Positive and negative sense coat protein gene-mediated protection against poplar mosaic carlavirus in Nicotiana benthamiana

Plants of Nicotiana benthamiana were transformed with four constructs based on the coat protein gene of a poplar mosaic carlavirus (PMV) isolate from the UK. The four constructs were: the capsid protein coding sequence plus a portion of the adjacent sequence encoding a protein with a molecular mass of 14 kDa (CP14k); the capsid protein coding sequence in the positive sense (CPP); a mutated capsid protein coding sequence (CPM) and the capsid protein coding sequence in the negative sense (CPN). Forty-one regenerated plants, after selection for their kanamycin resistance, were confirmed by PCR to contain the appropriate sequences. Virus coat protein was detected in small amounts in 50% of the plants transformed with the CP14k or CPP constructs. Primary transformants showed a range of reactions to challenge with two isolates of PMV. These varied from apparently no infection in inoculated or in later-formed young leaves, as assessed by ELISA, to typical systemic symptoms associated with large amounts of serologically detected virus. There was no correlation between the level of protection against virus infection and the observed accumulation of transgene protein product. Plants were protected whether transformed with the coat protein coding sequence in the positive or negative sense.     

Identification and Genomic Variability of Prune dwarf virus Variants Infecting Stone Fruit Trees in Turkey

Prune dwarf virus (PDV), a pathogen of stone fruit trees worldwide, shows a great variability in its biological, serological and molecular properties. The coat protein sequences of ten PDV variants from different stone fruit tree species in Turkey were determined. The sequence for each variant was 657 nucleotides in length. The phylogenetic analyses of the sequences of the Turkish variants and of additional sequences of other PDV variants from international databases indicate the existence of four groups of PDV variants: one contains cherry variants that differs in group specific conserved amino acids; a second contains one apricot and cherry variants, most members of the group being Turkish variants; a third contains mixed variants from cherry, peach, plum trees and an almond tree; and a fourth contains only variants from almond trees. Based on their hosts, we propose the nomenclature of these groups as cherry I, cherry II, mixed and almond groups, respectively.     

基于烟草脆裂病毒构建同时表达2种外源蛋白的载体及其应用

目的 构建在整个寄主植物中能同时表达两个非融合蛋白的病毒载体。方法 以烟草脆裂病毒（tobacco rattle virus，TRV）基因组RNA2的农杆菌侵染性克隆pYL156为材料，缺失TRV RNA2的2b基因5"端279 bp并将其起始密码子ATG改变为AGG、同时引入豌豆早枯病毒（pea early-browning virus，PEBV）外壳蛋白（coat protein，cp）基因启动子，获得pTRV2e²载体；在pTRV2e²载体的2b和PEBV的cp启动子下游插入不同的外源基因，测定病毒TRVe²表达外源蛋白的能力、携带外源基因后重组病毒的稳定性以及分析蛋白质的生物学功能。结果 病毒TRVe²能快速同时表达2个非融合的外源蛋白，且至少能表达70 ku外源蛋白，且该病毒携带~2.0 kb外源基因能稳定存活于寄主植物中；病毒TRVe²可用于分析蛋白质的生物学功能以及2个蛋白质间的相互作用。结论 本文构建的重组病毒TRVe²为快速有效地表达2个外源蛋白以及分析2个蛋白质间的相互作用提供技术工具。     

Production of the main surface antigen of Toxoplasma gondii in tobacco leaves and analysis of its antigenicity and immunogenicity

We adapted a previously described Agrobacterium-mediated transient expression system to test the expression level of three constructs carrying the surface antigen 1 (SAG1) of Toxoplasma gondii. Two constructs were based in a Potato virus X (PVX) amplicon. In one of them, the PVX movement protein genes were replaced by the sag1 gene. In the other, the sag1 gene was placed under the control of an additional coat protein subgenomic promoter. In the third construct, the sag1 gene was fused to an apoplastic peptide signal under the CaMV 35S promoter. Western blot analysis of leaf extracts infiltrated with each construct revealed a protein of 35 kDa. SAG1 accumulation in leaves ranged from 0.1 to 0.06% of total soluble protein (equivalent to 10 μg and 6 μg of SAG1 per gram of fresh leaf tissue, respectively). Three of five human seropositive samples reacted with tobacco-expressed SAG1 in Western blot analysis. The C3H mice were immunized with SAG-expressing leaf extracts and perorally challenged with a nonlethal dose of the T. gondii Me49 strain. Mice vaccinated with SAG1 showed significantly lower brain cyst burdens compared to those from the control group. Immunization with SAG1-expressing leaves elicited a specific humoral response with predominant participation of type IgG2a. In conclusion, a functional SAG1 version could be transiently expressed in tobacco leaves.     

Purification and properties of blackberry chlorotic ringspot, a new virus species in subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK

A mechanically transmissible virus was isolated from Bedford Giant blackberry plants showing chlorotic mottling and ringspot symptoms growing in Scotland. It infected several herbaceous test plants, many of them symptomlessly. This virus was also transmitted to several Rubus species and cultivars by graft inoculation with scions from the field‐infected Bedford Giant plant. Most grafted plants were infected symptomlessly, but Himalaya Giant blackberry and the hybrid berry Tayberry developed symptoms similar to those in the infected Bedford Giant plant. In the sap of infected Chenopodium quinoa, the virus lost infectivity when diluted 10^?4 but not 10^?3, after 6 h and 48 h when kept at 20°C and 4°C, respectively, but was infective for more than 8 days when kept at ?15°C. Preparations of purified virus from infected C. quinoa or spinach sedimented as three major nucleoprotein components and consisted of quasi‐isometric particles that varied in size from 24 to 32 nm in diameter and that were not penetrated by negative stain. Such virus particle preparations contained a major polypeptide of ca 28 kDa and three single‐stranded RNA species of estimated size 3.2, 2.8 and 2.1 kb. The complete sequence of the largest RNA (RNA 1, 3478 nt) and the partial sequence of the other RNAs (1863 and 2102 nt long, respectively) were determined and compared with sequences in databases. These findings, together with the biological and biochemical properties of this virus, indicate that it should be regarded as a distinct species in subgroup 1 of the genus Ilarvirus even though it was serologically unrelated to existing members of this subgroup. The virus showed a very distant serological relationship with prune dwarf virus (PDV) but differed significantly from it in the amino acid sequence of its coat protein, experimental host range and symptomatology and was unrelated to PDV at the molecular level. The virus, tentatively named blackberry chlorotic ringspot virus, is therefore a newly described virus and the first ilarvirus found naturally infecting Rubus in the UK.     

Regulation of the xylanase gene, cgxA, from Chaetomium gracile by transcriptional factors, XlnR and AnRP

The roles of XlnR and AnRP in regulating the expression of the xylanase gene, cgxA, from Chaetomium gracile were investigated using Aspergillus nidulansas an intermediate host. The XlnR consensus binding sequence –GGCTAA– in the promoter region was functional in vivo. The cgxA gene was induced when xylan was used as a carbon source but this inducibility was abolished when the XlnR binding sequence was mutated. Furthermore, the induction by xylan was increased when the AnRP binding sequence –TTGACAAAT– was mutated. Electrophoretic mobility shift assays using partially purified AnRP and an Aspergillus oryzae XlnR fusion protein, MalE-AoXlnR, provided evidence that the binding of the two proteins to their respective sites in the cgxA promoter region was mutually exclusive.     

Nucleotide sequence analysis of a cDNA clone encoding the 34K movement protein gene of odontoglossum ringspot virus,ORSV-Cy,the Korean isolate

The partial nucleotide sequence of the 3-terminal region of the Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) from cool-growing Cymbidium was determined. The sequence contained a full length open reading frame (ORF) coding for the viral cell-to-cell movement protein (MP). The ORF was located upstream of the coat protein gene and 105 nucleotides longer than that of tobacco mosaic virus (TMV). The ORF predicts a polypeptide chain of 303 amino acids with a molecular weight of 33573. The ORF contained a similar region of conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA was located at about 1,100 nucleotides away from the 3 end. The predicted amino acid sequence for the MP gene of ORSV-Cy is more closely related to pepper mild mottle virus (PMMV), TMV-vulgare and TMV-Rakkyo than to tobacco mild green mosaic virus (TMGMV), TMV-L, cowpea strain of TMV (SHMV), and cucumber green mottle mosaic virus (CGMMV).     

Nucleotide sequence of beet western yellows virus RNA.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).     

Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA 2.

The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.     

Pteromalus puparum venom impairs host cellular immune responses by decreasing expression of its scavenger receptor gene

Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Although there is a rich literature on these systems, parasitoid immune-disabling mechanisms have not been fully elucidated. Here we report on a newly discovered immune-disabling mechanism in the Pieris rapae/Pteromalus puparum host/parasitoid system. Because venom injections and parasitization suppresses host phagocytosis, we turned attention to the P. rapae scavenger receptor (Pr-SR), posing the hypothesis that P. puparum venom suppresses expression of the host Pr-SR gene. To test our hypothesis, we cloned a full-length cDNA of the Pr-SR. Multiple sequences alignment showed the deduced amino acid sequence of Pr-SR is similar to scavenger receptors of other lepidopterans. Bacterial and bead injections induced Pr-SR mRNA and protein expression, which peaked at 4 h post-bead injection. Venom injection inhibited Pr-SR expression. Pr-SR was specifically expressed in granulocytes compared to plasmatocytes. We localized the Pr-SR protein in cytoplasm and cellular membrane, with no evidence of secretion into host plasma. Double-strand RNA designed to Pr-SR mRNA silenced expression of Pr-SR and significantly impaired host phagocytosis and encapsulation reactions. Venom injections similarly silenced Pr-SR expression during the first 8 h post-treatment, after which the silencing effects gradually abated. We infer from these findings that one mechanism of impairing P. rapae hemocytic immune reactions is by silencing expression of Pr-SR.     

Interactions between the tomato spotted wilt virus movement protein and plant proteins showing homologies to myosin, kinesin and DnaJ-like chaperones

The non-structural protein encoded by the M RNA segment (NSm) of tomato spotted wilt virus (TSWV) has been implicated in cell-to-cell movement of nucleocapsids through modified plasmodesmata. Recently, DnaJ-like proteins from Nicotiana tabacum (tobacco) and Arabidopsis thaliana have been identified as NSm interacting host proteins, implying an involvement of molecular chaperones during systemic spread of the virus or other, presently unknown NSm-mediated virus functions. Examination of additional TSWV host plants and improvement of yeast two-hybrid interaction trap experiments led to the isolation of a DnaJ-like protein from Lycopersicon esculentum (tomato) and the identification of a protein from A. thaliana sharing some homologies with myosin and kinesin-like polypeptides. Sequence alignments of the tomato DnaJ-like protein unveiled the corresponding gene as an orthologue to the tobacco and A. thaliana DnaJ genes, substantiating that NSm interacting DnaJ-like polypeptides, identified from three different TSWV host species, apparently form a subgroup distinct from archetypical DnaJ chaperones. Increased levels of DnaJ-like proteins could be detected in TSWV systemically infected leaves and in plants exposed to heat shock, showing that the NSm interacting DnaJ-like chaperones are inducible upon biotic and abiotic stress. All together, the identification of DnaJ-like proteins and a protein resembling myosin and kinesin as NSm interacting plant proteins is in accordance with results accomplished for movement proteins from other plant attacking viruses showing an involvement of molecular chaperones and the cytoskeleton in at least intracellular trafficking.     

Complete Nucleotide Sequence of the RNA 3 of Bacopa chlorosis virus and Production of Polyclonal Antibodies to a Recombinant Coat Protein

The complete sequence of the RNA 3 of a virus causing chlorosis in Impatiens in Germany was determined and identified as an isolate of Bacopa chlorosis virus (BaCV, genus Ilarvirus). BaCV has previously only been reported from bacopa in the USA, but no coat protein (CP) sequence has been previously available. Both RNA 3 encoded proteins, CP and movement protein, showed highest sequence identity to Parietaria mottle virus, a subgroup 1 ilarvirus. Attempts to purify BaCV failed, so an antiserum was raised against a recombinant CP. The polyclonal antiserum so produced allowed specific detection of BaCV but showed no serological cross‐reaction with other ilarviruses and was unsuitable for immunoelectron microscopy. The host range includes many important flowering plant species, highlighting the potential threat BaCV might pose for the horticultural industry. This is the first report of BaCV occurring in Germany and outside the US.     

Determination of the complete amino acid sequence for the coat protein of brome mosaic virus by time-of-flight mass spectrometry. Evidence for mutations associated with change of propagation host

Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the "type" isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.     

Special origin of stem sequence influence the resistance of hairpin expressing plants against PVY

In this study, 16 hairpin RNA (hpRNA) vectors were constructed, each harboring 50 bp viral RNA sequence as the stem. They all targeted the coat protein (CP) gene of Potato virus Y (PVY). Virus resistance assay revealed that hairpin constructs targeting the anterior 200 bp regions of the CP gene were unable to induce virus resistance, while the 12 hpRNA constructs targeting posterior 600 bp regions induced high virus resistance up to 77.78 %. Northern blot analysis revealed that 50 bp-length hpRNA constructs could be transcribed efficiently and processed into siRNAs; however, no correlation between siRNA accumulation and degree of antiviral defense was observed. Results presented here indicated that the middle and 3′ end of the CP cDNA was important for hpRNA-mediated PVY resistance, improving the design of pathogen-derived hpRNA expression cassettes for transgenic plant against viruses.     

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium

Summary The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5 end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.