| Geminiviruses are a group of plant DNA viruses characterized by their unique twinned particles, and are currently emerging as a major threat to crop production worldwide. In southern China, the incidence and severity of begomovirus diseases have increased rapidly during the past several years. The current study was carried out for identification of tomato begomoviruses in Yunnan and elucidating molecular mechanism of pathogenicity of Tobacco curly shoot virus (TbCSV).23 tomato leaf curl samples were collected in Yunnan. They fell into several groups according to their reaction with a panel of monoclonal antibodies in TAS-ELISA and partial viral DNA sequences. The complete DNA A sequences of 4 isolates, corresponding to the four groups, were determined. Sequence comparisons and phylogenetic analysis revealed that they are most closely related to Tomato yellow leaf curl China virus (TYLCCNV), TbCSV, Tobacco leaf curl Yunnan virus (TbLCYNV) and Tomato yellow leaf curl Thailand virus (TYLCTHV), respectively. In addition, all TYLCCNV and TYLCTHV isolates were found to be associated with DNAβ molecules, while satellite DNA was not found in virus isolates of TbCSV and TbLCYNV. The complete DNAβ sequences of TYLCCNV isolate Y25, TYLCTHV isolates Y72, Y77 and Y79 were determined. Sequence analysis showed that Y25 DNAβ is most closely related to satellites associated with other TYLCCNV isolates, whereas DNAβ of Y72, Y77 and Y79 seem to be different from other characterized DNAβ, sharing the highest nucleotide sequence identity with DNAβ of TbCSV.Field survey demonstrated that only 13 isolates were associated with DNAβ among 42 TbCSV infected, field-collected samples. Infectivity showed an infectious clone of TbCSV isolate Y35 (TbCSV-Y35, an isolate associated with DNAβ) alone induced severe upward leaf curling in Nicotiana benthamiana and its natural host tobacco. In addition, TbCSV-Y35 can be transmitted between plants by whitefly vector, regardless of the presence or absence of DNAβ, indicating TbCSV-Y35 alone is sufficient for disease maintenance in the field. Co-inoculation of TbCSV-Y35 and itscognate DNAβ alters the symptom to a more severe downward leaf curling on Nicotiana spp. When inoculated on tomato, however, DNAβ failed to exacerbate the symptom phenotype induced by helper virus. In a significant number of tomato plants co-inoculated with clones of TbCSV-Y35 and DNAβ, the virus does not steadily maintain the satellite. In contrast to other begomovirus/DNAβ complexes, the satellite has no discernible effect on the DNA accumulation, infectivity, onset of symptoms and host range of helper virus. For a TbCSV isolate not associated with DNAβ (TbCSV-Yl), it induced symptoms resembling those of TbCSV-Y35 and retained the ability to functionally interact with DNAβ. Our data indicates that TbCSV DNAβ. is not necessary for infection but intensify symptoms in a host-dependent manner.TbCSV-Y35 mutant was constructed in vitro by Overlap Extension PCR, in which T was replaced with C at nucleotides 2422 and 2440, respectively, within the C4 open reading frame (ORP). The mutations destroy the two possible initiator ATG codons for the C4 ORF without disrupting the coding capacity of the C1 ORF, which entirely overlaps the C4 ORF. The mutant retained the capability of autonomous replication in leaf disc of N. benthamiana and was able to infect this host systemically although symptom development was dramatically attenuated and DNA levels were greatly reduced. Co-inoculation with DNAβ significantly enhanced the symptom severity in N. benthamiana plants as well as DNA accumulation in systemically infected tissue and in leaf disc. When tobacco and tomato plants were inoculated, however, the mutants were unable to move systemically, regardless of the presence or absence of DNAβ. The results suggest that the TbCSV C4 ORF encodes a polypeptide involved in symptom development and host-specific movement, while satellite DNAβ, which also encodes a pathogenicity determinant, can partially compensated the functional defect of C...
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