Effect Of The RNA5 Component On Pathogenicity Of Beet Necrotic Yellow Vein Virus By Infectious CDNAs

BNYVV isolates from Ningxia, Xinjiang, Heilongjiang, Gansu and Hebei provinces were screened by RT-PCR and the RNA5 component was identified in the isolates from Ningxia, Xinjiang and Heilongjiang among them, indicating that the BNYVV isolates containing RNA5 component were widely distributed in sugarbeet growing areas of China. The RNA5 components were cloned into pUCm-T vector and transformed in E. coli strain DH-5 a . Sequence analysis showed that the RNA5 components in the isolates from Ningxia, Xinjiang and Heilongjiang have 1347nts, 1348nts, 1346nts respectively. Comparing with the RNA5s in D5, F72, Bao and Hu isolates reported previously, identities shared between them were varied from 93.0%-98.7% in nucleotide sequences and 89.7%-98.7% in deduced amino acid sequences.The bacteria) expression vector of pET-Hu26 containing the coding region of BNYVV RNA5 was constructed and transformed into E.coli strain JM109 (DE3). With induction of IPTG, a fusion protein was over-expressed and used for prepation of antisum, which specifically binds to a protein of 26 kDa encoded by BNYVV RNA5 in Western blot.Full-length cDNAs of BNYVV RNA-5 for in vitro and in vivo transcription were synthesized by cloning into plasmid of pUC18 driven by a bacteriophage T7 promoter or pRT103 adjacent to a Cauliflower mosaic virus 35S promoter, respectively. Infectivities of the transcripts were determined by RT-PCR and Northern blotting after inoculation of plants.In vitro transcripts of RNA5 were co-inoculated to Tetragonia expansa and sugar beet seedlings with RNAs extracted from two BNYVV mutants of HuO containing RNA1 and 2 only, and Hu3 containing RNA1, 2 plus 3. By assay of symptom and viral RNA, it was indicated that the virus infection and accumulation in the hosts can be improved by presence of the RNA5. Cooperating with RNA3, RNAS seems to cause more severe symptom and yield losses in sugar beet. These results suggest that, besides of RNA3, RNA5 is another important element in BNYVV associated with its pathogenicity.In order to identify the motif responsible for the RNA5, four internal deletions and a point mutation at start codon of ATG within the coding region of RNA5 were constructed. In vitro transcripts of these mutants were co-inoculated to T. expansa together with the RNAs of Hu3 mutant It was demonstratedthat all these transcripts were infectious and protein expression encoded by RNA5 was not essential forBNYVV infection. Further experiments are being conducted to test the effects of these mutants on root yield and sugar content in sugar beet.