SYNERGISM IN REPLICATION OF CYMBIDIUM MOSAIC POTEXVIRUS (CYMMV) AND ODONTOGLOSSUM RINGSPOT TOBAMOVIRUS (ORSV) RNA IN ORCHID PROTOPLASTS

Abstract

Non-radioactive DIG-labeled sense and antisense cRNA probes were synthesized from cDNA clones of CymMV and ORSV for virus indexing in infected plants. A slot-blot hybridization assay was developed using either crude leaf extracts or total RNA from infected leaves of Nicotiana benthamiana. The assay could detect 50 pg and 250 pg of purified CymMV and ORSV RNA, respectively. As little as 30 mg of CymMV and ORSV infected N. benthamiana leaves was sufficient to provide positive detection. CymMV and ORSV were detected at 3125 and 625 times dilution of leaf extracts, respectively. The DIG-labeled cRNA probes are stable for more than a year. This method is sensitive, reliable and suitable for large-scale routine testing of plant viruses. By using the two DIG-labeled cRNA probes in situ, we could localize the viruses in systemically infected leaf, stem and petiole of N. benthamiana and orchids. An in vitro orchid protoplast system has been established to facilitate the study of CymMV and ORSV replication and interaction. In contrast to the protocol established before, a straightforward method to isolate viable and raphid-free petal protoplasts from petal of an orchid, Dendrobium Sonia (Dendrobium Caeser x Dendrobium Tomie Drake) was developed in this study. Inoculation of orchid protoplasts with viral RNA was achieved via electroporation. Sensitive detection of viral RNA was performed by northern blot analyses using non-radioactive DIG-labeled sense and antisense cRNA probes. Electroporated CymMV and ORSV RNA replicated in the orchid protopasts. The optimum field strength for both viral RNA to achieve good efficiency of electroporation was 750 V/cm and the optimum viral RNA concentration required was 1 µg and 4 µg per 2 X 106 protoplasts for CymMV and ORSV, respectively. Newly synthesized viral RNA accumulated to a maximum level around 18 h for CymMV and 24 h for ORSV. The autoradiograph pattern showing the viral genomic and subgenomic viral RNA in the extracts taken at various time intervals are referred to as "Replication Footprint Profiles" (RFPs) of both CymMV and ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, accumulation of positive and negative strand viral RNA increased by 6 to 12 times as compared with singly infected protoplasts. In addition, the RNA accumulation of ORSV reached its maximum at earlier hours. These results suggest that the observed synergism in replication of CymMV and ORSV co-infected orchid plants may be due to their ability to increase replication efficiency.