Modes of Viroid Transmission.

Studies on the ways in which viroids are transmitted are important for understanding their epidemiology and for developing effective control measures for viroid diseases. Viroids may be spread via vegetative propagules, mechanical damage, seed, pollen, or biological vectors. Vegetative propagation is the most prevalent mode of spread at the global, national and local level while further dissemination can readily occur by mechanical transmission through crop handling with viroid-contaminated hands or pruning and harvesting tools. The current knowledge of seed and pollen transmission of viroids in different crops is described. Biological vectors shown to transmit viroids include certain insects, parasitic plants, and goats. Under laboratory conditions, viroids were also shown to replicate in and be transmitted by phytopathogenic ascomycete fungi; therefore, fungi possibly serve as biological vectors of viroids in nature. The term "mycoviroids or fungal viroids" has been introduced in order to denote these viroids. Experimentally, known sequence variants of viroids can be transmitted as recombinant infectious cDNA clones or transcripts. In this review, we endeavor to provide a comprehensive overview of the modes of viroid transmission under both natural and experimental situations. A special focus is the key findings which can be applied to the control of viroid diseases.

Viroids, and the Legacy of Ricardo Flores (1947-2020).

Viroids were discovered by Diener in 1971 [...].

Taxonomy update for the family Alphasatellitidae: new subfamily, genera, and species.

Alphasatellites (family Alphasatellitidae) are circular, single-stranded DNA molecules (~1-1.4 kb) that encode a replication-associated protein and have commonly been associated with some members of the families Geminiviridae, Nanoviridae, and Metaxyviridae (recently established). Here, we provide a taxonomy update for the family Alphasatellitidae following the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021. The taxonomic update includes the establishment of the new subfamily Petromoalphasatellitinae. This new subfamily includes three new genera as well as the genus Babusatellite, which previously belonged to the subfamily Nanoalphasatellitinae. Additionally, three new genera and 14 new species have been established in the subfamily Geminialphasatellitinae, as well as five new species in the subfamily Nanoalphasatellitinae.

ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

ICTV Virus Taxonomy Profile: Pospiviroidae.

Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/pospiviroidae.

Analysis of DNAs associated with coconut foliar decay disease implicates a unique single-stranded DNA virus representing a new taxon.

The unique ecology, pathology and undefined taxonomy of coconut foliar decay virus (CFDV), found associated with coconut foliar decay disease (CFD) in 1986, prompted analyses of old virus samples by modern methods. Rolling circle amplification and deep sequencing applied to nucleic acid extracts from virion preparations and CFD-affected palms identified twelve distinct circular DNAs, eleven of which had a size of about 1.3 kb and one of 641 nt. Mass spectrometry-based protein identification proved that a 24 kDa protein encoded by two 1.3 kb DNAs is the virus capsid protein with highest sequence similarity to that of grabloviruses (family Geminiviridae), even though CFDV particles are not geminate. The nine other 1.3 kb DNAs represent alphasatellites coding for replication initiator proteins that differ clearly from those encoded by nanovirid DNA-R. The 641 nt DNA-gamma is unique and may encode a movement protein. Three DNAs, alphasatellite CFDAR, capsid protein encoding CFDV DNA-S.1 and DNA-gamma share sequence motifs near their replication origins and were consistently present in all samples analysed. These DNAs appear to be integral components of a possibly tripartite CFDV genome, different from those of any Geminiviridae or Nanoviridae family member, implicating CFDV as representative of a new genus and family.

ICTV Virus Taxonomy Profile: Avsunviroidae.

Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA-RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.

Silencing suppressor activity of a begomovirus DNA ß encoded protein and its effect on heterologous helper virus replication.

DNA ß satellites are circular single-stranded molecules associated with some monopartite begomoviruses in the family Geminiviridae. They co-infect with their helper viruses to induce severe disease in economically important crops. The ßC1 protein encoded by DNA ß is a pathogenicity determinant and has been reported to suppress post-transcriptional gene silencing (PTGS). The ßC1 proteins from various DNA ß molecules show low levels of amino acid sequence conservation. We show here that the ßC1 from DNA ß associated with Cotton leaf curl Multan virus (CLCuMV) is a suppressor of systemic PTGS. When this DNA ß satellite co-inoculated with a heterologous helper virus, Tomato leaf curl virus (ToLCV), reduced the level of ToLCV siRNA and this was associated with a higher level of virus accumulation in infected tobacco plants. This may be a mechanism by which ßC1 protects a heterologous virus from host gene silencing.

New Iranian and Australian peach latent mosaic viroid variants and evidence for rapid sequence evolution.

Peach latent mosaic viroid isolates from peach and plum in Iran have been compared with an Australian isolate from nectarine. Thirteen sequence variants 336-338 nt in size were obtained. All variants clustered phylogenetically with variants reported from several hosts and countries. A total nucleic acid extract, a slightly longer than full-length RT-PCR amplicon, and a recombinant plasmid clone from the Australian isolate were all infectious to, and symptomatic in, mechanically inoculated peach seedlings. The infectious clone generated two progeny viroid molecules, which each showed 10 different mutations compared with the parent clone inoculated 30 days previously.

A novel shaggy-like kinase interacts with the Tomato leaf curl virus pathogenicity determinant C4 protein.

Tomato leaf curl virus-Australia (ToLCV) C4 protein has been shown to be associated with virus pathogenesis. Here, we demonstrate that C4 acts as a suppressor of gene silencing. To understand the multifunctional role of C4, a novel shaggy-like kinase (SlSK) from tomato, which interacts with ToLCV C4 in a yeast two-hybrid assay, was isolated and interaction between these proteins was confirmed in vitro and in planta. Using deletion analysis of C4, a 12 amino acid region in the C-terminal part of C4 was identified which was shown to be essential for its binding to SlSK. We further demonstrate that this region is not only important for the interaction of C4 with SlSK, but is also required for C4 function to suppress gene silencing activity and to induce virus symptoms in a PVX system. The potential significance of ToLCV C4 and SlSK interaction is discussed.

Interaction with a host ubiquitin-conjugating enzyme is required for the pathogenicity of a geminiviral DNA beta satellite.

DNA beta is a single-stranded satellite DNA which encodes a single gene, betaC1. To better understand the role of betaC1 in the pathogenicity of DNA beta, a yeast two-hybrid screen of a tomato cDNA library was carried out using betaC1 from Cotton leaf curl Multan virus (CLCuMV) DNA beta as the bait. A ubiquitin-conjugating enzyme, designated SlUBC3, which functionally complemented a yeast mutant deficient in ubiquitin-conjugating enzymes was identified. The authenticity and specificity of the interaction between betaC1 and SlUBC3 was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro, using a protein-binding assay. Analysis of deletion mutants of the betaC1 protein showed that a myristoylation-like motif is required both for its interaction with SlUBC3 and the induction of DNA-beta-specific symptoms in host plants. The level of polyubiquitinated proteins in transgenic tobacco plants expressing betaC1 was found to be reduced compared with wild-type plants. These results are consistent with the hypothesis that interaction of betaC1 with SlUBC3 is required for DNA-beta-specific symptom induction, and that this is possibly due to downregulation of the host ubiquitin proteasome pathway.

Identification of sequence elements regulating promoter activity and replication of a monopartite begomovirus-associated DNA beta satellite.

DNA beta is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA beta contains a single gene, betaC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the beta-glucuronidase reporter gene driven by a betaC1 promoter-deletion series of the DNA beta associated with cotton leaf curl Multan virus identified a 68 nt region (between -139 and -207) which is important for betaC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the betaC1 start codon. Mutation of the G-box resulted in a significant reduction in betaC1 promoter activity and DNA beta replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA beta.

A monopartite begomovirus-associated DNA beta satellite substitutes for the DNA B of a bipartite begomovirus to permit systemic infection.

DNA beta is a circular single-stranded satellite DNA which co-infects with certain monopartite helper begomoviruses to cause economically important diseases, such as cotton leaf curl disease (CLCuD). DNA beta encodes a single protein, betaC1. Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus in which both DNA A and DNA B are required for systemic infection. Inoculation of tomato plants with ToLCNDV DNA A alone induced local but not systemic infection, whereas co-inoculation with DNA A and the DNA beta associated with CLCuD resulted in systemic infection. DNA beta containing a disrupted betaC1 open reading frame (ORF) did not mobilize DNA A systemically. Co-inoculation of plants with DNA A and a construct of the betaC1 ORF, under the control of the cauliflower mosaic virus 35S promoter, resulted in the systemic movement of DNA A. In inoculated tobacco and onion epidermal cells, betaC1 fused to GFP was localized at the cell periphery in association with punctate bodies, around and within the cell nucleus and with the endoplasmic reticulum. It is concluded that heterologous betaC1 protein can replace the movement function of the DNA B of a bipartite begomovirus. Evidence is also provided that tomato leaf curl virus-encoded C4 protein confers the same movement function to ToLCNDV DNA A. The intracellular distribution of betaC1 is consistent with the hypothesis that it has a role in transporting the DNA A from the nuclear site of replication to the plasmodesmatal exit sites of the infected cell.

Genomic regions of tomato leaf curl virus DNA satellite required for replication and for satellite-mediated delivery of heterologous DNAs.

Tomato leaf curl virus (TLCV) satellite DNA (sat-DNA) is a 682 nt, circular, single-stranded molecule that lacks an open reading frame (ORF) or an apparent promoter. It contains binding motifs for the TLCV replication-associated protein, but these are dispensable for replication. To identify the regions of the sat-DNA critical for replication, the entire sequence was scanned by deletion/replacement mutagenesis. Transient assays using Nicotiana benthamiana revealed that sequences within nt 296-35 (through nt 682) are essential for replication. Sequence deletions and replacements between nt 35 and 296 were tolerated but with a significant loss of infectivity, indicating that genome size strongly influences replication efficiency. Within the permissible region, inserts of 100-700 nt were retained in transient assays although with a slight reduction in replication. In addition, sat-DNA constructs containing short non-viral DNAs replicated and spread in tobacco plants, indicating their potential as gene-delivery vectors.

Identification and characterization of a host reversibly glycosylated peptide that interacts with the Tomato leaf curl virus V1 protein.

Monopartite geminiviruses of the genus Begomovirus have two virion-sense genes, V1 and V2. V2 encodes the viral coat protein, but the function of V1 is largely unknown, although some studies suggest that it may play a role in cell-to-cell movement. Yeast two-hybrid technology was used to identify possible host binding partners of V1 from Tomato leaf curl virus (TLCV) to better understand its function. A protein closely related to a family of plant reversibly glycosylated peptides, designated SlUPTG1, was found to interact with V1 in yeast and in vitro. SlUPTG1 may function endogenously in the synthesis of cell wall polysaccharides, since a bacterially expressed form of the protein acted as an autocatalytic glycosyltransferase in vitro, a SlUPTG1:GFP fusion protein localized to the cell wall, and expression of SlUPTG1 appeared to be highest in actively dividing tissues. However, expression of SlUPTG1 in a transient TLCV replication assay increased the accumulation of viral DNA, suggesting that this host factor also plays a role in viral infection. Together, these data provide new insight into the role of V1 in TLCV infection and reveal another host pathway which geminiviruses may manipulate to achieve an efficient infection.

Single-stranded DNA of Tomato leaf curl virus accumulates in the cytoplasm of phloem cells.

Geminiviruses have been reported to replicate in, and localize to, the nuclei of host plant cells. We have investigated the tissue and intracellular distribution of the monopartite Tomato leaf curl virus (TLCV) by in situ hybridization. Contrary to the current understanding of geminiviral localization, single-stranded (ss) DNA of TLCV accumulated in the cytoplasm. TLCV ssDNA was also found in the nucleus, as was lower levels of replicative form double-stranded (ds) DNA. Under the same conditions, Tomato golden mosaic virus (TGMV) ssDNA and dsDNA were found in nuclei. ssDNA of TLCV, TGMV, and Tomato yellow leaf curl Sardinia virus (TYLCSV) was detected in some xylem vessels under specific hybridization conditions. Tissue specificity of TLCV was partially released by co-infection with TGMV. Our observations suggest that the mechanism of TLCV movement may differ from that of bipartite begomoviruses.

A NAC domain protein interacts with tomato leaf curl virus replication accessory protein and enhances viral replication.

Geminivirus replication enhancer (REn) proteins dramatically increase the accumulation of viral DNA species by an unknown mechanism. In this study, we present evidence implicating SlNAC1, a new member of the NAC domain protein family from tomato (Solanum lycopersicum), in Tomato leaf curl virus (TLCV) REn function. We isolated SlNAC1 using yeast (Saccharomyces cerevisiae) two-hybrid technology and TLCV REn as bait, and confirmed the interaction between these proteins in vitro. TLCV induces SlNAC1 expression specifically in infected cells, and this upregulation requires REn. In a transient TLCV replication system, overexpression of SlNAC1 resulted in a substantial increase in viral DNA accumulation. SlNAC1 colocalized with REn to the nucleus and activated transcription of a reporter gene in yeast, suggesting that in healthy cells it functions as a transcription factor. Together, these results imply that SlNAC1 plays an important role in the process by which REn enhances TLCV replication.

A short insert in the leader sequence of RNA 3L, a long variant of Alfalfa mosaic virus RNA3, introduces two unidentified reading frames.

N20-RNA 3L, a large form of RNA 3 associated with Alfalfa mosaic virus (AMV) strain N20 comprises 2281 nt and has approximately 97% overall sequence similarity to the longest previously described RNA 3 of AMV strain YSMV (YSMV-RNA 3; 2188 nt). Compared with YSMV-RNA 3, N20-RNA 3L contains an additional 97 nt in the 5' leader upstream of the open reading frames for movement protein (MP) and coat protein (CP). Two overlapping unidentified reading frames (URF1 and URF2) result from this modification, each of which code for putative translation products of 21 amino acids. The URF1 putative peptide has a hydrophilic N-terminus and a hydrophobic C-terminus, indicating a possible association with both host cell membrane and cytosol whereas the putative URF2 product is predominantly hydrophobic. A further structural modification found in N20-RNA 3L is a new tandem repeat of 243 nts which overlaps with the MP open reading frame.

Host responses to transient expression of individual genes encoded by Tomato leaf curl virus.

The six open reading frames of Tomato leaf curl virus (TLCV) were expressed in host Nicotiana species using a Tobacco mosaic virus vector. Each of the genes, except that encoding the viral coat protein, produced a phenotypic effect when expressed in planta, but the corresponding untranslatable mutant genes were asymptomatic. The C1 (Rep) gene invoked a hypersensitive response in Nicotiana clevelandii that restricted the viral construct to sites of infection. Expression of the C2 gene in N. benthamiana produced necrotic lesions on inoculated leaves as well as severe veinal necrosis on systemically infected leaves. This gene was also able to suppress post-transcriptional gene silencing in N. tabacum. C4 induced viruslike symptoms in host plants tested, providing further evidence for the involvement of this gene in symptom expression. Expression of the V1 and C3 genes caused severe stunting of N. benthamiana plants, indicating they may also have a role in symptom development. These results reveal that a complex set of interactions between the TLCV gene products and host factors occurs in planta, and these are discussed in relation to our current understanding of TLCV gene function.