Identification And Functional Study Of Host Factors Involved In Begomovirus Replication And Of Cis-acting Elements Required For Betasatellite Replication

Viruses in the family Geminiviridae have small circle single-stranded DNA(ssDNA)genomes that have been widely distributed all over the world.Among which,the genus Begomovirus represents the largest and most important group and often cause severe damage.Begomoviruses encode a replication initiator protein(Rep)to recruit the host's DNA replication machine so as to amplify its genomic DNA in a rolling circle replication(RCR)manner,but intensive studies about the host factors and gene function remain obstacles.Besides,begomoviruses often associate with a kind of small molecules,called betasatellite,which is half the size of the helper begomovirus genome.Co-infection of begomovirus and its betasatellite is often the cause of triggering severe symptoms.To get a better insight into the mechanism of geminivirus replication and pathogenesis,as well as the mechanism of betasatellite replication and evolution,this study was focused on screening host factors that specifically affect begomoviruses replication and Rep-interacting proteins,as well as the betasatellite replication cis-elements identification and replication mechanism,and several progresses have been made:1 The identification of cis-elements essential for begomoviruses associated betasatellite replication and the studyof its replication mechanismBegomoviruses can trans-replicate both cognate and noncognate betasatellites,and seems to be lack of obvious specificity;when cognate and noncognate betasatellites were simultaneous in one plant,the helper virus prones to replicate and maintains the cognate betasatellite,but the detailed mechanism remains unclear.Previous study announced that the selective replication of cognate betasatellite by helper virus was determined by a satellite element called Rep binding motif(RBM),which specifically binds to cognate helper virus encoded Rep.Two tandem iteron-like cis-elements:GAGGACC were identified in TbCSB by sequece analysis,and we speculate that the iterons are involved in Rep affinity binding and satellite replication.Competitive EMSA assay combined with iterons mutation and substitution assays have drawn the conclusion that iterons in TbCSB RBM induce the high affinity binding with TbCSV Rep.Nicotiana benthamiana incoculation and leaf disc replication assays confirmed that the iterons were of high significance to TbCSB trans-replication,and the 3'-iteron plays pivotal role in replication,while 5'-iteron can enhance TbCSB replication.We have constructed the chimeric infectious clones of TYLCCNB and TbCSB,which the iterons were exchanged between each other.Infectious clones inoculation assay suggest that the iterons in betasatellites were the core elements that determine the selective replication of cognate betasatellite by its helper virus.Systematic Evolution of Ligands by Exponential enrichment(SELEX)was performed and we found that all the TbCSB DNA ligands contained a conserved GGACC or GAACC iteron-like element,which was of high identity to that of wild type TbCSB iteron(GAGGACC),and these ligands possess very high binding intensity with Rep.We compared the sequences of iterons in betasatellites and that of helper viruses,and we found that the iterons in both betasatellites and helper virues were highly conserved in location,size and nucleotides.Mutation analysis verified that the iterons in helper viruses were also indispensable for their replication.Further large scale of sequence analysis proved that the coincident iterons appear in lots of begomovirus/betsatellite viral complex.All these results indicate that betasatellite obtains the helper-virus analogous iterons in the long way of evolution,so as to adapt to the helper virus and replicate efficiently.Our study reports for the first time that betasatellites require the iterons homologous to the helper virus for effficient replication,and explains the replication specificity of betasatellite,that confused us for a long time,besides our study provides new opinions about the origin,evolution and prevalence of begomovirus/betasatellite disease complex.2 Screening and function study of begomovirus Rep interacting Nicotiana benthamiana host factorsGeminiviruses infect the differentiated host cells,and activate cell cycle by the interaction between lots of host factors and replication initiator protein(Rep),so as to create a favorable cell environment for DNA replication.In order to further investigate the mechanism of geminivirus replication,we screened the Nicotiana benthamiana cDNA library to identify new host factors that interact with TYLCCNV or TbCSV Rep by yeast two-hybrid(Y2H)and we ultimately obtained 15 putative N.benthamiana factors.When one of the N.benthamiana factor-Ferredoxin(NbFdn?)was downregulated by TRV VIGS,the downregulated plants display chlorisis symptoms.The virus caused symptoms alleviated a lot and viral DNA accumulated to almost undetectable level when inoculated with TbCSV,indicating that NbFdn ?participating in TbCSV replication and infection.NbFdn ? is reported to be a chloroplast localized protein and participates in photosynthetic electron transport during photosynthesis.Our subcellular localization study confirmed that NbFdn ?located on the surface of chloroplasts.The transcription level was upregulated when TbCSV infection,but the NbFdn ? mRNA level was significantly decreased when co-infected with TbCSB,however,the subcellular localization of NbFdn ? did not change in neither condition.Our previous results have shown that the mRNA level of COl 1,the receptor of JA was dramatically upregulated when TbCSV infection alone,while downregulated significantly when accompanied with TbCSB.In order to elucidate how the COI 1 and NbFdn ? response to TbCSV infection,we downregulate the endogenous NbFdn I by TRV silencing and find that the transcriptional level of COl 1 was also downregutaed dramatically,which indicates NbFdn ? may regulate the JA pathway by regulating the transcription of COI 1.Further,we confirmed that ?Cl to be the core viral factor that represses NbFdn I transcription and inhibits JA induced anti-virus defense.3 Identification of yeast factors involve in Mungbean yellow mosaic India virus replication and the study of gene function of MYMIV-AC5 proteinWe have constructed the Mungbean yellow mosaic India virus(MYMIV)-yeast replicating recombinant plasmid and transformed into BY4741 and W303-1B yeast trains,respectively.We found that MYMIV can replicate in yeast(Saccharomyces cerevisiae).We then screened the temperature-sensitive yeast mutant library,which contains 787 single essential-gene mutants and is on the background of BY4741 strain.The transformants were screened by nutrition and permissive or semi-permissive temperature,and ultimately 131 alternative yeast host factors were obtained that might affect MYMIV replication.These host factors involve in DNA replication,damage DNA repair,cell-division cycle regulation,pre-mRNA processing and other cell biology processes.By constructing the recombinant plasmids containing distinct size and mutated MYMIV genome,we found that MYMIV replicates in a non-typical Rolling circle replication(RCR)manner.Based on the MYMIV yeast replication system,we found that MYMIV-AC5m null mutant replicates in yeast strain W303-la with a relative lower efficiency when compared with the wild type MYMIV.MYMIV-AC5m mutant caused less severe yellow mosaic symptoms in mungbean plants,viral DNA accumulation were also less than that of wild type MYMIV in mungbean and Nicotiana benthamiana plants.Further,N.benthamiana leaf disc assay confirmed that AC5m decreased the viral DNA accumulation,suggesting that AC5 does enhance replication although it is not indispensable for MYMIV replication.Overexpressing AC5 by PVX vector can induce the burst of active oxygen and cause cell necrosis.AC5 transgenic N.benthamiana seedlings display dwarf and late flowering symptoms,and can significantly increase viral DNA accumulation when inoculated with MYMIV,indicating AC5 could be a pathogenicity determinant.Moreover,AC5 can suppress sense-RNA induced post-transcriptional gene silencing(PTGS)and downregulate the transcription of DOMAINS REARRANGED METHYTRANSFERASE 2(DRM2)so as to repress the hosts'transcriptional gene silencing(TGS)activity.