Functional Analysis Of Beet Necrotic Yellow Vein Virus RNA5 And The Screening Sugar Beet Cultivars For Resistance To BNYVV

Specific antiserum against BNYVV was successfully prepared by immunizing rabbit with a preliminarily purified virus, which titer was 3.6xl03 estimated by indirect ELISA, and can be used in western blot detection of sugar beet samples both inoculated with BNYVV and collected from the fields.Among the 13 sugar beet cultivars planted in rhiziomania fields of Baotou, there were 7 cultivars appeared to be strongly resistant to BNYVV, including Neitiankang 201, DF1028, KW0149, Nei101, KW9419, ZD204 and 01NSZdan. Dot hybridization analysis revealed that BNYVV could not be detected in the root samples of cultivars mentioned above except for KW9419, whereas positive results were obtained in some cultivars such as Neitiankang 201 and KW0149 by RT-PCR. It suggests that this kind of cultivars can inhibit virus multiplication but not prevent itself from virus infection.In vitro transcripts from RNA5 wild type clone were co-inoculated into Tetragonia expansa with the RNAs of Hu3 isolate containing RNA1, 2 and 3. Following RT-PCR and Northern blotting results shows RNA5 could be biologically replicated in the leaves. The virus concentration of the isolate containing RNA5 in T. expanxa was higher than that of the isolate Hu3 evaluated by ELISA detection. Furthermore, RNA5 in beet roots inoculated with BNYVV was confirmed by a simple rapid RT-PCR, and disease incidence of sugar beet inoculated with Hu3 plus RNA5 increase 10%. It suggests that the virus infection and accumulation in the hosts can be improved by presence of the RNA5. In the meantime, five open-reading-frame mutants of RNA5 could be transcribed and inoculated into T. expanxa leaves with Hu3. It seemed that all this transcripts could be replicated in inoculated plant while mutant RNA5 could not be detected in the second passage. Therefore, alignment of UTR in 5' -and 3' -proximal region in wild clone and one mutant clone of RNA5 indicates that there are two internal deletion (A350AAA353 and A364ATAAAA370) in the 5' terminal non-coding region of the mutant, and the rest sequence have no critical difference between them, which suggests that these deleted sequences have nothing with replication but play an important role in encapsidation and accordingly lead to the lost of infectiviry of the mutants in the next passage of mechanical inoculation.In order to investigate the subcellular location of P26 encoded by RNA5, two constructs of P26-eGFP fusion and eGFP instead of P26 were obtained based on RNA5 replicon.In addition, the systematic symptom displayed in the top non-inoculated Nicotiana benthamiana leaves 10 days post inoculation, and RT-PCR detection showed that BNYVV can systematically infected N. benthamiana. It will conveniently facilitate the research on multiplication and functional analysis of the virus gene.