| Geminiviruses are a group of plant viruses characterized by their unique twinned particles, which encapsidate a circular single-stranded DNA genome. They cause destructive diseases in many crops throughout the world and are emerging as great threat for crop production. In recent years, a novel satellite DNA, referred to as DNAP, was found to be associated with some monopartite geminiviruses and was required for inducing typical symptoms in natural hosts. All reported DNAP molecules posses a βClgene on the complementary-sense strand, which has a conserved position and size. However, the function of βC1 gene is unknown. To understanding the mechanisms of pathogenicity of DNAP associated with geminiviruses, the function of βC1 gene was investigated in this study.The βC1 genes of DNAP molecules associated with Tomato yellow leaf curl China virus (TYLCCNV) Y10(Y10β)and Tobacco curly shoot virus (TbCSV) Y35 (Y35β) were expressed in Escherichia coli strain BL21(DE3) pLys S, and recombinant βCI proteins of TYLCCNV Yl0β (Yl0βCl) and TbCSV Y35β (Y35βC1) were purified with Ni2 + -NTA agarose affinity chromatography. The polyclonal antibody against Y10βCl was produced in a rabbit. Then, the nucleic acid binding ability of recombinant PCI proteins were determined by UV crosslinking and Electrophoresis mobility shift assay (EMSA). The results showed that PCI protein could bind double-stranded DNA and single-stranded DNA, and its ability of DNA binding was related with the protein concentration. The DNA binding activity of PCI protein was influenced by salt concentration, and it lost the binding ability in the presence of high NaCl concentrations (over 1800mM). The results of EMSA and competitive binding assay also showed that the activity of PCI protein binding to DNA was not related with DNA size, form and sequence, suggesting that it binds DNA in a sequence non-specific manner.To determine the subcellular localization of βCI protein, the vectors expressing βCl gene fused with GUS or GFP gene for localization in plant or insect cells were constructed. The construct containing fid fused with GUS was introduced into the onion epidermis monolayer cells by particle bombardment. The results revealed by GUS staining showed that βCI fusion protein accumulated in the cell cytoplasm and nucleus, but had the high accumulation in the nucleic. Fusion protein of βCI with GFP expressed from recombinant baculovirus in sf21 insect cells, directed the accumulation of fluorescence to the nucleus as observed by confocal microscopy. Incontrast, similar construct with the mutated C1 gene or only GFP control accumulated within the nucleus and cytoplasm. TYLCCNV YlOpCl protein contains a putative nuclear localization sequence (NLS, 45PALAKKK51). The deletion mutants of Cl containing putative NLS had a high accumulation within the nucleus of cell, but the mutants having no putative NLS or mutated NLS (49KKK51AAA) accumulated equably within in the cytoplasm and nucleus, suggest that PCl protein maybe have a functional NLS.The leaf discs of Nicotiana benthamiana and N. tobaccum were transformed with Agrobacterium tumefaciens containing the C1 gene of TYLCCNV Y10 under the control of Cauliflower mosaic virus 35S promoter, 75 lines of transgenic N. benthamiana plants and 39 lines N. tobaccum plants were obtained. Some lines of both transgenic species developed virus-like symptoms, PCR and Southern blot analysis confirmed the C1 gene had integrated into the genomes of these plants. Nothern blot analysis showed the severity of symptoms in these plants paralleled the level of C1 transcripts, while transgenic plants transformed with the Cl gene containing a stop codon after the start codon remained symptomless. Thus, expression of a Cl gene of satellite DNAP is adequate for induction of virus-like symptoms in the absence of viral DNA-A.RNA silencing in plants is a natural immune system against virus infection. To determine if DNAP involved in suppression of RNA silencing, the effect of co-infection by TYLCCNV and...
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