Pathogenicities Of Three Begomoviruses And Their Associated Satellite DNA

Geminiviruses are a group of plant viruses characterized by their unique twinned particles,which encapsidated a circular single-stranded DNA genome. They cause destructive diseases in many crops throughout the world and have emerged as a great threat to crop production.Recently,many monopartite begomoviruses have been found to be associated with satellite DNAβmolecules, which are necessary for induction of the typical symptoms.Begomovirus-DNAβcomplexes have proven to be widespread and associated with many diseases throughout the Africa and Asia.In an attempt to better understand the pathogenesis of begomoviruses,the pathogenicities of the three begomoviruses and their associated DNAβmolecules were studied in this dissertation.1.Molecular variation of satellite DNAβmolecules associated with Malvastrum yellow vein virus and their role in pathogenicityPrevious analyses of the diversity of begomovirus-associated DNAβsatellites focused mainly on satellites originating from different hosts.In this dissertation,20 different isolates of DNAβmolecules associated with Malvastrum yellow vein virus (MYVV)isolated from Malvastrum coromandelianum plants in different geographical locations in Yunnan Province,China,were cloned and sequenced. Analyses of their molecular variation indicated that the satellites were clustered together according to their geographical location but that they had only limited sequence diversity.Infectivity tests using infectious clones of MYVV and its associated DNAβmolecule indicated that MYVV DNAβwas indispensable for symptom induction in Nicotiana benthamiana,N.glutinosa,Petunia hybrida,and M. coromandelianum plants.Furthermore,agrobacterium-mediated co-inoculation of N. benthamiana plants with MYVV and Tomato yellow leaf curl Thailand virus (TYLCYHV)DNAβor Tomato yellow leaf curl China virus(TYLCCNV)DNAβindicated that MYVV interacted functionally with heterologous DNAβmolecules in N.benthamiana plants and that the variation of the accumulation amounts of the viral DNA in infected plants correlated well with the severity of symptoms. 2.Molecular identification and pathogenicity of Tomato leaf curl China virus and its associated DNAβThree virus isolates G16,G17 and G18 were isolated from tomato plants showing leaf curl symptoms in Guangxi province,China.A specific 500 bp fragment was amplified from the DNA extracts of these isolates using the universal primers specific for members of the genus Begomovirus and these fragments were cloned and sequenced.The 500 bp fragments of the three samples have 96.6%to 98.5%nucleotide sequence identities,suggesting that they were different isolates of the same virus species.The complete nucleotide sequences of G16 and G18 were determined to be 2729 nucleotides(AJ704602)and 2733 nucleotides(AJ558119), respectively,with a typical genomic organization of begomoviruses.The complete nucleotide sequences of G16 and G18 shared 98.7%sequence identity,and were most closely related to Tomato leaf curl Vietnam virus(ToLCVV)(83.6%and 82.8%,respectively).According to the taxonomic criteria of Begomovirus,they were considered to be different isolates of a novel begomovirus species for which the name Tomato leaf curl China virus(ToLCCNV)was proposed.Phylogenetic and RDP analyses on the virus genomic sequences suggested that ToLCCNV may have arisen by recombination.Satellite DNAβmolecules were also found to be associated with the isolates and their complete nucleotide sequences were determined.Sequence analyses demonstrated that they had 1346 nts,1341 nts and 1342 nts long(AJ704610-AJ704612),respectively,with characteristic features of DNAβ.The complete nucleotide sequences of the three DNAβmolecules shared more than 92.8%sequence identity,and were closely related to DNAβof TYLCCNY Y8 isolate(49.1%,49.1%and 49.8%,respectively).To investigate the pathogenicity and biological role of DNAβin virus infection,the infectious clones of ToLCCNV and its associated DNAβwere constructed and their infectivities were tested in host plants.The results indicated that DNAβwas required for symptoms induction and that virus titer was increased in the presence of the satellite.TheβC1 protein of DNAβassociated with ToLCCNV was identified as a suppressor of RNA silencing.To understand the fundamental motif and/or region involved in silencing suppressor activity of theβC1 of ToLCCNV DNAβ,with the help of the predicted results by bioinformatics analyses,7 deletion mutants were constructed using overlap PCR method.The results of patch co-infiltration assay, northern blot and western blot showed that the central domain ofβC1 comprising the two key elements(amino acids 44 to 60 and amino acids 61 to74)was responsible for the suppressor activity ofβC1.Most of RNA silencing suppressors were also identified as pathogenicity determinants.Therefore,infectious clones of the above 7 deletion mutants were constructed.Infectivity and pathogenicity analyses showed that all of the mutants could systemically infect N.benthamiana in association with ToLCCNV,but failed to induce obvious symptoms,suggesting that the intactβC1 protein was responsible for inducing typical symptoms.However,all of the mutants accumulated at extremely low level in plants as compared with the wild-type ToLCCNV DNAβalthough the DNA accumulation level differed among the mutants.These results indicated that the suppressor activities ofβC1 did not have direct effect on the induction of symptoms.To determine the subcellur localization ofβC1 protein and its mutants,βC1 and its mutants were cloned into the pCHF3 vector to obtain constructs expressing fusion proteins of wild-type or mutatedβC1 and GFP.Agro-infiltrated N. benthamiana leaf cells were checked by confocal laser scanning microscope for subcellular localization.The results showed that the mutants ofβC1^dm44-60andβC1^dm61-74which could not suppress RNA silencing were also unable to target the nucleus,and that other fusion proteins of wild-type or mutatedβC1 and GFP localized in the nucleus and cytoplasm.These results collectively provided persuasive evidence that the nucleus localization capability of theβC1 was necessary for the suppression of RNA silencing.The results also indicated that the central domain was the key element indispensable for both nucleus localization and the suppression of RNA silencing.11 virus-derived or satellite-derived small RNAs were cloned from N. benthamiana plants infected by ToLCCNV alone or together with ToLCCNV DNAβ.Furthermore,analyses of their distributions along the viral genome showed that they were predominantly derived from the CP orβC1 transcripts of both polarities,suggesting hotspots for viral small-RNA generation. 3.Tomato yellow leaf curl Thailand virus was a monopartite begomovirus associated with a satellite DNAβTomato yellow leaf curl disease caused by Tomato yellow leaf curl Thailand virus(TYLCTHV)is one of the most important diseases,affecting tomato production in Thailand.In order to elucidate if TYLCTHV was a monopartite or bipartite begomovirus,infectious clones of TYLCTHV isolate Y72 and its associated DNAβwere constructed.Infectivity tests in host plants showed that TYLCTHV alone could induce typical symptoms in tested N.benthamiana,N. glutinosa and Solanum lycopersicum plants,while more severe symptoms and more viral DNA accumulation were observed when TYLCTHV was co-inoculated with its satellite DNAβ.Based on our results,we think that TYLCTHV was a monopartite begomovirus.4.Tomato leaf curl diseases in Guangxi province were caused by mixed infections of two begomoviruses and satellite DNA31 tomato samples showing typical leaf curl symptoms were collected from Guangxi Province.Mixed infections were tested by PCR,Southern blot,RCA-PCR in combination with sequencing to identify the causal agent of the tomato leaf curl disease.The results showed that all the samples were mix-infected by ToLCCNV and Papaya leaf curl China virus(PaLCCNV)and ToLCCNV DNAβor TYLCCNV DNAβ.