MOLECULAR BIOLOGICAL APPROACH TO THE CONTROL OF BEET YELLOWS VIRUS

Projektinformationen

ID Finanzhilfevereinbarung: BAP*0097

Projekt abgeschlossen

Startdatum 1 Juli 1986

Enddatum 31 Dezember 1989

Finanziert unter

• Multiannual research action programme (EEC) in the field of biotechnology (BAP), 1985-1989

Gesamtkosten € 0,00

EU-Beitrag Keine Daten

Koordiniert durch

JOHN INNES CENTRE
United Kingdom

Exploitable results

Molecular biology and the control of beet yellows virus

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.