First Report of Natural Infection of Pea (Pisum sativum) by Tomato spotted wilt virus in Hungary.

In June of 2009, stem vascular necrosis, interveinal necrosis of upper leaves, wilting of flowers, and necrotic spots on the pods were observed on garden pea (Pisum sativum L. 'Rajnai törpe') in northeast Hungary. A mechanical transmissible plant virus designated Ps091 was isolated from leaves of severely affected plants. Pathological investigations demonstrated that Ps091 had a host range very similar to that of Tomato spotted wilt virus (TSWV). It caused necrotic local lesions on Chenopodium spp. and induced systemic yellowing and necrosis on the upper leaves of Nicotiana benthamiana, N. clevelandii, and N. glutinosa by mechanical inoculation. Typical symptoms of TSWV infection appeared on the top leaves of pepper (Capsicum annuum L. 'Albaregia') and tomato (Solanum lycopersicum 'Kecskeméti 3') inoculated with Ps091. For molecular identification, total nucleic acids were extracted from Ps091-infected tobacco with a standard phenol-chloroform extraction method (2), and reverse transcription-PCR was conducted with TSWV N-gene specific, own designed primers (TSWV-S for: 5'-CCCAGCATTATGGCAAGCC-3', TSWV-S rev: 5'-TGATCTGGTCGAGGTTTTCCGCTAGCCC-3'). A tobacco plant infected with a reference pepper isolate, TSWV-Ca1 (1), and a healthy tobacco plant served as positive and negative controls, respectively. An approximately 300-bp DNA fragment was amplified from tobacco infected with Ps091 and TSWV-Ca1. The Ps091 amplicon was cloned, sequenced in both directions, and the sequence was deposited in GenBank (Accession No. HQ615692). Blast search analysis showed that TSWV-Ps091 had the highest identity (99%) with TSWV-P170RB strain (GenBank Accession No. DQ431238) in the cognate region. Since the latter isolate is a resistance breaking (RB) strain on pepper, pathogenicity of Ps091 on TSWV resistant pepper and tomato lines was studied. Mechanically inoculated pepper (C. annuum × C. chinense TSR F4 line) and tomato (S. lycopersicum 'Stevens') genotypes carrying the Tsw and Sw5 resistance genes, respectively, reacted with necrotic local lesions, but no systemic infections were detected by applying bioassays to N. clevelandii. These results demonstrate that Ps091 does not belong to the RB strains of TSWV. Back inoculations to pea ('Rajnai törpe') resulted in necrotic local spots as well as systemic stem and top necrosis, proving the causal relationship between TSWV-Ps091 and the pea disease observed in the field. Although TSWV has been known to cause epidemy in solanaceous crops and tobacco, to our knowledge, this is the first report of its natural occurrence on a legume plant, particularly on pea in Hungary. Because of the extreme severity of the disease caused on pea and high infection pressure, TSWV is a new threat to pea production in this country, where pea is a very important crop. References: (1) P. Salamon et al. Page 23 in: Plant Protection Days. Budapest, February, 2010. (2) J. L. White and J. M. Kaper. J. Virol. Methods 23:83, 1989.

Synthesis of (-)-strand RNA from the 3' untranslated region of plant viral genomes expressed in transgenic plants upon infection with related viruses.

When expressed in transgenic tobacco plants, transgene mRNA that includes the 3' untranslated region (3' UTR) of Lettuce mosaic virus served as template for synthesis of complementary (-)-strand RNA following an infection by Tobacco etch virus, Tobacco vein mottle virus or Pepper mottle virus, but not when infected with Cucumber mosaic virus. Deletion of the 3' UTR from the transgene abolished the synthesis of (-)-strand transcripts. Similar results were obtained in transgenic tobacco plants expressing mRNA that includes the RNA3 3' UTR of Cucumber mosaic virus when infected with Tomato aspermy virus. These results show that the viral RNA-dependent RNA polymerase of several potyviruses and Tomato aspermy virus have the ability to recognize heterologous 3' UTRs when included in transgene mRNAs, and to use them as transcription promoters.

Molecular evidence for the existence of two distinct subgroups in cucumber mosaic cucumovirus.

Infectious full-length cDNA clones from the genomic RNAs of a subgroup II cucumber mosaic cucumovirus strain (Trk7) were obtained. Sequence analysis of the whole genome revealed strong homology (99%) to the genome of Q-CMV, the only subgroup II strain whose entire genomic nucleotide sequence had been available in the database, and an overall 75% homology to those of subgroup I strains. We provide sequence comparisons of different parts of 1a, 2a and 2b proteins of Cucumovirus species, and propose phylogenetic trees based on these protein sequences.

Stunting induced by cucumber mosaic cucumovirus-infected Nicotiana glutinosa is determined by a single amino acid residue in the coat protein.

R-CMV, a subgroup II strain of cucumber mosaic cucumovirus (CMV) induces a very strong stunting response in Nicotiana glutinosa plants, while Trk7-CMV causes green mosaic in this host. The genetic determinant of this phenotype was mapped to a 534-nucleotide region at the 3' end of RNA3 with biologically active, full-length cDNA clones of R-CMV and Trk7-CMV and RNA3 chimeras of the two strains. Within this region, R-CMV differs from Trk7-CMV by a single amino acid at position 193 in the coat protein. Changing the codon for Lys at this position to Asn or Ser, by site-directed mutagenesis, also changed the phenotype of the viruses from green mosaic to induction of stunting. Profound differences in both the spread and the accumulation of the viruses causing stunting and green mosaic were observed, although these did not correlate with the host specificity of stunting. Since expression of R-CMV coat protein with the PVX vector did not cause stunting, the data suggest that the presence of other CMV components is necessary for the induction of this symptom.

Biological properties of pseudorecombinant and recombinant strains created with cucumber mosaic virus and tomato aspermy virus.

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) are closely related cucumoviruses. We have made pseudorecombinant viruses in which the RNAs 3 of these two viruses have been exchanged and recombinant viruses containing chimeric RNA 3 molecules, in which the coat proteins and the 3'-end regions of CMV and TAV have been exchanged, giving rise to recombinants designated RT3 and TR3. The replication properties and the cell-to-cell and long-distance movement patterns of these pseudorecombinant and recombinant viruses were examined in different hosts. All the viruses were able to replicate and accumulate RNA 4 in protoplasts. The pseudorecombinants and the R1R2RT3 recombinant infected tobacco systemically, but the R1R2TR3 recombinant was not detectable, even in the inoculated leaves. Comparison of the abilities of the viruses to replicate in protoplasts and intact cucumber plants suggests that cell-to-cell movement factors are also encoded by RNAs 1 and/or 2. Major determinants of symptom severity in Nicotiana glutinosa are localized on the 3' part of RNA 3, and in Nicotiana benthamiana, more severe symptoms were observed with the T1T2R3 strain than with the others tested.

Increase in the relative fitness of a plant virus RNA associated with its recombinant nature.

A recombination event at the 3' end of RNA 3 in a pseudorecombinant virus (C1C2T3) having RNAs 1 and 2 from TrK7-cucumber mosaic virus (CMV) and RNA 3 from P-tomato aspermy virus (TAV) was detected by ribonuclease protection assay (RPA). Sequence analysis of the 3' end of RNA 3 of C1C2T3 and of its parental CMV and TAV strains showed that RNA 3 of C1C2T3 had a hybrid nature in which most of the 3' noncoding region (3' ncr), including the whole 3' terminal tRNA-like structure, was derived from CMV RNA 2 by recombination. Recombination may have occurred in two steps. The first one would have caused the duplication of a large region 5' to the 3' end tRNA-like structure, derived from TAV and from CMV. The second one would have eliminated the TAV-derived sequences in this duplicated region. Competition experiments in tobacco plants showed that in a context of RNA 1 + 2 from CMV, RNA 3 from TAV is outcompleted by RNA 3 of CMV, but the recombinant TAV RNA 3 with the 3' end of CMV outcompetes both TAV and CMV RNA 3. This shows an increase in relative fitness associated with the recombinant nature of RNA 3. Our results document the potential importance of RNA-RNA recombination in the determination of the genetic structure of RNA viral populations.

Nucleotide sequence and infectious in vitro transcripts of RNA 3 of tomato aspermy virus pepper isolate.

The nucleotide sequence of a cDNA clone corresponding to the entire RNA 3 of a pepper isolate of tomato aspermy cucumovirus (P-TAV) was determined. The 2222 nucleotides (nt) long RNA 3 is dicistronic and codes a 3a (280 amino acids) and a coat protein (cp, 218 amino acids). The nucleotide sequence of P-TAV RNA 3 shows a high sequence identity (99%) with that of C-TAV RNA 3 and the amino acid sequences of the 3a and the coat proteins between these two TAV strains are also very similar. The transcription start site of the subgenomic RNA 4 was mapped to the G at the position of 1148 of RNA 3. A full-length cDNA copy of P-TAV RNA-3 was cloned downstream of the phage T7 promoter. Transcripts synthesized in vitro had no extra nucleotides at the 5' end, but there were nine non-viral nucleotides at the 3' terminus. The transcripts of TAV RNA 3 were infectious when coinoculated into test plants in the presence of purified RNA 1 and 2 of P-TAV or Trk7-CMV respectively. Northern blot analysis of RNA extracted from inoculated plants showed the expected genomic and subgenomic RNAs.

Complete nucleotide sequence of the RNA 3 from subgroup II of cucumber mosaic virus (CMV) strain: Trk7.

The complete nucleotide sequence (2209 nucleotides) of the RNA 3 of the cucumber mosaic virus strain Trk7 was determined. Trk7-CMV possesses two open reading frames which encode the 3a protein (279 amino acids) and the coat protein (218 amino acids). Sequence analysis proved the earlier biological and serological classification of the RNA 3 molecule of Trk7-CMV in subgroup II. A high degree of homology was found in the strains Q and Kin of CMV, while the average similarity between the two different subgroups is much lower.

Expression of homologous and heterologous viral coat protein-encoding genes using recombinant DI RNA from cymbidium ringspot tombusvirus.

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CyRSV) was tested as a potential RNA vector. The coat protein-encoding gene (CP) of the same virus or of the unrelated tomato aspermy cucumovirus (TAV) was inserted in a biologically active clone of CyRSV DI-3 RNA. Both homologous and heterologous CP genes were inserted into DI-3 cDNA clone in two different positions. The CyRSV CP was expressed only in the leaves which were inoculated with DI-3CPWt plus CP-less mutant helper virus (delta ABgII). Chimaeric DI RNA carrying a heterologous CP gene (DI-3HCPtav) was able to replicate and express the inserted TAV CP in the presence of a wild-type (wt) helper genome. Both constructs, which were stable and active for gene expression, carried the inserted CP genes in the same position, between the A and B blocks of DI-3 RNA. Other constructs in which the CP were cloned between the B and C blocks of DI-3 RNA were not able to direct the translation of the encoded CP. The expression level of CP derived from recombinant DI RNA was lower relative to expression of CP from wt virus infection.