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ABSTRACT: Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the (BYDV) - like translational enhancer (BTE) is present in (TNV-A), a member in the family. In this paper, an RNA stretch flanking the 5' proximal end of the TNV-A coat protein (CP) gene was shown to be essential for viral replication in plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5' or 3' sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (-3 to +6 of the CP initiation site) within TNV-A RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-A, ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses.
PLoS ONE 01/2013; 8(2):e57938. · 4.09 Impact Factor
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Xiuling Cao,
Yingui Lu,
Dianping Di,
Zhiyan Zhang,
He Liu,
Lanzhi Tian,
Aihong Zhang,
Yanjing Zhang,
Lindan Shi,
Bihong Guo,
Jin Xu,
Xifei Duan,
Xianbing Wang,
Chenggui Han,
Hongqin Miao, Jialin Yu,
Dawei Li
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ABSTRACT: Maize rough dwarf disease (MRDD), caused by several Fijiviruses in the family Reoviridae, is a global disease that is responsible for substantial yield losses in maize. Although some maize germplasm have low levels of polygenic resistance to MRDD, highly resistant cultivated varieties are not available for agronomic field production in China. In this work, we have generated transgenic maize lines that constitutively express rnc70, a mutant E. coli dsRNA-specific endoribonuclease gene. Transgenic lines were propagated and screened under field conditions for 12 generations. During three years of evaluations, two transgenic lines and their progeny were challenged with Rice black-streaked dwarf virus (RBSDV), the causal agent of MRDD in China, and these plants exhibited reduced levels of disease severity. In two normal years of MRDD abundance, both lines were more resistant than non-transgenic plants. Even in the most serious MRDD year, six out of seven progeny from one line were resistant, whereas non-transgenic plants were highly susceptible. Molecular approaches in the T12 generation revealed that the rnc70 transgene was integrated and expressed stably in transgenic lines. Under artificial conditions permitting heavy virus inoculation, the T12 progeny of two highly resistant lines had a reduced incidence of MRDD and accumulation of RBSDV in infected plants. In addition, we confirmed that the RNC70 protein could bind directly to RBSDV dsRNA in vitro. Overall, our data show that RNC70-mediated resistance in transgenic maize can provide efficient protection against dsRNA virus infection.
PLoS ONE 01/2013; 8(4):e60829. · 4.09 Impact Factor
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Mi Yeon Lee,
Lijie Yan,
Florien A Gorter,
Brian Y T Kim,
Yu Cui,
Yue Hu,
Cheng Yuan,
Jessica Grindheim,
Uma Ganesan,
Zhiyong Liu,
Chenggui Han, Jialin Yu,
Dawei Li,
Andrew O Jackson
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ABSTRACT: Barley stripe mosaic virus North Dakota 18 (ND18), Beijing (BJ), Xinjiang (XJ), Type and CV21 strains are unable to infect the Brachypodium distachyon Bd3-1 inbred line, which harbors a resistance gene designated Bsr1 (Cui et al., 2012), but the Norwich (NW) strain is virulent on Bd3-1. Analysis of ND18 and NW genomic RNA reassortants, and RNAβ mutants demonstrate that two amino acids within the helicase motif of the triple gene block 1 (TGB1) movement protein have major effects on their Bd3-1 phenotypes. Resistance to ND18 correlates with an arginine residue at TGB1 position 390 (R390) and a threonine at position 392 (T392), whereas the virulent NW strain contains lysines (K) at both positions. ND18 TGB1 R390K (NDTGB1R390K) and NDTGB1T392K single substitutions, and a NDTGB1R390K,T392K double mutation resulted in systemic infections of Bd3-1. Reciprocal NDTGB1 substitutions into NWTGB1 (NWTGB1K390R and NWTGB1K392T) failed to affect virulence, implying that K390 and K392 compensate for each other. In contrast, a NWTGB1K390R,K392T double mutant exhibited limited vascular movement in Bd3-1, but developed prominent necrotic streaks that spread from secondary leaf veins. This phenotype, combined with appearance of necrotic spots in ND18 certain mutants, and necrosis and rapid wilting of Bd3-1 plants after BJ strain (BJTGB1K390,T392) inoculations, show that Bd3-1 Bsr1 resistance is elicited by the TGB1 protein and suggest that it involves a hypersensitive response.
Journal of General Virology 09/2012; · 3.36 Impact Factor
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ABSTRACT: Spontaneous point mutations of viral genomes are important in RNA virus evolution and often result in modifications of their biological properties. Spontaneous variants of Beet black scorch virus (BBSV) and its satellite RNA were generated from cDNA clones by serial propagation in Chenopodium amaranticolor and Nicotiana benthamiana. Inoculation with recombinant RNAs synthesized in vitro revealed BBSV variants with divergent infectious phenotypes that affected either symptom expression or replication of satellite RNA variants. Sequence alignments showed a correlation between the phenotypes and distinct BBSV genomic loci in the 3' UTR or in the domain encoding the viral replicase. Comparative analysis between a virulent variant BBSV-m294 and the wild type (wt) BBSV by site-directed mutagenesis indicated that a single nucleotide (nt) substitution of a uridine to a guanine at 3477 nt in the 3' UTR was responsible for significant increases in viral pathogenicity. Gain-of-function analyses demonstrated that the ability of the BBSV variants to support replication of variant satRNAs was mainly determined by amino acid 516 in the P82 replicase. In this case, an arginine substitution for a glutamine residue was essential for high levels of replication, and alterations of other residues surrounding position 516 in the wtBBSV isolate led to only minor phenotypic effects. These results provide evidence that divergence of virus functions affecting pathogenicity and supporting parasitic replication can be determined by a single genetic site, either a nucleotide or an amino acid. The results suggest complex interactions occur between virus and associated satRNAs during virus evolution.
Journal of General Virology 09/2012; · 3.36 Impact Factor
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ABSTRACT: Beet black scorch virus (BBSV) encodes three movement proteins (P7a, P7b and P5') that facilitate its cell-to-cell movement. An arginine-rich motif of P7a N-terminus was found to determine nuclear and nucleolar localization. Amino acids substitution or deletion of the R-rich motif interfered with P7a nuclear and nucleolar localization. Bimolecular fluorescence complementation (BiFC) assays revealed that P7a protein interacted with Nicotiana benthamiana nuclear import factor importin α, suggesting that P7a is translocated into the nucleus by the classical importin α/β-dependent pathway. Moreover, P7a also interacted with the nucleolar protein fibrillarin. Mutations in the R-rich motif of P7a diminished P7a interactions with importin α and fibrillarin, influenced viral replication in Nicotiana benthamiana protoplasts and altered the symptom phenotype and viral RNA accumulation in Chenopodium amaranticolor plants. These results demonstrate that the R-rich motif of P7a is correlated with nuclear and nucleolar localization, viral replication and virus infection.
Virus Research 05/2012; 167(2):207-18. · 2.94 Impact Factor
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ABSTRACT: Full-length RNA4 of beet necrotic yellow vein virus (BNYVV) and its mutants, including a frame-shift and deletions within
the coding region, were inserted into a transcription plasmid. Transcripts derived from these plasmids and RNAs extracted
from a BNYVV isolate containing only RNAs 1, 2 and 3 were coinoculated to Tetragona expansa. Then these recombinant isolates were inoculated to sugar beet plants grown in a sand culture system containing Polymyxa betae that had been freed of virus particles to test efficiency of BNYVV transmission. The result showed that the efficiency of
BNYVV transmission among the plants by Polymyxa betae was highly decreased by different deletions in the coding region of RNA4, but not affected by the frame-shift mutant with
four nucleotides insertion. The observations suggest that at least some of the RNA4 sequence encompassing the deleted region
of 580 nucleotide acids (525 –1105 nt) are necessary for high efficient transmission of the virus by the fungus.
Chinese Science Bulletin 04/2012; 47(15):1281-1284. · 1.32 Impact Factor
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ABSTRACT: The transcripts of beet necrotic yellow virus RNA5 were prepared from the cloned cDNA in a bacteriophage T7in vitro run-off transcription system. The RNA5 transcripts and RNAs extracted from two BNYVV mutants, HuO containing RNA1, 2 and
Hu3 containing RNA1, 2, 3 respectively, were co-inoculated toTetragonia expansa and sugar beet seedlings. The results indicated that the virus infections and accumulation in the hosts could be improved
by presence of RNA5. Cooperating with RNA3, the RNA5 seems to cause more severe symptom and yield losses in sugar beet. These
results suggest that, in addition of RNA3, BNYVV RNA5 is another important factor associated with the virus pathogenicity.
KeywordsBNYVV RNA5-
in vitro transcription-pathogenicity
Chinese Science Bulletin 04/2012; 48(8):796-799. · 1.32 Impact Factor
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ABSTRACT: Functional analysis for gene silencing suppressor of P14 geneof Beet necrotic yellow vein virus and S6 gene ofRice black streak dwarf virus was carried out by agro-infiltration with recombinant vectors ofPotato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing
ofgfp transgenicNicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the
suppressed plants, thegfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more
severe, especially thegfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested
that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.
Chinese Science Bulletin 04/2012; 50(4):305-310. · 1.32 Impact Factor
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Yu Cui,
Mi Yeon Lee,
Naxin Huo,
Jennifer Bragg,
Lijie Yan,
Cheng Yuan,
Cui Li,
Sara J Holditch,
Jingzhong Xie,
Ming-Cheng Luo,
Dawei Li, Jialin Yu,
Joel Martin,
Wendy Schackwitz,
Yong Qiang Gu,
John P Vogel,
Andrew O Jackson,
Zhiyong Liu,
David F Garvin
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ABSTRACT: The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25 °C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F(6:7) recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F(2) population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.
PLoS ONE 01/2012; 7(6):e38333. · 4.09 Impact Factor
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ABSTRACT: Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.
PLoS ONE 01/2012; 7(9):e46451. · 4.09 Impact Factor
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ABSTRACT: Beet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far.
Systemic host N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3β and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom.
Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.
Virology Journal 07/2011; 8:335. · 2.34 Impact Factor
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ABSTRACT: Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, can infect several graminaceous plant species including rice, maize and wheat, and is transmitted by planthoppers. Although several RBSDV proteins have been studied in detail, functions of the nonstructural protein P6 are still largely unknown.
In the current study, we employed yeast two-hybrid assays, bimolecular fluorescence complementation and subcellular localization experiments to show that P6 can self-interact to form punctate, cytoplasmic viroplasm-like structures (VLS) when expressed alone in plant cells. The region from residues 395 to 659 is necessary for P6 self-interaction, whereas two polypeptides (residues 580-620 and 615-655) are involved in the subcellular localization of P6. Furthermore, P6 strongly interacts with the viroplasm-associated protein P9-1 and recruits P9-1 to localize in VLS. The P6 395-659 region is also important for the P6-P9-1 interaction, and deleting any region of P9-1 abolishes this heterologous interaction.
RBSDV P6 protein has an intrinsic ability to self-interact and forms VLS without other RBSDV proteins or RNAs. P6 recruits P9-1 to VLS by direct protein-protein interaction. This is the first report on the functionality of RBSDV P6 protein. P6 may be involved in the process of viroplasm nucleation and virus morphogenesis.
Virology Journal 01/2011; 8:24. · 2.34 Impact Factor
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ABSTRACT: Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.
PLoS ONE 01/2011; 6(10):e26468. · 4.09 Impact Factor
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ABSTRACT: Beet black scorch virus (BBSV) is a positive-sense, single-stranded RNA virus belonging to Necrovirus genus. In order to better understand the life cycle of BBSV, we have investigated the subcellular localization of BBSV capsid protein (CP) by its fusion with green fluorescent protein (GFP) agroinfiltrated into Nicotiana benthamiana leaves and by particle bombardment into onion (Allium cepa) epidermal cells. Confocal laser scanning microscopy (CLSM) showed that BBSV CP fused to GFP displayed enhanced fluorescence in nuclei and nuclear import of the CP was confirmed in BBSV-infected N. benthamiana leaves. Mutational analysis revealed that the N-terminal basic amino acid cluster (4)KRNKGGKKSR(13) of the CP is essential for nuclear localization. Bimolecular fluorescence complementation (BiFC) assays indicated that the CP could interact with the nuclear import factor importin α, suggesting that the CP is possibly imported into the nucleus via an importin α-dependent pathway. This is the first report of the nuclear localization of the CP encoded by a necrovirus.
Virus Research 11/2010; 155(1):307-15. · 2.94 Impact Factor
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ABSTRACT: Plant viruses show significant potential as expression vectors for producing foreign proteins in plants. In this study, codon-optimized VP6 gene (sVP6) of human rotavirus was engineered as a replacement to the coat protein (CP) open reading frame of Beet black scorch virus (BBSV). In vitro-generated RNA transcripts corresponding to the engineered virus were infectious when inoculated onto the leaves of Chenopodium amaranticolor. Molecular analysis revealed that sVP6 was efficiently expressed and accounted for 0.25% of the total soluble protein (TSP) in plant leaves on 7 dpi. On average, a high level 1.54 microg of sVP6 was expressed in each gram of infected leaves. Oral immunization of female BALB/c mice with the plant-based sVP6 protein induced high titers of anti-VP6 mucosal IgA and serum IgG. 60% of suckling pups born from immunized dams were protected against the virulent rotavirus challenge and those infected pups developed less severe diarrhea. These results suggested that it is feasible to induce lactogenic immunity against an enteric pathogen through oral vaccination, by using the antigen produced in a new BBSV-based plant protein expression system.
Vaccine 08/2010; 28(37):6021-7. · 3.77 Impact Factor
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ABSTRACT: Plant virus-based expression systems provide attractive alternatives for production of animal virus-originated antigenic peptides. In the present study, an infectious cDNA clone of Tobacco necrosis virus A Chinese isolate (TNV-A(C)) was used for expression of different peptides derived from Foot and mouth disease virus (FMDV) serotype O VP1 fused downstream of the coat protein (CP) open reading frame (ORF). Chenopodium amaranticolor inoculated with in vitro transcripts of the chimaeras developed symptoms similar to those caused by wild-type TNV-A(C). Western blot and RT-PCR detection of the infected leaves demonstrated that the chimaeras were infective, and a large number of self-assembled virions could be purified and observed under electron microscopy. Immunogold labelling revealed that highly expressed FMDV VP1 peptides could be displayed on the surfaces of virus particles. Additional immunoblotting and DNA sequence analyses showed that most of the chimaeras contained unmodified foreign peptides even after six successive passages in C. amaranticolor and three passages in Nicotiana benthamiana. Our results also suggest that the amino acid sequence and peptide length have a substantial influence on viral morphogenesis and systemic infections. Finally, animal experiments showed that purified chimaeric virus particles (CVPs) could induce a strong immune response against FMDV structural protein VP1 via an intramuscular route. And when inoculated nasally, CVPs could induce systemic and mucosal immune responses in mice.
Plant Biotechnology Journal 03/2010; 8(4):506-23. · 5.44 Impact Factor
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ABSTRACT: In order to suppress RNA silencing, many plant and some animal viruses encode RNA silencing suppressors to achieve infection. In this study, we report that B3 and B4, encoded by DNA3 and DNA4 of banana bunchy top virus (BBTV), exhibit RNA silencing suppression activity. B3 and B4 were able to increase the transient expression of green fluorescent protein (GFP) and dramatically enhanced the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana. B4 was able to reverse established gene silencing on an inoculated leaf or on an upper leaf. B3, however, was only active during infection of an inoculated leaf. Furthermore, B4, but not B3, was able to enhance GFP expression in the transgenic N. benthamiana line 16c. In conclusion, B3 and B4 are the RNA silencing suppressors of BBTV, and they may act at different steps in the RNA silencing pathways.
Archives of Virology 10/2009; 154(11):1775-83. · 2.11 Impact Factor
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ABSTRACT: Historical reports indicate that Cucumber mosaic virus (CMV) strain subgroup I is more prevalent than subgroup II in most parts of the world, but recent reports suggest that subgroup II isolates may be far more abundant than previously found in China. In order to evaluate the dominance of CMV subgroup I and subgroup II strains in co-infected tobacco plants, four isolates, NX and YQ in subgroup I, and ZL and AG in subgroup II, were tested in competition experiments. In these comparisons, the frequency of infection was assessed, and ratios between singly and doubly infected plants were calculated based on ELISA tests of tobacco leaves. In contrast to previous reports suggesting that subgroup I strains are usually more competitive than subgroup II strains in the field, the results from the present study indicate that the subgroup II ZL isolate was more competitive than the subgroup I YQ isolate, even though the ZL isolate caused milder symptoms than YQ in singly infected tobacco. In contrast, the subgroup I strains NX and YQ were more competitive than subgroup II AG. This information provides evidence for variation in the competitive abilities of subgroup II strains in tests with subgroup I strains, and suggests that direct competition during mixed infections may account in part for the recent spread of some subgroup II strains in China and elsewhere.
Journal of Phytopathology 03/2009; 157(7‐8):457 - 464. · 0.79 Impact Factor
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ABSTRACT: Lily mottle virus (LMoV), Potyvirus genus, is very difficult to purify for the preparation of diagnostic antisera. The coat protein (CP) gene of LMoV was amplified by RT-PCR from infected plants and cloned into the prokaryotic pET-30a vector to generate the recombinant plasmid pET-CP. The resulting carboxy-terminal His-tagged CP was over-expressed in Escherichia coli BL 21 cells by Isopropyl-β-d-thiogalactoside (IPTG) induction and purified over Ni-NTA affinity columns. The purified CP was used to elicit a polyclonal antiserum in rabbits. The antiserum had a titre of 1 : 128 in double diffusion tests, and specifically recognized LMoV in Western blots, Enzyme-Linked Immunosorbent Assays and Immuno-Electron Microscopy. The CP antiserum was also used to evaluate LMoV infection of lily bulbs used for commercial production in Yunnan, China. Substantial levels of infection were found in both imported and native bulbs, and provide the basis to implement flexible, rapid and large-scale virus indexing of lily plants for use in propagation and to meet virus-free quarantine regulations.
Journal of Phytopathology 12/2008; 157(6):362 - 369. · 0.79 Impact Factor
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ABSTRACT: Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.
Journal of Virology 06/2008; 82(10):4991-5006. · 5.40 Impact Factor
