Expression And Binding Studies Of Some Geminivirus Proteins

Geminiviruses are circular single-stranded DNA viruses and are classified into family Geminiviridae. The family consists of four genera Mastrevirus, Curtovirus, Topocuvirus and Begomovirus. Begomoviruses can be broadly divided into two groups, the Old World (eastern hemisphere, Europe, Africa, Australia, Asia) and the New World viruses (western hemisphere, the Americas). Their genomes have number of characteristics that distinguish Old World and New World viruses. All New World begomoviruses are bipartite, whereas both bipartite and monopartite begomoviruses are present in the Old World. Chilli leaf curl betasatellite (ChLCB) is a disease specific betasatellite and is found associated with monopartite as well as bipartite begomoviruses. On the other hand Tomato leaf curl New Delhi virus (ToLCNDV) is an Old World bipartite begomovirus whereas Cabbage leaf curl virus (CaLCuV) is New World bipartite begomovirus. The aim of research work presented here was to study the interaction of ChLCB with both the Old and New World bipartite begomoviruses and the identification of DNA A encoded proteins that contributed to symptoms induction, suppressor of gene silencing, and pathogenicity. The other objective was to find out the host genes that are up or down regulated due to the interaction of âC1 protein encoded by ChLCB. Differential display analysis was done to find out the host genes that are activated against âC1Chi, whereas gateway cloning technology and two expression systems i.e. prokaryotic (BL21 bacterial strain) and yeast (Pichia pastoris) were used for the expression analysis of geminivirus encoded proteins. Co-inoculation of the DNA ACa and DNA BCa yielded efficient infection, typical symptoms of leaf curling, leaf crinkling, mild yellow, leaf deformation and stunting after seven to eight days of inoculation and the levels of siRNA derived from CaLCuV correlated positively with the levels of viral DNA. It was observed that the inoculation of ACa + âChi showed mild symptoms and low levels of viral DNA ACa accumulation in systemic leaves as compared to both components of ACa + BCa. From this study it was concluded that betasatellite interacted with the New World begomovirus (CaLCuV), intensified symptoms and also helped in virus movement. These results confirmed that the interaction of betasatellites was not limited to monopartite begomoviruses and that betasatellites can complement movement of bipartite begomoviruses from both Old and New World.The role of DNA A encoded proteins of both Tomato leaf curl New Delhi virus and Cabbage leaf curl virus in viral pathogenicity was studied. For this study cloning and expression of AC2Ca, AC4Ca, and AV2To genes were carried out into pDONOR/Zeo entry vector and destination vectors p100, p201 and p202 having 35S promoter and different protein tags such as HA and FLAG through gateway technology. Floral dip transformation method was also used to express protein in Arabidopsis transgenic plants. It was found that AC2Ca was a suppressor of RNA silencing and its expression induced the hypersensitive response. Similarly, AC4Ca suppressed miRNA whereas AV2To was a weak suppressor of RNA silencing. Putative transgenic plants expressing AC2p201Ca, AC4p100Ca and AC4p201Ca showed distinct phenotype. AC4p100Ca and AC4p201Ca developed downward leaf curling, crumpling and stunted plants, whereas AC2p201Ca plants developed elongated leaves, rosette like pattern, slightly rolling and margins of the leaves were deformed. On the other hand, p202(AV2)To developed upward leaf curling and deformed leaves, AV2p100To and AV2p201To expression vectors showed scrunch and slightly deformed leaves. From all experimental data it can be concluded that these proteins are pathogenicity determinants and are involved in symptom induction and upon complementation of movement function by ChLCB were able to develop disease phenotype in the absence of DNA B Ca. In this regard efforts were made to express âC1Chi protein through both bacterial and yeast expression systems to understand their binding activity with DNA/RNA or protein-protein interaction through in vitro binding assays like pull down assay or co-immunoprecipitation. It was found that âC1Chi was lethal protein for bacterial and yeast expression system. The expression of âC1Chi from bacterial system was not detectable due to nucleotide mutations which resulted in failure of protein expression. Similarly, Pichia pastoris system was also failed to produce detectable expression and protein could not be purified. On the other hand AV2To was successfully expressed in BL21 system and indicated that bacterial system was working and the problem was occurred due to toxicity of âC1. Later âC1Chi was expressed in N. tabacum to isolate differentially expressed genes (DEGs) through differential display analysis during host protein interaction. It was observed that WRKY transcription factor IId-1 splice, putative Rieske iron-sulfur protein, NADH dehydrogenase subunit 1 and NADH dehydrogenase subunit 2, Quinonprotein alcohol dehydrogenase, trigger factor (chaperone in protein export), Arabidopsis thaliana calmodulin-binding receptor-like kinase, and Polyribonucleotide nucleotidyltransferase were isolated from N. tabacum in response of âC1Chi under 35 (CaMV) promoter after two and four days of infiltration. Delta, Livak and Pfaffi mathematical models of quantitative real time PCR gave an idea that these DEGs were up and down regulated. Interestingly these all DEGs are related to the chloroplast and mitochondrial genes, involved in plant development and growth processes, cell protection, replication mechanisms, act as carriers of ATP synthesis and electron transporters during photosynthesis and respiration processes. Kegg Orthology based annotation system indicated that Polynucleotide nucleotidyltransferase is a RNA binding protein, and involved in the purine and pyrimidine metabolic pathway that relates to RNA degradation. The data presented here show that the interaction of betasatellite is not limited to monopartite begomoviruses. The spread of begomovirus complexes that contain betasatellite thus pose a serious threat to global agriculture. It was concluded that AC2Ca, AC4Ca, AV2To, and âC1Chi proteins of both monopartite and bipartite begomoviruses involved in viral movement, symptom induction, and pathogenicity determinant.