Expression of functional elements inserted into the 35S promoter region of infectious cauliflower mosaic virus replicons.
We describe experiments directed towards development of cauliflower mosaic virus (CaMV) replicons for propagation of functional elements during infection of plants. Modifications and inserts were introduced into replaceable domains associated with the 35S promoter. The 35S enhancer (-208 to -56) was found to potentiate promoter activity when in reverse orientation sufficient to establish systemic infection. However, replacement of the 35S enhancer with that from the nos promoter caused loss of infectivity. A 31 bp oligonucleotide containing a polypurine tract specifying initiation of CaMV plus strand DNA synthesis was inserted into a 35S enhancer deletion mutant and propagated in plants. Analysis of progeny DNA showed the presence of an additional discontinuity at its new location in the 35S enhancer, indicating that the artificial primer had functioned correctly in an ectopic site. An intron and flanking sequences from the RNA leader of the Arabidopsis phytoene desaturase (pds) gene, when inserted into the 35S enhancer in forward orientation was very efficiently spliced during infection. The CaMV replicon carrying the pds gene fragment produced unusual infection characteristics, with plants showing early symptoms and then recovering. We conclude that infectious CaMV replicons can be used to carry a variety of elements that target both viral and host functions.
Noad
R J
RJ
Department of Virus Research, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
Turner
D S
DS
Covey
S N
SN
D017360:Arabidopsis / Q000821:virology; D001483:Base Sequence; D001937:Brassica / Q000821:virology; D017796:Caulimovirus / Q000235:genetics* / Q000502:physiology; D002678:Chimera; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000302:isolation & purification; D004742:Enhancer Elements, Genetic; D007438:Introns; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012093:Replicon; D014779:Virus Replication
D004279:DNA, Viral