provisionally named Polygonum ringspot virus. A feature distinguishing it from members of other Tospovirus species was the presence of a very short intergenic region on the S segment lacking the potential for formation of the predicted hairpin structure involved in subgenomic expression. Antibodies made against purified nucleocapsids allowed serological comparison with other tospovirus isolates and revealed a relationship with tomato yellow ring virus, and to a lesser extent, to iris yellow spot virus. Serological tests detected the virus in various locations in northern and central Italy. The experimental host range was wide, although in nature the virus appeared restricted to two Polygonum species.

Detection and Characterization of a Distinct Isolate of Tomato yellow fruit ring virus from Potato.

A distinct isolate (TY-PF36) of Tomato yellow fruit ring virus (TYFRV) was obtained from potato (Solanum tuberosum) in Iran. Chlorosis and necrotic spots on leaves associated with leaf and stem necrosis symptoms appear on the affected plants. Of 32 plant species and cultivars mechanically inoculated, 24 were susceptible to the virus isolate. The isolate strongly reacted with TYFRV antibodies in enzyme-linked immunosorbent assay (ELISA), but not with the specific antibodies of other tospoviruses tested. TYFRV-specific N gene primers described previously, however, failed to produce DNA fragments from the total RNA extracts of the infected plants in reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the complete N gene and partial L gene of this isolate revealed considerable differences to those reported for TYFRV with identities ranging from 83.9 to 84.2% and 84.9 to 85.4%, respectively. Two specific primers were designed for detecting TY-PF36 using RT-PCR; TY-PF36 was detected in symptomatic field samples of potato, peanut, soybean, and two weed species, Datura metel and D. stramonium.

Incidence and Distribution of Tomato yellow fruit ring virus on Soybean in Iran

Incidence and Distribution of Tomato yellow fruit ring virus on Soybean in Iran

Incidence and Distribution of Important Viral Pathogens in Some Iranian Potato Fields

From a total of 8,135 potato leaves collected from 132 fields in 11 provinces of Iran, the incidence and distribution of Alfalfa mosaic virus (AlMV), Eggplant mottled dwarf virus (EMDV), Potato leafroll virus (PLRV), Potato virus A (PVA), Potato virus M (PVM), Potato virus S(PVS), Potato virus X (PVX), Potato virus Y (PVY), and Tomato yellow fruit ring virus (TYFRV) were assessed using serological and biological methods. Based on enzyme-linked immunosorbent assay (ELISA) results, viruses in decreasing order of incidence in potato were PVS (35.9%), PVY (34.4%), PVA (27.0%), PVX (20.8%), PLRV (13.9%), PVM (9.0%), AlMV (7.0%), TYFRV (5.9%), and EMDV (5.1%). All 132 fields surveyed had some degree of virus infection, ranging from 28.8 to 98.6%, with an overall incidence of 75.2%. The highest and lowest incidence of virus infections among the surveyed provinces occurred in Kerman (93.2%) and Ardabil (56.7%), respectively. Overall, 25.0 and 50.2% of the collected potato samples had single or mixed infections, respectively. High levels of mixed infections were found between PVX and PVS (8.6%), and PVX and PVY (7.6%). Moreover, co-infection of samples with PVS and PVY, PVA and PVS, and PVA and PVY, the aphid-vectored virus/virus combinations, occurred at the highest incidence in almost all provinces surveyed, 15.3, 13.8, and 12.8%, respectively. In this study, Beet curly top virus was detected in symptomatic potato samples collected from some fields in the Kermanshah province.

Characterisation of cineraria strain ofTomato yellow ring virusfrom Iran

A tospovirus isolated from naturally infected cineraria plants in commercial greenhouses in Shiraz, Iran, induced rings and chlorotic and necrotic spots on leaves, growth reduction and death of the plants. The viruswas transmitted by three populations of Thrips tabaci. Antisera were produced against purified virions and viral nucleocapsids and used to study the virus host range and relationships. On the basis of enzyme-linked immunosorbent assay, tomato, anemone, Arum sp., dandelion and pepper were natural hosts of the virus. SDS-PAGE revealed three major structural proteins corresponding to the Tospovirus N, G1 and G2 proteins. Three RNA segments were resolved in a 1% agarose gel with molecular masses of 1.02×106 Da, 1.87×106 Da and 2.86×106 Da, corresponding to the virus S, M and L RNAs, respectively. No serological relationships were detected between the cineraria virus and Tomato spotted wilt virus, Tomato chlorotic spot virus and Groundnut ring spot virus using the virus nucleocapsid IgG in dot immunobinding assays. Further characterisation by nucleotide sequencing of the virus N gene revealed that it was a strain of Tomato yellow ring virus, although the cineraria isolate appears to have a different experimental host range compared to tomato isolates of the virus.

Molecular and biological comparison of two Tomato yellow ring virus (TYRV) isolates: challenging the Tospovirus species concept

Two strains of Tomato yellow ring virus (TYRV, genus Tospovirus), one from tomato (referred to as TYRV-t) and the other from soybean and potato (denoted TYRV-s), collected from different geographical regions in Iran, were compared. Their genomic S RNA segments differed in size by 55 nucleotides. Comparison of the S RNA intergenic regions revealed the absence of a stretch of 115 nucleotides within the S RNA segment of TYRV-s and, conversely, of 56 nts in that of TYRV-t, apparently a stable genetic difference as it was also found in another isolate of TYRV-s collected from potato. Sequence comparison of the N protein ORFs revealed an identity of 92% between the N proteins of both strains, and the observed strong cross-reaction of TYRV-s in DAS-ELISA with a polyclonal antiserum directed against the TYRV-t N protein confirmed this high identity. Host range analysis revealed several differences, e.g. TYRV-s, but not TYRV-t, being able to systemically infect Nicotiana species, and TYRV-s being localised in tomato. The observed molecular and biological differences of both viruses call into question the currently used criteria for Tospovirus species demarcation.

A New Tomato-Infecting Tospovirus from Iran

ABSTRACT A new tospovirus species serologically distinct from all other established tospoviruses was found in tomato in Iran. Typical disease symptoms observed include necrotic lesions on the leaves and yellow ring spots on the fruits, hence the name Tomato yellow ring virus (TYRV) was proposed. The S RNA of this virus was cloned and its 3,061 nucleotide long sequence showed features characteristic for tospoviral S RNA segments. The nucleocapsid (N) protein with a predicted Mr of 30.0 kDa showed closest relationship to the N protein of Iris yellow spot virus (74% sequence identity).