Biomedical subjects
N Grimsley
Publications and source records attributed to N Grimsley.
Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intermolecular recombination.
Intermolecular reconstitution of a plant virus has been detected in whole plants in a system using a defective cauliflower mosaic virus genome and transgenic host plants containing the missing viral gene. The information for the gene VI protein of the virus was integrated into the chromosome of host Brassica napus plants and leaves of these plants were inoculated with Agrobacterium tumefaciens containing the complementing viral sequences. In several cases, upper leaves contained replicating viral DNA which was able to incite CaMV symptoms on turnip plants. The sequence of the resultant recombinant viral molecules suggested that both DNA and RNA recombination events may have been involved in the production of functional virus, one event being gene targeting of the T-DNA.
Genomic homologous recombination in planta.
A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process.
Recovery of Agrobacterium tumefaciens T-DNA molecules from whole plants early after transfer.
A system for the analysis of independent T-DNA transfer events from Agrobacterium to plants is described. The complete T-DNA except for the 25 bp border sequences was replaced by one genome of a plant virus so that upon transfer to the plant, a viable replicon is produced by circularization. Rescue of virus from such infected plants allowed analysis of DNA sequences at or close to the ends of T-DNA molecules. A rather conserved right border remnant of three nucleotides was found, whereas the sequences remaining at the left end were more variable. A point deletion in the left 25 bp sequence results in even less precise processing at the left end. In addition, many rescued T-DNA molecules carry small direct repeats between the joined T-DNA ends; linear T-DNA molecules are therefore transported to the plant.
DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions.
DNA transfer from Agrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyle-donous plant Zea mays, was analysed using the recently developed technique of agroinfection. Agroinfection of Z. mays with maize streak virus using strains of A. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterial virC genes. In contrast, agroinfection of the control host Brassica rapa with cauliflower mosaic virus was less dependent on the virC gene products. In other respects, the basic mechanism of the plant-bacterium interaction was found to be similar. While intact virA, B, D and G functions were absolutely necessary, mutants in virE were attenuated. Agroinfection of maize was effective in the absence of an exogenously supplied vir gene inducer, and indeed wounded Z. mays tissues were found to produce substance(s) which induced the expression of A. tumefaciens vir genes. These findings are discussed in the light of current knowledge about the function of Agrobacterium vir genes.
Recombination in a plant virus: template-switching in cauliflower mosaic virus.
A hybrid plasmid, containing tandemly arranged pieces of two different but well-defined cauliflower mosaic virus (CaMV) genomes, was used to study the mechanism by which infectious viral DNA can escape from transforming DNA. Systemic viral infection followed inoculation of Brassica plants with a strain of Agrobacterium tumefaciens containing the hybrid plasmid in its T-DNA. Restriction mapping of uncloned viral DNA from these plants, and sequencing of relevant portions of cloned viral DNA, showed that the majority of viral progeny were probably descendants of DNA produced by transcription/reverse transcription of the viral genome, thus providing further evidence for the hypothesis that this process is normally involved in viral replication. The reverse transcriptase enzyme, which is thought to undergo an intramolecular template switch during viral replication, is shown to move very close to the 5' end of the terminal repeat on the 35S RNA molecule before switching templates. The remaining minority of viral genomes can best be explained as arising from products of recombination between homologous regions of CaMV DNA.
"Agroinfection," an alternative route for viral infection of plants by using the Ti plasmid.
Most plant viruses are transmitted by insect vectors. We present an alternative method for the introduction of infectious viral DNA that uses the ability of Agrobacterium to transfer DNA from bacterial cells to plants. Cauliflower mosaic virus was chosen to develop this method because it is the best characterized plant DNA virus and can be introduced into plants via aphids, virus particles, viral DNA, or suitably treated cloned DNA. We show that systemic infection of turnips results from wounding and inoculation with strains of Agrobacterium tumefaciens in which more than one genome of cauliflower mosaic virus have been placed tandemly in the T-DNA of the tumor-inducing plasmid. Thus such constructions allow escape of the viral genome from the T-DNA once inside the plants. The combined use of the tumor-inducing plasmid and viral DNA opens the way to molecular biological approaches that are not possible with either system alone.