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Chatterji et al 2001 4 homologous virus We based our choice of truncated Rep protein on the knowledge of overlapping sites for DNA cleavage domains for DNA binding and for protein oligomerization 13 14 We hypothesized that a truncated Rep protein that was competent for DNA binding and oligomerization domain might have a greater probability
Chatterji et al 2001, The minimal DNA binding domain of the Replication associated protein Rep of Tomato. leaf curl New Delhi virus was determined by electrophoretic mobility gel shift analysis. and co purification assays DNA binding activity maps to amino acids 1 to 160 Rep1 160. of the Rep protein and overlaps with the protein oligomerization domain Transient. expression of Rep protein Rep1 160 was found to inhibit homologous viral DNA. accumulation by 70 to 86 in tobacco protoplasts and in Nicotiana benthamiana plants. The results obtained showed that expression of N terminal sequences of Rep protein. could efficiently interfere with DNA binding and oligomerization activities during virus. Downloaded from http www jbc org by guest on May 19 2020. infection Surprisingly this protein reduced accumulation of the African cassava mosaic. virus Pepper huasteco yellow vein virus and Potato yellow mosaic virus by 22 48. EMSAs and co purification studies showed that Rep1 160 did not bind with high affinity in. vitro to the corresponding common region sequences of heterologous geminiviruses. However Rep1 160 formed oligomers with the Rep proteins of the other geminiviruses. These data suggest that the regulation of virus accumulation may involve binding of the. Rep to target DNA sequences and to the other Rep molecules during virus replication. Chatterji et al 2001,INTRODUCTION, Geminiviruses cause economically significant diseases in a wide range of cereal. vegetable and fiber crops 1 These viruses have a single stranded DNA genome that is. replicated in nuclei of infected cells by a rolling circle mechanism 2 3 Of the different. gene products encoded by the virus only AC1 the replication associated protein Rep is. essential for viral DNA replication The first step in the replication process involves. recognition of specific DNA sequences referred to as iterons 4 by the Rep protein in. the common region CR of the virus genome Most iteron sequences occur as direct. Downloaded from http www jbc org by guest on May 19 2020. repeat motifs of 6 12 bp between the TATA box and the start site of transcription of the. AC1 gene The iterons serve as high affinity binding sites of the Rep protein and. therefore function as the origin recognition sequences Specific regions on the N. terminus of Rep protein are involved in DNA binding and have been identified for. Tomato golden mosaic virus TGMV 5 6 African cassava mosaic virus ACMV 7. and Tomato yellow leaf curl virus TYLCV 8, The potential binding site sequences in the common region of the Tomato leaf. curl New Delhi virus ToLCNDV 9 genome were identified by site directed. mutagenesis 10 Further analyses using gel shift assays confirmed that the Rep protein. specifically binds to the iterated motifs GGTGTCTGGAGTC nts 2640 2653 in the. origin of replication 11 In the present study our objective was to identify the DNA. binding domain of the Rep protein and to determine the nature and contribution of DNA. binding and protein oligomerization properties of the Rep protein to limit viral DNA. accumulation in plants In two cases truncated Rep proteins have been shown to confer. resistance to other geminiviruses 7 12 and the resistance was specific and limited to the. Chatterji et al 2001, homologous virus We based our choice of truncated Rep protein on the knowledge of. overlapping sites for DNA cleavage domains for DNA binding and for protein. oligomerization 13 14 We hypothesized that a truncated Rep protein that was. competent for DNA binding and oligomerization domain might have a greater probability. to interfere with the virus replication and may be effective against both homologous and. heterologous viruses, In this study we mapped the minimal binding domain on the Rep protein by. electrophoretic mobility shift assays EMSAs We also tested the effect of truncated and. full length AC1 sequences on DNA replication of ToLCNDV and other geminiviruses in. Downloaded from http www jbc org by guest on May 19 2020. transient assays using BY2 protoplasts and N benthamiana plants These studies. revealed that transient expression of the ToLCNDV truncated Rep protein encoding the. DNA binding and the oligomerization domains could significantly inhibit replication of. ToLCNDV viral DNA and to some extent the replication of other geminiviruses having. similar iteron sequences,Chatterji et al 2001,MATERIALS AND METHODS. Plasmid constructs, The full length AC1 genes from the severe and the mild strains of ToLCNDV. were amplified by PCR from pMPA1 DNA A of the severe strain ToLCNDV and. pMPA2 DNA A of the mild strain ToLCNDV 15 and cloned in the bacterial. expression vector pGEX 4T 3 Pharmacia and overexpressed in E coli cells The. recombinant proteins were named according to the number of amino acids at the N or C. terminus of the Rep protein The C terminal truncations were made by inserting an in. Downloaded from http www jbc org by guest on May 19 2020. frame stop codon at positions 2436 pAC11 52 2250 pAC11 114 and 2110 pAC11 160. The truncated AC1 sequences were sub cloned as Bam HI to Xho I fragment in pGEX. 4T 3 vector generating pAC11 52 and pAC11 114 and pAC11 160 respectively At the N. terminus the first 21 amino acids of the protein were deleted and a Nhe I site was inserted. to create an in frame start codon The truncated fragment was cloned as a Nhe I to Xho I. fragment in the vector pGEX 4T 3 to produce pAC122 360 The plasmid pAC152 360 and. pAC1114 360 were produced similarly but had a deletion of the first 51 and 113 amino acids. respectively from the N terminus of the AC1 gene,Protein expression and analysis. The truncated Rep proteins were expressed from plasmids mentioned above in E. coli cells The glutathione S transferase GST tagged AC1 fusion proteins were purified. by glutathione affinity chromatography on glutathione Sepharose beads according to the. manufacturer s recommendations, Briefly the cells were grown to a density of 0 75 0 8 O D600 The cultures were. induced by addition of isopropyl D thiogalactoside IPTG at a final concentration of 1. Chatterji et al 2001, mM and grown further for 2 hours The cells were finally harvested at 4000 rpm. Beckman JS 10 5 rotor for 10 minutes The pellets were suspended in ice cold 1X. PBS 10 mM KH2PO4 100 mM NaCl and lysed by sonication The lysate was clarified. at 17 000 X g for 30 minutes The resulting supernatant was loaded on a glutathione. Sepharose 4B column Pharmacia WI previously equilibrated with 1X PBS After. repeated washing of the column with 1X PBS the protein was eluted with glutathione. elution buffer Pharmacia The eluted fractions were dialyzed against 1X PBS to. remove glutathione and concentrated using centricon filters Amicon Centricon Protein. concentrations were estimated using Bradford s reagent Bio Rad CA. Downloaded from http www jbc org by guest on May 19 2020. Protein extracts from E coli cells co expressing the untagged wild type AC1 and. GST fusion of truncated Rep proteins were tested for AC1 oligomerization by co. purification on glutathione Sepharose Co purification of proteins was monitored by. resolving the eluted fractions on SDS PAGE and by immunoblotting The full length and. truncated Rep proteins were detected using the polyclonal anti AC1 antibody A similar. procedure was used to assess the oligomerization of full length Rep proteins from other. geminiviruses with the truncated Rep protein of ToLCNDV pAC11 160 Protein extracts. from E coli cells co expressing wild type AC1 of Pepper huasteco yellow vein virus. PHYVV Potato yellow mosaic virus PYMV and African cassava mosaic virus. ACMV and the GST tagged pAC11 160 were incubated with GST Sepharose beads. washed thoroughly with 1 X PBS and eluted with glutathione elution buffer Pharmcia. The eluate was resolved on SDS PAGE gels transferred to nitrocellulose membranes and. detected by immunoblotting using polyclonal anti AC1 antibody and anti GST antibody. Chatterji et al 2001,Construction of expression cassettes. For expression of the truncated AC1 gene in plant cells the mutants described. above were sub cloned as Bam HI fragments in the plant expression vector pILTAB 350. This placed the DNA fragment 3 of the cassava vein mosaic virus CsVMV promoter. 16 upstream of the AC1 gene sequences to produce the gene expression cassettes. pILTAB 401 encoding AC11 52 pILTAB 402 encoding AC11 114 and pILTAB 403. encoding AC11 160 respectively, Constructions of infectious clones of plasmids containing full length DNA of. Downloaded from http www jbc org by guest on May 19 2020. ToLCNDV are named pMPA1 and pMPB1 and were previously described 15 John. Stanley John Innes Institute Norwich UK generously provided full length infectious. dimers of ACMV Kenya pCLV 1 3A and pCLV 2B 17 Infectious monomers of. PHYVV 18 were kindly provided by Riviera Bustamante CINVESTAV Irapuato. Mexico The PYMV clones have been described 19,Electrophoretic mobility shift assays EMSAs. The sequences of the synthetic oligonucleotides used as probes or competitors in. EMSAs are given in Table 3 In the case of the severe and mild strains of ToLCNDV the 18. mer oligonucleotides corresponding to the binding sites of the Rep protein were used as probe. 11 For the geminiviruses ACMV PHYVV and PYMV fragments of their CR sequences. were synthesized and used as competitors in EMSAs All oligonucleotides were synthesized. commercially by BRL, The single stranded 18 mer oligonucleotides containing the potential binding sites of. the Rep protein of ToLCNDV were annealed to their complementary strands The. Chatterji et al 2001, oligonucleotides were end labeled with 32P ATP using T4 polynucleotide kinase and. purified on polyacrylamide gels The final concentration of the probes was 500 pM 30 000. cpm The concentration of competitor DNA used was 50 nM per reaction Both the probe. and the competitor DNAs were purified on Sephadex G 25 columns quantified by. scintillation counting and diluted to 30 000 cpm for each binding reaction. The binding assays were performed using purified Rep protein Typically the binding. reactions contained 500 ng of pure protein 1 ng of labeled DNA and 0 2 g of poly dI dC. Binding buffer contained 20 mM HEPES pH 7 5 60 mM KCl 1 mM DTT and 15. Downloaded from http www jbc org by guest on May 19 2020. glycerol Reactions were incubated at 25 C for 30 minutes and the complexes were resolved. on 4 polyacrylamide gels in 0 25 X TBE buffer The gels were dried on Whatman paper. and exposed to X ray film Comparative efficiency of binding was analyzed by quantifying. the amount of radioactivity in the retarded bands using a phosphorimager Molecular. Transient replication assays in protoplasts and plants. Protoplasts derived from N tabacum BY 2 suspension cultures were used for. transfection with viral DNA 20 Protoplasts were collected from cultures 48 h post. inoculation for DNA isolation and analysis One million protoplasts were inoculated by. electroporation 250v 500 F with 2 g each of A and B component DNAs and 40 g of. sheared herring sperm DNA 10 For co inoculation experiments 2 g of the plasmid. DNA containing the expression cassettes with truncated AC1 gene sequences were used. Total DNA from the protoplasts was extracted 48 hours after transfection 21 22 Viral. DNA accumulation was analyzed by Southern blotting 10. Total proteins were extracted from the protoplasts 48h after transfection by. sonication of the cell pellets in ice cold 1X PBS 10 mM KH2PO4 100 mM NaCl The. Chatterji et al 2001, lysate was clarified at 17 000 X g for 15 minutes and the resulting supernatant was used. for immunoprecipitations Immunoprecipitations were done by incubating 50 g of total. protein extracts with polyclonal anti AC1 antiserum 1mg overnight at 4 C Protein. antibody complexes were incubated with protein A agarose for 2h at 4 C and then. washed with 1XPBS Bound proteins were eluted from the agarose beads in SDS PAGE. sample buffer by boiling at 100 C for 5 minutes Proteins resolved on the gel were. transferred on nitrocellulose membranes and analyzed by immunoblotting with. polyclonal anti AC1 antibody using the 3 3 Diaminobenzidine tetrahydrochloride DAB. for colorimetric quantitation of the expressed Rep levels in the cell extracts. Downloaded from http www jbc org by guest on May 19 2020. Two weeks old seedlings of N benthamiana were grown in magenta boxes and. inoculated with partial tandem dimers of viral DNA using a Bio Rad helium driven. particle gun 10 Ten plants were inoculated with each mutant using 0 5 g each of. DNA A and DNA B genomic components per plant Plants were observed for symptom. development and newly emerging leaves were harvested for Southern blot analysis 4. weeks post inoculation,Southern blot analysis, DNA extractions from systemically infected leaf samples were completed as. described in Dellaporta et al 21 and from protoplasts by following the procedure of. Mettler 22 Total DNA 4 g was fractionated on 1 agarose gels without ethidium. bromide and transferred to nylon membranes Viral DNA was detected by using a 900 bp. Afl II Pst I fragment of the A component containing sequences from the ORFs AC1. AC2 and AC3 genes or a probe specific for the B component 878 bp PCR amplified. BC1 gene The amount of viral DNA was quantified as previously described 10 In. Chatterji et al 2001, case of geminiviruses other than ToLCNDV fragments of their AC1 and BC1 genes were. amplified and used as probes to analyze the replication levels of viral DNA. Downloaded from http www jbc org by guest on May 19 2020. Chatterji et al 2001, Determination of a minimal binding domain of the Rep protein of ToLCNDV. The Rep protein binds specifically to a directly repeated DNA sequence motif in. the common region of the ToLCNDV genome 11 Purified Rep proteins truncated at. amino acids 160 114 and 52 were used to map the C terminal boundary of the Rep DNA. binding domain in vitro As a control full length Rep protein amino acids 1 360 was. used in all assays The truncated and full length Rep proteins were expressed in E coli. with a GST tag and affinity purified on a glutathione Sepharose 4B column The. Downloaded from http www jbc org by guest on May 19 2020. affinity purified proteins were highly enriched as determined by Coomassie staining. following electrophoresis on SDS PAGE gels The proteins were detected in. immunoblots using anti GST antibody data not shown. The purified Rep proteins were tested for their ability to bind a radiolabeled 18. mer nts 2632 2653 that contains the Rep binding site sequence 5 GGTGTCTGGAGTC. 3 DNA protein complexes that contained Rep1 360 and Rep1 160 were detected No. binding was observed for Rep1 52 or Rep1 114 Figure 1A lanes 1 to 4 These results. located the C terminal boundary of the DNA binding domain of the Rep protein between. amino acids 115 and 160, The N terminal boundary of the DNA binding domain was determined in vitro by. comparing the binding of full length Rep1 360 and Rep22 360 Rep52 360 and Rep114 360 to the 18. bp iteron sequence 5 GGTGTCTGGAGTC3 in EMSAs The DNA protein complexes. were observed in case of full length Rep protein while no DNA protein complexes were. detected for the Rep22 360 Rep52 360 or Rep114 360 Figure 1B lanes 2 3 and 4 These results. demonstrated that the sequences within the first 21 amino acids of the Rep protein are. Chatterji et al 2001, essential for protein DNA interactions Together these results placed the DNA binding. domain of ToLCNDV Rep protein between amino acids 1 160. To determine if truncations at the N and the C termini of Rep protein affect its. ability to oligomerize GST tagged truncated Rep proteins were co expressed with. untagged wild type full length Rep protein in bacterial cells and co purified on. glutathione Sepharose beads The bound fractions were eluted and analyzed in. immunoblots using polyclonal anti AC1 antiserum The wild type Rep1 360 co purified. with GST tagged truncated proteins Rep22 360 Rep52 360 and Rep114 360 Figure 1C lanes 1 to. 5 suggesting that truncations made at the N terminus in the Rep did not affect the ability. Downloaded from http www jbc org by guest on May 19 2020. of the Rep protein to oligomerize with itself even though each of the truncated protein. was deficient for DNA binding, ToLCNDV replication is inhibited by transiently expressed Rep protein. The effect of Rep protein on viral DNA replication was investigated by co. inoculating N tabacum BY2 protoplasts with DNA A and various cassettes that express. truncated AC1 gene sequences from the CsVMV promoter ToLCNDV DNA A. replicated in BY 2 cells and accumulated high levels of single stranded ss and. supercoiled sc DNA Figure 2A lane 1 In contrast there was a significant decrease in. the level of viral DNA replication 78 drop in the presence of Rep1 160 Figure 2A lane. 4 Table 1 Reduction in replication was estimated by quantifying the amount of. radioactivity using a phosphorimager Storm 860 Molecular Dynamics The reduction. in virus replication was not as dramatic in the presence of Rep1 52 Figure 2A lane 2 or. Rep1 114 Figure 2A lane 3 when compared with Rep1 160 Figure 2A lane 4 EMSAs. showed that the Rep1 52 and Rep1 114 do not bind DNA Figure 1A lanes 2 and 3 implying. Chatterji et al 2001, that an intact DNA binding domain and or a protein oligomerization domain is essential. for inhibition of replication, Similar experiments were conducted with the mild strain of ToLCNDV in which. analogous truncated mutations of the Rep gene were co introduced in tobacco protoplasts. with DNA A from the mild strain In these studies an analogous inhibition of viral. DNA accumulation in BY2 protoplasts was detected Figure 2A lanes 5 to 8 Table 2. To determine the relative expression levels of the three truncated Rep proteins in. transfected tobacco protoplasts total proteins were extracted from the tobacco protoplasts. 48h after infection and immuno precipitated with the anti AC1 antibody and the protein. Downloaded from http www jbc org by guest on May 19 2020. antibody complexes were resolved on SDS PAGE gels All the three truncated proteins. could be detected in immuno blots from the transfected protoplasts when developed. using 3 3 Diaminobenzidine tetrahydrochloride DAB DAB produces a brown. precipitate with the peroxidase and thereby provided a direct measure of the amount of. antibody bound to the expressed protein in the samples revealed that all the three. truncated Rep proteins were expressed stably and in equivalent amounts in the protoplasts. Figure 2B lanes 2 6,Infection of N benthamiana, Two weeks old seedlings of N benthamiana plants were co bombarded with 2 g. each of infectious dimers of ToLCNDV DNA A and DNA B in the presence or absence. of genes encoding Rep1 160 The plants were observed daily for symptom development. All of the plants inoculated only with the wild type virus DNAs developed severe. symptoms five days after inoculation In contrast plants co inoculated with the virus and. the genes encoding Rep1 160 developed milder symptoms of ToLCNDV infection Table. 1 About 55 of the plants were asymptomatic 30 showed mild chlorosis and 15 of. Chatterji et al 2001, plants expressed mild leaf curl symptoms Table 1 None of the plants showed severe. infection or stunted growth as in plants infected only with ToLCNDV Most of the plants. co inoculated with Rep1 52 and Rep1 114 developed severe symptoms by seven days post. inoculation Tables 1 and 2, The levels of viral DNA in ToLCNDV infected plants was analyzed by southern. blot analysis of young leaves sampled 28 days post inoculation using probes that detected. DNA A and DNA B see Materials and Methods Figure 3A and 3B respectively The. amount of viral DNA ranged from undetectable to very low an average of 15 of the. wild type levels in asymptomatic plants and the accumulation of both genomic. Downloaded from http www jbc org by guest on May 19 2020. components increased with increasing severity of symptom expression Plants co. inoculated with expression cassettes Rep1 52 and Rep1 114 developed intermediate to severe. symptoms in most of the plants and accumulated viral DNA between 85 to 92 of. wild type infection, Co infection with Rep1 160 of ToLCNDV severe reduces the viral DNA accumulation. of other geminiviruses, To investigate the potential of truncated Rep protein to inhibit the replication of. other geminiviruses we selected examples of viruses that belonged to the Old World. ACMV and New World geminiviruses PHYVV and PYMV TT We reasoned that for. the Rep to be able to interfere in replication of heterologous geminiviruses it must a. bind to the origin sequences of these viruses and b oligomerize with their Rep proteins. For the EMSA studies fragments of the intergenic region sequences of the selected. heterologous geminiviruses close to the TATA box were chosen The coordinates of. these sequences are given in Table 3 To determine if the putative iteron sequences of the. other geminiviruses could compete with the cognate iteron sequences of ToLCNDV for. Chatterji et al 2001, binding to ToLCNDV Rep protein synthetic oligonucleotides encoding the CR. sequences from each virus were synthesized and used as competitors in EMSAs None of. the CR sequences were effective competitors in EMSA with the ToLCNDV Rep protein. Figure 4A lanes 3 to 6 and did not affect binding of the Rep protein with its cognate. 13 mer iteron sequences to a significant degree, The crude lysates of E coli cells co expressing wild type Rep proteins from. ACMV PHYVV or PYMV and the GST tagged ToLCNDV Rep1 160 were tested for the. ability to bind to each other Crude protein extracts from bacterial cells co expressing the. target proteins were loaded on a GST Sepharose column washed extensively with 1 x. Downloaded from http www jbc org by guest on May 19 2020. PBS and eluted with glutathione elution buffer The resulting fractions were detected in. immunoblots using polyclonal anti AC1 and anti GST antibodies In immunoblots the. anti AC1 antibody detected wild type untagged Rep proteins from ACMV PHYVV and. PYMV TT that co purified with the ToLCNDV Rep1 160 Figure 4B lanes 2 to 6 When. the same blot was washed and reprobed with anti GST antibody only the truncated Rep. protein of ToLCNDV Rep1 160 in each of the samples was detected Figure 4C lanes 2 to. To determine if Rep1 160 could reduce accumulation of other geminiviruses in vivo. replication assays were conducted by co bombarding the N benthamiana plants with. partial tandem dimers of full length A and B components of ACMV PHYVV and. PYMV TT with genes encoding Rep 1 160 of ToLCNDV Most plants developed typical. symptoms of virus infection within 21 27 days post inoculation as opposed to 7 10 days. required for the symptoms on control plants to develop infection Southern blot analysis. of viral DNA extracted from the plants harvested 28days post inoculation showed a. minor reduction in the levels of virus accumulation as compared with the control plants.