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Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination

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PLANT JOURNAL 1999年第6期17卷 591-601页

作者： Kohli, A Griffiths, S Palacios, N Twyman, RM Vain, P Laurie, DA Christou, P John Innes Ctr Plant Sci Res Norwich NR4 7UH Norfolk England

The characterization of plasmid-genomic DNA junctions following plant transformation has established links between DNA double-strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid-plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end-ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine-rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid-plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine-rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the 'highly recombinogenic' region of the full-length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis-acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.

关键词：碱基序列花椰菜花叶病毒属/遗传学 DNA修复 DNA 病毒/化学 DNA 病毒/遗传学基因重排分子序列数据核酸构象水稻/遗传学水稻/代谢水稻/病毒学植物基因修饰质粒/遗传学启动区(遗传学) 重组遗传序列同源性核酸转化遗传

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Sequence-specific and methylation-dependent and -independent binding of rice nuclear proteins to a rice tungro bacilliform virus vascular bundle expression element

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第4期276卷 2644-2651页

作者： He, XY Fütterer, J Hohn, T Friedrich Miescher Inst CH-4002 Basel Switzerland Swiss Fed Inst Technol Inst Plant Sci CH-8092 Zurich Switzerland

Nuclear proteins from rice (Oryza sativa) were identified that bind specifically to a rice tungro bacilliform virus promoter region containing a vascular bundle expression element (VBE). One set of proteins of 29, 33, and 37 kDa, present in shoot and cell suspension extracts but hardly detectable in root extracts, bound to a site containing the sequence AGAAGGACCAGA within the VEE, which also contains two CpG and one CpNpG potential methylation motifs, Binding by these proteins was determined to be cytosine methylation-independent. However, a novel protein present in all analyzed extracts bound specifically to the methylated VEE. A region of at least 49 nucleotides overlapping the VEE and complete cytosine methylation of the three Cp(Np)G motifs was required for efficient binding of this methylated VBE-binding protein (MVBP).

关键词： 5-甲基胞嘧啶/类似物和衍生物杆状DNA病毒属/遗传学碱基序列 CpG岛胞嘧啶/类似物和衍生物 DNA结合蛋白质类/代谢分子序列数据核蛋白质类/代谢水稻/病毒学植物蛋白质类/代谢植物芽/代谢启动区(遗传学) 蛋白质结合组织分布

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In vivo and in vitro phosphorylation of rice dwarf phytoreovirus Pns12 cytoplasmic nonstructural protein

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ARCHIVES OF VIROLOGY 1999年第7期144卷 1371-1380页

作者： Suzuki, N Hosokawa, D Matsuura, Y Kikuchi, A Omura, T Akita Prefectural Coll Agr Inst Biotechnol Lab Plant Genet Engn Akita Japan Tokyo Univ Agr & Engn Fac Agr Plant Pathol Lab Tokyo Japan Natl Inst Infect Dis Dept Virol 2 Tokyo Japan Natl Agr Res Ctr Ibaraki Osaka Japan

In vivo and in vitro phosphorylation and intracellular location of rice dwarf phytoreovirus Pns12, which is encoded by one of the twelve dsRNA genome segments, S12, and comprises 312 amino acids, was investigated. When [P-32]phosphoric acid was incorporated into RDV-infected leafhopper cultured cells, labelled Pns12 was immunoprecipitated from the cells by a monospecific anti-Pns12 polyclonal antibody. Recombinant Pns12 was purified from Spodoptera frugiperda cells infected with AcRS12, a baculovirus recombinant carrying a full-length cDNA of RDV S12. Purified Pns12 was also demonstrated to be phosphorylated in vitro by a kinase activity present in host (rice, barley, wheat, leafhopper) cells and non-host (tobacco, spinach, white clover, S. frugiperda, mosquito, mammals) cells as well. Immunocytochemical studies showed Pns12 accumulated in the cytoplasm of infected cells, and frequently localized in a slightly electron-dense patch. These results demonstrated that RDV Pns12 was a cytoplasmic nonstructural phosphoprotein.

关键词：细胞系细胞质/化学水稻/病毒学磷酰化呼肠病毒科/化学斜纹夜蛾属病毒非结构蛋白质类/分析病毒非结构蛋白质类/代谢动物

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Molecular variability of geographically distinct isolates of Rice yellow mottle virus in Africa

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ARCHIVES OF VIROLOGY 2000年第8期145卷 1621-1638页

作者： Pinel, A N'Guessan, P Bousalem, M Fargette, D IRD CIRAD LPRC Inst Rech Dev F-34032 Montpellier France CNRA Bouake Cote Ivoire

The coat protein gene (ORF4) and the 3' untranslated region of a sample of 40 isolates of Rice yellow mottle virus (RYMV), 32 from West Africa and 8 from East Africa, have been sequenced. Five major strains were differentiated, three from West Africa (S1, S2, S3) and two from East Africa (S4, S5), with a spatial overlap of the strains within each of these two regions. Nucleotide and amino-acid divergence between strains was up to 11%. Although more isolates from West African were sequenced, variability was twofold lower than among East African isolates. Variability in ORF4 and in ORF2 coincided. Within strain and within isolate variations in nucleotide sequences were low. Bipartite nuclear targeting motif, Ca2+ binding sites and at least two stretches of amino-acids were conserved among the 40 RYMV isolates and the other sobemoviruses. Variants associating sequence motifs characteristic of different strains have been found, possibly resulting from recombination events. Differences in pathogenicity among isolates were associated with changes of amino-acids in the bipartite nuclear targeting motif of the R domain of the capsid protein, and around conserved positions 151-154 of the S domain. We hypothesise that the observed pattern of variation of RYMV reflects the effect of spatial isolation between East and West Africa coupled with adaptive changes associated to the original virus reservoirs of the different strains.

关键词： 3'非翻译区/遗传学非洲氨基酸序列病毒壳体/遗传学克隆分子共有序列基因组病毒分子序列数据花叶病病毒/遗传学开放读码框架水稻/病毒学种系发生重组蛋白质类/遗传学逆转录聚合酶链反应序列比对

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Inter- and intra-site genetic diversity of natural field populations of rice tungro bacilliform virus in the Philippines

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ARCHIVES OF VIROLOGY 2000年第2期145卷 275-289页

作者： Arboleda, M Azzam, O Int Rice Res Inst Entomol & Plant Pathol Div Makati 1271 Philippines

The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines. Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates. In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity. Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations. However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations. The number of genotypes detected at a site varied from two to nine with a few genotypes dominating. In general the isolates at a site persisted from season to season indicating a genetic stability for the Focal virus population. Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures. Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host/environmental shifts.

关键词：农业杆状DNA病毒属/分类杆状DNA病毒属/遗传学杆状DNA病毒属/分离和提纯杆状DNA病毒属/生理学 DNA探针 DNA 病毒/遗传学 DNA 病毒/分离和提纯 DNA 病毒/代谢脱氧核糖核酸酶类位点特异性Ⅱ型/代谢基因型半翅目/病毒学昆虫媒介/病毒学水稻/病毒学菲律宾植物疾病/病毒学多态现象遗传多态性限制性片段长度变异(遗传学) 动物

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Genetic diversity of rice tungro spherical virus in tungro-endemic provinces of the Philippines and Indonesia

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ARCHIVES OF VIROLOGY 2000年第6期145卷 1183-1197页

作者： Azzam, O Yambao, MLM Muhsin, M McNally, KL Umadhay, KML Int Rice Res Inst Makati Philippines Res Inst Food Crops Biotechnol Bogor Indonesia

The two adjacent genes of coat protein 1 and 2 of rice tungro spherical virus (RTSV) were amplified from total RNA extracts of serologically indistinguishable field isolates from the Philippines and Indonesia, using reverse transcriptase polymerase chain reaction (RT-PCR). Digestion with HindIII and BstYI restriction endonucleases differentiated the amplified DNA products into eight distinct coat protein genotypes. These genotypes were then used as indicators of virus diversity in the field. Inter- and intra-site diversities were determined over three cropping seasons. At each of the sites surveyed, one or two main genotypes prevailed together with other related minor or mixed genotypes that did not replace the main genotype over the sampling time. The cluster of genotypes found at the Philippines sites was significantly different from the one at the Indonesia sites, suggesting geographic isolation for virus populations. Phylogenetic studies based on the nucleotide sequences of 38 selected isolates confirm the spatial distribution of RTSV virus populations but show that gene flow may occur between populations. Under the present conditions, rice varieties do not seem to exert selective pressure on the virus populations. Based on the selective constraints in the coat protein amino acid sequences and the virus genetic composition per site, a negative selection model followed by random-sampling events due to vector transmissions is proposed to explain the inter-site diversity observed.

关键词：病毒壳体/遗传学印度尼西亚昆虫/病毒学分子序列数据水稻/病毒学菲律宾种系发生限制性内切酶图谱法逆转录聚合酶链反应序列分析 DNA 变异(遗传学)/遗传学矮化病毒属/分类矮化病毒属/遗传学矮化病毒属/生理学动物

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Detection of rice grassy stunt tenuivirus nonstructural proteins p2, p5 and p6 from infected rice plants and from viruliferous brown planthoppers

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ARCHIVES OF VIROLOGY 2002年第12期147卷 2291-2300页

作者： Chomchan, P Miranda, GJ Shirako, Y Univ Tokyo Asian Ctr Bioresources & Environm Sci ANESC Bunkyo Ku Tokyo 1138657 Japan Univ Tokyo Grad Sch Agr & Life Sci Tokyo 1130033 Japan Int Rice Res Inst Manila Philippines

The genome of Rice grassy stunt virus (RGSV) consists of 6 ambisense RNA segments, among which RNAs 1, 2, 5 and 6 are equivalent to RNAs 1, 2, 3 and 4, respectively, of Rice stripe virus, the type species of the genus Tenuivirus. The RGSV 36-kDa nucleocapsid protein (N) is encoded on the complementary strand of RNA 5. Here, we studied accumulation of three nonstructural proteins, a 23-kDa p2 protein encoded on vRNA 2 (virus genomic strand), a 22-kDa p5 protein encoded on vRNA 5, and a 21-kDa p6 protein encoded on vRNA 6, from RGSV-infected rice leaf tissues and from viruliferous vector insects (brown planthopper, Nilaparvata lugens) by Western blot analyses. p2, p5 and p6 were detected from RGSV-infected rice leaf homogenates;p2 was detected mostly in the cytoplasmic soluble fraction but also a small amount was detected in the cell-wall, organelle-enriched and crude membrane fractions;p5 and p6 were detected from the cytoplasmic soluble fraction in large amounts. Among individual nymphs of N. lugens raised on RGSV-infected rice leaves, only 20% of insects were positive with the N protein. A large amount of p5 was detected from all the N-positive insects. Small amounts of p2 and p6 were detected only from a subset of the Nand p5-positive insects. p5 may have an essential role in virus infection in both plant and insect hosts, whereas p2 may function in plants such as a cell-to-cell movement protein.

关键词：印迹法蛋白质昆虫媒介昆虫/病毒学分子量核壳蛋白质类/分析若虫/病毒学水稻/病毒学植物疾病/病毒学植物提取物植物叶/病毒学亚细胞部分/病毒学纤细病毒属/代谢病毒非结构蛋白质类/分析病毒非结构蛋白质类/化学动物

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Infection of barley protoplasts with rice hoja blanca tenuivirus - Brief Report

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ARCHIVES OF VIROLOGY 1999年第11期144卷 2247-2252页

作者： Nguyen, M Kormelink, R Goldbach, R Haenni, AL Wageningen Univ Agr Dept Virol Wageningen Netherlands Inst Jacques Monod F-75251 Paris 05 France

A barley protoplast system has been established that supports replication of Rice hoja blanca tenuivirus (RHBV). Following polyethylene glycol-mediated RHBV inoculation of barley protoplasts, newly synthesized viral RNAs and proteins could be detected. Time course analyses revealed de novo synthesis of genome length viral RNA4, as well as subgenomic-sized RNA4 molecules of both polarities. Two proteins, N and NS4, encoded by viral complementary RNA3 and viral RNA4 respectively, were detected by Western immunoblot analysis. The barley protoplast system thus constitutes a promising tool for in vivo studies of the sequential steps involved in the multiplication cycle of RHBV.

关键词：大麦属/病毒学核壳蛋白质类/生物合成核壳蛋白质类/遗传学水稻/病毒学植物病毒/遗传学植物病毒/生长和发育原生质体/病毒学 RNA病毒/遗传学 RNA病毒/生长和发育 RNA 病毒/生物合成病毒非结构蛋白质类/生物合成病毒非结构蛋白质类/遗传学

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Comparison of nucleotide sequences between northern and southern Philippine isolates of rice grassy stunt virus indicates occurrence of natural genetic reassortment

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VIROLOGY 2000年第1期266卷 26-32页

作者： Miranda, GJ Azzam, O Shirako, Y Univ Tokyo ANESC Asian Ctr Bioresources & Environm Sci Bunkyo Ku Tokyo 1138657 Japan Univ Tokyo Grad Sch Agr Life Sci Bunkyo Ku Tokyo 1138657 Japan Int Rice Res Inst Makati 1271 Philippines

Rice grassy stunt virus is a member of the genus Tenuivirus, is persistently transmitted by a brown planthopper, and has occurred in rice plants in South, Southeast, and East Asia (similar to North and South America). We determined the complete nucleotide (nt) sequences of RNAs 1 (9760 nt), 2 (4069 nt), 3 (3127 nt), 4 (2909 nt), 5 (2704 nt), and 6 (2590 nt) of a southern Philippine isolate from South Cotabato and compared them with those of a northern Philippine isolate from Laguna (Toriyama et al., 1997 1998). The numbers of nucleotides in the terminal untranslated regions and open reading frames were identical between the two isolates except for the 5' untranslated region of the complementary strand of RNA 4. Overall nucleotide differences between the two isolates were only 0.08% in RNA 1, 0.58% in RNA 4, and 0.26% in RNA 5, whereas they were 2.19% in RNA 2, 8.38% in RNA 3, and 3.63% in RNA 6. In the intergenic regions, the two isolates differed by 9.12% in RNA 2, 11.6% in RNA 3, and 6.86% in RNA 6 with multiple consecutive nucleotide deletion/insertions, whereas they differed by only 0.78% in RNA 4 and 0.34% in RNA 5. The nucleotide variation in the intergenic region of RNA 6 within the South Cotabato isolate was only 0.33%. These differences in accumulation of mutations among individual RNA segments indicate that there was genetic reassortment in the two geographical isolates;RNAs 1, 4, and 5 of the two isolates came from a common ancestor, whereas RNAs 2, 3, and 6 were from two different ancestors. (C) 2000 Academic Press.

关键词：氨基酸序列 DNA 互补/遗传学分子序列数据突变水稻/病毒学菲律宾植物病毒/遗传学植物病毒/分离和提纯 RNA病毒/遗传学 RNA病毒/分离和提纯 RNA 病毒/遗传学 RNA 病毒/分离和提纯再装配病毒/遗传学再装配病毒/分离和提纯序列比对序列分析 DNA 变异(遗传学) 病毒蛋白质类/化学病毒蛋白质类/遗传学

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Molecular analysis of two complete rice tungro bacilliform virus genomic sequences from India

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ARCHIVES OF VIROLOGY 2002年第6期147卷 1173-1187页

作者： Nath, N Mathur, S Dasgupta, I Univ Delhi Dept Plant Mol Biol New Delhi 110021 India

The complete genomic sequences of two geographically distinct isolates of rice tungro bacilliform virus (RTBV) from India were determined. Both the sequences showed equal divergence from previously reported Southeast Asian isolates. Numerous insertions, deletions and substitutions, mostly in the intergenic regions, were found. The genome sizes were 7907 and 7934 by respectively, 95 and 68 residues short of an infectious clone reported earlier. Between them, both the isolates showed high homology all along the genome, except for a 30-nucleotide insertion/deletion close to the 3' end of ORF III in one of them. Both the isolates indicated an unconventional start codon in ORF I, similar to the type isolate. In addition, as novel features, both the Indian isolates showed an unconventional start codon for ORF IV. Considering the low amounts of genome variability noticed in other RTBV isolates, the Indian isolates show that they have diverged sufficiently from the rest and should be considered belonging to a distinct strain.

关键词：氨基酸序列杆状DNA病毒属/化学杆状DNA病毒属/分类杆状DNA病毒属/遗传学克隆分子 DNA 基因间/遗传学基因组病毒印度分子序列数据开放读码框架/遗传学水稻/病毒学种系发生植物疾病/病毒学限制性内切酶图谱法序列分析 DNA

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