Abstract
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.
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Amasino RM, Powell ALG, Gordon MP: Changes in T-DNA methylation and expression are associated with phenotypic variation and plant regeneration in a crown gall tumor line. Mol Gen Genet 197: 437–446 (1984).
Bisaro DM, Hamilton WDO, Coutts RHA, Buck KW: Molecular cloning and characterisation of the two DNA components of tomato golden mosaic virus. Nucl Acids Res 10: 4913–4922 (1982).
Fraley RT, Rogers SG, Horsch RB, Eichholtz D, Flick JS, Fink CL, Hoffmann NL, Sanders PR: The SEV system: A new disarmed Ti plasmid vector for plant transformation. Bio/Technology 3: 629–635 (1985).
Grimsley N, Hohn B, Hohn T, Walden R: “Agroinfection,” an alternative route for viral infection of plants by using ti plasmid. Proc Natl Acad Sci USA 83: 3282–3286 (1986).
Grimsley N, Hohn T, Davies JW, Hohn B: Agrobacterium-mediated delivery of infectious maize streak virus into maize plants. Nature 325: 177–179 (1987).
Grimsley N, Bisaro DM: Agroinfection. In: Hohn B, Hohn T, King P, Schell J, Verma DPS (eds) Advances in Plant Gene Research, Vol IV, Springer-Verlag, Vienna, New York (1987) in press.
Haber S, Ikegami M, Bajet NB, Goodman RM: Evidence for a divided genome in bean golden mosaic virus, a geminivirus. Nature 289: 324–326 (1981).
Hamilton WDO, Bisaro DM, Buck KW: Identification of novel DNA forms in tomato golden mosaic virus infected tissue: Evidence for a two component viral genome. Nucl Acids Res 10: 4910–4912 (1982).
Hamilton WDO, Bisaro DM, Coutts RHA, Buck KW: Demonstration of the bipartite nature of the genome of a single-stranded plant virus by infection with the cloned DNA components. Nucl Acids Res 11: 7387–7396 (1983).
Hamilton WDO, Stein VE, Coutts RHA, Buck KW: Complete nucleotide sequence of the infectious cloned DNA components of tomato golden mosaic virus: Potential coding regions and regulatory sequences. EMBO J 3: 2197–2205 (1984).
Hepburn AG, Clarke LE, Pearson L, White J: The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J Mol Appl Genet 2: 315–328 (1983).
Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).
Horsch RB, Klee HJ: Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: Role of the T-DNA borders in the transfer process. Proc Natl Acad Sci USA 83: 4428–4432 (1986).
Lazarowitz SG: Molecular characterization of geminiviruses. Plant Mol Biol Reporter 4: 177–192 (1987).
McKnight TD, Lillis MT, Simpson RB: Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol 8: 439–445 (1987).
Rogers SG, Horsch RB, Fraley RT: Gene transfer in plants: production of transformed plants using Ti plasmid vectors. In: Weissbach H, Weissbach A (eds) Methods in Enzymology, Vol. 118 (1986) pp 627–640.
Rogers SG, Bisaro DM, Horsch RB, Fraley RT, Hoffmann NL, Brand L, Elmer JS, Lloyd AM: Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants. Cell 45: 593–600 (1986).
Stachel SE, Timmerman B, Zambryski P: Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322: 706–713 (1986).
Stanley J, Davies JW: Structure and function of the DNA genome of geminiviruses. In: Davies JW (ed.) Molecular Plant Virology, Vol II, Replication and Gene Expression. CRC Press, Boca Raton, Florida (1985) pp 191–218.
Stein VE, Coutts RHA, Buck KW: Serological studies of tomato golden mosaic virus, a geminivirus. J Gen Virology 64: 2493–2498 (1983).
Sunter G, Gardiner WE, Rushing AE, Rogers SG, Bisaro DM: Independent encapsidation of tomato golden mosaic virus A component DNA in transgenic plants. Plant Mol Biol 8: 477–484 (1987).
Townsend R, Watts J, Stanley J: Synthesis of viral DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with cassava latent virus (CLV); evidence for the independent replication of one component of the CLV genome. Nucl Acids Res 14: 1253–1265 (1986).
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Elmer, J.S., Sunter, G., Gardiner, W.E. et al. Agrobacterium-mediated inoculation of plants with tomato golden mosaic virus DNAs. Plant Mol Biol 10, 225–234 (1988). https://doi.org/10.1007/BF00027399
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DOI: https://doi.org/10.1007/BF00027399