Search

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.)

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

RNA viruses and viroids replicate with high mutation rates, forming quasispecies, population of variants centered around dominant sequences. The mechanisms governing quasispecies remain unclear. Plasmodesmata regulate viroid movement and were hypothesized to impact viroid quasispecies. Here, we sequenced the progeny of potato spindle tuber viroid intermediate (PSTVd-I) strain from mature guard cells lacking plasmodesmal connections and from in vitro-cultivated mesophyll cell protoplasts from systemic leaves of early-infected tomato (Solanum lycopersicum) plants. Remarkably, more variants accumulated in guard cells compared to whole leaves. Similarly, after extended cell culture, we observed more variants in cultivated mesophyll protoplasts. Coinfection and single-cell sequencing experiments demonstrated that the same plant cell can be infected multiple times by the same or different PSTVd sequences. To study the impact of initial population composition on PSTVd-I quasispecies, we conducted coinfections with PSTVd-I and variants. Two inoculum ratios (10:1 or 1:10) established quasispecies with or without PSTVd-I as the master sequence. In the absence of the master sequence, the percentage of novel variants initially increased. Moreover, a 1:1 PSTVd-I/variant RNA ratio resulted in PSTVd-I dominating (>50%), while the variants reached 20%. After PSTVd-I-only infection, the variants reached around 10%, while after variant-only infection, the variants were significantly more than 10%. These results emphasize the role of cell-to-cell communication and initial population composition in shaping PSTVd quasispecies., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tobacco mosaic virus movement protein (TMV MP) is essential for virus spread between cells. To accomplish its task, TMV MP binds viral RNA, interacts with components of the cytoskeleton, and increases the size exclusion limit (SEL) of plasmodesmata. Plasmodesmata are gated intercellular channels that allow passage of small molecules and macromolecules, including RNA and protein, between plant cells. Moreover, plasmodesmata are diverse and those connecting different cell types appear to have unique mechanisms to regulate macromolecular trafficking, which likely contributes to the establishment of distinct cell boundaries. Consequently, TMV MP might be competent to mediate RNA transport through some but not all plasmodesmal gates. Due to a lack of viral mutants defective for movement between specific cell types, the ability of TMV MP in this regard is incompletely understood. In contrast, a number of trafficking impaired Potato spindle tuber viroid (PSTVd) mutants have been identified. PSTVd is a systemically infectious non-coding RNA that nevertheless can perform all functions required for replication as well as cell-to-cell and systemic spread. Previous studies have shown that PSTVd employs different structure and sequence elements to move between diverse cell types in host plants, and mutants defective for transport between specific cell types have been identified. Therefore, PSTVd may serve as a tool to analyze the functions of MPs of viral and cellular origin. To probe the RNA transport activity of TMV MP, transgenic plants expressing the protein were inoculated with PSTVd mutants. Remarkably, TMV MP complemented a PSTVd mutant defective for mesophyll entry but could not support two mutants impaired for phloem entry, suggesting it fails to productively interface with plasmodesmata at the phloem boundary and that additional viral and host factors may be required. Consistent with this idea, TMV co-infection, but not the combination of MP and coat protein (CP) expression, was able to complement one of the phloem entry mutants. These observations suggest that phloem loading is a critical impediment to establishing systemic infection that could involve the entire ensemble of TMV proteins. They also demonstrate a novel strategy for analysis of MPs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Wu, Bisaro. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Accurate calculation of mutation rates for viruses and viroids is necessary for evolutionary studies and to evaluate adaptation potential. However, estimation of in vivo mutation rates is complicated by selection, which leads to loss or proliferation of certain mutations. To minimize this concern, lethal mutations, including nonsense and non-synonymous mutations, have been used to determine mutation rates for several viruses and viroids, including Potato spindle tuber viroid (PSTVd). However, this approach has limitations, including focus on a relatively small number of genome sites and the possibility that mutations may not actually be lethal or may be maintained by wild type individuals. To avoid selection bias altogether, we sequenced minus-strand PSTVd dimers from concatemeric replication intermediates. The underlying rationale is that mutations found in only one of the monomers were likely generated de novo during RNA polymerase II (Pol II) transcription of the circular plus-strand RNA genome. This approach yielded an apparent Pol II error rate of ~1/1837 nucleotides per transcription cycle, and an estimated mutation rate of ~1/919 nucleotides for a single replication cycle. Remarkably, de novo mutations were nearly absent from the most conserved, replication-critical regions of the PSTVd genome, suggesting that sequence conservation is a consequence of both essential function and template optimization for greater Pol II fidelity. Such biased fidelity may constitute a novel strategy to ensure population success while allowing abundant sampling of sequence space in other genome regions. Comparison with variants in progeny populations derived from a cloned, wild type PSTVd master sequence revealed that most de novo mutations were lost through selection., Competing Interests: The authors have declared that no competing interests exist.

Geminiviruses are a significant group of emergent plant DNA viruses causing devastating diseases in food crops worldwide, including the Southern United States, Central America and the Caribbean. Crop failure due to geminivirus-related disease can be as high as 100%. Improved global transportation has enhanced the spread of geminiviruses and their vectors, supporting the emergence of new, more virulent recombinant strains. With limited coding capacity, geminiviruses encode multifunctional proteins, including the AC2/C2 gene that plays a central role in the viral replication-cycle through suppression of host defenses and transcriptional regulation of the late viral genes. The AC2/C2 proteins encoded by mono- and bipartite geminiviruses and the curtovirus C2 can be considered virulence factors, and are known to interact with both basal and inducible systems. This review highlights the role of AC2/C2 in affecting the jasmonic acid and salicylic acid (JA and SA) pathways, the ubiquitin/proteasome system (UPS), and RNA silencing pathways. In addition to suppressing host defenses, AC2/C2 play a critical role in regulating expression of the coat protein during the viral life cycle. It is important that the timing of CP expression is regulated to ensure that ssDNA is converted to dsDNA early during an infection and is sequestered late in the infection. How AC2 interacts with host transcription factors to regulate CP expression is discussed along with how computational approaches can help identify critical host networks targeted by geminivirus AC2 proteins. Thus, the role of AC2/C2 in the viral life-cycle is to prevent the host from mounting an efficient defense response to geminivirus infection and to ensure maximal amplification and encapsidation of the viral genome., (Copyright © 2020 Guerrero, Regedanz, Lu, Ruan, Bisaro and Sunter.)

While G/U pairs are present in many RNAs, the lack of molecular studies to characterize the roles of multiple G/U pairs within a single RNA limits our understanding of their biological significance. From known RNA 3D structures, we observed that the probability a G/U will form a Watson-Crick (WC) base pair depends on sequence context. We analyzed 17 G/U pairs in the 359-nucleotide genome of Potato spindle tuber viroid (PSTVd), a circular non-coding RNA that replicates and spreads systemically in host plants. Most putative G/U base pairs were experimentally supported by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). Deep sequencing PSTVd genomes from plants inoculated with a cloned master sequence revealed naturally occurring variants, and showed that G/U pairs are maintained to the same extent as canonical WC base pairs. Comprehensive mutational analysis demonstrated that nearly all G/U pairs are critical for replication and/or systemic spread. Two selected G/U pairs were found to be required for PSTVd entry into, but not for exit from, the host vascular system. This study identifies critical roles for G/U pairs in the survival of an infectious RNA, and increases understanding of structure-based regulation of replication and trafficking of pathogen and cellular RNAs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177-182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure., Competing Interests: The authors have declared that no competing interests exist.

Regulation of protein synthesis is critical for maintaining cellular homeostasis. In mammalian systems, translational regulatory networks have been elucidated in considerable detail. In plants, however, regulation occurs through different mechanisms that remain largely elusive. In this study, we present evidence that the Arabidopsis thaliana energy sensing kinase SnRK1, a homologue of mammalian AMP-activated kinase and yeast sucrose non-fermenting 1 (SNF1), inhibits translation by phosphorylating the cap binding proteins eIF4E and eIFiso4E. We establish that eIF4E and eIFiso4E contain two deeply conserved SnRK1 consensus target sites and that both interact with SnRK1 in vivo. We then demonstrate that SnRK1 phosphorylation inhibits the ability of Arabidopsis eIF4E and eIFiso4E to complement a yeast strain lacking endogenous eIF4E, and that inhibition correlates with repression of polysome formation. Finally, we show that SnRK1 over-expression in Nicotiana benthamiana plants reduces polysome formation, and that this effect can be counteracted by transient expression of eIF4E or mutant eIF4E containing non-phosphorylatable SnRK1 target residues, but not by a phosphomimic eIF4E. Together, these studies elucidate a novel and direct pathway for translational control in plant cells. In light of previous findings that SnRK1 conditions an innate antiviral defense and is inhibited by geminivirus pathogenicity factors, we speculate that phosphorylation of cap binding proteins may be a component of the resistance mechanism., (© 2019 Federation of European Biochemical Societies.)

Histone posttranslational modifications (PTMs) impart information that regulates chromatin structure and activity. Their effects are mediated by histone reader proteins that bind specific PTMs to modify chromatin and/or recruit appropriate effectors to alter the chromatin landscape. Despite their crucial juxtaposition between information and functional outcome, relatively few plant histone readers have been identified, and nothing is known about their impact on viral chromatin and pathogenesis. We used the geminivirus Cabbage leaf curl virus (CaLCuV) as a model to functionally characterize two recently identified reader proteins, EMSY-LIKE 1 (EML1) and EML3, which contain Tudor-like Agenet domains predictive of histone PTM binding function. Here, we show that mutant Arabidopsis plants exhibit contrasting hypersusceptible ( eml1 ) and tolerant ( eml3 ) responses to CaLCuV infection and that EML1 deficiency correlates with RNA polymerase II (Pol II) enrichment on viral chromatin and upregulated viral gene expression. Consistent with reader activity, EML1 and EML3 associate with nucleosomes and with CaLCuV chromatin, suggesting a direct impact on pathogenesis. We also demonstrate that EML1 and EML3 bind peptides containing histone H3 lysine 36 (H3K36), a PTM usually associated with active gene expression. The interaction encompasses multiple H3K36 PTMs, including methylation and acetylation, suggesting nuanced regulation. Furthermore, EML1 and EML3 associate with similar regions of viral chromatin, implying possible competition between the two readers. Regions of EML1 and EML3 association correlate with sites of trimethylated H3K36 (H3K36me3) enrichment, consistent with regulation of geminivirus chromatin by direct EML targeting. IMPORTANCE Histone PTMs convey information that regulates chromatin compaction and DNA accessibility. Histone reader proteins bind specific PTMs and translate their effects by modifying chromatin and/or by recruiting effectors that alter chromatin structure or activity. In this study, CaLCuV was used to characterize the activities of two Arabidopsis Agenet domain histone readers, EML1 and EML3. We show that eml1 mutants are hypersusceptible to CaLCuV, whereas eml3 plants are more tolerant of infection than wild-type plants. We also demonstrate that EML1 and EML3 associate with histones and viral chromatin in planta and that both proteins bind peptides containing H3K36, a PTM associated with active gene expression. Consistent with antiviral activity, EML1 suppresses CaLCuV gene expression and reduces Pol II access to viral chromatin. By linking EML1 and EML3 to pathogenesis, these studies have expanded our knowledge of histone reader proteins and uncovered an additional level of viral chromatin regulation., (Copyright © 2018 American Society for Microbiology.)

Plants employ RNA-directed DNA methylation (RdDM) and dimethylation of histone 3 lysine 9 (H3K9me2) to silence geminiviruses and transposable elements (TEs). We previously showed that canonical RdDM (Pol IV-RdDM) involving RNA polymerases IV and V (Pol IV and Pol V) is required for Arabidopsis thaliana to recover from infection with Beet curly top virus lacking a suppressor protein that inhibits methylation (BCTV L2 - ). Recovery, which is characterized by reduced viral DNA levels and symptom remission, allows normal floral development. Here, we used formaldehyde-assisted isolation of regulatory elements (FAIRE) to confirm that >90% of BCTV L2 - chromatin is highly compacted during recovery, and a micrococcal nuclease-chromatin immunoprecipitation assay showed that this is largely due to increased nucleosome occupancy. Physical compaction correlated with augmented cytosine and H3K9 methylation and with reduced viral gene expression. We additionally demonstrated that these phenomena are dependent on Pol V and by extension the Pol IV-RdDM pathway. BCTV L2 - was also used to evaluate the impact of viral infection on host loci, including repressed retrotransposons Ta3 and Athila6A Remarkably, an unexpected Pol V-dependent hypersuppression of these TEs was observed, resulting in transcript levels even lower than those detected in uninfected plants. Hypersuppression is likely to be especially important for natural recovery from wild-type geminiviruses, as viral L2 and AL2 proteins cause ectopic TE expression. Thus, Pol IV-RdDM targets both viral and TE chromatin during recovery, simultaneously silencing the majority of viral genomes and maintaining host genome integrity by enforcing tighter control of TEs in future reproductive tissues. IMPORTANCE In plants, RdDM pathways use small RNAs to target cytosine and H3K9 methylation, thereby silencing DNA virus genomes and transposable elements (TEs). Further, Pol IV-RdDM involving Pol IV and Pol V is a key aspect of host defense that can lead to recovery from geminivirus infection. Recovery is characterized by reduced viral DNA levels and symptom remission and thus allows normal floral development. Studies described here demonstrate that the Pol V-dependent enhanced viral DNA and histone methylation observed during recovery result in increased chromatin compaction and suppressed gene expression. In addition, we show that TE-associated chromatin is also targeted for hypersuppression during recovery, such that TE transcripts are reduced below the already low levels seen in uninfected plants. Thus, Pol IV-RdDM at once silences the majority of viral genomes and enforces a tight control over TEs which might otherwise jeopardize genome integrity in future reproductive tissue., (Copyright © 2018 American Society for Microbiology.)

Unlabelled: In plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirus Beet curly top virus (BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formed de novo in infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to trigger de novo deposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin., Importance: In plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a reevaluation of this view. We used BCTV to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naive viral chromatin and further that Pol IV-RdDM is responsible., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR-Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR-Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses., Competing Interests: Competing interests N.J.B., A.W.H. and D.F.V. are inventors on a patent application (WO2015048707A2) for the technology described in this work.

Unlabelled: Both posttranscriptional and transcriptional gene silencing (PTGS and TGS, respectively) participate in defense against the DNA-containing geminiviruses. As a countermeasure, members of the genus Begomovirus (e.g., Cabbage leaf curl virus) encode an AL2 protein that is both a transcriptional activator and a silencing suppressor. The related L2 protein of Beet curly top virus (genus Curtovirus) lacks transcription activation activity. Previous studies showed that both AL2 and L2 suppress silencing by a mechanism that correlates with adenosine kinase (ADK) inhibition, while AL2 in addition activates transcription of cellular genes that negatively regulate silencing pathways. The goal of this study was to clarify the general means by which these viral proteins inhibit various aspects of silencing. We confirmed that AL2 inhibits systemic silencing spread by a mechanism that requires transcription activation activity. Surprisingly, we also found that reversal of PTGS and TGS by ADK inactivation depended on whether experiments were conducted in vegetative or reproductive Nicotiana benthamiana plants (i.e., before or after the vegetative-to-reproductive transition). While AL2 was able to reverse silencing in both vegetative and reproductive plants, L2 and ADK inhibition were effective only in vegetative plants. This suggests that silencing maintenance mechanisms can change during development or in response to stress. Remarkably, we also observed that AL2 lacking its transcription activation domain could reverse TGS in reproductive plants, revealing a third, previously unsuspected AL2 suppression mechanism that depends on neither ADK inactivation nor transcription activation., Importance: RNA silencing in plants is a multivalent antiviral defense, and viruses respond by elaborating multiple and sometimes multifunctional proteins that inhibit various aspects of silencing. The studies described here add an additional layer of complexity to this interplay. By examining geminivirus AL2 and L2 suppressor activities, we show that L2 is unable to suppress silencing in Nicotiana benthamiana plants that have undergone the vegetative-to-reproductive transition. As L2 was previously shown to be effective in mature Arabidopsis plants, these results illustrate that silencing mechanisms can change during development or in response to stress in ways that may be species specific. The AL2 and L2 proteins are known to share a suppression mechanism that correlates with the ability of both proteins to inhibit ADK, while AL2 in addition can inhibit silencing by transcriptionally activating cellular genes. Here, we also provide evidence for a third AL2 suppression mechanism that depends on neither transcription activation nor ADK inactivation. In addition to revealing the remarkable versatility of AL2, this work highlights the utility of viral suppressors as probes for the analysis of silencing pathways., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Arabidopsis encodes five double-stranded RNA binding (DRB) proteins. DRB1 and DRB2 are involved in microRNA (miRNA) biogenesis, while DRB4 functions in cytoplasmic posttranscriptional small interfering RNA (siRNA) pathways. DRB3 and DRB5 are not involved in double-stranded RNA (dsRNA) processing but assist in silencing transcripts targeted by DRB2-associated miRNAs. The goal of this study was to determine which, if any, of the DRB proteins might also participate in a nuclear siRNA pathway that leads to geminivirus genome methylation. Here, we demonstrate that DRB3 functions with Dicer-like 3 (DCL3) and Argonaute 4 (AGO4) in methylation-mediated antiviral defense. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, using an RNA-directed DNA methylation (RdDM) pathway similar to that used to suppress endogenous invasive DNAs such as transposons. Chromatin methylation inhibits virus replication and transcription, and methylation-deficient host plants are hypersusceptible to geminivirus infection. Using a panel of drb mutants, we found that drb3 plants uniquely exhibit a similar hypersensitivity and that viral genome methylation is substantially reduced in drb3 compared to wild-type plants. In addition, like dcl3 and ago4 mutants, drb3 plants fail to recover from infection and cannot accomplish the viral genome hypermethylation that is invariably observed in asymptomatic, recovered tissues. Small RNA analysis, bimolecular fluorescence complementation, and coimmunoprecipitation experiments show that DRB3 acts downstream of siRNA biogenesis and suggest that it associates with DCL3 and AGO4 in distinct subnuclear compartments. These studies reveal that in addition to its previously established role in the miRNA pathway, DRB3 also functions in antiviral RdDM., Importance: Plants use RNA-directed DNA methylation (RdDM) as an epigenetic defense against geminiviruses. RNA silencing pathways in Arabidopsis include five double-stranded RNA binding proteins (DRBs) related to Drosophila R2D2 and mammalian TRBP and PACT. While DRB proteins have defined roles in miRNA and cytoplasmic siRNA pathways, a role in nuclear RdDM was elusive. Here, we used the geminivirus system to show that DRB3 is involved in methylation-mediated antiviral defense. Beginning with a panel of Arabidopsis drb mutants, we demonstrated that drb3 plants uniquely show enhanced susceptibility to geminiviruses. Further, like dcl3 and ago4 mutants, drb3 plants fail to hypermethylate the viral genome, a requirement for host recovery. We also show that DRB3 physically interacts with the RdDM pathway components DCL3 and AGO4 in the nucleus. This work highlights the utility of geminiviruses as models for de novo RdDM and places DRB3 protein in this fundamental epigenetic pathway.

SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5'-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5'-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK in vitro by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for in vivo linkage but also revealing that in vivo regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.

In plants, post-transcriptional gene silencing (PTGS) is a sequence-specific mechanism of RNA degradation induced by double-stranded RNA (dsRNA), which is processed into small interfering RNAs (siRNAs). siRNAs are methylated and, thereby, stabilized by the activity of the S-adenosylmethionine-dependent RNA methyltransferase HEN1. PTGS is amplified by host-encoded RNA-dependent RNA polymerases (RDR), which generate dsRNA that is processed into secondary siRNAs. To counteract this RNA silencing-mediated response of the host, plant viruses express proteins with silencing suppression activity. Here, we report that the coat protein (CP) of crinivirus (family Closteroviridae, genus Crinivirus) Tomato chlorosis virus, a known suppressor of silencing, interacts with S-adenosylhomocysteine hydrolase (SAHH), a plant protein essential for sustaining the methyl cycle and S-adenosylmethionine-dependent methyltransferase activity. Our results show that, by contributing to an increased accumulation of secondary siRNAs generated by the action of RDR6, SAHH enhances local RNA silencing. Although downregulation of SAHH prevents local silencing, it enhances the spread of systemic silencing. Our results also show that SAHH is important in the suppression of local RNA silencing not only by the crinivirus Tomato chlorosis virus CP but also by the multifunctional helper component-proteinase of the potyvirus Potato virus Y.

Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA β. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, βC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2⁻ mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or βC1 expression. We also demonstrate that while TYLCCNB or βC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that βC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that βC1 protein inhibits SAHH activity in vitro. That βC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by βC1 stabilizes geminivirus/betasatellite complexes.

It is well-established that plants use cytoplasmic, post-transcriptional gene silencing (PTGS) as a defense against RNA viruses and DNA virus transcripts. More recently, it has become clear that small RNA-directed methylation leading to transcriptional gene silencing (TGS) is also used as a defense against DNA virus chromatin. Here we use the DNA-containing geminiviruses as models to discuss what is currently known about both types of antiviral silencing, and viral suppression of PTGS and TGS as a counterdefense., (2010 Elsevier B.V. All rights reserved.)

Background: Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems., Results: The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS), transketolase, the sulfur allele of magnesium chelatase (ChlI), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant., Conclusion: The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

RNA-induced silencing is a potent innate antiviral defense strategy in plants, and suppression of silencing is a hallmark of pathogenic plant viruses. However, the impact of silencing as a mammalian antiviral defense mechanism and the ability of mammalian viruses to suppress silencing in natural host cells have remained controversial. The ability of herpes simplex virus type 1 (HSV-1) to suppress silencing was examined in a transient expression system that employed an imperfect hairpin to target degradation of transcripts encoding enhanced green fluorescent protein (EGFP). HSV-1 infection suppressed EGFP-specific silencing as demonstrated by increased EGFP mRNA levels and an increase in the EGFP mRNA half-life. The increase in EGFP mRNA stability occurred despite the well-characterized host macromolecular shutoff functions of HSV-1 that globally destabilize mRNAs. Moreover, mutant viruses defective in these functions increased the stability of EGFP mRNA even more than did the wild-type virus in silenced cells compared to results in control cells. The importance of RNA silencing to HSV-1 replication was confirmed by a significantly enhanced virus burst size in cells in which silencing was knocked down with small inhibitory RNAs directed to Argonaute 2, an integral component of the silencing complex. Given that HSV-1 encodes several microRNAs, it is possible that a dynamic equilibrium exists between silencing and silencing suppression that is capable of modulating viral gene expression to promote replication, to evade host defenses, and/or to promote latency.

Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS.

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.

The DNA genomes of geminiviruses have a limited coding capacity that is compensated for by the production of small multifunctional proteins. The AL2 protein encoded by members of the genus Begomovirus (e.g., Tomato golden mosaic virus) is a transcriptional activator, a silencing suppressor, and a suppressor of a basal defense. The related L2 protein of Beet curly top virus (genus Curtovirus) shares the pathogenicity functions of AL2 but lacks transcriptional activation activity. It is known that AL2 and L2 can suppress local silencing by interacting with adenosine kinase (ADK) and can suppress basal defense by interacting with SNF1 kinase. However, how the activities of these viral proteins are regulated remains an unanswered question. Here, we provide some answers by demonstrating that AL2, but not L2, interacts with itself. The zinc finger-like motif (CCHC) is required but is not sufficient for AL2 self-interaction. Alanine substitutions for the invariant cysteine residues that comprise the motif abolish self-interaction or cause aberrant subnuclear localization but do not abolish interaction with ADK and SNF1. Using bimolecular fluorescence complementation, we show that AL2:AL2 complexes accumulate primarily in the nucleus, whereas AL2:ADK and L2:ADK complexes accumulate mainly in the cytoplasm. Further, the cysteine residue mutations impair the ability of AL2 to activate the coat protein promoter but do not affect local silencing suppression. Thus, AL2 self-interaction correlates with nuclear localization and efficient activation of transcription, whereas AL2 and L2 monomers can suppress local silencing by interacting with ADK in the cytoplasm.

Recombinant green fluorescent protein (GFP) with a molecular mass of 29 kDa was transiently expressed in Agrobacterium-inoculated leaf-disks prepared from Nicotiana benthamiana plants. Expression of GFP from the Cauliflower mosaic virus (CaMV) 35 S promoter within a replicating vector based on the geminivirus Beet curly top virus (BCTV) was more than 3 times higher than from a control, non-replicating vector. Use of the Cassava vein mosaic virus (CsVMV) promoter in the BCTV replicating vector increased the expression of recombinant GFP 320% at the transcript level, compared to use of the control CaMV 35 S promoter. Expression of recombinant GFP from Agrobacterium-inoculated leaf-disks of N. benthamiana was further enhanced up to 240% in the presence of post-transcriptional gene silencing suppressor p19.

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.

Most plant viruses are initiators and targets of RNA silencing and encode proteins that suppress this adaptive host defense. The DNA-containing geminiviruses are no exception, and the AL2 protein (also known as AC2, C2, and transcriptional activator protein) encoded by members of the genus Begomovirus has been shown to act as a silencing suppressor. Here, a three-component, Agrobacterium-mediated transient assay is used to further examine the silencing suppression activity of AL2 from Tomato golden mosaic virus (TGMV, a begomovirus) and to determine if the related L2 protein of Beet curly top virus (BCTV, genus Curtovirus) also has suppression activity. We show that TGMV AL2, AL2(1-100) (lacking the transcriptional activation domain), and BCTV L2 can all suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene when silencing is induced by a construct expressing an inverted repeat GFP RNA (dsGFP). We previously found that these viral proteins interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance. Using the GFP-dsGFP system, we demonstrate here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins. In addition, AL2/L2 suppression phenotypes and nucleic acid binding properties are shown to be different from those of the RNA virus suppressors HC-Pro and p19. These findings provide strong evidence that ADK activity is required to support RNA silencing, and indicate that the geminivirus proteins suppress silencing by a novel mechanism that involves ADK inhibition. Further, since AL2(1-100) is as effective a suppressor as the full-length AL2 protein, activation and silencing suppression appear to be independent activities.

Recombinant endostatin was transiently expressed in Agrobacterium-inoculated leaf disks of Nicotiana tabacum var. Xanthi with a molecular size of 23 kDa. Expression of endostatin from a replicating vector based on tomato golden mosaic virus (TGMV) was 170% higher at the transcript level and double higher at the protein level than from a control vector of a non-replicating construct. Purified recombinant endostatin from tobacco leaf-disks has an anti-proliferative effect on bovine endothelial cells.

AL2 and L2 are related proteins encoded by geminiviruses of the Begomovirus and Curtovirus genera, respectively. Both are pathogenicity determinants that cause enhanced susceptibility when expressed in transgenic plants. To understand how geminiviruses defeat host mechanisms that limit infectivity, we searched for cellular proteins that interact with AL2 and L2. Here, we present evidence that the viral proteins interact with and inactivate adenosine kinase (ADK), a nucleoside kinase that catalyzes the salvage synthesis of 5'-AMP from adenosine and ATP. We show that the AL2 and L2 proteins inactivate ADK in vitro and after coexpression in Escherichia coli and yeast. We also demonstrate that ADK activity is reduced in transgenic plants expressing the viral proteins and in geminivirus-infected plant tissues. By contrast, ADK activity is increased after inoculation of plants with diverse RNA viruses or a geminivirus lacking a functional L2 gene. Consistent with its ability to interact with multiple cellular kinases, we also demonstrate that AL2 is present in both the nucleus and the cytoplasm of infected plant cells. These data indicate that ADK is targeted by viral pathogens and provide evidence that this "housekeeping" enzyme might be a part of host defense responses. In previous work, we showed that AL2 and L2 also interact with and inactivate SNF1 kinase, a global regulator of metabolism that is activated by 5'-AMP. Together, these observations suggest that metabolic alterations mediated by SNF1 are an important component of innate antiviral defenses and that the inactivation of ADK and SNF1 by the geminivirus proteins represents a dual strategy to counter this defense. AL2 proteins also have been shown to act as suppressors of RNA silencing, an adaptive host defense response. A possible relationship between ADK inactivation and silencing suppression is discussed.

Geminivirus AL2 and L2 proteins cause enhanced susceptibility, characterized primarily by an increase in viral infectivity, when expressed in transgenic plants. Here, we present genetic and biochemical evidence that enhanced susceptibility is attributable to the interaction of AL2 and L2 with SNF1 kinase, a global regulator of metabolism. Specifically, we show that AL2 and L2 inactivate SNF1 in vitro and in vivo. We further demonstrate that expression of an antisense SNF1 transgene in Nicotiana benthamiana plants causes enhanced susceptibility similar to that conditioned by the AL2 and L2 transgenes, whereas SNF1 overexpression leads to enhanced resistance. Transgenic plants expressing an AL2 protein that lacks a significant portion of the SNF1 interaction domain do not display enhanced susceptibility. Together, these observations suggest that the metabolic alterations mediated by SNF1 are a component of innate antiviral defenses and that SNF1 inactivation by AL2 and L2 is a counterdefensive measure. They also indicate that geminiviruses are able to modify host metabolism to their own advantage, and they provide a molecular link between metabolic status and inherent susceptibility to viral pathogens.

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.

Vascular puncture inoculation (VPI) is an effective technique for transmission of maize viruses without using arthropods or other biological vectors. It involves using a jeweler's engraving tool to push minuten pins through a droplet of virus inoculum toward the major vascular bundle in the scutellum of germinating kernels. Here, VPI is shown to be useful for introducing RNA and DNA viral genomes into maize. Maize dwarf mosaic potyvirus (MDMV) virions, MDMV genomic RNA, foxtail mosaic potexvirus (FoMV) genomic RNA and maize streak geminivirus (MSV) DNA were introduced into kernels by VPI, and infection rates determined. At high concentrations, both MDMV virion and genomic RNA preparations produced 100% infection of susceptible maize. However, MDMV genomic RNA was transmitted with about 100-fold lower efficiency than virions. FoMV genomic RNA and MSV DNA were transmitted at lower efficiency than the MDMV RNA, and the highest transmission rates were about 50%. Ribonuclease A pretreatment prevented genomic MDMV and FoMV RNA transmission, but not MDMV virion transmission indicating the viral RNA was the infectious entity. Proteinase K (ProK) pretreatment reduced transmission of MDMV RNA suggesting that integrity of the viral genomic protein bound covalently to the viral RNA may be important for efficient transmission.

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 infection of susceptible plant hosts by single-stranded DNA viruses in the geminivirus group depends on the interaction of host and viral factors for the replication of viral DNA, the expression of viral genes, and the movement of virus throughout the plant. This paper reports that two strains of the geminivirus, beet curly top virus (BCTV) differ in their ability to infect certain ecotypes of Arabidopsis thaliana. Symptoms appeared on susceptible plants approximately 2-3 weeks after inoculation with BCTV-Logan and after 10-15 days with BCTV-CFH. Symptoms were more severe in BCTV-CFH-infected plants and included leaf curling, the formation of stunted, deformed inflorescence structures and the accumulation of anthocyanin pigments in symptomatic tissues. Analysis of viral DNA accumulation indicated that symptom development and severity were correlated with the amount of viral DNA present in the plants. Viral DNA was undetectable in two ecotypes that were phenotypically resistant to BCTV-Logan. Studies of viral DNA replication in excised inflorescence pieces demonstrated that BCTV-Logan could replicate in tissues from these resistant ecotypes, suggesting that resistance was due to a block in viral movement. Genetic studies of these two ecotypes indicate that resistance is due to a single, recessive locus. This is the first example of a single resistance locus to any geminivirus. The identification of resistant and susceptible interactions between different BCTV strains and A. thaliana ecotypes provides an excellent model system for the genetic and molecular analysis of the interaction of a plant host with this important group of plant pathogens.

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 compared the fate of U.G mispairs or analogous T.G mispairs in DNA heteroduplexes transfected into tobacco protoplasts. The heteroduplex DNA consisted of tomato golden mosaic virus DNA sequences in the Escherichia coli vectors pUC118 or pUC119. After transfection, the mismatched U residues were lost with an efficiency of greater than 95%, probably as a result of the uracil-DNA glycosylase pathway for excision of U residues in any sequence context. In contrast to the preferential removal of the mispaired U residues, biased removal of T residues from analogous heteroduplexes was not seen in the transfected plant cells. Also, we investigated the effect of extensively methylating one strand of the heteroduplex DNA used for transfection. Surprisingly, such methylation resulted in highly biased loss of the mismatched base from the 5-methylcytosine-rich strand of T.G-containing heteroduplexes.

Tomato golden mosaic virus is a bipartite geminivirus whose genome is divided between two circular DNA molecules. DNA A encodes functions necessary for viral DNA replication and encapsidation, whereas DNA B provides functions needed for movement in the host. Previous studies have shown that the viral AL2 gene product transactivates expression of the coat protein gene (AR1). We have investigated the role of the AL2 protein in the regulation of B component gene expression and examined the transcriptional and post-transcriptional components of this regulation. We found that AL2 protein is required for efficient expression of both the AR1 and BR1 genes, but not the BL1 gene. A comparison of steady state transcript levels and transcript levels determined by nuclear run-on analysis showed that activation of AR1 and BR1 gene expression by the AL2 protein occurs primarily at the level of transcription. These results provide an explanation for the lack of infectivity demonstrated by AL2 mutants, and suggest that the AL2 protein interacts with the cellular transcription machinery to activate the expression of rightward viral genes.