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1.
J Virol ; 74(23): 11073-80, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11070002

ABSTRACT

Most plant viruses rely on the production of subgenomic RNAs (sgRNAs) for the expression of their genes and survival in the plant. Although this is a widely adopted strategy among viruses, the mechanism(s) whereby sgRNA production occurs remains poorly defined. Turnip yellow mosaic tymovirus (TYMV) is a positive-stranded RNA virus that produces an sgRNA for the expression of its coat protein. Here we report that the subgenomic promoter sequence of TYMV is located on a 494-nucleotide fragment, containing previously identified highly conserved sequence elements, which are shown here to be essential for promoter function. After duplication, the subgenomic promoter can be inserted into the coat protein open reading frame, giving rise to the in vivo production of a second sgRNA. It is suggested that this promoter can function when contained on a different molecule than viral genomic RNA. This interesting trait may be of general use for plant and plant virus research.


Subject(s)
Promoter Regions, Genetic , Tymovirus/genetics , Capsid/physiology , Open Reading Frames , RNA, Viral/analysis , Virus Replication
2.
J Insect Physiol ; 44(3-4): 263-270, 1998 Mar.
Article in English | MEDLINE | ID: mdl-12769960

ABSTRACT

The resistance of a transgenic line of oilseed rape expressing constitutively the cysteine proteinase inhibitor oryzacystatin I (OCI) was assessed against Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). The levels of OCI expression in the transformed line averaged 0.2% and 0.05% of total soluble protein in leaves and petioles respectively. In vitro analyses showed that P. chrysocephala larvae use both cysteine and serine proteinases for protein digestion, and that all the cysteine proteolytic activity is OCI-sensitive. However, bioassays showed that adults fed identically on leaf discs from control or transformed plants. When larvae were reared on transgenic plants expressing OCI, they showed an increase in weight gain compared to those reared on control plants. Furthermore, those larvae from transgenic plants exhibited a 2-fold increase in both cysteine and serine proteolytic activity as a reponse to the presence of OCI. The plasticity of insect digestive physiology and feeding behaviour are discussed, as well as the relevance of engineering a genotype expressing both types of proteinase inhibitors.

4.
Gene ; 136(1-2): 87-94, 1993 Dec 22.
Article in English | MEDLINE | ID: mdl-8294045

ABSTRACT

In order to evaluate new possibilities for protecting plants against virus infection by interference with viral replication, two chimeric genes were constructed in which the (+) strand 3'-terminal 100 nucleotides (nt) of the noncoding region of the turnip yellow mosaic virus (TYMV) genome were placed downstream from the sense or antisense cat coding region. The two chimeric genes were then introduced into the genome of rapeseed (Brassica napus) using an Agrobacterium rhizogenes vector system. Plants expressing high levels of either chimeric gene showed partial protection against infection by TYMV RNA or virions. One interesting feature of the protection is that a proportion of the inoculated transgenic plants does not become infected. Protection was overcome when the inoculum concentration was increased. RNA complementary to the initial transcript was detected after infection.


Subject(s)
Brassica/microbiology , Genome, Viral , Plant Diseases/microbiology , Tymovirus/genetics , Base Sequence , Chloramphenicol O-Acetyltransferase/genetics , DNA Primers , Genes, Plant , Molecular Sequence Data , Plants, Genetically Modified , RNA, Viral/biosynthesis , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , Rhizobium/genetics , Transformation, Genetic , Tymovirus/physiology
5.
J Gen Virol ; 74 ( Pt 9): 1911-7, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8376966

ABSTRACT

We describe here a convenient and efficient system for studying turnip yellow mosaic virus (TYMV) replication in leaf protoplasts. Inoculation of rapeseed (Brassica napus) or Chinese cabbage (B. sinensis) protoplasts was achieved via electroporation, and sensitive detection of viral RNA products was performed by Northern blot analyses using a non-radioactive digoxigenin-labelled cDNA probe. Virus replication was detected when 1.5 x 10(6) rapeseed protoplasts were inoculated with 20 ng of TYMV RNA. Electrotransfection of TYMV RNA was more efficient in rapeseed than in Chinese cabbage protoplasts, and gave somewhat higher signals than those of TYMV virions. TYMV RNA appeared to replicate equally well whether the protoplasts were incubated in the dark or under constant light.


Subject(s)
Brassica/microbiology , Mosaic Viruses/physiology , RNA, Viral/metabolism , Blotting, Northern , Electric Stimulation , Mosaic Viruses/metabolism , Protoplasts/microbiology , RNA, Viral/biosynthesis , RNA, Viral/isolation & purification , Transfection , Virus Replication
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