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Molecular Mechanism Of Pathogenicity Of Tomato Yellow Leaf Curl China Virus And Viral Satellite DNA Induced Gene Silencing

Posted on:2005-06-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:X R TaoFull Text:PDF
GTID:1103360122991109Subject:Biochemistry and Molecular Biology
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Geminiviruses are a group of single-stranded circular DNA viruses occurred worldwide, and caused severe diseases in several economically important crops. In China, several kinds of begomoviruses were identified in tomato, tobacco and squash in Yunnan and Guangxi province. Here, a begomo virus isolate Y10 from Honghe in Yunnan Pro vice was identified, and its genome structure, function and pathogenecity were studied in this dissertation.The complete sequence of DNA-A of Y10 isolate was determined, comprising 2737 nucleotides. The Y10 DNA-A exhibits the typical genome structure of begomoviruses originating from the Old World. Sequence comparison analysis reveals that Y10 DNA-A has more than 89% identity with Tomato yellow leaf curl China virus (TYLCCNV), indicating that Y10 is an isolate of TYLCCNV (TYLCCNV-Y10). Attempts to isolate DNA-B component were unsuccessful, however, a novel molecule (designated as DNAP) was found to be associated with TYLCCNV-Y10. The complete sequence of TYLCCNV-Y10 DNAp consists of 1336 nucleotides. TYLCCNV-Y10 DNAP shows negligible sequence homology with DNA-A and it shares high sequence identity with DNAp of other TYLCCNV but low sequence identity with DNAp of different begomovirus species. Association of DNAP with TYLCCNV DNA-A was confirmed in the samples collected from Yunnan Province. All twenty-five TYLCCNV samples were found to be associated with DNAp.In order to investigate the pathogenecity and biological role of TYLCCNV DNA-A and DNAp, the infectious clones of DNA-A and DNAp. of TYLCCNV-Y10 were constructed. We found that TYLCCNV-Y10 DNA-A alone could systemically infect Nicotiana benthamiana, N. glutinosa, N. tabacum Samsun NN/nn, Lycopersicon esculentum and Petunia hybrida plants but produced no symptoms. TYLCCNV-Y10 DNAP is required for the induction of the typical symptoms including severe leaf curling, vein darkening, enation and stunting in these host plants. The Southern blot analysis shows that TYLCCNV DNAP could dramatically enhance the viral DNA-A level in the plant. The time course TAS-ELISA demonstrates that DNAP could accelerrate the virus titer to anextreme high level in a short time. Analysis of viral DNA replication in leaf disks indicates that DNAP could not replicate automatically, but depends DNA-A for replication. DNAp was also found to be encapsidated in the coat protein encoded by DNA-A and transmitted by Bemisia tabaci. Therefore, TYLCCNV DNA-A and DNAP are mutually dependent.The function of Cl gene, A-Rich region and an 115bp high conserved region on the genome of TYLCCNV DNAp were identified, respectively. When the Cl gene was mutated, the mutants failed to induce leaf curl symptoms, indicating that C1 is a key determinant in symptom induction. Southern blot analysis showed that the C1 mutants could be replicated in the plants. The result suggests that though the C1 is required for symptom induction, it is not essential for the replication of DNAp. When the A-Rich region was deleted, the A-Rich deletion mutant could be replicated and infected plants systemically, indicating that A-Rich region is not required for the replication of DNAp. The immunotrapping-PCR demonstrated that A-Rich deletion mutant could be encapsided in the coat protein encoded by DNA-A, suggesting that A-Rich region does not determine the encapsidation of DNAp. The 115bp high conserved region and upstream region with several five nucleotides motif (GGNiN2N3) were fused with an episome, respectively, to identify the essential region for DNAP replication. We found that the 115 bp high conserved region is sufficient for replication of DNAP, while the upstream region contain several GGNiN2N3 motif is not required for the trans-replication of DNAp.Several natural recombinants (RecDNA-Ap) between TYLCCNV DNA-A and DNAP were isolated and its pathogenecity were identified. The full-length sequences of several RecDNA-A of TYLCCNV-Y10 were determined and they have the same size of DNAp. The sequence analysis reveals that the RecDNA-Ap have three common region: the common r...
Keywords/Search Tags:Geminivirus, Tomato yellow leaf curl China virus, DNA#, #C1, Virus induced gene silencing
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