Rearranged Endogenized Plant Pararetroviruses as Evidence of Heritable RNA-based Immunity.

Eukaryotic genomics frequently revealed historical spontaneous endogenization events of external invading nucleic acids, such as viral elements. In plants, an extensive occurrence of endogenous plant pararetroviruses (EPRVs) is usually believed to endow hosts with an additional layer of internal suppressive weaponry. However, an actual demonstration of this activity remains speculative. We analyzed the EPRV component and accompanying silencing effectors of Solanum lycopersicum, documenting that intronic/intergenic pararetroviral integrations bearing inverted-repeats fuel the plant's RNA-based immune system with suitable transcripts capable of evoking a silencing response. A surprisingly small set of rearrangements explained a substantial fraction of pararetroviral-derived endogenous small-interfering (si)RNAs, enriched in 22-nt forms typically associated with anti-viral post-transcriptional gene silencing. We provide preliminary evidence that such genetic and immunological signals may be found in other species outside the genus Solanum. Based on molecular dating, bioinformatics, and empirical explorations, we propose that homology-dependent silencing emerging from particular immuno-competent rearranged chromosomal areas that constitute an adaptive heritable trans-acting record of past infections, with potential impact against the unlocking of plant latent EPRVs and cognate-free pararetroviruses.

Synthetic sub-genomic transcript promoter from Horseradish Latent Virus (HRLV).

MAIN CONCLUSION: We characterized an efficient chimeric sub-genomic transcript promoter from Horseradish Latent Virus, FHS4, active in both dicot and monocot plants, and it could be a potential tool for plant biotechnology. Plant pararetroviruses are a rich source of novel plant promoters widely used for biotechnological applications. Here, we comprehensively characterized a unique sub-genomic transcript (Sgt) promoter of Horseradish Latent Virus (HRLV) and identified a fragment (HS4; - 340 to + 10; 351 bp) that showed the highest expression of reporter genes in both transient and transgenic assays as evidenced by biochemical, histochemical GUS reporter assay and transcript analysis of uidA gene by qRT-PCR. Phylogenetic analysis showed that the HSgt promoter was closely related to the sub-genomic promoter of the Cauliflower Mosaic Virus (CaMV19S). We found that the as-1 element and W-box played an important role in the transcriptional activity of the HS4 promoter. Furthermore, the HS4 promoter was also induced by salicylic acid. Alongside, we enhanced the activity of the HS4 promoter by coupling the enhancer region from Figwort Mosaic Virus (FMV) promoter to the upstream region of it. This hybrid promoter FHS4 was around 1.1 times stronger than the most commonly used promoter, 35S (Cauliflower Mosaic Virus full-length transcript promoter), and was efficient in driving reporter genes in both dicot and monocot plants. Subsequently, transgenic tobacco plants expressing an anti-microbial peptide BrLTP2.1 (Brassica rapa lipid transport protein 2.1), under the control of the FHS4 promoter, were developed. The in vitro anti-fungal assay revealed that the plant-derived BrLTP2.1 protein driven by an FHS4 promoter manifested increased resistance against an important plant fungal pathogen, Alternaria alternata. Finally, we concluded that the FHS4 promoter can be used as an alternative to the 35S promoter and has a high potential to become an efficient tool in plant biotechnology.

Disturbance of floral colour pattern by activation of an endogenous pararetrovirus, petunia vein clearing virus, in aged petunia plants.

White areas of star-type bicolour petals of petunia (Petunia hybrida) are caused by post-transcriptional gene silencing (PTGS) of the key enzyme of anthocyanin biosynthesis. We observed blotched flowers and a vein-clearing symptom in aged petunia plants. To determine the cause of blotched flowers, we focused on an endogenous pararetrovirus, petunia vein clearing virus (PVCV), because this virus may have a suppressor of PTGS (VSR). Transcripts and episomal DNAs derived from proviral PVCVs accumulated in aged plants, indicating that PVCV was activated as the host plant aged. Furthermore, DNA methylation of CG and CHG sites in the promoter region of proviral PVCV decreased in aged plants, suggesting that poor maintenance of DNA methylation activates PVCV. In parallel, de novo DNA methylation of CHH sites in its promoter region was also detected. Therefore, both activation and inactivation of PVCV occurred in aged plants. The accumulation of PVCV transcripts and episomal DNAs in blotched regions and the detection of VSR activity support a mechanism in which suppression of PTGS by PVCV causes blotched flowers.

Broken, silent, and in hiding: tamed endogenous pararetroviruses escape elimination from the genome of sugar beet (Beta vulgaris).

BACKGROUND AND AIMS: Endogenous pararetroviruses (EPRVs) are widespread components of plant genomes that originated from episomal DNA viruses of the Caulimoviridae family. Due to fragmentation and rearrangements, most EPRVs have lost their ability to replicate through reverse transcription and to initiate viral infection. Similar to the closely related retrotransposons, extant EPRVs were retained and often amplified in plant genomes for several million years. Here, we characterize the complete genomic EPRV fraction of the crop sugar beet (Beta vulgaris, Amaranthaceae) to understand how they shaped the beet genome and to suggest explanations for their absent virulence. METHODS: Using next- and third-generation sequencing data and genome assembly, we reconstructed full-length in silico representatives for the three host-specific EPRVs (beetEPRVs) in the B. vulgaris genome. Focusing on the endogenous caulimovirid beetEPRV3, we investigated its chromosomal localization, abundance and distribution by fluorescent in situ and Southern hybridization. KEY RESULTS: Full-length beetEPRVs range between 7.5 and 10.7 kb in size, are heterogeneous in structure and sequence, and occupy about 0.3 % of the beet genome. Although all three beetEPRVs were assigned to the florendoviruses, they showed variably arranged protein-coding domains, different fragmentation, and preferences for diverse sequence contexts. We observed small RNAs that specifically target the individual beetEPRVs, indicating stringent epigenetic suppression. BeetEPRV3 sequences occur along all sugar beet chromosomes, preferentially in the vicinity of each other and are associated with heterochromatic, centromeric and intercalary satellite DNAs. BeetEPRV3 members also exist in genomes of related wild species, indicating an initial beetEPRV3 integration 13.4-7.2 million years ago. CONCLUSIONS: Our study in beet illustrates the variability of EPRV structure and sequence in a single host genome. Evidence of sequence fragmentation and epigenetic silencing implies possible plant strategies to cope with long-term persistence of EPRVs, including amplification, fixation in the heterochromatin, and containment of EPRV virulence.

Evidence of Rubus Yellow Net Virus Integration into the Red Raspberry Genome.

Rubus yellow net virus (RYNV) infects Rubus spp., causing a severe decline when present in mixed infections with other viruses. RYNV belongs to the family Caulimoviridae, also known as plant pararetroviruses, which can exist as episomal or integrated elements (endogenous). Most of integrated pararetroviruses are noninfectious; however, a few cases have been reported where they excised from the plant genome and formed infectious particles. Graft transmission onto indicator plants R. occidentalis "Munger" has been the standard test method for RYNV detection in certification programs. Previously, it was noticed that some RYNV PCR-positive plants did not induce symptoms on "Munger", suggesting an integration event. In this study, bio-indexing and different molecular techniques were employed to differentiate between integrated and episomal RYNV sequences. Reverse transcription-PCR using RYNV-specific oligonucleotides after DNase treatment generated positive results for the virus in graft transmissible isolates (episomal) only. To confirm these results, rolling circle amplification on DNA preparations from the same samples resulted in amplicons identified as RYNV only from plants with graft transmissible RYNV. High-throughput sequencing was used to identify the RYNV-like sequences present in the host DNA. These results indicate the integration of RYNV into the red raspberry genome and highlight the necessity to recognize this phenomenon (integration) in future Rubus quarantine and certification programs.

Identification and characterization of water chestnut Soymovirus-1 (WCSV-1), a novel Soymovirus in water chestnuts (Eleocharis dulcis).

BACKGROUND: A disease of unknown etiology in water chestnut plants (Eleocharis dulcis) was reported in China between 2012 and 2014. High throughput sequencing of small RNA (sRNA) combined with bioinformatics, and molecular identification based on PCR detection with virus-specific primers and DNA sequencing is a desirable approach to identify an unknown infectious agent. In this study, we employed this approach to identify viral sequences in water chestnut plants and to explore the molecular interaction of the identified viral pathogen and its natural plant host. RESULTS: Based on high throughput sequencing of virus-derived small RNAs (vsRNA), we identified the sequence a new-to-science double-strand DNA virus isolated from water chestnut cv. 'Tuanfeng' samples, a widely grown cultivar in Hubei province, China, and analyzed its genomic organization. The complete genomic sequence is 7535 base-pairs in length, and shares 42-52% nucleotide sequence identity with viruses in the Caulimoviridae family. The virus contains nine predicated open reading frames (ORFs) encoding nine hypothetical proteins, with conserved domains characteristic of caulimoviruses. Phylogenetic analyses at the nucleotide and amino acid levels indicated that the virus belongs to the genus Soymovirus. The virus is tentatively named Water chestnut soymovirus-1 (WCSV-1). Phylogenetic analysis of the putative viral polymerase protein suggested that WCSV-1 is distinct to other well established species in the Soymovirus genus. This conclusion was supported by phylogenetic analyses of the amino acid sequences encoded by ORFs I, IV, VI, or VII. The sRNA bioinformatics showed that the majority of the vsRNAs are 22-nt in length with a preference for U at the 5'-terminal nucleotide. The vsRNAs are unevenly distributed over both strands of the entire WCSV-1 circular genome, and are clustered into small defined regions. In addition, we detected WCSV-1 in asymptomatic and symptomatic water chestnut samples collected from different regions of China by using PCR. RNA-seq assays further confirmed the presence of WCSV-1-derived viral RNA in infected plants. CONCLUSIONS: This is the first discovery of a dsDNA virus in the genus Soymovirus infecting water chestnuts. Data presented also add new information towards a better understanding of the co-evolutionary mechanisms between the virus and its natural plant host.

The proposed new species, cacao red vein virus, and three previously recognized badnavirus species are associated with cacao swollen shoot disease.

BACKGROUND: Cacao swollen shoot virus (CSSV), Cacao swollen shoot CD virus (CSSCDV), and Cacao swollen shoot Togo A virus (CSSTAV) cause cacao swollen shoot disease (CSSD) in West Africa. During 2000-2003, leaf and shoot-swelling symptoms and rapid tree death were observed in cacao in Cote d'Ivoire and Ghana. Molecular tests showed positive infection in only ~50-60% of symptomatic trees, suggesting the possible emergence of an unknown badnavirus. METHODS: The DNA virome was determined from symptomatic cacao samples using Illumina-Hi Seq, and sequence accuracy was verified by Sanger sequencing. The resultant 14, and seven previously known, full-length badnaviral genomic and RT-RNase H sequences were analyzed by pairwise distance analysis to resolve species relationships, and by Maximum likelihood (ML) to reconstruct phylogenetic relationships. The viral coding and non-coding sequences, genome organization, and predicted conserved protein domains (CPDs) were identified and characterized at the species level. RESULTS: The 21 CSSD-badnaviral genomes and RT-RNase H sequences shared 70-100% and 72-100% identity, respectively. The RT-RNase H analysis predicted four species, based on an ≥80% species cutoff. The ML genome sequence tree resolved three well-supported clades, with ≥70% bootstrap, whereas, the RT-RNase H phylogeny was poorly resolved, however, both trees grouped CSSD isolates within one large clade, including the newly discovered Cacao red vein virus (CRVV) proposed species. The genome arrangement of the four species consists of four, five, or six predicted open reading frames (ORFs), and the CPDs have similar architectures. By comparison, two New World cacao-infecting badnaviruses encode four ORFs, and harbor CPDs like the West African species. CONCLUSIONS: Three previously recognized West African cacao-infecting badnaviral species were identified, and a fourth, previously unidentified species, CRVV, is described for the first time. The CRVV is a suspect causal agent of the rapid decline phenotype, however Koch's Postulates have not been proven. To reconcile viral evolutionary with epidemiology considerations, more detailed information about CSSD-genomic variability is essential. Also, the functional basis for the multiple genome arrangements and subtly distinct CPD architectures among cacao-infecting badnaviruses is poorly understood. New knowledge about functional relationships may help explain the diverse symptomatologies observed in affected cacao trees.

Complete genome sequence of a putative new caulimovirus which exists as endogenous pararetroviral sequences in Angelica dahurica.

A virus isolate designated Angelica bushy stunt virus (AnBSV), provisionally representing a new species in the genus Caulimovirus, was discovered in the medicinal plant Angelica dahurica. The complete 8,300-nt genomic DNA of AnBSV had seven putative open reading frames containing conserved domains/motifs, which are typical features of caulimoviruses, and showed the greatest nucleotide sequence identity (74% identity and 27% query coverage) to a lamium leaf distortion virus isolate. Interestingly, the new caulimovirus exists as endogenous pararetroviral sequences in the host plant and is considered to have multiple defective plant genome-integrated copies that may lead to the generation of subgenomic DNA species.

How endogenous plant pararetroviruses shed light on Musa evolution.

BACKGROUND AND AIMS: Banana genomes harbour numerous copies of viral sequences derived from banana streak viruses (BSVs) - dsDNA viruses belonging to the family Caulimoviridae.These viral integrants (eBSVs) are mostly defective, probably as a result of 'pseudogenization' driven by host genome evolution. However, some can give rise to infection by releasing a functional viral genome following abiotic stresses. These distinct infective eBSVs correspond to the three main widespread BSV species (BSOLV, BSGFV and BSIMV), fully described within the Musa balbisiana B genomes of the seedy diploid 'Pisang Klutuk Wulung' (PKW). METHODS: We characterize eBSV distribution among a Musa sampling including seedy BB diploids and interspecific hybrids with Musa acuminate exhibiting different levels of ploidy for the B genome (ABB, AAB, AB). We used representative samples of the two areas of sympatry between M. acuminate and M. balbisiana species representing the native area of the most widely cultivated AAB cultivars (in India and in East Asia, ranging from the Philippines to New Guinea). Seventy-seven accessions were characterized using eBSV-related PCR markers and Southern hybridization approaches. We coded both sets of results to create a common dissimilarity matrix with which to interpret eBSV distribution. KEY RESULTS: We propose a Musa phylogeny driven by the M. balbisiana genome based on a dendrogram resulting from a joint neighbour-joining analysis of the three BSV species, showing for the first time lineages between BB and ABB/AAB hybrids. eBSVs appear to be relevant phylogenetic markers that can illustrate theM. balbisiana phylogeography story. CONCLUSION: The theoretical implications of this study for further elucidation of the historical and geographical process of Musa domestication are numerous. Discovery of banana plants with B genome non-infective for eBSV opens the way to the introduction of new genitors in programmes of genetic banana improvement.

Detection of herbaceous-plant pararetrovirus in lichen herbarium samples.

Cauliflower mosaic virus (CaMV) - a plant pararetrovirus that naturally causes diseases in Brassicaceae and Solanaceae plant hosts worldwide - has been detected by PCR for the first time in herbarium samples of Usnea sp. lichens. The virus's presence in these lichens did not result in any micro- or macromorphological changes, and the herbarium records were classified as representative for the distinct species. Sequence analyses classified all the detected viruses into one lineage of CaMV isolates. We have shown here that herbarium samples could be a good source for virus study, especially where a longer time span is involved.

Endogenous pararetrovirus sequences associated with 24 nt small RNAs at the centromeres of Fritillaria imperialis L. (Liliaceae), a species with a giant genome.

Endogenous pararetroviral sequences are the most commonly found virus sequences integrated into angiosperm genomes. We describe an endogenous pararetrovirus (EPRV) repeat in Fritillaria imperialis, a species that is under study as a result of its exceptionally large genome (1C = 42 096 Mbp, approximately 240 times bigger than Arabidopsis thaliana). The repeat (FriEPRV) was identified from Illumina reads using the RepeatExplorer pipeline, and exists in a complex genomic organization at the centromere of most, or all, chromosomes. The repeat was reconstructed into three consensus sequences that formed three interconnected loops, one of which carries sequence motifs expected of an EPRV (including the gag and pol domains). FriEPRV shows sequence similarity to members of the Caulimoviridae pararetrovirus family, with phylogenetic analysis indicating a close relationship to Petuvirus. It is possible that no complete EPRV sequence exists, although our data suggest an abundance that exceeds the genome size of Arabidopsis. Analysis of single nucleotide polymorphisms revealed elevated levels of C→T and G→A transitions, consistent with deamination of methylated cytosine. Bisulphite sequencing revealed high levels of methylation at CG and CHG motifs (up to 100%), and 15-20% methylation, on average, at CHH motifs. FriEPRV's centromeric location may suggest targeted insertion, perhaps associated with meiotic drive. We observed an abundance of 24 nt small RNAs that specifically target FriEPRV, potentially providing a signature of RNA-dependent DNA methylation. Such signatures of epigenetic regulation suggest that the huge genome of F. imperialis has not arisen as a consequence of a catastrophic breakdown in the regulation of repeat amplification.

Analysis of plant pararetrovirus promoter sequence(s) for developing a useful synthetic promoter with enhanced activity in rice, pearl millet, and tobacco plants.

Promoters are one of the most important components for many gene-based research as they can fine-tune precise gene expression. Many unique plant promoters have been characterized, but strong promoters with dual expression in both monocot and dicot systems are still lacking. In this study, we attempted to make such a promoter by combining specific domains from monocot-infecting pararetroviral-based promoters sugarcane bacilliform virus (SCBV) and banana streak virus (BSV) to a strong dicot-infecting pararetroviral-based promoter mirabilis mosaic virus (MMV). The generated chimeric promoters, MS, SM, MB, and BM, were tested in monocot and dicot systems and further validated in transgenic tobacco plants. We found that the developed chimeric promoters were species-specific (monocot or dicot), which depended on their respective core promoter (CP) region. Furthermore, with this knowledge, deletion-hybrid promoters were developed and evaluated, which led to the development of a unique dual-expressing promoter, MSD3, with high gene expression efficiency (GUS and GFP reporter genes) in rice, pearl millet, and tobacco plants. We conclude that the MSD3 promoter can be an important genetic tool and will be valuable in plant biology research and application.

Cauliflower mosaic virus: Virus-host interactions and its uses in biotechnology and medicine.

Cauliflower mosaic virus (CaMV) was the first discovered plant virus with genomic DNA that uses reverse transcriptase for replication. The CaMV 35S promoter is a constitutive promoter and thus, an attractive driver of gene expression in plant biotechnology. It is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. In the last century, producing food for the world's population while preserving the environment and human health is the main topic of agriculture. The damage caused by viral diseases has a significant negative economic impact on agriculture, and disease control is based on two strategies: immunization and prevention to contain virus spread, so correct identification of plant viruses is important for disease management. Here, we discuss CaMV from different aspects: taxonomy, structure and genome, host plants and symptoms, transmission and pathogenicity, prevention, control and application in biotechnology as well as in medicine. Also, we calculated the CAI index for three ORFs IV, V, and VI of the CaMV virus in host plants, the results of which can be used in the discussion of gene transfer or antibody production to identify the CaMV.

Discovery and Analyses of Caulimovirid-like Sequences in Upland Cotton (Gossypium hirsutum).

Analyses of Illumina-based high-throughput sequencing data generated during characterization of the cotton leafroll dwarf virus population in Mississippi (2020-2022) consistently yielded contigs varying in size (most frequently from 4 to 7 kb) with identical nucleotide content and sharing similarities with reverse transcriptases (RTases) encoded by extant plant pararetroviruses (family Caulimoviridiae). Initial data prompted an in-depth study involving molecular and bioinformatic approaches to characterize the nature and origins of these caulimovirid-like sequences. As a result, here, we report on endogenous viral elements (EVEs) related to extant members of the family Caulimoviridae, integrated into a genome of upland cotton (Gossypium hirsutum), for which we propose the provisional name "endogenous cotton pararetroviral elements" (eCPRVE). Our investigations pinpointed a ~15 kbp-long locus on the A04 chromosome consisting of head-to-head orientated tandem copies located on positive- and negative-sense DNA strands (eCPRVE+ and eCPRVE-). Sequences of the eCPRVE+ comprised nearly complete and slightly decayed genome information, including ORFs coding for the viral movement protein (MP), coat protein (CP), RTase, and transactivator/viroplasm protein (TA). Phylogenetic analyses of major viral proteins suggest that the eCPRVE+ may have been initially derived from a genome of a cognate virus belonging to a putative new genus within the family. Unexpectedly, an identical 15 kb-long locus composed of two eCPRVE copies was also detected in a newly recognized species G. ekmanianum, shedding some light on the relatively recent evolution within the cotton family.

Emerging and Re-Emerging Diseases Caused by Badnaviruses.

New and emerging plant diseases are caused by different pathogens including viruses that often cause significant crop losses. Badnaviruses are pararetroviruses that contain a single molecule of ds DNA genome of 7 to 9 kb in size and infect a large number of economically important crops such as banana and plantains, black pepper, cacao, citrus, grapevine, pineapple, sugarcane, sweet potato, taro, and yam, causing significant yield losses. Many of the species in the genus have a restricted host range and several of them are known to infect a single crop. Combined infections of different virus species and strains offer conditions that favor the development of new strains via recombination, especially in vegetatively propagated crops. The primary spread of badnaviruses is through vegetative propagating materials while for the secondary spread, they depend on insects such as mealybugs and aphids. Disease emerges as a consequence of the interactions between host and pathogens under favorable environmental conditions. The viral genome of the pararetroviruses is known to be integrated into the chromosome of the host and a few plants with integrants when subjected to different kinds of abiotic stress will give rise to episomal forms of the virus and cause disease. Attempts have been made to develop management strategies for badnaviruses both conventionally and using precision breeding techniques such as genome editing. Until 2016 only 32 badnavirus species infecting different crops were known, but in a span of six years, this number has gone up to 68. The current review highlights the emerging disease problems and management options for badnaviruses infecting economically important crops.

Endogenous Caulimovirids: Fossils, Zombies, and Living in Plant Genomes.

The Caulimoviridae is a family of double-stranded DNA viruses that infect plants. The genomes of most vascular plants contain endogenous caulimovirids (ECVs), a class of repetitive DNA elements that is abundant in some plant genomes, resulting from the integration of viral DNA in the chromosomes of germline cells during episodes of infection that have sometimes occurred millions of years ago. In this review, we reflect on 25 years of research on ECVs that has shown that members of the Caulimoviridae have occupied an unprecedented range of ecological niches over time and shed light on their diversity and macroevolution. We highlight gaps in knowledge and prospects of future research fueled by increased access to plant genome sequence data and new tools for genome annotation for addressing the extent, impact, and role of ECVs on plant biology and the origin and evolutionary trajectories of the Caulimoviridae.

Genome-wide identification of Reverse Transcriptase domains of recently inserted endogenous plant pararetrovirus (Caulimoviridae).

Endogenous viral elements (EVEs) are viral sequences that have been integrated into the nuclear chromosomes. Endogenous pararetrovirus (EPRV) are a class of EVEs derived from DNA viruses of the family Caulimoviridae. Previous works based on a limited number of genome assemblies demonstrated that EPRVs are abundant in plants and are present in several species. The availability of genome sequences has been immensely increased in the recent years and we took advantage of these resources to have a more extensive view of the presence of EPRVs in plant genomes. We analyzed 278 genome assemblies corresponding to 267 species (254 from Viridiplantae) using tBLASTn against a collection of conserved domains of the Reverse Transcriptases (RT) of Caulimoviridae. We concentrated our search on complete and well-conserved RT domains with an uninterrupted ORF comprising the genetic information for at least 300 amino acids. We obtained 11.527 sequences from the genomes of 202 species spanning the whole Tracheophyta clade. These elements were grouped in 57 clusters and classified in 13 genera, including a newly proposed genus we called Wendovirus. Wendoviruses are characterized by the presence of four open reading frames and two of them encode for aspartic proteinases. Comparing plant genomes, we observed important differences between the plant families and genera in the number and type of EPRVs found. In general, florendoviruses are the most abundant and widely distributed EPRVs. The presence of multiple identical RT domain sequences in some of the genomes suggests their recent amplification.

The essential role of the quasi-long terminal repeat sequence for replication and gene expression of an endogenous pararetrovirus, petunia vein clearing virus.

Petunia vein clearing virus (PVCV) is a type member of the genus Petuvirus within the Caulimoviridae family and is defined as one viral unit consisting of a single open reading frame (ORF) encoding a viral polyprotein and one quasi-long terminal repeat (QTR) sequence. Since some full-length PVCV sequences are found in the petunia genome and a vector for horizontal transmission of PVCV has not been identified yet, PVCV is referred to as an endogenous pararetrovirus. Molecular mechanisms of replication, gene expression and horizontal transmission of endogenous pararetroviruses in plants are elusive. In this study, agroinfiltration experiments using various PVCV infectious clones indicated that the replication (episomal DNA synthesis) and gene expression of PVCV were efficient when the QTR sequences are present on both sides of the ORF. Whereas replacement of the QTR with another promoter and/or terminator is possible for gene expression, it is essential for QTR sequences to be on both sides for viral replication. Although horizontal transmission of PVCV by grafting and biolistic inoculation was previously reported, agroinfiltration is a useful and convenient method for studying its replication and gene expression.

Metagenomic Analysis of Ampelographic Collections of Dagestan Revealed the Presence of Two Novel Grapevine Viruses.

In this study, we analyzed the virome of 73 grape samples from two Dagestan ampelographic collections in Russia using high-throughput sequencing of total RNAs. Fourteen viruses and four viroids were identified, with one to eleven of them detected in each plant. For the first time in Russia, we identified grapevine leafroll-associated virus 7 and grapevine Kizil Sapak virus. A total of 206 genomes of viruses and viroids were obtained, and their phylogenetic analysis was carried out. The de novo assembly and tblastx analysis allowed us to obtain contigs of a novel (+) ssRNA genome of a plant virus from the genus Umbravirus, which was tentatively named grapevine umbra-like virus (GULV), as well as contigs of a novel dsDNA pararetrovirus from the genus Caulimovirus, which was tentatively named grapevine pararetrovirus (GPRV). Complete genomes of these viruses were obtained and used for Sequence Demarcation Tool (SDT) analysis and phylogeny studies. GULV and GPRV were detected in 16 and 33 germplasm samples from the Dagestan collections, respectively.

Cauliflower mosaic virus P6 inclusion body formation: A dynamic and intricate process.

During an infection, Cauliflower mosaic virus (CaMV) forms inclusion bodies (IBs) mainly composed of viral protein P6, where viral activities occur. Because viral processes occur in IBs, understanding the mechanisms by which they are formed is crucial. FL-P6 expressed in N. benthamiana leaves formed IBs of a variety of shapes and sizes. Small IBs were dynamic, undergoing fusion/dissociation events. Co-expression of actin-binding polypeptides with FL-P6 altered IB size distribution and inhibited movement. This suggests that IB movement is required for fusion and growth. A P6 deletion mutant was discovered that formed a single large IB per cell, which suggests it exhibited altered fusion/dissociation dynamics. Myosin-inhibiting drugs did not affect small IB movement, while those inhibiting actin polymerization did. Large IBs colocalized with components of the aggresome pathway, while small ones generally did not. This suggests a possible involvement of the aggresome pathway in large IB formation.