| Replication of genomic RNA is one of the fundamental steps in viral infection cycles. Cis-acting elements play key roles in the regulation of viral replication, which located mostly in untranslated regions(UTR) of viral genomes, so studies on the relationship between viral UTR and its infection are important to dissect the cis-acting elements in the UTRs and understand the whole process of virus replication. In this study, tobacco vein banding mosaic virus(TVBMV) infectious cloning was used as materials, and the cis-acting elements were identified to responsible for viral infection by deleting different sequences in 5′ and 3′-UTR.Except for powerful means of investigating the viral replication, infectious clones of plant virus could be developed into over-expression vectors for functional protein production, and virus induced gene silencing(VIGS) vectors for plant gene function study. In this study, we reported the construction of agroinfiltrated infectious clone of p Ca PVX100 derived from PVX isolate 1985, which has been modified as expression vectors of p Ca PVX440 and p Ca PVX760, and a gene silencing vector of p Ca PVX440-LIC. The results were as follows:(1) 5′-Δ1-7, 5′-Δ1-25, 5′-Δ1-64, 5′-Δ1-150 and 5′-Δ1-171 were obtained by 5′-to-3′ progressive deletions of 7, 25, 64, 150 and 171 nt within TVBMV 5′-UTR(171 nt). N. benthamiana plants were infiltrated with Agrobacterium harboring those mutants separately, and plants infiltrated with that of wild type p Ca TVBMV-GFP(wt) were used as control. The result shows the plants were infected systemically with mutants of 5′-Δ1-7, 5′-Δ1-25, 5′-Δ1-64, 5′-Δ1-150, however, the more nucleotides were deleted, the longer time was needed for systemic infection and the lower viral relative RNA accumulation was obtained. Mutant 5′-Δ1-171 has no infectivity. N. benthamiana plants infiltrated with Agrobacterium harboring 5′-Δ1-171 were not infected systemically.Except for partial progeny viral RNA of 5′-Δ1-7, progeny viral RNA of 5′-Δ1-25, 5′-Δ1-64 and 5′-Δ1-150 were not recovered to wt, however, a series of AU-rich sequences were found at the 5′-proximal of the progeny viral genomes. The number and order of added AU-rich sequence were not identical to each other even in the progeny of the same parental RNA, however, the progeny viral RNAs of all mutants have sequence of A4-6U similar to that of wt at their utmost 5′-end of the genomes.In order to determine whether the AU-rich sequences at the utmost 5′-end of the genome will continue to change, The virus were passaged by rub inoculation of systemic leaves of 5′-Δ1-64 and 5′-Δ1-150-infected to N. benthamiana plants, and found that the sequence of the fifth generation were not identical to the first generation, and the dominant sequences were emerged. Two derivatives of Δ1-64-5 and Δ1-150-5 were constructed by adding the dominant sequences to the utmost of 5′-end, and the result shows time for systemic infection of those derivatives were shorter than that of Δ1-64 and Δ1-150, indicating the dominant sequence were favourable for viral infection.N. benthamiana plants were infiltrated with the mixtrue Agrobacterium harboring 5′-Δ1-25, 5′-Δ1-64 and 5′-Δ1-150. The result shows progeny viral RNA were all the progeny viral RNA of 5′-Δ1-25, indicating the progeny of mutant that loss less nucleotides had advantage in viral infection.5′-Δ26-64, 5′-Δ65-100, 5′-Δ101-125, 5′-Δ126-150, 5′-Δ151-160 and 5′-Δ161-171 were constructed by partition deletions of 26-171 nt within TVBMV 5′-UTR. The results show time for systemic infection and relative RNA accumulation of 5′-Δ101-125, 5′-Δ126-150 were identical to wt. Fluorescence in plants infected with 5′-Δ26-64 were dimmer and relative RNA accumulation were lower than that of wt, however, fluorescence in plants infected with 5′-Δ151-160 were brighter and relative RNA accumulation were higher than that of wt. The sequencing results indicate the progeny viral genome of 5′-Δ26-64, 5′-Δ65-100, 5′-Δ101-125, 5′-Δ126-150, 5′-Δ151-160 were identical to their parental genome. N. benthamiana plants were infiltrated with Agrobacterium harboring 5′-Δ161-171, and time for systemic infection was longer than that of wt, and the progeny viral sequence were various at the parental deletion site. Three of 32 clones retained the same sequence as that of Δ161-171, whereas 29 of 32 clones obtained 11 nt sequences, similar to 172-182 nt next to the deletion site. The data indicate that the deletion of 161-171 nt in TVBMV 5′-UTR was not lethal, but delayed the speed of viral infection.The RNA dependent RNA polymerase(Rd Rp) activity of the mutant 5′-Δ1-25 was lost by mutating the GDD sequence to GAA, obtained the mutant Δ1-25-GAA. Transcripts in Δ1-25-GAA-infiltrated leaves were subjected to 5′-RACE and sequencing. The result shows no nucleotides were added to the utmost of the 5′-end of Δ1-25-GAA, indicating the activity of Rd Rp was essential for viral genomes repair. N. benthamiana plants but not N. tabacum plants were systemically infected with mutant 5′-Δ1-150. The result shows viral genome repair are also related to host factors.(2) 3′-△1-20 and 3′-△1-60 were constructed by 5′-to-3′ progressive deletions within TVBMV 3′-UTR(184 nt). N. benthamiana plants were infiltrated with Agrobacterium harboring those mutants separately, and the plants were infected systemically only with of 3′-Δ1-20. The result shows two-14 nt-sequences were inserted to the deletion site in the progeny viral RNA of 3′-△1-20. Sequence of 8-42 nt in TVBMV 3′-UTR was predicted to form an imperfect stem-loop structure by Mfold analysis, and new inserted sequence of 14 nt help TVBMV genome forming a perfect stem-loop structure in 5′-end of 3′-UTR. In order to determine the importance of 8-42 nt in 3′-UTR during viral infection, mutants 3′-△8-42, 3′-△8-14, 3′-△15-35, 3′-△36-42 were constructed through deletion mutagenesis. The result shows the plants were infected systemically with mutants of 3′-△8-14, 3′-△15-35, 3′-△36-42 and the plants were not infected systemically with mutants of 3′-△8-42. The sequencing results show the stem-loop structure is very important for viral infection. Further predicted analysis shows the stem-loop structure were conserved in members of potyvirus containing tobacco vein mottle virus and zucchini yellow mosaic virus et al.3′-△43-60, 3′-△61-100, 3′-△101-141, 3′-△142-162 and 3′-△163-174 were constructed by deletion 43-175 nt within TVBMV 3′-UTR. N. benthamiana plants were infiltrated with Agrobacterium harboring those mutants separately, and the plants were infected systemically only with mutants of 3′-Δ142-162, indicating the deletion of 142-162 nt in TVBMV 3′-UTR was dispensable for viral infection, but the relative RNA accumulation was lower than that of wt.N. benthamiana plants were infected systemically with mutants that deletions up to 10 nt from the 3′-terminus of TVBMV genome, however, systemic symptoms are delayed remarkably. Other mutants losing more nucleotides had lost their infectivity. The progeny viral RNA of deletion mutants lacking 2-10 nt were repaired in planta, and only part of them recovered to wt.An infectious clone of PVX was reported to construct successfully, and was modified to over-expression and VIGS vectors. The full length 35 S promoter and the PVX1985 sequence were then inserted into p Cambia0390. The resulting vector is referred to as p Ca PVX100. The plasmid can be inoculated by agroinfiltration, and the disease symptoms caused by p Ca PVX100 in tobacco cv. NC89, tomato cv. Micro-Tom, and potato cv. Zaodabai. were similar to that caused by the wild type PVX.PVX CP promoter and TMV CP promoter following corresponding cloning sites were introduced to the region between TGB and CP ORF, The resultant plasmid was designated as p Ca PVX440, which was designed to express two foreign genes simultaneously in the same cells. vector p Ca PVX760 was produced by replacing most of the PVX CP sequence in the p Ca PVX440 with a multiple cloning sites. The cp-less vector has the ability of producing heterologous proteins in high level. Vector p Ca PVX440-LIC was produced by inserting a LIC sequence in the p Ca PVX440. A fragment was inserted, in the sense or antisense orientation, into p Ca PVX440-LIC to produce p Ca PVX440-LIC-PDS or p Ca PVX440-LIC-PDSas. Plants infiltrated with those plasmids developed photo-bleaching phenotype in their systemic leaves. Relative accumulation of pds m RNA in those leaves was detected by semi-quantitative RT-PCR. the pds m RNA level accumulated in plants inoculated with p Ca PVX440-LIC-PDSas and p Ca PVX440-LIC-PDS were significantly lower than that inoculated with p Ca PVX440-LIC, indicating that p Ca PVX440-LIC can be used as a viral vector to silence genes in N. benthamiana plants. |
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