| I have studied the Beet black scorch virus, Xinjiang (BBSV-X) and Ningxia (BBSV-N) isolates and have found pathogenicity differences between these two strains. The complete cDNA clone of BBSV-X was obtained by RT-PCR of the Xinjiang RNA. Comparisons between BBSV-X and BBSV-N revealed a nucleotide sequence identity of 98.9%, and the genomic structure was almost uniform. However, three additional nucleotides were interspersed between nucleotides 889 and 975 within ORF2 in BBSV-X. These three nucleotide differences resulted in a frameshift difference in comparisons with the BBSV-N strain that affected amino acids 285 to 312 of the p82 protein, which may be the replicase-associated protein. The amino acid sequence identity of the p82 proteins of BBSV-X and BBSV-N was 95.4%. I also constructed an infectious clone of BBSV-X by placing the cDNA under the control of the T7 promoter. Based on the infectious cDNAs of BBSV-N and BBSV-X, I exchanged partial fragments of the p82 gene. After inoculating Chenopodium amaranticolor and Vigna sinensis with in vitro transcripts, mutants resulting from the p82 fragment exchanges and wild type virus were observed for phenotypic changes, but the symptoms and host range were not altered. Thus, there appears to be no direct relationship between the variation observed in p82 and the pathogenicity of the two BBSV isolates.An infectious clone of the sat-RNA of BBSV-X was also constructed by placing the cDNA under the control of the 35S promoter. This plasmid could infect Chenopodium amaranticolor with the help of in vitro transcripts of BBSV-X. Six different deletion and insertion mutants of BBSV-X were constructed and these were inoculated to Chenopodium amaranticolor. The preliminary results show that the sat-RNA could enhance symptom of BBSV-X and its insertion mutants on Chenopodium amaranticolor, but the pathogenicity of the CP deletion mutants was reduced.In order to determine the relationship between BBSV and other Necrovirus members, including the type species Tobacco necrosis virus (TNV), I also studied the biological, physical, chemical and serological properties of the China soybean isolate of TNV. The data presented below indicate that this isolate is a new strain of TNV-A which I have designated TNV-AC. The TNV-AC isolate has a high stability in vitro and also has a broad host range including 29 species in 8 families. TNV-AC generally induces local lesions in most hosts, but soybean and N. benthamiana are systemic hosts. I purified the virus and then obtained a high titer TNV-AC anti-serum. The results of serological studies showed that TNV-AC is closely related to the TNV willow isolate, but fails to react with TNV-D and BBSV.Based on TNV sequences reported previously, I designed degenerate primers and obtained subclones representing portions of the TNV-AC genome. The complete sequence of the isolate was determined by 5'-RACE and 3'-RACE. The data reveal that the virus encodes five ORFs in a 3682 nt genome, and that the nucleotide sequence identity is respectively 86.4%, 43.7% and 44.3% between the TNV-AC isolate and those of TNV-A, TNV-D and TNV-DH, whose sequences have been reported previously. Thus, according to the biological properties and nucleotide sequences of these strains, I postulate that the soybean isolate is a new strain of TNV-A.To produce infectious cDNA clones, TNV-AC cDNA was placed under the control of the T7 and the 35S promoters, and deletion mutants in ORF1, ORF3, ORF4, and ORF5 were generated. Through analyzing the symptoms and genome RNA replication, I conclude that the ORF1 gene is the replicase-associated gene, and that ORF3 and ORF4 are cell-to-cell movement genes. The protein encoded by ORF5 affects long distance virus movement, which results in a two day delay of lesion appearance in Chenopodium amaranticolor.
|
PDF Full Text Request