Geminivirus Beet Curly Top Virus Research Articles

Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin.

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. 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.

Mutation in Arabidopsis CSN5A partially complements the lack of Beet curly top virus pathogenicity factor L2

Ubiquitination, a post-translational modification that controls multiple cellular processes, has proven to be a common target for viruses. Recently, geminivirus C2/L2 protein has been shown to interact with the plant CSN5, catalytic subunit of the CSN complex, and possibly disrupt the activity of this complex over CULLIN1, thus interfering with the function of the CULLIN1-based SCF ubiquitin E3 ligases. With the aim of shedding light on the genetic interaction between geminivirus C2/L2 and the plant CSN5, we designed an infection experiment using a combination of mutant plant and virus. Our results indicate that the geminivirus Beet curly top virus (BCTV) infects the Arabidopsis CSN5a mutant less efficiently; moreover, mutation in BCTV L2 can be partially complemented by mutation in Arabidopsis CSN5a. These results suggest that the inhibition of the CSN5 activity might be an important role of geminivirus C2/L2 during the establishment of the infection, and raises the possibility that geminiviral infection might require CSN activity.

Development of a gene silencing DNA vector derived from a broad host range geminivirus

BackgroundGene 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.ResultsThe 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.ConclusionThe 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.

Improved expression of recombinant GFP using a replicating vector based on Beet curly top virus in leaf-disks and infiltrated Nicotiana benthamiana leaves

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.

Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor.

The complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other geminiviruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72-74 percent amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98-100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (Cl, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed.

Strain-Specific Determinants of Beet Curly Top Geminivirus DNA Replication

The Logan and CFH strains of the geminivirus beet curly top virus (BCTV) possess cis- and trans-DNA replication factors which exhibit specificity and are not functionally interchangeable. We demonstrate that the cis -acting replication specificity element is entirely contained within a 82- to 97-bp fragment which includes most of the viral DNA origin of replication We also demonstrate that the strain specific trans-acting replication determinant is located within amino acid residues 3-89 of the BCTV C1 replication protein. Transient replication assays indicated that chimeric BCTV genomes containing reciprocally exchanged regions of the CFH and Logan genomes were replication competent when the cis- and trans -replication specificity elements were derived from the same strain. Two reciprocal chimeric viral genomes with heterologous cis- and trans -replication elements were incapable of self-replication, yet could trans -replicate one another in a coinoculation experiment. Only chimeric genomes possessing the Logan trans-replication element were capable of mobilizing and amplifying a transgenic Logan derived D1-DNA. D1-DNA mobilization and amplification occurred in transient replication assays even when the helper virus genome was incapable of self-replication, providing that the trans-replication element was derived from the Logan strain. These results genetically define specific regions of the BCTV C1 replication protein determining vital DNA replication origin recognition and provide clear evidence that strains of BCTV have evolved specific cis- and trans-replication factors which functionally define the Logan and CFH strains as distinct viral agents.

Strain-Specific Mobilization and AmpLification of a Transgenic Defective-Interfering DNA of the Geminivirus Beet Curly Top Virus

Transgenic Nicotiana benthamiana plants have been constructed which bear integrated, tandemly repeated copies of a beet curly top virus (BCTV) defective-interfering (DI) DNA derived from the Logan strain. Transgenic DI-DNA plant lines challenge-inoculated with BCTV-Logan exhibited delayed and attenuated symptoms compared to nontransgenic plants. Infection of transgenic plants with the Logan strain resulted in the mobilization of the integrated DI-DNA sequence, which was subsequently amplified as an episome. The accumulation of Logan helper virus DNA forms was reduced in transgenic plants, relative to nontransgenic plants. In contrast, no delay or attenuation of symptoms was observed for transgenic plants challenge-inoculated with the BCTV strains CFH and Worland. Infection by the CFH and Worland strains did not result in mobilization or amplification of the integrated Logan DI-DNA sequence, and no consistent differences in the accumulation of CFH or Worland genomic viral DNA forms were observed among transgenic and nontransgenic plants. These results, and a comparison of putative DNA replication origin sequences, suggest that BCTV strains display specificity with respect to recognition of heterologous DNA replication origin cis-elements.

Recombinant Beet Curly Top Virus Genomes Exhibit Both Parental and Novel Pathogenic Phenotypes

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.

Transcriptional Analysis of the Virion-Sense Genes of the Geminivirus Beet Curly Top Virus

The genome of the geminivirus beet curly top virus (BCTV) consists of a single circular DNA containing overlapping open reading frames (ORFs) located on both the virion-sense and complementary-sense DNA strands. To investigate the expression of these ORFs, RNA extracted from infected Nicotiana benthamiana and Beta vulgaris has been examined for the presence of viral transcripts. An abundant 1.1-kb virion-sense polyadenylated RNA and four complementary-sense polyadenylated RNAs of 1.7, 1.5, 1.3, and 0.7 kb have been identified by northern blot hybridization, confirming the bidirectional transcription strategy implied by the arrangement of ORFs. We previously demonstrated that two overlapping virion-sense ORFs are involved in coat protein synthesis (ORF V1) and viral single-stranded DNA accumulation (ORF V2). Mutants of a third virion-sense ORF (ORF V3), located upstream and overlapping ORFs V1 and V2, retain the ability to replicate efficiently in N. benthamiana leaf discs but produce an asymptomatic infection in N. benthamiana and B. vulgaris at low frequency, associated with reduced levels of viral DNA compared to wild-type infection. Our data support the recent suggestion that ORF V3 participates in virus movement. The 1.1 kb virion-sense RNA comprises a population of overlapping transcripts with 5′ termini suitably positioned for the expression of ORFs V1, V2, and V3. The overlapping arrangement of the transcripts and juxtaposition of putative regulatory elements could provide a means for the temporal control of virion-sense gene expression.

Mutational analysis of the monopartite geminivirus beet curly top virus

Mutants of the monopartite geminivirus beet curly top virus have been screened for infectivity and symptom development in Nicotiana benthamiana and Beta vulgaris, for replication competence in N. benthamiana leaf discs, and for transmission by the leafhopper Circulifer tenellus. Disruption of open reading frame (ORF) V2 by the introduction of a termination codon resulted in symptomless infection of N. benthamiana associated with low levels of virus and reduced single-stranded (as) DNA and prevented systemic infection of B. vulgaris. Reduced levels of ssDNA were produced by the mutant in N. benthamiana leaf discs, suggesting that V2 affects the synthesis or accumulation of this viral DNA form. Mutants in which ORF C2 had been truncated by the introduction of termination codons or by frame-shifting remained highly infectious and induced severe symptoms in both N. benthamiana and B. vulgaris. Similarly, a mutant containing a termination codon within ORF C3 was highly infectious and induced severe symptoms in N. benthamiana although infectivity in B. vulgaris was greatly reduced, symptoms were extremely mild, and virus levels were low. A synergistic effect of a double mutation in ORFs C2 and C3, manifested by the inability of mutants to systemically infect N. benthamiana and the production of reduced amounts of ssDNA in N. benthamiana leaf discs, suggests that both ORFs are functional in this host. A mutant containing a termination codon within the 5′ terminus of ORF C4 produced severe symptoms in both N. benthamiana and B. vulgaris resembling those induced by wild-type virus. Comparison with the phenotypes of previously characterized ORF C4 mutants suggests that a conserved core sequence of this ORF is an important symptom determinant. ORF C2, C3, and C4 mutants produced virus particles and were transmitted by C. tenellus, eliminating agroinoculation as a contributory factor to the mutant phenotypes. Our results are compared with those derived from mutagenesis studies on related bipartite geminiviruses.

Characterization of beet curly top virus subgenomic DNA localizes sequences required for replication

Subgenomic viral DNA is accumulated in Nicotiana benthamiana and Beta vulgaris plants agroinoculated with the geminivirus beet curly top virus. The subgenomic DNA is more abundant in N. benthamiana and is distributed between two broad size groups in this host. Six unique examples, ranging in size from 887 to 1311 nucleotides, have been cloned from viral double-stranded DNA purified from N. benthamiana and analyzed by sequence determination. Deletions are distributed throughout most of the genome and only nucleotides 2946-410 are represented in all subgenomic DNAs. Comparison with a previously characterized subgenomic DNA suggests that cis-acting signals necessary for viral DNA replication are located in a predominantly intergenic region between nucleotides 2946-308.

A number of subgenomic DNAs are produced following agroinoculation of plants with beet curly top virus

In addition to ss and ds genomic DNA, agroinoculation of Nicotiana benthamiana plants with the Logan strain of the geminivirus beet curly top virus (BCTV) consistently resulted in de novo production of subgenomic DNAs on initial passage. Single-stranded and dsDNA forms representing at least seven size classes (0.8 to 1.8 kb) of subgenomic DNA were observed in total DNA extracts from inoculated plants. Extracts from infected sugar beet and tomato contained variable but usually smaller amounts of subgenomic DNAs, suggesting that their production may be influenced by the host species. Restriction endonuclease mapping and partial nucleotide sequencing of three independent clones of a 1.5 kb size class indicated that this subgenomic DNA is produced from the standard viral genome by two separate deletion events. One deletion of 941 bp includes portions of the leftward open reading frames (ORFs) L1, L2 and L3, while the other deletion of 579 bp encompasses portions of the intergenic region and the rightward ORFs R1, R2 and R3. The data indicate that the 1.5 kb BCTV subgenomic DNA is a defective DNA that has retained cis-elements essential for replication.

Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA.

Agrobacterium-mediated inoculation of Nicotiana benthamiana plants with Ti plasmids containing tandem genome repeats derived from different strains of the gemini-virus beet curly top virus (BCTV) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants. When two putative plus-strand origins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant genotype. The genotype was dependent upon the arrangement of repeated parental genomes in the inocula. Sequencing across the junction between parental BCTV strains in the recombinant progeny allowed mapping of the plus-strand origin of replication to a 20-base-pair sequence within the conserved hairpin found in all geminivirus genomes. In contrast, when inocula contained tandemly repeated BCTV genome sequences but only a single conserved hairpin, a number of different progeny genotypes were simultaneously replicated in infected plants, a result expected if unit-length viral genomes were generated by random intramolecular recombination events. These results and other considerations indicate that geminivirus DNA replication occurs by a rolling-circle mechanism.

The nucleotide sequence of an infectious clone of the geminivirus beet curly top virus

A number of infectious clones of a Californian isolate of the leafhopper-transmitted geminivirus beet curly top virus (BCTV) have been constructed from virus-specific double-stranded DNA isolated from infected Beta vulgaris and used to demonstrate a single component genome. The nucleotide sequence of one infectious clone has been determined (2993 nucleotides). Comparison with other geminiviruses has shown that the organisation of the genome closely resembles DNA 1 of the whitefly-transmitted members. The four conserved coding regions of DNA 1 have highly homologous counterparts in BCTV with the exception of the putative coat protein which is more closely related to those of the leafhopper-transmitted geminiviruses suggesting a strong interrelationship between coat protein and insect vector. A BCTV component equivalent to DNA 2 is not required for virus infection or transmission and has not been isolated from infected plants.