Identification of novel DNA forms in tomato golden mosaic virus infected tissue. Evidence for a two component viral genome.

Extracts obtained from cells infected with the geminivirus tomato golden mosaic (TGMV) are shown to contain, in addition to viral single-stranded DNA, several novel species of virus-specific single- and double- stranded DNA (ss and ds DNA). The results of nuclease studies and electron microscopy suggest that three of the intracellular DNAs are unit-genome length duplexes of closed circular, relaxed circular, and linear form. The remaining ds DNA species are of high molecular weight and appear to be concatamers consisting of two or more unit-length circular ds TGMV DNA resulted in fragments whose combined size is twice the unit-genome length. Thus ds TGMV is composed of two components of nearly identical size but different nucleotide sequence.     

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.     

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.     

Molecular cloning and characterisation of the two DNA components of tomato golden mosaic virus.

We report the molecular cloning of the tomato golden mosaic virus (TGMV) genome in the E. coli plasmid pAT 153. The results of this work conclusively show that TGMV DNA consists of two components (designated A and B) of almost, but not exactly, the same size. Four different recombinant plasmids are described, two containing component A in opposite orientation and two containing component B in opposite orientation. Southern blot analysis revealed little sequence homology between A and B and showed both components to be equally represented in viral and intracellular DNA forms. Detailed restriction maps of the cloned DNAs are presented, and a comparison of these with digests of intracellular viral dsDNA indicates that the former are full-length faithful copies of TGMV DNA. This is the first report of the cloning of a geminivirus genome.     

Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants

Phenotypically normal petunia plants carrying chromosomal inserts of either the tomato golden mosaic virus (TGMV) A or the B component DNA, as single or tandem inserts, were obtained using an Agrobacterium tumefaciens Ti plasmid-based transformation system. Southern hybridization analysis revealed that the tandem, direct-repeat A plants contained free single and double stranded A component DNAs. No free B component DNA was detected in plants carrying tandem repeats of the B component. Progeny of self-fertilized plants appeared normal. In contrast, one-quarter of the progeny from tandem A by tandem B plant crosses showed chlorotic lesions on their leaves similar to virus symptoms. The significance of these results and the use of this method for the study of virus functions involved in TGMV replication and symptom production are 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.     

Agrobacterium-mediated inoculation of plants with tomato golden mosaic virus DNAs

We have adapted the agroinfection procedure of Grimsley and co-workers [4,5] to develop a simple, efficient, reproducible infectivity assay for the insect-transmitted, split-genome geminivirus, tomato golden mosaic virus (TGMV). Agrobacterium T-DNA vectors provide efficient delivery of both components of TGMV when used in mixed inoculation of wild-type host plants. A greater increase in infection efficiency can be obtained by Agrobacterium delivery of the TGMV A component to permissive transgenic plants. These permissive plants contain multiple tandem copies of the B component integrated into the host genome. An inoculum containing as few as 2000 Agrobacterium cells can produce 100% infection under these conditions. Further, our results show that there is a marked effect of the configuration of the TGMV A components within the T-DNA vector on time of symptom development. We have also found that transgenic plants carrying tandem copies of the A component do not complement the B component. Possible mechanisms to explain these results and the potential use of this system to further study the functions of the geminivirus components in infection are discussed.     

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.     

Molecular characterisation of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus.

A subgenomic single-stranded DNA present in particles of the geminivirus, tomato golden mosaic virus, has been shown by electron microscope heteroduplex mapping and Southern hybridisation analysis to consist of circular molecules, ca. 1.2 kb in size, derived from the smaller of the two genomic DNA components, DNA B, by deletion of open reading frame (ORF) BR1 and the C-terminal portion of ORF BL1. A covalently closed circular, supercoiled, double-stranded form of the subgenomic DNA has been isolated from virus-infected plants and cloned into pEMBL9. Analysis of the sequence of 22 clones across the deletion boundaries revealed only four different deletion boundaries, derived from four different left hand borders and three different right hand borders. Each border was within a region of 11 nucleotides and gave rise to a narrow size range (1248-1261 nucleotides) for the population of 22 subgenomic DNAs. However apparently smaller subgenomic DNAs were sometimes formed when plants were inoculated with cloned subgenomic DNA, or a construct derived from a subgenomic DNA in which a neomycin phosphotransferase gene had been inserted, together with the genomic DNA components. Mechanisms to account for the size, specificity and formation of the subgenomic DNA are discussed.     

The behaviour of tomato golden mosaic virus DNA in cultured cells isolated from systemically infected tobacco leaves

When callus tissue was cultured from leaf pieces taken from a Nicotiana tabacum cv. Xanthi nc. plant systemically infected with tomato golden mosaic virus (TGMV), TGMV-specific DNA persisted for up to 6 months in culture. Analysis of TGMV-specific intracellular DNA forms indicated a decrease in double-stranded relative to single-stranded forms and an increase in sub-genomic relative to genomic single-stranded DNA species in the callus tissue compared to those in the original leaf explant. The implications of the results with regard to TGMV replication are discussed.     

Total nucleotide sequences of the infectious cloned DNAs of bean golden mosaic virus

Complete nucleotide sequences of the infectious cloned DNA components (DNA A and B) of bean bolden mosaic virus were determined. The DNA A (2585 nucleotides) and DNA B (2647 nucleotides) have little sequence homology with each other, but both A and B contain a common region of 205 nucleotides. A possible large hairpin structure is detected in the common region. Nucleotide sequences of DNAs A and B revealed the presence of 8 potential coding regions for proteins (m.w. greater than 10,000). Among them, four open reading frames (ORFs 1-4) encode proteins of m.w. 30,000 or greater, and are individually coded in virion DNA A and B senses (+) and their complementary senses (-), respectively. The other four ORFs 5-8 are in virion DNA B(+) and its complementary sense (-). All of the ORFs 1-4 have regulatory signals for RNA synthesis (TATAA/T) in the region 5' upstream from a potential start codon ATG.     

Nucleotide sequence of the brome mosaic virus genome and its implications for viral replication

The nucleotide sequences of brome mosaic virus (BMV) RNAs 1 (3234 bases) and 2 (2865 bases) have been determined, completing the primary structure of the 8200 base tripartite BMV genome. cDNA clones covering 99% of BMV RNA1 and a full-length cDNA clone of BMV RNA2 were isolated in the course of this work. Extensive sequence homology and known interaction with several proteins suggest that the 3' ends of the BMV RNAs are the major regulatory regions of the genome. Smaller regions at the 5' ends of RNAs 1 and 2 show strong homology to each other and lesser homology to RNA3. These and other features of the sequences are discussed in relation to replication, regulation and evolution of the BMV genome.