A Novel Distinct Genetic Variant of Tomato Torrado Virus with Substantially Shorter RNA1-Specific 3'Untranslated Region (3'UTR).

Tomato torrado virus (ToTV) induces severe systemic necrosis in Solanum lycopersicum. This work aimed at describing the genetic variability of necrosis-inducing ToTV-Wal'17 collected in 2017, derived from the ToTV-Wal'03 after long-term passages in plants. Sequence analyses of the ToTV-Wal'17 indicated twenty-eight single nucleotide substitutions in coding sequence of both RNAs, twelve of which resulted in amino acid changes in viral polyproteins. Moreover the sequencing data revealed that the 3'UTR of ToTV-Wal'17 RNA1 was 394 nts shorter in comparison to Wal'03. The performed sequence analyses revealed that 3'UTR of RNA1 of ToTV-Wal'17 is the most divergent across all previously described European isolates.

Analysis of the Role of Bradysia impatiens (Diptera: Sciaridae) as a Vector Transmitting Peanut Stunt Virus on the Model Plant Nicotiana benthamiana.

Bradysia species, commonly known as fungus gnats, are ubiquitous in greenhouses, nurseries of horticultural plants, and commercial mushroom houses, causing significant economic losses. Moreover, the insects from the Bradysia genus have a well-documented role in plant pathogenic fungi transmission. Here, a study on the potential of Bradysia impatiens to acquire and transmit the peanut stunt virus (PSV) from plant to plant was undertaken. Four-day-old larvae of B. impatiens were exposed to PSV-P strain by feeding on virus-infected leaves of Nicotiana benthamiana and then transferred to healthy plants in laboratory conditions. Using the reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR (RT-qPCR), and digital droplet PCR (RT-ddPCR), the PSV RNAs in the larva, pupa, and imago of B. impatiens were detected and quantified. The presence of PSV genomic RNA strands as well as viral coat protein in N. benthamiana, on which the viruliferous larvae were feeding, was also confirmed at the molecular level, even though the characteristic symptoms of PSV infection were not observed. The results have shown that larvae of B. impatiens could acquire the virus and transmit it to healthy plants. Moreover, it has been proven that PSV might persist in the insect body transstadially. Although the molecular mechanisms of virion acquisition and retention during insect development need further studies, this is the first report on B. impatiens playing a potential role in plant virus transmission.

Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants.

Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTVpJL-Kra) that could infect Nicotiana benthamiana and Solanum lycopersicum. Importantly, a modified variant of the viral RNA2-with inserted sGFP (forming, together with virus RNA1, into ToTVpJL-KraGFP)-was engineered as well. RNA2 of ToTVpJL-KraGFP was modified by introducing an additional open reading frame (ORF) of sGFP flanked with an amino acid-coding sequence corresponding to the putative virus protease recognition site. Our further analysis revealed that sGFP-tagged ToTV-Kra was successfully passaged by mechanical inoculation and spread systemically in plants. Therefore, the clone might be applied in studying the in vivo cellular, tissue, and organ-level localization of ToTV during infection. By performing whole-plant imaging, followed by fluorescence and confocal microscopy, the presence of the ToTVpJL-KraGFP-derived fluorescence signal was confirmed in infected plants. All this information was verified by sGFP-specific immunoprecipitation and western blot analysis. The molecular biology of the torradovirus-plant interaction is still poorly characterized; therefore, the results obtained here opened up new possibilities for further research. The application of sGFP-tagged virus infectious clones and their development method can be used for analyzing plant-virus interactions in a wide context of plant pathology.

Construction of infectious clones of tomato torrado virus and their delivery by agroinfiltration.

The first biologically active infectious clones of tomato torrado virus (ToTV) were generated and delivered into Nicotiana benthamiana and Solanum lycopersicum plants via Agrobacterium tumefaciens. The engineered constructs consisted of PCR-amplified complementary DNAs derived from the ToTV RNA1 and RNA2 components, individually inserted into an engineered pGreen binary vector between the CaMV 35S promoter and nopaline synthase terminator. These constructs were introduced into the plant hosts by means of A. tumefaciens-mediated infiltration. In the presence of the progeny virus, typical symptoms of ToTV infection developed in N. benthamiana and S. lycopersicum. Moreover, the virus was sap-transmissible when isolated from agroinfiltrated plants and induced symptoms similar to those caused by the wild-type virus. The presence of viral particles and viral genetic material was confirmed by electron microscopy and re-inoculation to S. lycopersicum and N. benthamiana, as well as by reverse transcription polymerase chain reaction and high-resolution melt analysis.

Genetic variability within the Polish tomato torrado virus Kra isolate caused by deletions in the 3'-untranslated region of genomic RNA1.

Tomato torrado virus (ToTV) is in the genus Torradovirus in the family Secoviridae. ToTV contains a single-stranded, positive-sense, bipartite RNA genome encapsidated in icosahedral particles. It is a serious tomato pathogen causing significant crop reductions. Its occurrence has been reported from many countries worldwide. However, the state of knowledge of ToTV epidemiology, sequences and phylogeny is still rather poor. In this study we found that the Polish ToTV isolates are characterized by significant genetic variability of the 3'-untranslated region (UTR) of RNA1. The high resolution melting real-time PCR approach showed the presence of genetic variants within Polish ToTV isolates purified from Nicotiana benthamiana. Further sequencing of Kra ToTV revealed five genetic variants of RNA1 within the isolate differing in the 3'-untranslated region length resulting from deletions ranging from 6 to 163 nucleotides. In light of the published studies, the genetic variability of ToTV associated with large deletions within an isolate may not necessarily be rare and may influence the virus evolution and adaptation.

How can plant virus satellite RNAs alter the effects of plant virus infection? A study of the changes in the Nicotiana benthamiana proteome after infection by peanut stunt virus in the presence or absence of its satellite RNA.

Peanut stunt virus (PSV), which belongs to the Cucumovirus genus, is a pathogen of legumes. Certain PSV strains associated with a satellite RNA (satRNA) modify the symptoms of infected plants and interfere with plant metabolism. We used PSV-P genomic transcripts (GTs) with and without PSV-P satRNA and a comparative proteomic 2D-DIGE/MS study to assess their effects on Nicotiana benthamiana infection. When the proteomes of the PSV-P genomic transcripts-infected (no satRNA present) and mock-inoculated plants were compared 29 differentially regulated proteins were found. When comparisons were made for plants infected with PSV-P-GT in the presence or absence of satRNA, and for mock-infected plants and those infected with the satRNA-associated PSV-P-GT, 40 and 60 such proteins, respectively, were found. The presence of satRNA mostly decreased the amounts of the affected host proteins. Proteins involved in photosynthesis and carbohydrate metabolism, for example ferredoxin-NADP-reductase and malate dehydrogenase, are among the identified affected proteins in all comparisons. Proteins involved in protein synthesis and degradation were also affected. Such proteins include chaperonin 60ß--whose abundance of the proteins changed for all comparisons--and aminopeptidase that is a satRNA- or PSV-P-GT/satRNA-responsive protein. Additionally, the levels of the stress-related proteins superoxide dismutase and acidic endochitinase Q increased in the PSV-P-GT- and PSV-P-GT/satRNA-infected plants. This study appears to be the first report on plant proteome changes in response to a satRNA presence during viral infection and, as such, may provide a reference for future studies concerning the influence of satRNAs during viral infections.

Analysis of two strains of Peanut stunt virus: satRNA-associated and satRNA free.

Peanut stunt virus (PSV) is a pathogen of legumes, vegetables, trees, and weeds occurring worldwide. The species is characterized by significant genetic variability. PSV strains are classified into four subgroups on the basis of their nucleotide sequence homology. Here, we are presenting two further, fully sequenced PSV strains-PSV-Ag and PSV-G, that could be considered as I subgroup representatives. However, their sequence homology with other typical I subgroups members, similarly as another strain-PSV-P, characterized by our group previously, is lower than 90%. This lead us to propose further subdivision of the I subgroup into IA, IB, and IC units, and to classify PSV-Ag and PSV-G strains to the last one. In this article, we are showing that identity level of PSV-Ag and PSV-G is very high and apart from the presence of satRNA in the first one, they differ only by a few nucleotides in their genomic RNAs. Nevertheless, symptoms they cause on host plants might differ significantly, just as the levels in infected plants. Effect of single amino acid changes between strains on the three-dimensional structure of viral proteins was analyzed. Differences occur mainly on the protein surfaces which can possibly affect protein-protein interaction in infected cells, which is discussed.

The nucleotide sequence of a Polish isolate of Tomato torrado virus.

A new virus was isolated from greenhouse tomato plants showing symptoms of leaf and apex necrosis in Wielkopolska province in Poland in 2003. The observed symptoms and the virus morphology resembled viruses previously reported in Spain called Tomato torrado virus (ToTV) and that in Mexico called Tomato marchitez virus (ToMarV). The complete genome of a Polish isolate Wal'03 was determined using RT-PCR amplification using oligonucleotide primers developed against the ToTV sequences deposited in Genbank, followed by cloning, sequencing, and comparison with the sequence of the type isolate. Phylogenetic analyses, performed on the basis of fragments of polyproteins sequences, established the relationship of Polish isolate Wal'03 with Spanish ToTV and Mexican ToMarV, as well as with other viruses from Sequivirus, Sadwavirus, and Cheravirus genera, reported to be the most similar to the new tomato viruses. Wal'03 genome strands has the same organization and very high homology with the ToTV type isolate, showing only some nucleotide and deduced amino acid changes, in contrast to ToMarV, which was significantly different. The phylogenetic tree clustered aforementioned viruses to the same group, indicating that they have a common origin.