Based on a full-length infectious cDNA clone, gene modifications of Tobacco necrosis virus A Chinese isolate (TNV-AC) were made by site-directed mutagenesis or nucleotide deletions for in vitro transcrip-tion of mutan...Based on a full-length infectious cDNA clone, gene modifications of Tobacco necrosis virus A Chinese isolate (TNV-AC) were made by site-directed mutagenesis or nucleotide deletions for in vitro transcrip-tion of mutant viral RNAs. Mechanical inoculations of Chenopodium amaranticolor with in vitro tran-scripts, containing a single nucleic acid substitution at the presumed transcriptional start sites for the two subgenomic (sg) RNAs, showed that the sgRNA1 and sgRNA2 of TNV-AC were initiated at G2184 or G2460, respectively. Mutagenesis of the translational initiation-codons for the open-reading frame (ORF) P8 or P6 encoded by sgRNA1 indicated that each of the two genes was essential for formation of local lesions on C. amaranticolor leaves, perhaps by blocking virus cell-to-cell movement, but were not necessary for viral RNA replication in the protoplast of tobacco cell BY-2. Results of prokaryotic expression showed that the ORF coding for coat protein on TNV-AC sgRNA2 was initiatively translated by the first AUG codon at nucleotides 2612―2614. Site-directed mutation of translational start codons, and deletion of the entire coding region, showed that the intact TNV-AC coat protein was dispensable for establishment of TNV-AC infection in C. amaranticolor, otherwise the numbers of local lesions and the viral RNA accumulation level were reduced, or the time to symptom appearance significantly delayed. These results suggested that the nucleotide sequence around the translational start codon coding for TNV-AC coat protein gene may play an important role in the local symptoms. Aspects of the involve-ment of the coat protein in the TNV-AC life cycle were discussed.展开更多
以烟草坏死病毒A中国分离物(Tobacco necrosis virus A Chinese isolate,TNV-AC)侵染性cDNA克隆为基础,通过基因替换、基因插入策略构建获得多种重组TNV-AC,比较了外源基因片段插入位置、插入形式及接种植物培养温度对TNV-AC诱导的基因...以烟草坏死病毒A中国分离物(Tobacco necrosis virus A Chinese isolate,TNV-AC)侵染性cDNA克隆为基础,通过基因替换、基因插入策略构建获得多种重组TNV-AC,比较了外源基因片段插入位置、插入形式及接种植物培养温度对TNV-AC诱导的基因沉默的影响.外源基因片段替换CP基因19～828 nt的重组TNV-AC丧失了在本生烟中的系统移动能力,也不能有效诱导相应基因发生明显的沉默,说明替换策略不适合于TNV-AC.向CP基因终止密码子UAG附近插入外源基因片段后,TNV-AC仍可进行复制,但最适的插入位点位于UAG之后,且容纳外源片段的长度约为120 nt.当外源片段以反向重复的形式插入UAG之后,诱导基因沉默的效率较高.接种植物的培养温度也会显著影响基因沉默的效率以及插入片段的稳定性,低温(18℃)条件下诱导NbPDS基因沉默的效率明显高于高温(24℃)条件,且沉默表型可持续110天以上.除了本生烟PDS基因,TNV-AC沉默载体还可诱导本生烟sulfur基因Su和镁离子螯合酶H亚基基因ChlH发生沉默,以上结果说明,TNV-AC具有开发为本生烟基因功能鉴定的新VIGS载体的潜力.展开更多
The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has no...The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional(3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus(CMV) or tobacco necrosis virus A Chinese isolate(TNV-AC), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1 a and 2 a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast.Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant(+) RNA virus replication complexes.展开更多
基金the National Natural Science Foundation of China (Grants Nos. 30470077 and 30325001).
文摘Based on a full-length infectious cDNA clone, gene modifications of Tobacco necrosis virus A Chinese isolate (TNV-AC) were made by site-directed mutagenesis or nucleotide deletions for in vitro transcrip-tion of mutant viral RNAs. Mechanical inoculations of Chenopodium amaranticolor with in vitro tran-scripts, containing a single nucleic acid substitution at the presumed transcriptional start sites for the two subgenomic (sg) RNAs, showed that the sgRNA1 and sgRNA2 of TNV-AC were initiated at G2184 or G2460, respectively. Mutagenesis of the translational initiation-codons for the open-reading frame (ORF) P8 or P6 encoded by sgRNA1 indicated that each of the two genes was essential for formation of local lesions on C. amaranticolor leaves, perhaps by blocking virus cell-to-cell movement, but were not necessary for viral RNA replication in the protoplast of tobacco cell BY-2. Results of prokaryotic expression showed that the ORF coding for coat protein on TNV-AC sgRNA2 was initiatively translated by the first AUG codon at nucleotides 2612―2614. Site-directed mutation of translational start codons, and deletion of the entire coding region, showed that the intact TNV-AC coat protein was dispensable for establishment of TNV-AC infection in C. amaranticolor, otherwise the numbers of local lesions and the viral RNA accumulation level were reduced, or the time to symptom appearance significantly delayed. These results suggested that the nucleotide sequence around the translational start codon coding for TNV-AC coat protein gene may play an important role in the local symptoms. Aspects of the involve-ment of the coat protein in the TNV-AC life cycle were discussed.
文摘以烟草坏死病毒A中国分离物(Tobacco necrosis virus A Chinese isolate,TNV-AC)侵染性cDNA克隆为基础,通过基因替换、基因插入策略构建获得多种重组TNV-AC,比较了外源基因片段插入位置、插入形式及接种植物培养温度对TNV-AC诱导的基因沉默的影响.外源基因片段替换CP基因19～828 nt的重组TNV-AC丧失了在本生烟中的系统移动能力,也不能有效诱导相应基因发生明显的沉默,说明替换策略不适合于TNV-AC.向CP基因终止密码子UAG附近插入外源基因片段后,TNV-AC仍可进行复制,但最适的插入位点位于UAG之后,且容纳外源片段的长度约为120 nt.当外源片段以反向重复的形式插入UAG之后,诱导基因沉默的效率较高.接种植物的培养温度也会显著影响基因沉默的效率以及插入片段的稳定性,低温(18℃)条件下诱导NbPDS基因沉默的效率明显高于高温(24℃)条件,且沉默表型可持续110天以上.除了本生烟PDS基因,TNV-AC沉默载体还可诱导本生烟sulfur基因Su和镁离子螯合酶H亚基基因ChlH发生沉默,以上结果说明,TNV-AC具有开发为本生烟基因功能鉴定的新VIGS载体的潜力.
基金supported by grants from the National Transgenic Science and Technology Program (2019ZX08010-003)the National Natural Science Foundation of China (31872637)+2 种基金Chinese Universities Scientific Fund (2020TC181)the Research Grants Council of Hong Kong (GRF14126116, GRF14121019, C4012-16E, C4002-17G, and Ao E/M-05/12)Cooperative Research Program for Agriculture Science & Technology Development (0109532019) Rural Development Administration, Republic of Korea。
文摘The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional(3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus(CMV) or tobacco necrosis virus A Chinese isolate(TNV-AC), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1 a and 2 a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast.Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant(+) RNA virus replication complexes.
