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Tomato is cultivated in Greece for more than 200 years, even though is not native. Along with a favorable environment for the crop all the year round, Greece has become a major competitor in tomato production around Europe. However, there is an increasing demand to improve tomato crop. People have devoted a significant effort for crop improvement via phenotypic screening resulting in a large number of tomato ecotypes. An increasing demand to clarify the relationships among local tomato genotypes utilizing the most preferred molecular markers the Simple Sequence Repeats (SSR-markers) is the main objective of this study. Twenty-nine tomato ecotypes cultivated in Crete, Greece were genotyped utilizing eleven simple sequence repeat (SSR) along with the Structure analysis of the germplasm. A neighbor-joining dendrogram of the 29 ecotypes was produced. The statistical analysis proved that 58% of the genetic variation (p

Viroids are considered the most minimalistic group of pathogens. Despite their presumed inability to encode for proteins, viroids induce several diseases in plants of primary economic importance. The production of viroid derived siRNAs (vd-siRNAs) of 21-24 nt, accompanies viroid infections in plants and results from the activation of the RNA silencing mechanism and specifically the function of Dicer endonucleases. A comprehensive set of experiments for the study and thorough analysis of viroid-infected plants has been developed. Here we present a detailed experimental plan including optimized protocols for plant infection by agroinfiltration, RNA extraction, and northern blot hybridization for the detection of both viroid genomic RNA and vd-siRNAs.

Viroids are considered the most minimalistic group of pathogens. Despite their presumed inability to encode for proteins, viroids induce several diseases in plants of primary economic importance. The production of viroid derived siRNAs (vd-siRNAs) of 21-24 nt, accompanies viroid infections in plants and results from the activation of the RNA silencing mechanism and specifically the function of Dicer endonucleases. A comprehensive set of experiments for the study and thorough analysis of viroid-infected plants has been developed. Here we present a detailed experimental plan including optimized protocols for plant infection by agroinfiltration, RNA extraction, and northern blot hybridization for the detection of both viroid genomic RNA and vd-siRNAs.

[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.

In 2017 and 2018, grapevine leaves and stems were collected from symptomatic and symptomless vines from various cultivars of prefecture of Heraklion and the vineyard of the Greek Institute of Grapevine in Likovrisi (Attica). Total RNA extractions were performed according to Maliogka et al. (2015) and multiplex RT-PCR method (Hajizadeh et al. 2012) revealed the presence of Australian grapevine viroid (AGVd) in 13 out of 298 samples tested (11 from Crete and 2 from Attica). The full-length genome of the viroid was verified with Sanger sequencing for two isolates from Crete, AGVd S122 (369 bp; GenBank accession No. MT227864) and AGVd Her75 (370 bp; MT227865). In symptomatic AGVd positive plants showing bright yellow and chlorotic discolorations, pathogens such as grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus-3 (GLRaV-3) and grapevine yellow speckle viroid-2 (GYSVd-2) were detected, in different combinations in each plant, making it difficult for the observed symptoms to be directly correlated with the AGVd presence.

Citrus plants are infected by at least seven viroid species belonging to the Pospiviroidae family (Murcia et al., 2015). In Chania prefecture (Crete island), Citrus spp. are considered as important cultivated species and especially the Persian (or Tahiti) lime (Citrus latifolia) has become a new dynamic and expanding crop. Viroids have been reported in other citrus species in Greece (Boubourakas et al., 2010; Wang et al, 2013), but no research has been conducted in lime. In the spring of 2018, in a small lime orchard of forty 5-years old trees (grafted on C. aurantium), yellow fruit sectoring and shoot bark cracking symptoms were observed in six trees. A total of eight samples, six symptomatic and two asymptomatic trees, were collected. All of them were tested for the presence of the most prevalent citrus viroid, citrus exocortis viroid (CEVd) by reverse transcription-polymerase reaction (RT-PCR), amplifying the entire viroid genome as previously described (Bernad and Duran-Villa, 2006). The results revealed the presence of CEVd in all the symptomatic plants, whereas the viroid was not detected in the asymptomatic trees. In an effort to study the virome of lime species, one symptomatic sample (Cl-0) was subjected to high-throughput sequencing (HTS) of total RNA extracted from phloem tissue, on an Illumina NextSeq 500 platform, generating ~12.5 million of 150 bp pair-end reads. The de novo assembly of these reads and the BLASTN analysis of the produced contigs resulted in the confirmation of CEVd presence in addition to the identification of hop stunt viroid (HSVd), citrus bark cracking viroid (CBCVd) and citrus dwarfing viroid (CDVd). A recently discovered virus was also detected, which will be described elsewhere. The complete viroid genomes of 370, 302, 286 and 291 nucleotides in length for CEVd, HSVd, CBCVd and CDVd, respectively, were reconstructed and the genomes shared 99% (CEVd, CDVd) and 100% (HSVd, CBCVd) nucleotide identity with other available sequences of isolates from different countries. To confirm the results obtained from the HTS experiment, the Cl-0 sample in addition with all symptomatic (six in total) and two asymptomatic trees were further tested for the presence of HSVd, C?CVd and CDVd using specific primers to amplify the full viroid genome, as previously reported (Bernad and Duran-Villa, 2006). The RT-PCR results revealed the presence of HSVd also in all six symptomatic trees, whereas CDVd and CBCVd were detected in five and four symptomatic samples, respectively. The mixed infections in the symptomatic trees were with three or four viroids. No viroid was detected in the asymptomatic trees. Direct Sanger sequencing of the PCR amplicons confirmed the presence of the four viroids in the HTS analyzed Cl-0 sample. The retrieved nucleotide sequences were identical to those recovered from the HTS, and were deposited in GenBank with accession numbers MN136643, MN136644, MN136645 and MN136646. To our knowledge, this is the first report of citrus viroids infecting lime in Greece associated with bark cracking symptoms and ongoing studies with larger surveys and HTS are in progress to elucidate a possible link, if any, between symptoms and presence of a specific viroid.

RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana, an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next-generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.

Grapevine yellow speckle viroid-2 is a small, circular, single-stranded RNA molecule that is classified in the genus Apscaviroid (family Pospiviroidae). Grapevine yellow speckle viroid-2 (GYSVd-2) has been identified in many grapevine-producing countries such as India, Australia, China, Thailand (Singhal et al. 2019; Tangkanchanapas et al. 2017), Turkey, and Italy (Gambino et al. 2014). Until now, no information regarding its presence and spread in Greek vineyards was available. For this purpose, a random survey was conducted, and 327 samples of leaves and stems were collected from a large range of cultivars from Crete, Attiki, Peloponnese, Northern Greece, and Dodecanese. Total RNA was extracted according to the protocol of Maliogka et al. (2015), and reverse transcription PCR (RT-PCR) was performed using a new primer pair, GYSVd-2 up (5′-GACCTGCAGAGAAAAGAAGAAGG-3′)/GYSVd-2 do (5′-GCTCGACTAGCGGAGGC-3′). GYSVd-2 was frequently identified in the tested samples (39/327). The full viroid genomes of two isolates from Cretan vines (cv. Soultanina) and two from Peloponnese (cv. Superior) were amplified and sequenced using two different pairs of primers: GYSVd-2up 2 (5′-CCTAAGATGCCTCCGCTAGT-3′)/GYSVd-2do 2 (5′-AGACAGGCCCGCGTTTCG-3′) and GYSVd-2up3 (5′-AGAAGGGCCCAGAGGGGAAT-3′)/GYSVd-2do3 (5′-TCTTTTCTCTGCAGGTCCGC-3′). Sanger sequencing of both amplicons was performed in order to confirm the primer annealing sites. The 363-bp amplified genomes of the Cretan isolates (deposited in ENA under accession nos. LR735992 and LR735993) revealed high identity with GYSVd-2 isolates deposited in GenBank (98.63 to 100% nt identity) and 100% nt identity among them, whereas the isolates from Peloponnese (accession nos. LR735995 and LR735996) revealed 98.9 to 100% nt identity to deposited viroid isolates. At the same time the viroid was detected during a high-throughput sequencing analysis of rRNA depleted total RNA from a grapevine (cv. Mavro Nemeas) from Peloponnese. Sequencing was conducted on an Illumina NovaSeq6000 platform yielding ∼60 million 100-bp paired-end reads. After extraction of the Vitis vinifera genome, the de novo assembly of the reads using SPAdes produced 20,365 contigs. The 1,411 contigs exhibiting a size over 240 nt were screened against the nt/nr databases using BLAST. Among the identified viral sequences, the 363-nt-long genome of GYSVd-2 (LR735994) was retrieved, showing 100% nt identity to the cultivar Superior isolate from Peloponnese as well as to isolates from Croatia, Pakistan, and China (MF979530, KY978405, and FJ490172, respectively). The presence of the viroid in this sample was confirmed with the aforementioned RT-PCRs. To our knowledge, this is the first report of GYSVd-2 in Greece, which is expanding our knowledge on its worldwide distribution. Our initial data indicate that the viroid is dispersed in several regions of Greece, and it shows rather low genetic variability. Additional work is needed to elucidate its spread and impact on Greek grapevines.

Viroids are plant infecting, non - coding RNA molecules of economic importance. Potato spindle tuber viroid (PSTVd), the type species of Pospiviroidae family, has been shown to be affected by specific RNA silencing pathways. Dicer like 1 (DCL1), a key player in micro RNA (miRNA) pathway has been previously linked with PSTVd infectivity. In this report we aim to further dissect the interaction between the miRNA pathway and Pospiviroid virulence. We mainly focused on the Zinc-finger protein SERRATE (SE) a co-factor of DCL1 and core component of miRNA pathway. We generated Nicotiana tabacum and Nicotiana benthamiana SE knock-down plants exhibiting considerable miRNA reduction and strong phenotypic abnormalities. PSTVd infection of SE suppressed plants resulted in a significant viroid reduction, especially at the initial infection stages. This positive correlation between SE levels and viroid infectivity underlines its role in PSTVd life cycle and reveals the importance of the miRNA pathway upon viroid infection.

Long non protein coding RNAs (lncRNAs) constitute a large category of the RNA world, able to regulate different biological processes. In this review we are focusing on infectious lncRNAs, their classification, pathogenesis and impact on the infected organisms. Here they are presented in two separate groups: 'dependent lncRNAs' (comprising satellites RNA, Hepatitis D virus and lncRNAs of viral origin) which need a helper virus and 'independent lncRNAs' (viroids) that can self-replicate. Even though these lncRNA do not encode any protein, their structure and/or sequence comprise all the necessary information to drive specific interactions with host factors and regulate several cellular functions. These new data that have emerged during the last few years concerning lncRNAs modify the way we understand molecular biology's 'central dogma' and give new perspectives for applications and potential therapeutic strategies.

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The HCV viral particle is composed of a capsid containing the genome, surrounded by an endoplasmic reticulum (ER)-derived lipid bilayer where E1 and E2 are assembled as heterodimers. However, different forms of viral particles have been identified in the serum of HCV-infected patients, including non-enveloped particles. Previous reports have demonstrated that HCV non-enveloped capsid-like particles (HCVne) can be generated by HCV core protein sequence. This sequence possesses a highly conserved ΥΧΧΦ motif and distal di-leucine motifs that confer primary endocytosis signals, enabling HCVne to enter hepatic cells via clathrin-mediated endocytosis. Although HCV core's primary function is to encapsidate the viral genome, it also interacts with a variety of cellular proteins in order to regulate host cell functions such as gene transcription, lipid metabolism, apoptosis and several signaling pathways. In this report, we demonstrate that the YXXΦ motif of HCV core protein is crucial for the architectural integrity of the particulate form of HCVne. Moreover, we show that the YXXΦ motif in the HCV core sequence plays a pivotal role in the signaling events following HCVne clathrin-mediated endocytosis by inducing the AP-2 clathrin adaptor protein, which in turn redirect HCVne trafficking to the lipid droplets (LDs) via the endosomal-lysosomal pathway. HCVne and LDs co-localization affects the HCV life cycle by enhancing viral replication.

Viruses are essentially composed of a nucleic acid (segmented or not, DNA, or RNA) and a protein coat. Despite their simplicity, these small pathogens are responsible for significant economic and humanitarian losses that have had dramatic consequences in the course of human history. Since their discovery, scientists have developed different strategies to efficiently detect viruses, using all possible viral features. Viruses shape, proteins, and nucleic acid are used in viral detection. In this review, the development of these techniques, especially for plant and mammalian viruses, their strengths and weaknesses as well as the latest cutting-edge technologies that may be playing important roles in the years to come are described.

Hepatitis C virus (HCV) has been shown to actively replicate in cells of the immune system, altering both their function and cytokine expression. Naked nucleocapsids have been reported in the serum of infected patients. We investigated interference of recombinant non-enveloped capsid-like particles with signaling pathways in T cells. HCV non-enveloped particles (HCVne) internalization was verified in Jurkat and Hut 78 T cells, as well as primary human peripheral blood and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs-p38 signaling pathway. Using specific phosphoantibodies, signaling pathways inhibitors, and chemical agents, it was demonstrated that p38 activation in T cells correlated with IL-2 transcriptional activation and was accompanied by a parallel increase of IL-2 cytokine secretion. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found to be elevated. We propose that HCVne uptake by T lymphocytes results in increased MAPKs-p38 activity and IL-2 expression, thus altering the host immune response. Cellular and Molecular Life Sciences

Although HCV is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients. The HCV core particle serves as a protective capsid shell for the viral genome and recombinant in vitro assembled HCV core particles induce strong specific immunity. We investigated the post-binding mechanism of recombinant core particle uptake and its intracellular fate. In hepatic cells, these particles are internalized, most likely in a clathrin-dependent pathway, reaching early to late endosomes and finally lysosomes. The endocytic acidic milieu is implicated in trafficking process. Using specific phosphoantibodies, signaling pathway inhibitors and chemical agents, ERK(1/2) was found to be activated in a sustained way after endocytosis, followed by downstream immediate early genes (c-fos and egr-1) modulation. We propose that the intriguing properties of cellular internalization of HCV non-enveloped particles can induce specific ERK(1/2)-MAPKs events that could be important in HCV life cycle and pathogenesis of HCV infection.

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.

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., Author Summary Viroids consist of a peculiar type of highly structured small circular RNAs, capable of infecting crop plants and ornamentals. They do not encode any protein, yet they manage to replicate, move through the plant and often cause severe symptoms. In order to achieve this, viroids hijack plant cellular machinery. In addition, they manage to overcome plant RNAi response, which is the major antiviral defense mechanism of plants. DCL proteins have a central role in the RNAi pathway. We have used Nicotiana benthamiana plants, an experimental host of some viroids, and produced plants suppressed for DCL proteins (individually or in combination). By infecting DCL knockdown plants with Potato spindle tuber viroid we were able to identify which DCL proteins are mainly involved in the antiviroid response. We found that it is the combined activity of DCL2 and DCL3 pathways which most potently suppress viroid infectivity. In contrast, DCL4, the main antiviral DCL, seemed to obscure the DCL2-DCL3 effect on viroid infectivity. This lead us to the hypothesis that viroids may have evolved to be primarily processed by DCL4, as it seems to be the DCL protein with less detrimental effects on viroid infectivity. Our findings may aid understanding the complex interaction of viroids with the plant defense machinery.

Circulating ‘free’ non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV-infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core 173 -eGFP, eGFP-core 191 and eGFP-core 173 can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells – the main target of HCV – but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance.

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The viral particle is composed of a capsid containing the genome, surrounded by E1 and E2 proteins, however different forms of viral particles have been observed including non-enveloped particles. Previous reports have proposed that hepatitis C non-enveloped capsid-like particles (HCVne) enter cells of hepatic origin via clathrin-mediated endocytosis, during which different signaling events occur. In this report we show that HCVne particles are capable of inducing the recently discovered ERK5 pathway, in a dose dependent way. The ERK5 pathway can be activated by growth factors and other extracellular signals. This specific activation occurs through a well characterized upstream kinase, MEK5, and is capable of inducing gene regulation of mef2. In contrast, when HCV core structural and NS5A non-structural proteins were expressed endogenously no activation of this pathway was detected. These cell signaling events could be of critical importance and might give clues for the elucidation of cellular manifestations associated with HCV infection.

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 Rosea1 to obtain recombinant clone ZYMV-Ros1. While in some plant-virus combinations Rosea1 induces accumulation of anthocyanins in infected tissues, ZYMV-Ros1 infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmentation in infected tissues of the model plant Nicotiana benthamiana. In this species, ZYMV-Ros1 multiplied efficiently in local inoculated tissue but only a few progeny particles established 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-Ros1 local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Ros1 systemic accumulation 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-Ros1 systemic accumulation dramatically 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., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) through grants BIO2014-54269-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.