DNA sequences essential for replication of the B genome component of tomato golden mosaic virus.

The genome of the geminivirus tomato golden mosaic virus (TGMV) is divided between two DNA components, designated A and B, which differ in sequence except for a 230-nucleotide common region. The A genome component is known to encode viral functions necessary for viral DNA replication, while the B genome component specifies functions necessary for spread of the virus through the infected plant. To identify cis-acting sequences required for viral DNA replication, several mutants were constructed by the introduction of small insertions into TGMV B at selected sites within and just outside the common region. Other mutants had the common region inverted or deleted. All of the mutants were tested for their effects on infectivity and DNA replication in whole plants and leaf discs. Our results indicate that the common region in its correct orientation is required for infectivity and for replication of TGMV B. Furthermore, the conserved hairpin loop sequence located within the TGMV common region and found in all geminiviruses is necessary for DNA replication, and may be part of the viral replication origin.     

The Nucleotide Sequence and Genome Structure of Mung Bean Yellow Mosaic Geminivirus

Complete nucleotide sequences of the infectious cloned DNA components (DNA 1 and DNA 2) of mung bean yellow mosaic virus (MYMV) were determined. MYMV DNA 1 and DNA 2 consists of 2,723 and 2,675 nucleotides respectively. DNA 1 and DNA 2 have little sequence similarity except for a region of approximately 200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed nine potential coding regions for proteins of mol. wt. > 10,000, six in DNA 1 and three in DNA 2. The nucleotide sequence of MYMV DNA was compared with that of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV). The 200-base region common to the two DNAs of each virus had little sequence similarity, except for a highly conserved 33-36 base sequence potentially capable of forming a stable hairpin structure. The potential coding regions in the MYMV DNAs had counterparts in the BGMV, TGMV and ACMV, suggesting an overall similarity in genome organization, except for absence of 1L3 in MYMV DNA 1. The most highly conserved ORFs, MYMV 1R1, BGMV 1R1, TGMV 1R1 and ACMV 1R1, are the putative genes for the coat proteins of MYMV, BGMV, TGMV and ACMV, respectively. MYMV 1L1 has also a high degree of sequence similarity with BGMV 1L1, TGMV 1L1 and ACMV 1L1.     

A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.     

Transgenic cassava resistance to <Emphasis Type="Italic">African cassava mosaic virus</Emphasis> is enhanced by viral DNA-A bidirectional promoter-derived siRNAs

Expression of double-stranded RNA (dsRNA) homologous to virus sequences can effectively interfere with RNA virus infection in plant cells by triggering RNA silencing. Here we applied this approach against a DNA virus, African cassava mosaic virus (ACMV), in its natural host cassava. Transgenic cassava plants were developed to express small interfering RNAs (siRNA) from a CaMV 35S promoter-controlled, intron-containing dsRNA cognate to the common region-containing bidirectional promoter of ACMV DNA-A. In two of three independent transgenic lines, accelerated plant recovery from ACMV-NOg infection was observed, which correlates with the presence of transgene-derived siRNAs 21–24 nt in length. Overall, cassava mosaic disease symptoms were dramatically attenuated in these two lines and less viral DNA accumulation was detected in their leaves than in those of wild-type plants. In a transient replication assay using leaf disks from the two transgenic lines, strongly reduced accumulation of viral single-stranded DNA was observed. Our study suggests that a natural RNA silencing mechanism targeting DNA viruses through production of virus-derived siRNAs is turned on earlier and more efficiently in transgenic plants expressing dsRNA cognate to the viral promoter and common region.     

Selection for wild type size derivatives of tomato golden mosaic virus during systemic infection.

A chimeric tomato golden mosaic virus (TGMV) A component DNA, which results from replacement of the coding region of the viral coat protein gene (CP) with the larger bacterial beta-glucuronidase coding sequence (GUS), can replicate in agroinoculated leaf discs but is unstable in systemically infected plants (1). We have made similar replacements of the TGMV CP gene with the GUS coding sequence in both the sense and antisense orientations. Both derivatives replicated in leaf discs inoculated via Agrobacterium. However, systemic movement of the GUS substituted vectors was not detected in agroinoculated Nicotiana benthamiana plants. The only TGMV A derivatives detected in systemically infected leaves of inoculated plants were similar in size to the wild type viral component. Sequence analysis of derivatives from six independently inoculated plants revealed that they did not result from internal deletions of the larger replicons detected in leaf discs but, instead, were generated by fusion events occuring within the original T-DNA insert. These results indicate that systemic movement of TGMV in N. benthamiana plants provides a strong selective pressure favoring viral derivatives similar in size to the wild type virus components.     

Independent encapsidation of tomato golden mosaic virus A component DNA in transgenic plants

Tomato golden mosaic virus (TGMV), a member of the geminivirus group, has a genome consisting of two DNA molecules designated the A and B components. Both are required for infectivity in healthy plants, although the former has been shown to replicate independently in transgenic plants containing tandem direct repeats of the A genome component. In the studies presented here, petunia plants transgenic for either both components (A×B hybrids) or the A component alone were examined for the presence of virus particles and encapsidated, single stranded viral DNA. The results of DNase protection experiments and direct observation of extracts from transgenic plants by electron microscopy indicate that single stranded TGMV DNA is in both cases packaged into paired particles identical to those obtained from virus-infected plants. DNase-treated virions isolated from A×B hybrid petunia are infectious when inoculated onto healthy Nicotiana benthamiana. Likewise, virions obtained from transgenic A petunia are infectious for plants transgenic for the B component.Our observations of TGMV replication in transgenic plants indicate that TGMV A DNA encodes all viral functions necessary for the replication and encapsidation of viral DNA. The possible role of the B component in TGMV replication is discussed.     

Genetic analysis of the tomato golden mosaic virus. II. The product of the AL1 coding sequence is required for replication.

Tomato golden mosaic virus (TGMV) belongs to the geminivirus subgroup that is characterized by a split genome consisting of two single-stranded circular DNAs. The TGMV A genome component encodes the virus coat protein as well as all of the functions necessary for viral DNA replication. Analysis of the nucleotide sequence indicates that the TGMV A component has, in addition to the coat protein encoding ORF, four overlapping open reading frames (ORFs) with the potential to encode proteins of greater than 10 kD. We have investigated the functions of these putative proteins in both symptom formation and DNA replication by creating mutations in each of the ORFs. Our results show that the AL4 ORF, which is encoded within the N-terminal region of ORF AL1, is not essential for normal virus infection. In contrast, we find that disruption of the AL3 ORF results in delay and attenuation of symptom formation. We also report that the products of the AL1 and AL2 ORFs are absolutely required for symptom formation. Studies of DNA replication show that only the AL1 open reading frame is essential for viral DNA synthesis. The significance of these results for the development of vectors from the geminiviruses is discussed.     

A geminivirus replication protein is a sequence-specific DNA binding protein.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component encodes the only viral protein, AL1, that is required for viral replication. We showed that AL1 interacts specifically with TGMV A and B DNA by using an immunoprecipitation assay for AL1:DNA complex formation. In this assay, a monoclonal antibody against AL1 precipitated AL1:TGMV DNA complexes, whereas an unrelated antibody failed to precipitate the complexes. Competition assays with homologous and heterologous DNAs established the specificity of AL1:DNA binding. AL1 produced by transgenic tobacco plants and by baculovirus-infected insect cells exhibited similar DNA binding activity. The AL1 binding site maps to 52 bp on the left side of the common region, a 235-bp region that is highly conserved between the two TGMV genome components. The AL1:DNA binding site does not include the putative hairpin structure that is conserved in the common regions or the equivalent 5' intergenic regions of all geminiviruses. These studies demonstrate that a geminivirus replication protein is a sequence-specific DNA binding protein, and the studies have important implications for the role of this protein in virus replication.     

Peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells

The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses.     

Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development

The geminiviruses are a unique group of higher plant viruses that are composed of twin isometric particles which contain circular, single-stranded DNA. Tomato golden mosaic virus (TGMV), a whitefly-transmitted agent, belongs to the subgroup of geminiviruses whose members possess a bipartite genome. The TGMV A genome component has the capacity to encode at least four proteins. One of these is the viral coat protein, as inferred by homology with coat-protein, genes of other geminiviruses and by the observation of typical geminate particles in transgenic plants that contain inserts of TGMV A DNA. We have investigated the role of the coat protein in TGMV replication and report here that its coding sequence may be interrupted or substantially deleted without loss of infectivity. However, certain coat-protein mutants showed reproducible delays in time of symptom appearance as well as reduced symptom development, when inoculated onto transgenic Nicotiana benthamiana plants containing the TGMV B component. The most attenuated symptoms were seen with a mutant in which the coat-protein coding sequence was almost entirely deleted. The significance of these findings for the development of plant vectors from TGMV DNA is discussed.     

Beet curly top virus DI DNA-mediated resistance is linked to its size

Beet curly top virus (BCTV) infection is associated with the de novo synthesis of a heterogeneous population of subgenomic viral DNAs. Nicotiana benthamiana plants transformed with a partial repeat of one such subgenomic DNA remained susceptible to infection but produced ameliorated symptoms when agroinoculated with BCTV. Symptom amelioration is associated with the mobilization of subgenomic DNA from the integrated copy. In an attempt to improve the resistance, N. benthamiana has been transformed with a partial repeat of a much smaller subgenomic DNA. However, transgenic plants showed almost no resistance although subgenomic DNA was mobilised from the host genome. To further understand the molecular basis of the interference phenomenon, we compared the ability of BCTV to replicate and accumulate in leaf discs derived from resistant and non-resistant transgenic plants. Both subgenomic DNAs were able to interfere with virus replication but only in case of resistant plants the DI DNA efficiently suppressed viral accumulation.     

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

Genome affinities and epitope profiles of whitefly-transmitted geminiviruses from the Americas

The relationships among fifteen isolates of whitefly-transmitted geminiviruses (WTGs) from North, Central and South America and six from other continents were assessed (a) in nucleic acid hybridisation tests with sulphonated DNA probes for eight of the viruses, and/or (b) in triple-antibody-sandwich ELISA with panels of monoclonal antibodies (MAbs) to particles of African cassava mosaic virus (ACMV) and Indian cassava mosaic virus (ICMV). Probes specific for DNA-A of four American viruses, abutilon mosaic (AbMV), bean golden mosaic (BGMV), squash leaf curl (SLCV) and tomato golden mosaic (TGMV), detected virtually all the American viruses but reacted weakly if at all with ICMV, ACMV or tomato yellow leaf curl virus from Thailand (TYLCV-T). Conversely, the probe for ACMV DNA-A did not detect any of the American viruses, and that for TYLCV-T DNA-A reacted weakly with SLCV and TGMV0020but did not detect the others. In contrast, probes specific for DNA-B of the four American viruses or ACMV detected only the homologous virus, except for slight reactions between the AbMV DNA-B probe and both chino del tomate virus (CdTV)-DNA and SLCV-DNA. However, a probe for DNA-B of bean calico mosaic virus (BCMoV) reacted weakly with BGMV-PR DNA, and a probe for DNA-B of CdTV from Mexico detected several American viruses. Six out of 17 MAbs specific for ACMV and six out of 10 MAbs specific for ICMV reacted with one or other of the 14 American virus isolates tested. Two and-ACMV MAbs reacted with all, and one anti-ACMV MAb and two anti-ICMV MAbs reacted with nearly all the American viruses, one anti-ACMV MAb reacted with about half the American viruses and six other MAbs reacted with only one or two of them. Of the American viruses, CdTV and AbMV were the least closely related to the others. The epitope profiles of BCMoV, BGMV, cotton leaf crumple virus, serrano golden mosaic virus and SLCV were virtually indistinguishable. TGMV, potato yellow mosaic virus (PYMV) and an euphorbia virus had profiles intermediate between those of the BGMV cluster and AbMV-CdTV. In general, the epitope profiles and the results of hybridisation tests with DNA-A probes show that the similarities among the American viruses are greater than those between the American viruses and the viruses from other continents; the hybridisation tests with DNA-B probes show that substantial differences exist between individual American viruses. In America, geminivirus evolution seems to have proceeded convergently from different progenitor viruses, or divergently from one ancestral form, with DNA-B diverging to a greater extent than DNA-A and its particle-protein gene.     

Computer analysis between nucleotide and amino acid sequences of bean golden mosaic virus and those of maize streak, wheat dwarf, chloris striate mosaic, and beet curly top viruses

Bean golden mosaic virus (BGMV) DNA 1 and 2 have little sequence homology with maize streak virus (MSV), wheat dwarf virus (WDV), and chloris striate mosaic virus (CSMV) DNAs. BGMV DNA 1 and beet curly top virus (BCTV) DNA are closely related, whereas BGMV DNA 2 and BCTV DNA are not related. Direct amino acid homologies of predicted proteins between BGMV ORFs and MSV ORFs, WDV ORFs or CSMV ORFs were 40-50%. BGMV 1L1 and BCTV L1, and BGMV IL3 and BCTV L4 were highly conserved. The sequence TAATATTAC was detected in the loops of hairpin structures of 5 gemini-viruses.     

High-frequency reversion of geminivirus replication protein mutants during infection

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.     

Comparative Analysis of Tissue Tropism of Bipartite Geminiviruses

Abutilon mosaic virus (AbMV), a bipartite geminivirus of the genus Begomovirus, has been vegetatively propagated for many years in Abutilon sellovianum in which it is strictly phloem-restricted. Using in situ hybridization and immunological analyses, the tissue tropism of AbMV in the laboratory host Nicotiana benthamiana was compared with that of two other bipartite begomoviruses, African cassava mosaic virus (ACMV) and tomato golden mosaic virus (TGMV). Analysis of the first systemically infected leaves and longitudinal sections of axillary and flower buds revealed that all three viruses are initially confined to the vascular traces, although both ACMV and TGMV are later detectable in nearly all tissue types. In contrast, AbMV remained strictly phloem-limited in this host throughout the course of infection. The ability of ACMV and TGMV to move out of N. benthamiana phloem tissues is correlated with the development of severe symptoms in comparison with the mild symptoms associated with AbMV infection. It was also demonstrated that Sida micrantha mosaic virus, a virus that is closely related to AbMV, is phloem-limited in Malva parviflora even though it induces severe leaf curl, stunting and necrosis in this host. The present data demonstrate that bipartite begomoviruses can exhibit strikingly different patterns of tissue tropism.