Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008
| Identifer | oai:union.ndltd.org:ADTP/280340 |
| Date | January 2008 |
| Creators | Eini Gandomani, Omid |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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