Studies on transcription and replication of viral genomes in infected plant cells by situ hybridization

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In situ hybridization is potentially a very powerful technique for detecting the localization of viral gemones in infected cells. Unfortunatelly until now, it have no wide application to plant virus-infected cells. We have developed convenient method of in situ hybridization for tobacco mosaic virus (TMV)-infected cells using biotinylated RNA probes.To prepare sense-strand RNA probes, TMV-RNA were fragmented (200-600 nucleoties long) by alkali treatment in the presence of Mg^<2+>. The RNA fragments were labeled with photobiotin by mercury vapour lamp irradiation. To prepare antisense-strand RNA probes, the cloned cDNA of TMV coat protein gene was inserted into a transcription vector, pGITC, that carries the SP 6 promoter at the transcription initiation site. The transcription vector was linearized just downstream of the cDNA insert with Bam H I, and was transcribed to obtain biotin labeled antisense RNA probe, using the SP 6 polymerase in the presence of biotin-11-UTP.When these probes were hybridized with TMV-RNA isolated from virions or doublestrand TMV-RNA extracted from TMV-infected tobacco plant on nylon membrane, hybridization reactions were detected by tagging avidin-al^kaline phosphatase followed by incubation in nitro blue tetrazolium.By in situ hybridization with these probes, viral nucleic acids were detected in the TMV-infected tobacco protoplasts spread on slides.In situ hybridization at electron microscope levels are in progress.