Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus

[EN] In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.This work was supported by grant BFU2015--70261 from the Ministerio de Economia y Competitividad (MINECO, Spain)--FEDER and PROMETEO/2019/012 from Generalitat Valenciana (GVA) (to C. H). M.P.-C. was the recipient of contracts from MINECO--FEDER and GVA, and E.H. was the recipient of a contract from MINECO--FEDER. K.K. was supported by the grant `Emblematic Action for Research in the Cretan Agrofood sector: Four Institutions, Four References' (AGRO4CRETE -2018S.01300000) held by the General Secretary for Research and Technology of Greece.Pérez-Cañamás, M.; Hevia, E.; Hernandez Fort, C.; Katsarou, K. (2021). Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus. Journal of General Virology. 102(10):1-9. https://doi.org/10.1099/jgv.0.001656191021

PSTVd infection in Nicotiana benthamiana plants has a minor yet detectable effect on CG methylation

Viroids are small circular RNAs infecting a wide range of plants. They do not code for any protein or peptide and therefore rely on their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study, we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses, we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study genomic DNA methylation of 5-methylcytosine (5mC) in CG sites upon viroid infection. In this study we identified a limited number of target loci differentially methylated upon PSTVd infection. These results enhance our understanding of the epigenetic host changes as a result of pospiviroid infection

Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana

[EN] Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Roseal to obtain recombinant clone ZYMV-Rosl. While in some plant-virus combinations Roseal induces accumulation of anthocyanins in infected tissues, ZYMV-Rosl infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmen-tation in infected tissues of the model plant Nicotiana ben-thamiana. In this species, ZYMV-Rosl multiplied efficiently in local inoculated tissue but only a few progeny particles estab-lished infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Rosl local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Rosl systemic accumula-tion was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Rosl systemic accumulation dra-matically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.We thank V. Aragones for excellent technical assistance. This work was supported by the Spanish Ministerio de Economia y Competitividad (MINECO) through grants BI02014-54269-R and AGL2013-49919-EXP and by the Greek Ministry for Education and Religious Affairs (Program Aristeia II, 4499, ViroidmiR; ESPA 2007-2013). A. Carbonell was supported by an Individual Fellowship from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 655841.Cordero-Cucart, MT.; Cerdán García, L.; Carbonell Olivares, A.; Katsarou, K.; Kalantidis, K.; Daros Arnau, JA. (2016). Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana. Molecular Plant-Microbe Interactions. 30(1):63-71. https://doi.org/10.1094/MPMI-11-16-0239-RS637130

Alpha-1 antitrypsin deficiency impairs lung antibacterial immunity in mice

Alpha-1 antitrypsin (AAT) is a major inhibitor of serine proteases in mammals. Therefore, its deficiency leads to protease-antiprotease imbalance and a risk for developing lung emphysema. Although therapy with human plasma-purified AAT attenuates AAT deficiency-related emphysema, its impact on lung antibacterial immunity is poorly defined. Here, we examined the effect of AAT therapy on lung protective immunity in AAT-deficient (KO) mice challenged with Streptococcus pneumoniae. AAT-KO mice were highly susceptible to S. pneumoniae, as determined by severe lobar pneumonia and early mortality. Mechanistically, we found that neutrophil-derived elastase (NE) degraded the opsonophagocytically important collectins, surfactant protein A (SP-A) and D (SP-D), which was accompanied by significantly impaired lung bacterial clearance in S. pneumoniae-infected AAT-KO mice. Treatment of S. pneumoniae-infected AAT-KO mice with human AAT protected SP-A and SP-D from NE-mediated degradation and corrected the pulmonary pathology observed in these mice. Likewise, treatment with Sivelestat, a specific inhibitor of NE, also protected collectins from degradation and significantly decreased bacterial loads in S. pneumoniae-infected AAT-KO mice. Our findings show that NE is responsible for the degradation of lung SP-A and SP-D in AAT-KO mice affecting lung protective immunity in AAT deficiency

Molecular dissection of the prototype foamy virus (PFV) RNA 5′-UTR identifies essential elements of a ribosomal shunt

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5′-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5′-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A′ or sORF B, and on the translation event at the corresponding 5′-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs

Insight on genes affecting tuber development in potato upon <i>Potato spindle tuber viroid</i> (PSTVd) infection

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection

Revisiting the Non-Coding Nature of Pospiviroids

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study

Μελέτη της τροποποίησης της σηματοδότησης των MAPKs - ERKs παρουσία των HCV δομικών πρωτεϊνών

An estimated 3% of the world population is infected by hepatitis C (HCV). HCV is an enveloped positive-strand RNA virus, with a genome of approximately 9.600 nucleotides, encoding for structural (Core, E1, E2, p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins. In most infected individuals, this virus evades the immune system and establishes a chronic infection that can progress to cirrhosis, and hepatocellular carcinoma. HCV proteins are involved in the development of hepatocellular carcinoma as they modulate MAPK signalling pathway. It has been proposed that capsids from a number of viruses like the Ebola virus, can produce signalling events. The HCV nucleocapsid is surrounded by a lipid layer containing glycoproteins E1 and E2. However, different forms of HCV particles may exist in the circulation of infected individuals and among them naked capsids have also been reported. Furthermore, novel HCV subgenomes with in-frame deletions of both envelope proteins (E1 and E2), were identified in the liver, as well as in the serum of HCV infected individuals. Recently Tsitoura et al. reported the generation of recombinant non-enveloped HCV core particles in the absence of other HCV proteins. More importantly, this report demonstrated that these naked capsids can be taken up by cells and induce cell-signalling phenomena. Based on the previous, we aimed in developing a strategy for the labelling of the HCV non-enveloped capsids by using the enhanced green fluorescent protein (GFP). GFP has been fused to a great number of proteins in order to study their intracellular trafficking and localization. We used the baculovirus expression system to generate recombinant fluorescent non-enveloped capsid-like particles (fluorescent HCVne) that possess identical properties to the one already described. The chimeric proteins core₁₇₃-GFP, GFP-core₁₉₁ and GFP- core₁₇₃ produced can be efficiently expressed and self assembled to form fluorescent non-enveloped capsid-like particles that can efficiently be taken up by human cells. In addition, we observed the trafficking of these fluorescent HCVne particles in live-microscopy, providing evidence of an attractive tool for viral tracking. To understand the biological significance of naked HCVne particles, we investigated the mechanism of entry of these particles. Recent studies have revealed a surprising variety of endocytic routes for enveloped as well as non-enveloped viral capsids including clathrin-mediated and caveolin-mediated endocytosis. Hepatitis C enveloped particles use the clathrin-mediated endocytosis, but to our knowledge no information about the entry mechanism of ΗCV non-enveloped particles exist. Data presented here, suggest that HCVne particles penetrate into hepatic cells via pH dependent clathrin-mediated endocytosis. During this process different MAPK pathways become activated. Entry process starts with the attachment of HCVne particles at the cell surface which is followed by a clathrin-mediated internalization, and localization of particles to early endosomes. Internalization occurs relatively fast, with the majority of entering viral particles internalized in early endosomes between 9 to 15 minutes During the endocytosis of the particles from the cellular surface to early endosome the pH changes in endosomes seem to be implicated. In addition, phosphorylated proteins and particularly tyrosine phosphoproteins, as well as MAPK-p38 protein are shown to be important during this trafficking. After entering early endosomes, HCVne particles moved along from endosomal to lysosomal compartments, through involvement of microtubules network. Data presented here shows a colocalization of HCVne particles with late endosomes at 1 hour post incubation reaching maximum colocalization with lysosomes at 4 hours. During early endosome-lysosome transport, different phosphorylated proteins are showed to be essential, such proteins are the serine/threonine and tyrosine phosphoproteins, as well as phospho-ERK₁/₂ and possibly phospho-p38. Previously described data showed specific ERF translocation from the nucleus to the cytoplasm after HCVne particles uptake, suggesting a possible activation of ERK₁/₂ pathway. In this report we described that ERK₁/₂ present a maximum activation 30 min after internalization that disappears when cells are treated with specific MEK₁/₂ inhibitors and is shown to be endocytosis-dependent. In addition, HCVne particles endocytosis produce a sustained ERK₁/₂ activation and a nuclear ERK₁/₂ accumulation. Magnitude and duration of ERK₁/₂ phosphorylation is important for the immediate early genes (IEG) promoter activation as well as for the stability of the produced proteins. In this study we show an increased activation of IEG c-fos and egr-1, totally or partially attributed to the ERK₁/₂ pathway. This constitutes an interesting observation as c-fos and egr-1 has been found to be overexpressed with high frequency in aggressive and invasive hepatocarcinomas making these genes an important target for putative therapy. We have also investigated another important signalling molecule of the MAPK pathway, the ERK5. ERK5, also known as BMK1, is a MAPK that presents approximately 80% of similarities with ERK₁/₂. This kinase contains the typical ERK phosphorylation motif (TEY) and a unique C-terminal region that is believed to be responsible for its distinct biological activities. ERK5 activity is increased in response to growth factors, oxidative stress and hyperosmolarity, via a direct phosphorylation by MEK5. It is important to note that ERK5 has been implicated in different types of cancers and angiogenesis. We were able to show that ERK5 can be activated by HCVne particles, via MEK5 protein, but not from endogenously expressed core or NS5A proteins. In addition, a well known downstream target of this pathway, mef2 also seems to be affected. Although our knowledge about HCV mechanism of entry, replication and infectivity are progressing a great number of aspects are still left unanswered. The evaluation of the impact of cellular environmental modifications produced by signalling events similar to those produced by the endocytotic properties of naturally occurring HCVne could potentially be of major importance for HCV life cycle and severity of the disease.Ο ιός της ηπατίτιδας C (HCV) ταυτοποιήθηκε το 1989 και υπάρχουν παγκοσμίως περίπου 170 εκατομμύρια φορείς του ιού. Ποσοστό που κυμαίνεται στο 80 με 85% καταλήγει σε χρόνια ηπατίτιδα και από αυτούς, το 20% αναπτύσσει κίρρωση του ήπατος. Ετησίως το 3- 5% των κιρρωτικών ασθενών καταλήγει σε ηπατοκυτταρικό καρκίνο. Η θεραπεία βασίζεται στην ταυτόχρονη χορήγηση πεγκυλιωμένης ιντερφερόνης-α και ριμπαβιρίνης, αλλά ποσοστό μεγαλύτερο από το 50% των ασθενών δεν ανταποκρίνεται στη θεραπεία. Ο HCV ανήκει στο γένος των Hepacivirus, στην οικογένεια των φλαβιιών (flaviviridae). Το ιικό του γονιδίωμα συνίσταται σε ένα μονόκλωνο, θετικής πολικότητας RNA μόριο μήκους 9,6 Kb το οποίο κωδικοποιεί μια πολυπρωτεΐνη μήκους περίπου 3000 αμινοξέων η οποία πρωτεολύεται με την βοήθεια κυτταρικών και ιικών πρωτεασών σε τουλάχιστον τρία δομικά (core, E1, E2) και επτά μη δομικά πολυπεπτίδια (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). Από αυτές τις πρωτεΐνες η core είναι υπεύθυνη, μεταξύ άλλων, για την δημιουργία του καψιδίου, ενώ οι πρωτεΐνες Ε1 και Ε2 σχηματίζουν τον φάκελο του ιού. Στον ορό των ασθενών είναι κοινώς αποδεκτή η ύπαρξη καψιδίων όχι μόνο με φάκελο αλλά και χωρίς, των οποίων ο ρόλος παραμένει ακόμα αδιευκρίνιστος. Η παρούσα εργασία επικεντρώθηκε στην κατασκευή ανασυνδυασμένων βακουλοϊών, μέσω των οποίων επιτεύχθηκε απομόνωση γυμνών core καψιδίων με ή χωρίς την ιδιότητα του φθορισμού. Ακολούθησε λεπτομερής μελέτη για τον χαρακτηρισμό των κατασκευασθέντων καψιδίων με την οποία αποδείχτηκε η λειτουργικότητα τους. Στη συνέχεια μελετήθηκε ο τρόπος εισόδου τους σε κύτταρα, κυρίως ηπατικής προέλευσης, και αποδείχτηκε ότι τα καψίδια χρησιμοποιούν την ενδοκύτωση μέσω κλαθρίνης για να εισχωρήσουν στο κύτταρο-ξενιστή και να φτάσουν στα πρώιμα και όψιμα ενδοσώματα και τελικά στα λυσοσώματα. Επιπλέον, αποδείχτηκε πως κατά την διάρκεια της συγκεκριμένης πορείας εμπλέκονται το χαμηλό pH, οι καθεψίνες B και L καθώς και διάφορες φωσφορυλιωμένες πρωτεΐνες όπως η p38, οι ERK₁/₂ και διαφόρων ειδών φωσφατάσες. Στη συνέχεια, τα γυμνά core καψίδια που απομονώθηκαν επωάστηκαν εξωγενώς με διάφορα είδη κυττάρων και μελετήθηκε η ενεργοποίηση των σηματοδοτικών μονοπατιών MAPKs και ειδικότερα τα μονοπάτια των ERK₁/₂ και ERK5. Παρατηρήθηκε πως ενεργοποιούν και τα δυο μονοπάτια, φωσφορυλιώνοντας τις ανάλογες πρωτεΐνες, μετά από επώαση τριάντα λεπτών. Το φαινόμενο αυτό είναι ποσοτικά εξαρτώμενο ενώ οι μάρτυρες που χρησιμοποιήθηκαν δεν εμφάνισαν καμία ενεργοποίηση. Με τη χρήση διαλυτής core ή GFP πρωτεΐνης, ακόμα και σε μεγάλες συγκεντρώσεις, καθώς και με τη χρήση διαφόρων χημικών αναστολέων, δεν ενεργοποιήθηκε κανένα από τα δύο μονοπάτια υποδηλώνοντας τη σημασία της καψιδιακής δομής και της διαδικασίας εισόδου του καψιδίου. Ακολούθησε, μελέτη για τον κυτταρικό εντοπισμό των ERK₁/₂ και ERK5 φωσφο-πρωτεϊνών, καθώς και της ιδιότητας ενεργοποίησης χαρακτηριστικών καθοδικών στόχων. Τα core καψίδια προκαλούν την πυρηνική μετατόπιση των ERK₁/₂ πρωτεϊνών, επηρεάζοντας τη μεταγραφή των γονιδίων πρώιμης απόκρισης c-fos και egr-1. Επιπλέον, αποδείχτηκε πως η παρατεταμένη ενεργοποίηση του μονοπατιού οδηγούσε στην σταθεροποίηση της πρωτεΐνης c-Fos. Αντίθετα, δεν παρατηρήθηκε πυρηνική μετακίνηση ή παρατεταμένη φωσφορυλίωση της ERK5 πρωτεΐνης, και επομένως η παρατηρούμενη ενεργοποίηση του γονιδίου mef2, γνωστού καθοδικού στόχου του μονοπατιού, φαίνεται να επιτυγχάνεται με έμμεσο τρόπο. Η ανάλυση των αποτελεσμάτων στο σύνολό τους αποτελούν σημαντική συμβολή στην κατανόηση των μηχανισμών παθογένειας που είναι πιθανό να διέπουν την λοίμωξη από τον ιό

Combined activity of DCL2 and DCL3 is crucial in the defense against potato spindle tuber viroid

Viroids are self replicating non-coding RNAs capable of infecting a wide range of plant hosts. They do not encode any proteins, thus the mechanism by which they escape plant defenses remains unclear. RNAi silencing is a major defense mechanism against virus infections, with the four DCL proteins being principal components of the pathway. We have used Nicotiana benthamiana as a model to study Potato spindle tuber viroid infection. This viroid is a member of the Pospiviroidae family and replicates in the nucleus via an asymmetric rolling circle mechanism. We have created knock-down plants for all four DCL genes and their combinations. Previously, we showed that DCL4 has a positive effect on PSTVd infectivity since viroid levels drop when DCL4 is suppressed. Here, we show that PSTVd levels remain decreased throughout infection in DCL4 knockdown plants, and that simultaneous knockdown of DCL1, DCL2 or DCL3 together with DCL4 cannot reverse this effect. Through infection of plants suppressed for multiple DCLs we further show that a combined suppression of DCL2 and DCL3 has a major effect in succumbing plant antiviral defense. Based on our results, we further suggest that Pospoviroids may have evolved to be primarily processed by DCL4 as it seems to be a DCL protein with less detrimental effects on viroid infectivity. These findings pave the way to delineate the complexity of the relationship between viroids and plant RNA silencing response

First Report of Southern Tomato Virus from Tomato (Solanum lycopersicum) in Greece

Southern tomato virus is a member of the genus Amalgavirus in the family Amalgaviridae. Members of this family are characterized by double-stranded RNA genomes of about 3.4 kbp (Sabanadzovic et al. 2009). Southern tomato virus (STV) was first detected in tomato (Solanum lycopersicum L.) plants in the United States and Mexico and since has been reported from many other countries. Although STV has been reported to cause asymptomatic infection, in some cultivars symptoms include interveinal necrosis, stunting, fruit discoloration, and reduced fruit size (Harju et al. 2021; Sabanadzovic et al. 2009). Transmission of STV occurs vertically through infected seeds (Sabanadzovic et al. 2009), and to date no virions have been identified in infected plants. In 2019, tomato seedlings from a breeding collection in Crete (Greece) were assessed for the presence of viruses using high-throughput sequencing (HTS). A total of 40 plant lines were grown, with plants pooled into two groups for HTS (pool 1 with 22 samples and pool 2 with 18 samples). RNA was extracted from leaf tissue using TRIzol (Katsarou et al. 2022), and 2 μg of RNA from each sample was pooled for HTS (Macrogen, the Netherlands). Assembly of raw reads was carried out using metaSPAdes (Nurk et al. 2017). BLASTn analysis against RVDBv20.0 (Goodacre et al. 2018) of the contigs from pool 1 did not result in any sequences matching plant viruses. In contrast, BLASTn of the assembled contigs from pool 2 (NCBI SRA: BioProject PRJNA818693) identified three small contigs of 336, 307, and 230 nt, respectively, with 98 to 100% nucleotide sequence identity to STV. Mapping to the STV reference genome sequence (GenBank accession no. NC_011591) using Geneious R7 confirmed 54 reads that mapped to the STV genome. To confirm the presence of STV in the tomato plants from pool 2, reverse transcription PCR was carried out using the RNA extracts previously prepared for HTS. Two sets of PCR primers were designed, STV_F (5′-TATATTGGAGGAGGAGGCGGT-3′) and STV_R (5′-ATATTCCTTCACCCTGCGCC-3′), which were predicted to amplify a 658-nt region of the RdRP gene, and a second set of nested primers, STVnF (5′-TGGAGATGAGGTGCTCGAAGA-3′) and STVnR (5′-TGGCTATGATGTATCTGTGCTTGA-3′), which amplify 458 nt within the first-round target region. Complementary DNA was synthesized using M-MuLV reverse transcriptase (Minotech, Greece), and two rounds of PCR were subsequently carried out using Taq DNA polymerase (EnzyQuest, Greece) as per the manufacturer’s recommendations. Analysis of the PCR products confirmed the presence of amplicons in three samples. The second-round PCR amplicons from the three samples were excised, gel-purified, and Sanger-sequenced (Genewiz, Germany). The trimmed reads (351 nt) were identical to each other, and BLASTn analysis confirmed their identity as STV, with the Crete sequences identical to three isolates from Germany (MK948545) and Switzerland (MF422617 and MF422618). This is the first report of STV in Greece. STV is seed transmitted but often causes no apparent symptoms in infected plants. This is the likely explanation for infected plants to be present in the breeding collection assessed in this study. Knowledge that these plants are not coinfected with other viruses may assist further work to identify symptoms associated with STV infection, as mixed infections are common in previous reports, as well as further investigate plant-host interactions between STV and tomato. Further work is now required to assess the field occurrence, yield effects, or other impacts of STV infections in tomato crops in Crete