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Viroids are circular, single-stranded non-coding RNAs that rely entirely on their sequences and structures for activity. Decades of research have uncovered molecular mechanisms of viroid infection, replication, and their interactions with host factors. Notably, viroid-derived small RNAs (vd-RNAs) activate host defenses, while essential host factors and RNA motifs linked to trafficking and quasispecies evolution have been well studied. In this review, we examine key aspects of viroid biology, including the structural motifs and host factors that influence the replication cycle, as well as the mechanisms behind intra- and intercellular movement. We explore the role of vd-RNAs in activating host defense responses. Additionally, we present current perspectives on viroid quasispecies evolution and address the emergence of viroid-like RNAs across various kingdoms. These insights are crucial for deepening our understanding of the viroid replication cycle and their complex interactions with host plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Variations in infection progression with concurrent or prior infections by different viruses, viroids, or their strains are evident, but detailed investigations into viroid variant interactions are lacking. We studied potato spindle tuber viroid intermediate strain (PSTVd-I) to explore variant interactions. Two mutants, U177A/A182U (AU, replication- and trafficking-competent) and U178G/U179G (GG, replication-competent but trafficking-defective) on loop 27 increased cell-to-cell movement of wild-type (WT) PSTVd without affecting replication. In mixed infection assays, both mutants accelerated WT phloem unloading, while only AU promoted it in separate leaf assays, suggesting that enhancement of WT infection is not due to systemic signals. The mutants likely enhance WT infection due to their loop-specific functions, as evidenced by the lack of impact on WT infection seen with the distantly located G347U (UU) mutant. This study provides the first comprehensive analysis of viroid variant interactions, highlighting the prolonged phloem unloading process as a significant barrier to systemic spread., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

RNA silencing is an RNA-directed gene regulatory system that is present in a wide range of eukaryotes, and which functions as an antiviral defense in plants. Silencing pathways are complex and partially overlapping, but at least three basic classes can be distinguished: cytoplasmic RNA silencing (or post-transcriptional gene silencing; PTGS) mediated by small interfering RNAs (siRNAs), silencing mediated by microRNAs (miRNAs), and transcriptional gene silencing (TGS) mediated by siRNA-directed methylation of DNA and histone proteins. Recent advances in our understanding of different geminivirus silencing suppressors indicate that they can affect all three pathways, suggesting that multiple aspects of silencing impact geminivirus replication.

Transient expression studies using a Nicotiana benthamiana suspension cell-derived protoplast system have identified a minimal sequence that is necessary and sufficient for activation of the tomato golden mosaic virus coat protein (CP) promoter by the viral TrAP protein (also called AL2). The sequence has a bipartite arrangement in which elements located between -125 to -107 and -96 to -60 from the transcription start site are both required for TrAP-mediated activation. One of the sequences (-96 to -60) also appears to interact with a repressor, as its deletion increases basal promoter activity in the absence of TrAP. That competition experiments using the -107 to -60 sequence to titrate the repressor also resulted in increased basal transcription is consistent with this idea. Thus, in a protoplast system which models mesophyll, regulation of the minimal CP promoter involves both activation and derepression by TrAP.

The AL2 gene of the geminivirus tomato golden mosaic virus (TGMV) encodes a transcriptional activator protein (TrAP) that is required for efficient expression of the viral coat protein (CP) and BR1 gene promoters. In contrast, L2, the positional homolog of AL2 in the related beet curly top virus (BCTV), is not required for CP expression, raising questions about the functional relationship between the AL2 and L2 gene products. In this study, transgenic Nicotiana benthamiana and N. tabacum var. Samsun plants expressing a truncated AL2 gene (AL2(1-100), lacking the activation domain) or full-length L2 were prepared. These transgenic plants showed a novel enhanced susceptibility (ES) phenotype following inoculation with TGMV, BCTV, or tobacco mosaic virus (TMV), an unrelated RNA virus. ES is characterized by a reduction in the mean latent period (from 1 to 9 days) and by a decrease in the inoculum concentration required to infect transgenic plants (ID50 reduced 6- to 60-fold). However, ES does not result in an enhancement of disease symptoms, and viral nucleic acids do not accumulate to substantially greater levels in infected transgenic plants. That both viral transgenes condition ES suggests that their products share the ability to suppress a host stress or defense response that acts against DNA and RNA viruses. The data further indicate that the transcriptional activation activity of AL2 protein is not required for suppression. The nature of the response targeted by the AL2 and L2 gene products is discussed., (Copyright 2001 Academic Press.)

The AL2 gene found in members of the genus Begomovirus of the Geminiviridae encodes a transcriptional activator protein (TrAP; also known as AL2, AC2, or C2 protein). TrAP activates expression from the viral coat protein (CP) and BR1 movement gene promoters in mesophyll cells and protoplasts and acts to derepress the CP promoter in vascular tissue. The experiments presented here were designed to elucidate some of the biochemical properties of this multifunctional regulatory protein and to define its activation domain. The results indicate that TrAP from tomato golden mosaic virus (TGMV) binds single-stranded DNA in a sequence nonspecific manner and only weakly interacts with double-stranded DNA, confirming earlier results obtained with TrAP from other begomoviruses. In addition, evidence is presented that indicates that TrAP binds zinc and that zinc is necessary for optimal interaction with ssDNA. We also show that TrAP is phosphorylated when expressed in insect cells and that it contains a transcriptional activation domain of the acidic type. The minimal activation domain is quite small; the region comprising only the 15 C-terminal amino acids of the protein is capable of activating transcription in mouse fibroblasts (NIH3T3 cells) when fused to a heterologous DNA-binding domain., (Copyright 1999 Academic Press.)

Tomato golden mosaic virus (TGMV) is a bipartite member of the subgroup III Geminiviridae. Like all geminiviruses, TGMV replicates in the nucleus of susceptible cells by rolling circle replication (RCR). Double-stranded replicative form DNA generated during RCR serves as template for the transcription of viral genes by RNA polymerase II and the associated cellular transcription machinery. Previous studies in tobacco protoplasts and Nicotiana benthamiana leaf discs have shown that the viral AL2 gene product transactivates expression of the coat protein (CP) and BR1 movement protein genes, and that activation occurs at the level of transcription. Because of its function and properties, we propose the name TrAP, transcriptional activator protein, for the AL2 gene product. Using transgenes consisting of complete and truncated versions of the CP promoter fused to the GUS reporter gene, we show in the studies presented here that TrAP is required for CP gene expression in both mesophyll and phloem tissues. Surprisingly, TrAP appears to induce CP expression by different mechanisms in different cell types: it may activate the CP promoter in mesophyll cells, and acts to derepress the promoter in phloem tissue. In addition, TrAP is clearly capable of inducing the expression of responsive chromosomal promoters and could, in principle, activate host genes. Distinct viral sequence elements mediate expression and derepression in phloem and activation in mesophyll, suggesting that TrAP interacts with different components of the cellular transcription machinery to accomplish CP gene expression in different cell types, and underscoring the intricacy and complexity of virus-host interactions.

The monopartite DNA genome of beet curly top geminivirus (BCTV, strain Logan) contains four leftward (complementary sense) open reading frames (ORFs) designated L1, L2, L3, and L4. We investigated the functions of the L2 and L3 ORFs by mutational analysis. We found that in Nicotiana benthamiana and sugarbeet plants, neither a functional L2 nor a functional L3 gene is required for infectivity. Double mutants were also infectious, and no evidence for a synergistic effect of these genes was evident. However, while sugarbeet plants inoculated with L2 or L3 mutants showed symptoms that were indistinguishable from those elicited by wild type virus, mutant-inoculated N. benthamiana plants displayed a novel phenotype in which recovery of the plant from initially severe disease symptoms was greatly enhanced. Enhanced recovery was associated with a large reduction in viral DNA levels. Our studies did not provide evidence for functional homology between the BCTV L2 gene and its presumed homologue (AL2) in the bipartite geminiviruses. In contrast, mutants with lesions in the L3 ORF accumulated three- to five-fold less DNA than wild type virus in a protoplast replication assay, consistent with the interpretation that the BCTV L3 gene is a homologue of the bipartite geminivirus AL3 gene which is known to function as a replication enhancer. Functional homology was directly confirmed in experiments which demonstrated that the BCTV L3 gene can complement a tomato golden mosaic virus AL3 mutant, and vice versa.

The geminivirus group is diverse and contains viruses which can be placed into three distinct subgroups on the basis of their genome organization and biological properties. However, most dicot-infecting gemiviruses possess AL1, AL2, and AL3 open reading frames. AL1 encodes the only viral protein that is absolutely required for replication, AL2 codes for a protein that transactivates the expression of virion sense promoters, and AL3 specifies a protein that enhances viral DNA replication. In the studies presented here, we examined the functional specificity of the AL1, AL2, and AL3 specifies gene products of subgroup II and subgroup III geminiviruses. Surprisingly, we found that all viruses tested were able to produce a gene product which complemented the reduced DNA replication phenotype of a tomato golden mosaic virus (TGMV) AL3 mutant. We also found that all bipartite subgroup III viruses tested produced a protein that could transactivate the virion sense promoters of a TGMV AL2 mutant, although a subgroup II virus did not. In contrast, the replication activity of AL1 protein proved to be virus specific. The data are discussed with regard to the functions of these proteins in viral replication and their practical significance for the development of crop protection strategies.

Recombinant genomes derived from the Logan and CFH strains of the geminivirus beet curly top virus (BCTV) have been constructed and analyzed for pathogenicity on Nicotiana benthamiana and sugar beet (Beta vulgaris L.). Infectivity assays indicated that the latent period on N. benthamiana was primarily determined by a DNA fragment bearing the leftward open reading frames (ORFs) L1, L2, L3, and L4. Recombinants bearing leftward ORFs from the CFH strain were characterized as having a short latent period (mean = 6-11 days), while the reciprocal recombinants bearing leftward ORFs from the Logan strain had latent periods defined as long (mean = 16-22 days). Infectivity assays on sugar beet indicated that certain recombinant BCTV genomes exhibited novel pathogenic properties not common to either wild type strain, including the loss of systemic movement and replication competency, or asymptomatic systemic infection of sugar beet. The results indicate that N. benthamiana is a more permissive host than sugar beet with respect to heterologous combinations of BCTV genes, and that pathogenicity and virulence of BCTV in sugar beet requires the interaction of certain viral gene products and/or cis-elements that have coevolved in the same strain.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two DNA components, designated DNA A and DNA B. DNA A encodes AL1, the only viral protein required for DNA replication. AL1 protein interacts specifically with sequences in the common region that is conserved between the two genome components, near sequences involved in the transcription of complementary sense genes encoding BL1 protein and the AL1 protein itself. In the experiments described here, we replaced the AL1 and BL1 open reading frames with the beta-glucuronidase (GUS) reporter gene and used the gene replacement constructs to examine AL1 and BL1 gene expression in tobacco protoplasts. We found that expression of the GUS reporter in the AL1 replacement construct was reduced to background levels when transfections included a plasmid expressing AL1 protein from the cauliflower mosaic virus 35S promoter, indicating that AL1 gene expression is autoregulated. Surprisingly, a similar repression of BL1 gene expression by AL1 protein was not observed. Plasmids expressing the TGMV AL2 or AL3 proteins had no significant effect on AL1 or BL1 gene expression. In the course of these studies, we showed for the first time that the product of the AL3 ORF alone is sufficient to complement the replication-deficient phenotype of a TGMV AL3 mutant. The results are discussed in light of the multiple activities of AL1 protein.

The monopartite DNA genome of beet curly top geminivirus (BCTV, strain Logan) contains four leftward, complementary sense open reading frames (ORFs) designated L1, L2, L3, and L4 and three rightward, virion sense ORFs designated R1, R2, and R3 (R1 encodes the coat protein). The R3 ORF has not been reported previously in the BCTV genome, and evidence for three functional virion sense genes on one genome component has not been presented before for any geminivirus. We investigated the functions of the virion sense ORFs by introducing mutations into each of them. We found that in Nicotiana benthamiana plants, BCTV genomes containing mutations in ORF R1 were not infectious, whereas an R3- mutant was very weakly infectious. The small proportion of plants infected by the R3- mutant remained asymptomatic and contained greatly reduced amounts of viral DNA. An R2- mutant was highly infectious but asymptomatic, and in infected plants it accumulated mostly the double-stranded DNA (dsDNA) replicative form in nearly wild-type amounts. All of the mutants replicated in tobacco protoplasts, although R1- and R2- mutants accumulated reduced amounts of genomic single-stranded DNA (ssDNA) relative to wild-type virus. In the case of R2- mutants, the reduction was large (approx. ninefold) and was accompanied by a similar increase in dsDNA levels. The results suggest that the R1 and R3 gene products are required for efficient movement of the virus in the infected plant, whereas the R2 gene product may be involved in the regulation of ssDNA vs dsDNA levels.

We have analyzed the replication kinetics of the DNA A and DNA B genome components of the geminivirus tomato golden mosaic virus (TGMV) in protoplasts derived from Nicotiana tabacum suspension culture. In addition, the kinetics of TGMV coat protein promoter activity, as measured by expression of a beta-glucuronidase (GUS) reporter, have been examined. In our protoplast system, double-stranded DNA forms of both viral genome components appeared by 18 hr post-transfection, while single-stranded DNA accumulated to detectable levels after 18-24 hr. Expression of GUS from the TGMV coat protein promoter did not require viral DNA replication, nor was it dependent on expression of AL1, the only viral gene necessary for DNA replication. However, maximal expression was achieved following AL1-mediated replication of DNA A. GUS activity from replicating templates exceeded that from nonreplicating templates by 60- to 90-fold. Expression of the GUS reporter gene from nonreplicating viral DNA templates was similar to GUS expression from the 35S promoter of cauliflower mosaic virus in N. tabacum protoplasts.

The beta-glucuronidase (GUS) reporter gene was used to replace the coat protein gene (open reading frame AR1) of tomato golden mosaic virus (TGMV) and transiently expressed in tobacco protoplasts. While these TGMV/GUS genomes gave a high level of GUS activity, genomes which also contained a mutation in the AL2 open reading frame (TGMV/GUS/AL2-) did not express GUS. GUS activity could be restored by cotransfecting protoplasts with the TGMV/GUS/AL2- genome and a wild-type TGMV genome. Thus, the AL2 gene product transactivates expression of TGMV coat protein gene.

Tomato golden mosaic virus (TGMV) is a geminivirus whose genome is divided between two DNA components, designated A and B. The TGMV genome contains six open reading frames (ORFs) which can encode proteins of greater than 10 kDa. We have used a protoplast transfection system to determine the effects of viral proteins, as defined by these ORFs, on the accumulation of viral DNA in infected cells. The accumulation of cost protein was also examined in leaf discs. Our results indicate that mutations in ORFs AR1 and AL2 do not affect viral double-stranded DNA (dsDNA) levels, although AR1 and AL2 mutants accumulate only small amounts of single-stranded viral DNA (ssDNA). In contrast, a large reduction in both ss- and dsDNA levels is observed when a mutation is introduced into ORF AL3. Mutations within either of the two DNA B ORFs do not affect DNA replication. The AL3, BR1, and BL1 mutants are capable of synthesizing coat protein; however, coat protein is not detected in leaf discs inoculated with AR1 or AL2 mutants. Testable models are proposed to explain the influence of AL2 protein on coat protein accumulation and to account for the stimulation of viral DNA synthesis mediated by the AL3 gene product.

In a previous study, the bipartite genome of tomato golden mosaic virus (TGMV) was shown to be transcribed into at least six polyadenylated RNAs (G. Sunter, W.E. Gardiner, and D. M. Bisaro, 1989, Virology 170, 243-250). Two of these, a 1.3-kb complementary sense and a 0.9-kb viral sense transcript, were mapped to the B genome component of this geminivirus. The results of more detailed primer extension and S1 nuclease protection experiments presented here define the limits of the single transcription unit corresponding to the 0.9-kb RNA which spans the BR1 open reading frame (ORF). The data also demonstrate that complementary sense TGMV RNAs are more complex than indicated by our earlier studies. Analysis of the 1.3-kb BL1-specific RNA indicates that it is actually a family of distinct transcripts with different start sites. Three transcripts have 5' ends that map near the common region of DNA B and all of these start sites lie upstream of the BL1 ORF. Similar analysis of the 1.6-kb complementary sense AL1 RNA indicates that a complex set of transcripts also map to the analogous region of genome component A. Four transcripts have 5' ends that map near the common region but only one of these start sites is upstream of the initiation codon for the AL1 open reading frame (ORF). None of the transcripts appear to be processed. The possible significance of multiple transcripts in these regions of the TGMV genome is discussed, and the common region-proximal transcription units of the A and B genome components are compared.

The bipartite genome of the geminivirus tomato golden mosaic virus (TGMV) contains at least six open reading frames (ORFs) with the potential to code for proteins of greater than 100 amino acids. In order to investigate the expression of these coding regions, RNA preparations from plants infected with TGMV have been examined for the presence of viral transcripts. We have identified six polyadenylated, virus-specific RNAs which correspond in size, polarity and map location to the six ORFs. Primer extension and S1 nuclease analysis of an RNA which maps to the viral coat protein gene (ORF AR1) has shown that this transcription unit begins at nucleotide 319 or 320 and ends in the vicinity of nucleotide 1090 of the TGMV A sequence, in agreement with a previous report (I.T.D. Petty, R.H.A. Coutts, and K.W. Buck, 1988, J. Gen. Virol. 69, 1359-1365). The data presented here confirm the bidirectional transcription strategy implied by the arrangement of ORFs on both strands of double-stranded TGMV DNA intermediates and lay the ground-work for further studies of viral transcription and its control.

Tobacco rattle virus (CAM strain)-infected tissue contains at least one unique species of virus-specific RNA in addition to the two virion RNAs. The new species (RNA-3; MW 0.5 x 10(6)) was characterized as follows: (1) Its synthesis and presence were detected by electrophoresis and fluorographic analysis of an extract of infected tissue that had been treated with actinomycin D and exposed to [3H]uridine; (2) its genesis from RNA-2 was determined by immobilization on specially treated diazotized paper and hybridization to cDNAs prepared to TRV RNAs 1 and 2; and (3) its template activity for a 30,000 MW protein was determined with the aid of a wheat germ-derived cell-free translation system.

Cells infected with the CAM strain of tobacco rattle virus (TRV) contain at least five species of virus-specific RNA in addition to the two genomic RNAs. These species were detected by hybridizing [(32)P]cDNA prepared to TRV RNAs-1 and -2 with electrophoretically separated infected cell RNA species immobilized on diazotized paper. The new viral RNA species have the following apparent molecular weights: RNA-3, 0.5 x 10(6); RNA-4, 0.22 x 10(6); RNA-5, 1.1 X 10(6); RNA-6,0.36 x 10(6); RNA-7, 0.16 x 10(6). RNA-5 is generated from RNA-1 since it hybridizes with RNA-1 cDNA but not with cDNA prepared to RNA-2. The rest hybridize with RNA-2 cDNA and thus are generated from the smaller genomic species.