Study On Double Stranded RNA Viruses Infecting Radish

Radish (Raphanus sativus) is a popular edible root vegetable of the Brassicaceaae family, while its leaves and seeds have culinary and medicinal uses. A double stranded RNA(dsRNA)virus, namely radish yellow edge virus (RYED) was firstly discovered in Japan by Natsuaki, for inducing yellow edge symptoms, and the viruses was classified to Alphacryptovirus of the family Partitiviridea.In our lab, three dsRNA viruses, namely raphanus sativus virus 1(RasV1), Raphanus sativus virus 2(RasV2) and Raphanus sativus virus 3(RasV3) were detected and genome-structure-analysed from leaves of Raphanus sativus cv. Yidianhong, when the yellowing-edge-phenomenon was commonly noticed in Hangzhou, Zhejiang. Seeds of fourteen radish cultivars were collected from different areas in China, and then pot-cultivated in the greenhouse. The seedlings presented light symptoms, such as light or dark green chlorosis, an occasional yellowing edge or edge chlorosis, leaf rolling and uneven leaves. Segments of dsRNA were commonly detected from all of the plants and their seedlings gernerated from seeds. In the present study, cell and molecular biology were investigated with plant seedlings containing the above viruses, after tissue culture system was set up for infected radish, for obtaining unique plant materials. The following results were obtained.⑴Tissue culture system for mass and rapid propagation of radish was established, to obtain plant materials with identical genetics and infction backgrouds, since the peculiar properties of plant crytovirus which are not graft or mechanically transmissible but only transmitted through pollen and seed. The adventitious shoots were induced from embryos explants, and propagation was achieved by using MS+0.01mg/L NAA+6.0mg/L 6-BA+3.0mg/L AgNO3 as differentiating medium and MS or 1/2MS +1.0mg/L NAA as rot inducing medium. The hardening period using 1/3 perlite +2/3 soil or 100% soil was found to be important for getting adult plants.⑵Double stranded RNA extraction, and dot-blot hybirdyzation from tissue cultured plant seedlings were used for combining detection of dsRNA viruses infecting radish culture-lines. Based on this, three series of tissue-culture-plantlet lines, including those infected with RasV1 alone, with both RasV1 and RasV2, and with RasV3 alone were respectively screened out.⑶Ultrastructural alterations in regenerated plantlets of R. sativus cv. Yidianhong infected with different dsRNA viruses were observed, after thin sections of radish leaves were prepared. Electron microscopy showed that normal cell structure and chloroplasts lamellar development and no vacuolated region. However, swollen chloroplasts and swollen stromas were observed from RasV1 infected plant leaf tissue, with crystalline substances in some chloroplasts. The same crystals were also observed in the RasV3 infected chloroplasts and were conjectured to be viroplasts or some kind protein of plant induced by virus infection. Chlorophyll (Chl) content, photosynthetic gas exchange, and Chl fluorescence in leaf tissues of R. sativus cv. Yidianhong infected with different dsRNA viruses were examined. The Chl content showed no remarkable difference between the radish seedlings infected with different dsRNA viruses, but net photosynthetic rate (Pn) in radish infected with RasV1 was higher than those of the plants infected either with both RasV1 and RasV2 or with RasV3, and the intercellular CO2 (Ci) was the lowest. The transpiration rate (Gs) with transpirayion rates (E) showed no significant difference. For the Chl fluorescence parameters Fv/Fm, Fv'/Fm',ΦPSII, qp and NPQ, there were no significant difference, which may suggested that infection by different dsRNA viruses did no or similar level disruption on the hosts. Based the above results, whether the crystalline substances in chloroplasts affect photosynthesis or not is undetermined.⑷Using a plant miRNA microarray, conventional miRNAs of rsdish were firstly detected. A total of 545 mature miRNAs and miRNA*s across 31 plant species were detected, using 1450 plant miRNA probes based on the Sanger miRbase15.0. Amongst, 198 of them were from the three species of Brassicaceaae family, including Arabidopsis thaliana, Brassica napus and B. Rapa. The above data provide potential information for understanding functional roles played by miRNAs among dsRNA infection and gene regulation of radish as virus host.