Margaret I. Boulton

The nucleotide sequence of maize streak virus DNA.

The nucleotide sequence of the DNA of maize streak virus (MSV) has been determined. The data were accommodated into one DNA circle of 2687 nucleotides, in contrast to previously characterised geminiviruses which have been shown to possess two circles of DNA. Comparison of the nucleotide sequences of the DNA of MSV with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) showed no detectable homology. Analysis of open reading frames revealed seven potential coding regions for proteins of mol. wt. greater than or equal to 10 000, three in the viral (+) sense and four in the complementary (‐) sense. The position of likely transcription signals on the MSV DNA sequence would suggest a bidirectional strategy of transcription as proposed for CLV and TGMV. Nine inverted repeat sequences which have a potential of forming hairpin structures of delta G greater than or equal to ‐14 kcal/mol have been detected. Three of these hairpin structures are in non‐coding regions and could be involved in the regulation of transcription and/or replication.

Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.

Rice tungro bacilliform virus DNA independently infects rice after Agrobacterium-mediated transfer

In nature, rice tungro disease is caused by an RNA and a DNA virus complex, but we have obtained an independently infectious clone of rice tungro bacilliform virus (RTBV) DNA. Infectivity could be demonstrated only when a more than unit-length copy was cloned in the Agrobacterium binary vector Bin 19 and agroinoculated into rice plants. Rice plants thus agroinfected with cloned RTBV DNA showed typical symptoms of tungro disease, presence of viral DNA and bacilliform particles, and could be used as a source of virus to infect healthy plants by the green leafhopper (Nephotettix virescens). The importance of this infectious clone in understanding the molecular biology of RTBV and the rice tungro disease is discussed.

The nucleotide sequence of a geminivirus from Digitaria sanguinalis

The encapsidated single-stranded circular DNA of a geminivirus isolated from Digitaria sanguinalis has been sequenced. The data obtained are consistent with there being one DNA circle of 2701 nucleotides. Comparison of the nucleotide sequence with those of maize streak virus (MSV) and wheat dwarf virus showed 64 and 47% DNA homology, respectively. The sequence has four potential coding regions for proteins of greater than 10 kDa, two in the viral (+) sense and two in the complementary (-) sense. Each of these potential coding regions has a highly homologous counterpart among the seven open reading frames previously described for MSV. Virion DNA contained, in addition to the circular single-stranded DNA, a population of small DNA molecules similar to those associated with MSV particles. A comparison with MSV DNA of the region complementary to these small DNA molecules revealed conserved sequences, which may have a role in defining the limits of these primer-like molecules.

A putative primer for second-strand DNA synthesis of maize streak virus is virion-associated.

We have isolated, from maize streak virus (MSV) preparations, a population of ‘nested’ DNA molecules. These molecules have ribonucleotides covalently linked to the DNA species’ discrete 5′ deoxyribonucleotide terminus. The major species has a DNA sequence of 80 nucleotides which is complementary to a region 5′ of two hairpin structures on the MSV genome, almost exclusively in an intergenic region. These molecules have been used to prime the synthesis in vitro of a complementary strand to virion DNA, initiating this reaction at one site on the genome.

Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA.

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.

Agrobacterium-mediated infectivity of cloned digitaria streak virus DNA

A monomeric clone of double-stranded DNA synthesized in vitro DNA of the geminivirus Digitaria streak (DSV) was subcloned as a tandem dimeric unit into a binary vector of Agrobacterium tumefaciens, creating a plasmid pDS2. Inoculation of digitaria sanguinalis with A. tumefaciens carrying pDS2 resulted in viral infection. The symptoms, virus particles, and DNA forms obtained were indistinguishable from those of a natural DSV infection of D. sanguinalis. Inoculations have also induced infections in Zea mays and Avena sativa. The sequence of the Agrobacterium-mediated infectious clone of DSV has been determined.

Polyethylene glycol-mediated infection of cucumber protoplasts by cucumber mosaic virus and virus RNA.

Summary In attempts to infect cucumber protoplasts with cucumber mosaic virus (CMV) RNA, the use of polyethylene glycol as an uptake inducer proved successful. This method involves resuspending protoplasts in high concentrations of polyethylene glycol in the presence of RNA, followed by rapid dilution. Levels of infection obtained with CMV-RNA were more reproducible than those using virus nucleoprotein. This method was also suitable for the inoculation of protoplasts with tobacco ringspot virus RNA.

Detection of a non-structural protein of M r 11 000 encoded by the virion DNA of maize streak virus.

A polypeptide of approximately 11 000 daltons (11 kDa protein) encoded by an open reading frame (10.9 ORF) from the virion sense of maize streak virus (MSV) DNA has been detected among the products of in vitro translation reactions programmed with RNA from infected maize plants and also in total protein extracts from infected leaves. The 11 kDa protein has not been detected in virions and is therefore proposed to have a nonstructural role.Viral DNA with an additional in-frame translation stop codon in the 10.9 ORF was not infectious when transmitted to maize plants via Agrobacterium tumefaciens “agroinfection”, suggesting that the 10.9 ORF may be essential for virus function. Computer comparison data show that equivalent ORFs in wheat dwarf virus (WDV) and digitaria streak virus (DSV) have some sequences in common with the 10.9 ORF of MSV. Further-more, the absence of similar sequences in geminiviruses which infect dicotyledonous plants suggests that the 11 kDa protein and its putative homologs in WDV and DSV have a function necessary only for those geminiviruses which infect the Gramineae.The significance of the 11 kDa protein in relation to expression of the virion sense DNA of MSV is discussed.

An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into plants.

Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ, that is induced by the plant‐wound signal molecule, acetosyringone (AS). virJ lies between virA and virB, and is transcribed in the same direction. The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB, required for virulence. virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis. Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains. While virJ is regulated by AS under the control of the virA/virG two‐component regulatory system, acvB is not induced by AS. VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction. The strain lacking both acvB and virJ had an impaired ability to transfer T‐DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T‐DNA transfer from A. tumefaciens to plant cells. acvB is the first chromosomal gene implicated in T‐DNA transfer, but whether it functions specifically for this process is not clear. We hypothesize that virJ evolved from acvB, presumably for a more specialized role in tumorigenesis.