BEET YELLOWS IS ON OF THE MOST IMPORTANT DISEASES OF THE SUGAR BEET CROP IN TEMPERATE COUNTRIES. IF AN EFFECT OF ANTISENSE RNA ON THE REPLICATION OF BEET YELLOWS VIRUS COULD BE CONFIRMED, THEN SUGAR BEET VARIETIES COULD TENTATIVELY BE ENGINEERED TO PRODUCE, CONSTITUTIVELY OR UPON SPECIFIC INDUCTIONS, THE INHIBITING FACTOR WHICH WOULD RENDER THIS CROP SPECIES RESISTANT TO INFECTIONS.
There is no known genetic source of resistance to beet yellow virus (BYV) and control is effected by spraying insecticides against the aphid vector. Recently, new concepts have been developed in which viral sequences, inserted into the plant genome, interfere with virus replication.
BYV has long flexuous particles which are broken by conventional virus purification methods. Therefore, a new purification protocol, which eliminated steps where particle damage could occur, was developed. In vitro translation of BYV ribonucleic acid (RNA) indicated that it expresses each gene from a subgenomic messenger ribonucleic acid (mRNA) rather than producing all its information as a single polyprotein which is subsequently cleaved. Using serology and complementary deoxyribonucleic acid (cDNA) probes, isolates of BYV were shown to vary in some parts of their sequence by probably not very much in their coat protein. cDNA clones expression coat protein were isolated and characterized.
Various methods for introducing deoxyribonucleicacid (DNA) constructs, which expressed chloramphenicol acetyl transferase, into beet protoplasts were investigated. 2 basic systems for making temporary holes in the protoplast plasma membrane enabling DNA to enter were studied: electroporation and sonication. A simple method involving the use of alternating current (AC) directly from the mains, the pulse being controlled by a fuse, was developed. This was as effective as or more effective than the conventional electroporation systems of shaped pulses of direct current (DC). Various parameters of electroporation were optimized and it was found that the cell lines from which the protoplasts were prepared was very important. Both plasmid DNA and particles of beet necrotic yellow vein virus (BNYVV) were expressed after being introduced into beet by mild sonication. The expression of BNYVV showed that molecules introduced by this means were still biologically active.
THE AIM OF THE PROJECT IS TO DETERMINE IF RNA COMPLEMENTARY TO INFECTIOUS RNA FROM BEET YELLOWNS VIRUS (ANTISENSE RNA) WILL INHIBIT THE REPLICATION OF THE VIRUS AND COULD THEREBY BE USED AS A MEANS OF CONTROLLING BYV INFECTION. AS A FIRST STEP, THE GENOME STRUCTURE AND THE MOLECULAR BIOLOGY OF BYV REPLICATION/EXPRESSION WILL BE STUDIED BY THE LABORATORY IN NORWICH IN WHOLE PLANTS, IN ISOLATED BEET PROTOPLASTS AND IN IN VITRO SYSTEMS. IN PARALLEL, THE LABORATORY IN COPENHAGEN WILL DEVELOP A SYSTEM FOR TRANSFORMATION OF BEET PROPOPLASTS AND/OR CELLS BY USING VARIOUS VECTORS DERIVED FROM AGROBACTERIUM TUMEFACIENS TI-PLASMID, OR DIRECT TRANSFORMATION OF PROTOPLASTS ALTERNATIVELY.
CONSTRUCTS WILL ALSO BE MADE WHICH WILL GIVE ANTISENSE RNA TO VARIOUS BEET GENES CURRENTLY BEING ISOLATED IN COPENHAGEN. FINALLY, CONSTRUCTS WILL BE MADE WHICH WILL TRANSCRIBE ANTISENSE RNA TO VARIOUS PARTS OF BYV GENOME.