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molecular cloning and characterization of wheat calreticulin (crt) gene involved in drought-stressed responses.calreticulin (crt) is a highly conserved and ubiquitously expressed ca(2+)-binding protein in multicellular eukaryotes. crt plays a crucial role in many cellular processes including ca(2+) storage and release, protein synthesis, and molecular chaperone activity. to elucidate the function of crts in plant responses against drought, a main abiotic stress limiting cereal crop production worldwide, a full-length cdna encoding calreticulin protein namely tacrt was isolated from wheat (triticum aestiv ...200818349049
the f-box protein acre189/acif1 regulates cell death and defense responses activated during pathogen recognition in tobacco and tomato.virus-induced gene silencing identified the avr9/cf-9 rapidly elicited gene acre189 as essential for the cf-9- and cf-4-mediated hypersensitive response (hr) in nicotiana benthamiana. we report a role for acre189 in disease resistance in tomato (solanum lycopersicum) and tobacco (nicotiana tabacum). acre189 (herein renamed avr9/cf-9-induced f-box1 [acif1]) encodes an f-box protein with a leu-rich-repeat domain. acif1 is widely conserved and is closely related to f-box proteins regulating plant h ...200818375657
conserved c-terminal motifs required for avirulence and suppression of cell death by phytophthora sojae effector avr1b.the sequenced genomes of oomycete plant pathogens contain large superfamilies of effector proteins containing the protein translocation motif rxlr-deer. however, the contributions of these effectors to pathogenicity remain poorly understood. here, we show that the phytophthora sojae effector protein avr1b can contribute positively to virulence and can suppress programmed cell death (pcd) triggered by the mouse bax protein in yeast, soybean (glycine max), and nicotiana benthamiana cells. we ident ...200818390593
regulatory network of microrna399 and pho2 by systemic signaling.recently, we showed that microrna399s (mir399s) control inorganic phosphate (pi) homeostasis by regulating the expression of pho2 encoding a ubiquitin-conjugating e2 enzyme 24. arabidopsis (arabidopsis thaliana) plants overexpressing mir399 or the pho2 mutant overaccumulate pi in shoots. the association of pi translocation and coexpression of mir399s and pho2 in vascular tissues suggests their involvement in long-distance signaling. in this study, we used reciprocal grafting between wild-type an ...200818390805
the arabidopsis class viii myosin atm2 is involved in endocytosis.members of the class xi of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and golgi vesicle trafficking comparable to yeast and animal class v myosins. the tasks of the second class of myosins of higher plants, class viii, are unclear. in this study the class viii myosin atm2 from the model plant arabidopsis thaliana was selected for the examination of cargo specificity in vivo. fluorescent protein-fusion plasmid construc ...200818393384
role of a geminivirus av2 protein putative protein kinase c motif on subcellular localization and pathogenicity.virus-derived genes or genome fragments are increasingly being used to generate transgenic plants with resistance to plant viruses. there is need to rapidly investigate these genes in plants using transient expression prior to using them as transgenes since they may be pathogenic to plants. in this study, we investigated the av2 protein encoded by east african cassava mosaic cameroon virus, a virus associated with a cassava disease epidemic in western africa. for subcellular localization, av2 wa ...200818405995
wolman disease/cholesteryl ester storage disease: efficacy of plant-produced human lysosomal acid lipase in mice.lysosomal acid lipase (lal) is an essential enzyme that hydrolyzes triglycerides (tgs) and cholesteryl esters (ces) in lysosomes. genetic lal mutations lead to wolman disease (wd) and cholesteryl ester storage disease (cesd). an lal-null (lal(-/-)) mouse model resembles human wd/cesd with storage of ces and tgs in multiple organs. human lal (hlal) was expressed in nicotiana benthamiana using the geneware expression system (g-hlal). purified g-hlal showed mannose receptor-dependent uptake into ma ...200818413899
functional analysis of bipartite begomovirus coat protein promoter sequences.we demonstrate that the al2 gene of cabbage leaf curl virus (calcuv) activates the cp promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for tomato golden mosaic virus (tgmv). binding studies indicate that sequences mediating repression and activation of the tgmv and calcuv cp promoter specifically bind different nuclear factors common to nicotiana benthamiana, spinach and tomato. however, chromatin immunoprecipitation demonstrates that tgmv al2 ...200818430450
multicolor fluorescence imaging of leaves--a useful tool for visualizing systemic viral infections in plants.multicolor fluorescence induced by uv light is a sensitive and specific tool that may be used to provide information about the primary and secondary metabolism of plants by monitoring signals of the chlorophyll fluorescence (chl-f) and blue-green fluorescence (bgf), respectively. we have followed the systemic infection of nicotiana benthamiana plants with the pepper mild mottle virus (pmmov) by means of a multicolor fluorescence-imaging system, to detect differences between two strains of pmmov ...200818435702
quantifying fungal infection of plant leaves by digital image analysis using scion image software.a digital image analysis method previously used to evaluate leaf color changes due to nutritional changes was modified to measure the severity of several foliar fungal diseases. images captured with a flatbed scanner or digital camera were analyzed with a freely available software package, scion image, to measure changes in leaf color caused by fungal sporulation or tissue damage. high correlations were observed between the percent diseased leaf area estimated by scion image analysis and the per ...200818466990
effects of viral silencing suppressors on tobacco ringspot virus infection in two nicotiana species.this study investigated the effects of silencing suppressors derived from six different viruses (p1, p19, p25, hcpro, ac2 and 2b), expressed in transgenic nicotiana tabacum and nicotiana benthamiana plants, on the infection pattern of tobacco ringspot virus (trsv) potato calico strain. in n. benthamiana, this virus produced an initial infection with severe systemic symptoms, but the infection was strongly reduced within a few weeks as the plant recovered from the infection. p25 and hcpro silenci ...200818474567
generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus 3.pathogen-specific recombinant antibodies have been used to characterize pathogen infections and to engineer resistance in crops. we selected a single-chain antibody fragment (scfvlr3cp-1) specific for the coat protein of grapevine leafroll-associated virus 3 (glrav-3), one of the agents of grapevine leafroll (glr) disease, from a phage display library. the antibody binds specifically to the entire length of glrav-3 particles and has a high binding affinity value (k(d)) of 42 nm. the amino acid m ...200818478181
symptomless infectious cdna clone of a norwegian isolate of poinsettia mosaic virus.an infectious cdna clone of a norwegian isolate of poinsettia mosaic virus (pnmv) was generated. it consisted of 6,098 nucleotides and encoded a polyprotein of 219.5 kda. sequence comparisons indicated that this isolate shared 98.6% (nucleotide) and 97.1% (amino acid) identity with the previously sequenced isolate from germany. rna transcripts derived from this cdna were infectious in nicotiana benthamiana. however, plants did not present typical pnmv symptoms. furthermore, rna transcripts from ...200818504521
three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths.three heterologous proteins were simultaneously expressed from a chimeric potyvirus potato virus a (pva) in nicotiana benthamiana. the genes for green fluorescent protein of aequoria victoriae ("g"; 714 nucleotides, nt), luciferase of renilla reniformis ("l", 933 nt) and beta-glucuronidase of escherichia coli ("u", 1806 nt) were inserted into the engineered cloning sites at the n-terminus of the p1 domain, the junction of p1 and helper component protein (hc-pro), and the junction of the viral re ...200818511144
physiological function of nbranbp1 in nicotiana benthamiana.this study addresses the physiological functions of the ran-binding protein homolog nbranbp1 in nicotiana benthamiana. virus-induced gene silencing (vigs) of nbranbp1 caused stunted growth, leaf yellowing, and abnormal leaf morphology. the nbranbp1 gene was constitutively expressed in diverse tissues and an nbranbp1:gfp fusion protein was primarily localized to the nuclear rim and the cytosol. bifc analysis revealed in vivo interaction between nbranbp1 and nbran1 in the nuclear envelope and the ...200818511878
increased organophosphate scavenging in a butyrylcholinesterase mutant.nicotiana benthamiana plant lines expressing a reengineered human butyrylcholinesterase (bche) with enhanced cocaine hydrolase activity were created. subsequent purification and biochemical analysis revealed that compared to wild-type butyrylcholinesterase, the cocaine hydrolase displayed increased affinity to the organophosphate (op) pesticides paraoxon (6.8 4x 10(-10)m vs. 1.11 x 10(-8)m) and malaoxon (9.81 x 10(-8)m vs. 5.99 x 10(-7)m). furthermore, the cocaine hydrolase retained identical an ...200818514178
mapk signaling regulates nitric oxide and nadph oxidase-dependent oxidative bursts in nicotiana benthamiana.nitric oxide (no) and reactive oxygen species (ros) act as signals in innate immunity in plants. the radical burst is induced by inf1 elicitin, produced by the oomycete pathogen phytophthora infestans. no associated1 (noa1) and nadph oxidase participate in the radical burst. here, we show that mitogen-activated protein kinase (mapk) cascades mek2-sipk/ntf4 and mek1-ntf6 participate in the regulation of the radical burst. no generation was induced by conditional activation of sipk/ntf4, but not b ...200818515503
influence of viral genes on the cell-to-cell spread of rna silencing.the turnip crinkle virus-based vector tcv-gfp delta cp had been devised previously to study cell-to-cell and long-distance spread of virus-induced rna silencing. tcv-gfp delta cp, which had been constructed by replacing the coat protein (cp) gene with a green fluorescent protein (gfp) coding sequence, was able to induce rna silencing in single epidermal cells, from which rna silencing spread from cell-to-cell. using this unique local silencing assay together with mutagenesis analysis, two tcv ge ...200818515824
agrobacterium tumefaciens-induced bacteraemia does not lead to reporter gene expression in mouse organs.agrobacterium tumefaciens is the main plant biotechnology gene transfer tool with host range which can be extended to non-plant eukaryotic organisms under laboratory conditions. known medical cases of agrobacterium species isolation from bloodstream infections necessitate the assessment of biosafety-related risks of a. tumefaciens encounters with mammalian organisms. here, we studied the survival of a. tumefaciens in bloodstream of mice injected with bacterial cultures. bacterial titers of 10(8) ...200818523638
a versatile assay for the identification of rna silencing suppressors based on complementation of viral movement.the cell-to-cell movement of turnip crinkle virus (tcv) in nicotiana benthamiana requires the presence of its coat protein (cp), a known suppressor of rna silencing. rna transcripts of a tcv construct containing a reporter gene (green fluorescent protein) (tcv-sgfp) in place of the cp open reading frame generated foci of three to five cells. tcv cp delivered in trans by agrobacterium tumefaciens infiltration potentiated movement of tcv-sgfp and increased foci diameter, on average, by a factor of ...200818533829
coat protein-mediated resistance against an indian isolate of the cucumber mosaic virus subgroup ib in nicotiana benthamiana.coat protein (cp) -mediated resistance against an indian isolate of the cucumber mosaic virus (cmv) subgroup ib was demonstrated in transgenic lines of nicotiana benthamiana through agrobacterium tumefaciens-mediated transformation. out of the fourteen independently transformed lines developed, two lines were tested for resistance against cmv by challenge inoculations. the transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumula ...200818535359
in vitro replication of bamboo mosaic virus satellite rna.an in vitro system was applied to analyze the replication of a satellite rna of bamboo mosaic virus (bamv), designated satbamv rna, using solubilized membrane-bound rna-dependent rna polymerase (rdrp) complexes isolated from bamv-infected nicotiana benthamiana. after removal of endogenous templates, the rdrp complexes of bamv catalyzed rna synthesis upon the addition of the full-length positive (+)- or negative (-)-strand satbamv rna transcripts used as templates. both (+)- and (-)-satbamv rna p ...200818538884
inhibition of 3' modification of small rnas in virus-infected plants require spatial and temporal co-expression of small rnas and viral silencing-suppressor proteins.plant viruses are inducers and targets of rna silencing. viruses counteract with rna silencing by expressing silencing-suppressor proteins. many of the identified proteins bind sirnas, which prevents assembly of silencing effector complexes, and also interfere with their 3' methylation, which protects them against degradation. here, we investigated the 3' modification of silencing-related small rnas in nicotiana benthamiana plants infected with viruses expressing rna silencing suppressors, the p ...200818539609
transreplication of a tomato yellow leaf curl thailand virus dna-b and replication of a dnabeta component by tomato leaf curl vietnam virus and tomato yellow leaf curl vietnam virus.the genomes of two tomato-infecting begomoviruses from vietnam were cloned and sequenced. a new variant of tomato leaf curl vietnam virus (tolcvv) consisting of a dna-a component and associated with a dnabeta molecule as well as an additional begomovirus tentatively named tomato yellow leaf curl vietnam virus (tylcvv) consisting also of a dna-a component were identified. to verify if monopartite viruses occurring in vietnam and thailand are able to transreplicate the dna-b component of tomato ye ...200818550192
the 5' untranslated region of the maize alcohol dehydrogenase gene contains an internal ribosome entry site.adh1, the maize gene encoding alcohol dehydrogenase adh1, mrna is efficiently translated in o2-deprived roots of maize, whereas many normal cellular mrnas are poorly translated. it has been shown that adh, the 5' untranslated region of adh1 mrna, provides effective translation of mrna under hypoxia and heat shock conditions in nicotiana benthamiana plants. we found that adh contains the internal ribosome entry site (ires) active both in vivo, in n. benthamiana cells, and in vitro, in rabbit reti ...200818556149
functional characterization and subcellular localization of the 16k cysteine-rich suppressor of gene silencing protein of tobacco rattle virus.the 16 kda cysteine-rich protein (16k) of tobacco rattle virus (trv) is known to partially suppress rna silencing in drosophila cells. in this study, we show that 16k suppresses rna silencing in green fluorescent protein (gfp)-transgenic nicotiana benthamiana plants using an agrobacterium-mediated transient assay. 16k slightly reduced the accumulation of short interfering rnas (sirna) of gfp, suggesting that the protein may interfere with the initiation and/or maintenance of rna silencing. delet ...200818559946
diversity of begomoviruses associated with mosaic disease of cultivated cassava (manihot esculenta crantz) and its wild relative (manihot glaziovii mull. arg.) in uganda.cassava (manihot esculenta) growing in uganda during 2001-2002 has been screened for the presence of begomoviruses using pcr-rflp, cloning full-length genomic components and nucleotide sequence analysis. in contrast with a recent survey in neighbouring kenya, which identified three distinct strains of east african cassava mosaic virus (eacmv, eacmv-ug and eacmv-ke2) as well as east african cassava mosaic zanzibar virus and the new species east african cassava mosaic kenya virus, only eacmv-ug an ...200818559947
mutations that alter a repeated acca element located at the 5' end of the potato virus x genome affect rna accumulation.the repeated acca or ac-rich sequence and structural (sl1) elements in the 5' non-translated region (ntr) of the potato virus x (pvx) rna play vital roles in the pvx life cycle by controlling translation, rna replication, movement, and assembly. it has already been shown that the repeated acca or ac-rich sequence affect both grna and sgrna accumulation, while not affecting minus-strand rna accumulation, and are also required for host protein binding. the functional significance of the repeated a ...200818589472
viroid-induced symptoms in nicotiana benthamiana plants are dependent on rdr6 activity.viroids are small self-replicating rnas that infect plants. how these noncoding pathogenic rnas interact with hosts to induce disease symptoms is a long-standing unanswered question. recent experimental data have led to the suggestive proposal of a pathogenic model based on the rna silencing mechanism. however, evidence of a direct relation between key components of the rna silencing pathway and symptom expression in infected plants remains elusive. to address this issue, we used a symptomatic t ...200818599649
a novel type of thioredoxin dedicated to symbiosis in legumes.thioredoxins (trxs) constitute a family of small proteins in plants. this family has been extensively characterized in arabidopsis (arabidopsis thaliana), which contains six different trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. a detailed study of this family in the model legume medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. as no possible orthologs were furt ...200818614707
disassembly of african cassava mosaic virus.the plant-infecting geminiviruses encapsidate their single-stranded dna genome in characteristic twinned particles that are unique among viruses. these particles are formed by joining two incomplete t=1 icosahedra. african cassava mosaic virions were purified by density-gradient centrifugation from infected nicotiana benthamiana plants and analysed for their stability with respect to ph changes and heat treatment by using electron microscopy. negative staining and rotary shadowing revealed stabl ...200818632975
hc-pro protein of sugar cane mosaic virus interacts specifically with maize ferredoxin-5 in vitro and in planta.symptom development of a plant viral disease is a result of molecular interactions between the virus and its host plant; thus, the elucidation of specific interactions is a prerequisite to reveal the mechanism of viral pathogenesis. here, we show that the chloroplast precursor of ferredoxin-5 (fd v) from maize (zea mays) interacts with the multifunctional hc-pro protein of sugar cane mosaic virus (scmv) in yeast, nicotiana benthamiana cells and maize protoplasts. our results demonstrate that the ...200818632977
peanut stunt virus 2b cistron plays a role in viral local and systemic accumulation and virulence in nicotiana benthamiana.to analyze the role of the 2b protein (2bp) of peanut stunt virus (psv) in the viral infection cycle, we constructed psv mutants that express either no 2bp or n-terminal-truncated 2bp. the accumulation of wild-type and mutant viruses in tobacco protoplasts indicated that the 2b cistron is not essential for viral replication. viral accumulation in nicotiana benthamiana plants suggested that the 2b cistron is responsible for viral accumulation in inoculated and upper leaves and has a role in virul ...200818663407
nblrk1, a lectin-like receptor kinase protein of nicotiana benthamiana, interacts with phytophthora infestans inf1 elicitin and mediates inf1-induced cell death.phytophthora infestans inf1 elicitin causes the hypersensitive response (hr) in nicotiana benthamiana (kamoun et al. in plant cell 10:1413-1425, 1998). to identify n. benthamiana proteins that interact with inf1, we carried out a yeast two-hybrid screen. this screen resulted in the isolation of a gene nblrk1 coding for a novel lectin-like receptor kinase. nblrk1 interacted with inf1 through its vib kinase subdomain. purified inf1 and nblrk1 proteins also interacted in vitro. inf1 treatment of n. ...200818682978
characterization of resistance mechanism in transgenic nicotiana benthamiana containing turnip crinkle virus coat protein.two transgenic lines, of nicotiana benthamiana expressing turnip crinkle virus (tcv)-coat protein (cp) gene with contrasting phenotype, the highest (#3) and the lowest (#18) cp expressers, were selected and challenged with the homologous tcv. the former, the highest expresser, showed nearly five times more cp expression than the latter. progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. the infected progenies of #3 line showed mild and delayed symptom with tcv. this ...200818704429
role of a xyloglucan-specific endo-beta-1,4-glucanase inhibitor in the interactions of nicotiana benthamiana with colletotrichum destructivum, c. orbiculare or pseudomonas syringae pv. tabaci.a xyloglucan-specific endo-beta-1,4-glucanase inhibitor cdna, nbxegip1, was amplified from diseased leaves of nicotiana benthamiana. the sequence was similar to the tomato xyloglucan-specific endo-beta-1,4-glucanase inhibitor (xegip) and tobacco nectarin iv genes that have been described as binding and inactivating fungal family 12 xyloglucan-specific endo-beta-1,4-glucanases. expression of nbxegip1 was not detected in healthy leaves, but the gene was induced during the later stages of infection ...200818705851
high-throughput in planta expression screening identifies an adp-ribosylation factor (arf1) involved in non-host resistance and r gene-mediated resistance.to identify positive regulators of cell death in plants, we performed a high-throughput screening, employing potato virus x-based overexpression in planta of a cdna library derived from paraquat-treated nicotiana benthamiana leaves. the screening of 30,000 cdna clones enabled the identification of an adp-ribosylation factor 1 (arf1) that induces cell death when overexpressed in n. benthamiana. overexpression of the guanosine diphosphate (gdp)-locked mutant of arf1 did not trigger cell death, sug ...200818705881
hibiscus chlorotic ringspot virus coat protein inhibits trans-acting small interfering rna biogenesis in arabidopsis.many plant and animal viruses have evolved suppressor proteins to block host rna silencing at various stages of the rna silencing pathways. hibiscus chlorotic ringspot virus (hcrsv) coat protein (cp) is capable of suppressing the transiently expressed sense-rna-induced post-transcriptional gene silencing (ptgs) in nicotiana benthamiana. here, constitutively expressed hcrsv cp from transgenic arabidopsis was found to be able to rescue expression of the silenced gus transgene. the hcrsv cp-transge ...200818753245
bah1/nla, a ring-type ubiquitin e3 ligase, regulates the accumulation of salicylic acid and immune responses to pseudomonas syringae dc3000.salicylic acid (sa) is a primary factor responsible for exerting diverse immune responses in plants and is synthesized in response to attack by a wide range of pathogens. the arabidopsis (arabidopsis thaliana) sid2 mutant is defective in a sa biosynthetic pathway involving isochorismate synthase1 (ics1) and consequently contains reduced levels of sa. however, the sid2 mutant as well as ics-suppressed tobacco (nicotiana benthamiana) still accumulate a small but significant level of sa. these obse ...200818753285
ntbzip60, an endoplasmic reticulum-localized transcription factor, plays a role in the defense response against bacterial pathogens in nicotiana tabacum.a spermine-based signal transduction pathway plays a defensive role against incompatible pathogens. we identified a novel spermine-responsive cdna from nicotiana tabacum that encodes a basic region/leucine zipper protein with a putative transmembrane domain. identity to arabidopsis thaliana atbzip60 was sufficiently high to name the novel cdna ntbzip60. expression analysis revealed that ntbzip60 is a component of the spermine-signal pathway, and is also involved in the unfolded protein response ...200818758894
recognition of the hyaloperonospora parasitica effector atr13 triggers resistance against oomycete, bacterial, and viral pathogens.phytopathogenic oomycetes cause some of the most devastating diseases affecting agricultural crops. hyaloperonospora parasitica is a native oomycete pathogen of arabidopsis and is related to other oomycete phytopathogens that include several species of phytophthora, including the causal agent of potato late blight. recently, four oomycete effector genes have been isolated, and several oomycete genomes have been sequenced. we have developed an efficient and genetically amenable system to test put ...200818198274
class viii myosins are required for plasmodesmatal localization of a closterovirus hsp70 homolog.the hsp70 homolog (hsp70h) of beet yellows virus (byv) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (a. i. prokhnevsky, v. v. peremyslov, and v. v. dolja, j. virol. 79:14421-14428, 2005). myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. plants have two classes of myosins, viii and xi, whose specific functions are poo ...200818199648
salicylic acid production in response to biotic and abiotic stress depends on isochorismate in nicotiana benthamiana.salicylic acid (sa) is an important signal involved in the activation of defence responses against abiotic and biotic stress. in tobacco, benzoic acid or glucosyl benzoate were proposed to be precursors of sa. this is in sharp contrast with studies in arabidopsis thaliana, where sa derives from isochorismate. we have determined the importance of isochorismate for sa biosynthesis in nicotiana benthamiana using virus-induced gene silencing of the isochorismate synthase (ics) gene. plants with sile ...200818201575
overexpression of osisap8, a member of stress associated protein (sap) gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice.we describe here the isolation and characterization of osisap8, a member of stress associated protein (sap) gene family from rice characterized by the presence of a20 and an1 type zinc finger domains. osisap8 is a multiple stress inducible gene, induced by various stresses, namely heat, cold, salt, desiccation, submergence, wounding, heavy metals as well as stress hormone abscisic acid. osisap8 protein fused to gfp was localized towards the periphery of the cells in the epidermal cells of infilt ...200818205020
optimized cdna libraries for virus-induced gene silencing (vigs) using tobacco rattle virus.abstract:200818211705
nuclear targeting by fragmentation of the potato spindle tuber viroid genome.transient expression of engineered reporter rnas encoding an intron-containing green fluorescent protein (gfp) from a potato virus x-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide potato spindle tuber viroid (pstvd) rna genome. to further delimit the putative nuclear-targeting signal, pstvd subgenomic fragments were embedded within the intron, and recombinant reporter rnas were inoculated onto nicotiana benthamiana plants. appearance of gre ...200818211806
heat shock 70 protein interaction with turnip mosaic virus rna-dependent rna polymerase within virus-induced membrane vesicles.tandem affinity purification was used in arabidopsis thaliana to identify cellular interactors of turnip mosaic virus (tumv) rna-dependent rna polymerase (rdrp). the heat shock cognate 70-3 (hsc70-3) and poly(a)-binding (pabp) host proteins were recovered and shown to interact with the rdrp in vitro. as previously shown for pabp, hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both rdrp interactors were co-immunoprecipitated from a membrane-enriched extract u ...200818222516
molecular variation of satellite dna beta molecules associated with malvastrum yellow vein virus and their role in pathogenicity.previous studies have found that the diversity of begomovirus-associated dna beta satellites is related to host and geographical origin. in this study, we have cloned and sequenced 20 different isolates of dna beta molecules associated with malvastrum yellow vein virus (myvv) isolated from malvastrum coromandelianum plants in different geographical locations of yunnan province, china. analyses of their molecular variation indicate that the satellites are clustered together according to their geo ...200818245261
tobacco mosaic virus (tmv) replicase and movement protein function synergistically in facilitating tmv spread by lateral diffusion in the plasmodesmal desmotubule of nicotiana benthamiana.virus spread through plasmodesmata (pd) is mediated by virus-encoded movement proteins (mps) that modify pd structure and function. the mp of tobacco mosaic virus ((tmv)mp) is an endoplasmic reticulum (er) integral membrane protein that binds viral rna (vrna), forming a vrna:mp:er complex. it has been hypothesized that (tmv)mp causes pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vrna:mp:er complex through pd; in the absence of mp, by contrast, the er cannot move through ...200818257683
the expression of a mountain cedar allergen comparing plant-viral apoplastic and yeast expression systems.jun a 3, a major allergenic protein in mountain cedar pollen, causes seasonal allergic rhinitis in hypersensitive individuals. recombinant jun a 3 was expressed in nicotiana benthamiana interstitial fluid (300 microg/g leaf material) and pichia pastoris (100 microg/ml media). polyclonal anti-jun a 3 and ige antibodies from the sera of allergic patients both reacted with the recombinant protein. of the two systems, recombinant protein from the plant apoplast contained fewer contaminating proteins ...200818270658
expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice.transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins including antibodies, antigens and hormones. here, we report expression of a full-length nucleoprotein gene of rabies virus in transgenic tomato plants. the nucleoprotein was also transiently expressed in nicotiana benthamiana plants by agroinfiltration. in both cases, the nucleoprotein was expressed at high levels, 1-5% of total soluble protein in tomato and 45% in n. b ...200818270708
supervirulent pseudorecombination and asymmetric synergism between genomic components of two distinct species of begomovirus associated with severe tomato leaf curl disease in india.isolates of two distinct begomovirus species, the severe strain of the species tomato leaf curl new delhi virus (tomato leaf curl new delhi virus-[india:new delhi:severe:1992]; tolcndv-[in:nd:svr:92], bipartite) and the varanasi strain of the species tomato leaf curl gujarat virus (tomato leaf curl gujarat virus-[india:varanasi:2001]; tolcgv-[in:var:01], mono/bipartite) infect tomato (lycopersicon esculentum) and cause severe yield losses in northern india. this study investigated the infectivit ...200818272774
salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by agrobacterium tumefaciens.we investigated the effects of salicylic acid (sa) and systemic acquired resistance (sar) on crown gall disease caused by agrobacterium tumefaciens. nicotiana benthamiana plants treated with sa showed decreased susceptibility to agrobacterium infection. exogenous application of sa to agrobacterium cultures decreased its growth, virulence, and attachment to plant cells. using agrobacterium whole-genome microarrays, we characterized the direct effects of sa on bacterial gene expression and showed ...200818156296
cis- and trans-acting functions of brome mosaic virus protein 1a in genomic rna1 replication.rna viruses employ a combination of mechanisms to regulate their gene expression and replication. brome mosaic virus (bmv) is a tripartite positive-strand rna virus used to study the requirements for virus infection. bmv genomic rna1 encodes protein 1a, which contains a methyltransferase (mt) domain and a helicase domain that are required for replication. 1a forms a complex with the 2a rna-dependent rna polymerase for the replication and transcription of all bmv rnas. rna1 expressed with 2a from ...200818160434
characterization of a defective rna derived from rna 3 of the y strain of cucumber mosaic virus.a defective (d) rna 3 naturally generated from the y strain of cucumber mosaic virus (cmv-y) was characterized using a biologically active cdna clone. sequencing of the clone revealed that the d rna 3, named d rna 3yalpha, was derived from cmv-y rna 3 and contained a 10 nt deletion in the 5' untranslated region and a 162 nt deletion within the 3a open reading frame. co-inoculation of d rna 3yalpha with cmv-y derived from in vitro transcripts facilitated propagation of cmv-y containing d rna 3yal ...200818175038
infectivity of the cloned components of a begomovirus: dna beta complex causing chilli leaf curl disease in india.the full-length genome of a begomovirus and its cognate dna-beta satellite component associated with chilli leaf curl disease (chlcd), originating from varanasi, india, were cloned. sequence analysis revealed that the viral genome (ef190217) is 2,750 bp and the dna-beta satellite (ef190215) is 1,361 bp in length. agroinoculation with partial tandem repeats of the viral genome along with the satellite induced symptoms typical of chlcd in chilli and nicotiana benthamiana. however, symptom expressi ...200818175042
a genomic map of viroid rna motifs critical for replication and systemic trafficking.rna replication and systemic trafficking play significant roles in developmental regulation and host-pathogen interactions. viroids are the simplest noncoding eukaryotic rna pathogens and genetic units that are capable of autonomous replication and systemic trafficking and offer excellent models to investigate the role of rna structures in these processes. like other rnas, the predicted secondary structure of a viroid rna contains many loops and bulges flanked by double-stranded helices, the bio ...200818178767
physiological function of ispe, a plastid mep pathway gene for isoprenoid biosynthesis, in organelle biogenesis and cell morphogenesis in nicotiana benthamiana.isoprenoid biosynthesis in plants occurs by two independent pathways: the cytosolic mevalonate (mva) pathway and the plastidic methylerythritol phosphate (mep) pathway. in this study, we investigated the cellular effects of depletion of ispe, a protein involved in the mep pathway, using virus-induced gene silencing (vigs). the ispe gene is preferentially expressed in young tissues, and induced by light and methyl jasmonate. the gfp fusion protein of ispe was targeted to chloroplasts. reduction o ...200818180879
phenotypes and functional effects caused by various viral rna silencing suppressors in transgenic nicotiana benthamiana and n. tabacum.rna silencing suppressor genes derived from six virus genera were transformed into nicotiana benthamiana and n. tabacum plants. these suppressors were p1 of rice yellow mottle virus (rymv), p1 of cocksfoot mottle virus, p19 of tomato bushy stunt virus, p25 of potato virus x, hcpro of potato virus y (strain n), 2b of cucumber mosaic virus (strain kin), and ac2 of african cassava mosaic virus (acmv). hcpro caused the most severe phenotypes in both nicotiana spp. ac2 also produced severe effects in ...200818184062
biosynthesis of circular proteins in plants.plant cyclotides are a large family of naturally occurring circular proteins that are produced from linear precursors containing one, two or three cyclotide domains. the mechanism of excision of the cyclotide domains and ligation of the free n- and c-termini to produce the circular peptides has not been elucidated. here, we investigate production of the prototypic cyclotide kalata b1 from the precursor oak1 from the african plant oldenlandia affinis. immunoprecipitation experiments and maldi-tof ...200818086282
partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (fragaria vesca).in addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (oga), are essential components for elicitation of defence responses. to investigate how modifications of pectin alter defence responses, we expressed the fruit-specific fragaria x ananassa pectin methyl esterase fape1 in the wild strawberry fragaria vesca. pectin from transgenic ripe fruits differed from the wild-type with regard to the degree an ...200818088306
pathogenicity of a naturally occurring recombinant dna satellite associated with tomato yellow leaf curl china virus.recombinant dna beta molecules (recdna-abeta) comprising parts of dna a and dna beta associated with tomato yellow leaf curl china virus (tylccnv) have been identified in naturally infected tobacco plants. several examples of the recombinant dna have been cloned and characterized by sequence analysis. all are approximately half the size of tylccnv genomic dna, and all contain the betac1 gene and the a-rich region from tylccnv dna beta as well as intergenic region sequences and the 5' terminus of ...200818089755
calcium efflux as a component of the hypersensitive response of nicotiana benthamiana to pseudomonas syringae.using a model plant nicotiana benthamiana, we have demonstrated that initial calcium uptake in response to the hr (hypersensitive response)-causing pathogen pseudomonas syringae pv syringae 61 is followed by net calcium efflux initiated at about 12 h after the bacterial challenge and sustained for at least 48 h. our data suggest that calcium not only acts as an important second messenger in the activation of resistance responses but may also be a downstream mediator of later cell death accelerat ...200818048411
transient expression of hpv16 e7 peptide (aa 44-60) and hpv16 l2 peptide (aa 108-120) on chimeric potyvirus-like particles using potato virus x-based vector.the optimized expression of recombinant potato virus a coat protein (acp) carrying two different epitopes from human papillomavirus type 16 (hpv16) was developed. epitope derived from minor capsid protein l2 was expressed as n-terminal fusion with acp while an epitope derived from e7 oncoprotein was fused to its c-terminus. the construct was cloned into potato x potexvirus (pvx) based vector and transiently expressed in plants using agrobacterium tumefaciens mediated inoculation. to increase the ...200817980618
one-step cloning approach for construction of agroinfectious begomovirus clones.most begomoviruses have a bipartite genome containing two circular ssdna segments (dna-a and dna-b). routine infectious clone construction relies upon cloning of the whole genome, which is then subcloned as a tandem one-and-half or two genome- (containing two replication origins) cassette into a vector prior to agro-inoculation. the construction of cassettes containing two replication origins is, however, a time-consuming process. here an improved method for rapid construction of agroinfectious ...200818022703
intracellular targeting of a hordeiviral membrane-spanning movement protein: sequence requirements and involvement of an unconventional mechanism.the membrane-spanning protein tgbp3 is one of the three movement proteins (mps) of poa semilatent virus. tgbp3 is thought to direct other viral mps and genomic rna to peripheral bodies located in close proximity to plasmodesmata. we used the ectopic expression of green fluorescent protein-fused tgbp3 in epidermal cells of nicotiana benthamiana leaves to study the tgbp3 intracellular trafficking pathway. treatment with inhibitors was used to reveal that the targeting of tgbp3 to plasmodesmata doe ...200818032484
replication-coupled packaging mechanism in positive-strand rna viruses: synchronized coexpression of functional multigenome rna components of an animal and a plant virus in nicotiana benthamiana cells by agroinfiltration.flock house virus (fhv), a bipartite rna virus of insects and a member of the nodaviridae family, shares viral replication features with the tripartite brome mosaic virus (bmv), an rna virus that infects plants and is a member of the bromoviridae family. in bmv and fhv, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand rna viruses of diverse origin. to unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we h ...200818032497
point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of rna-silencing suppression activity and symptom expression in broad bean.helper component protease (hc-pro) is a potyvirus-encoded multifunctional protein and a major determinant of symptom expression in a susceptible plant. here, we show the involvement of clover yellow vein virus (clyvv) hc-pro in necrotic symptom expression in broad bean (vicia faba cv. wase). in this host, lethal necrosis was induced by clyvv no. 30, from which a spontaneous, mosaic-inducing mutant (mm) was obtained. mapping with chimeric viruses between clyvv no. 30 and mm attributed the symptom ...200817955160
oligonucleotide microarray-based detection and genotyping of plum pox virus.plum pox virus (ppv) is the most damaging viral pathogen of stone fruits. the detection and identification of its strains are therefore of critical importance to plant quarantine and certification programs. existing methods to screen strains of ppv suffer from significant limitations such as the simultaneous detection and genotyping of several strains of ppv in samples infected with different isolates of the virus. a genomic strategy for ppv screening based on the viral nucleotide sequence was d ...200817920703
a stress-inducible resveratrol o-methyltransferase involved in the biosynthesis of pterostilbene in grapevine.stilbenes are considered the most important phytoalexin group in grapevine (vitis vinifera) and they are known to contribute to the protection against various pathogens. the main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exh ...200818799660
development of a new cucumber mosaic virus-based plant expression vector with truncated 3a movement protein.we have developed a cucumber mosaic virus (cmv)-based expression vector for the production of heterologous proteins in plants. cell-to-cell movement of cmv is dependent on the presence of coat protein (cp). previous studies have shown that deletion of 33 amino acids (aa) from the carboxy-terminus of the 3a movement protein facilitates cell-to-cell movement that is independent of cp. the cmv-based expression vector that we have designed utilizes this truncated 3a protein, allowing the expression ...200818804833
identification of a movement protein of the tenuivirus rice stripe virus.rice stripe virus (rsv) is the type member of the genus tenuivirus. rsv has four single-stranded rnas and causes severe disease in rice fields in different parts of china. to date, no reports have described how rsv spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. we investigated functions of six rsv-encoded proteins using trans-complementation experiments and biolistic bombardment. we demonstrate that nsvc4, encoded by rsv rna4, supports the interce ...200818818319
molecular divergence of grapevine virus a (gva) variants associated with shiraz disease in south africa.shiraz disease (sd) is a highly destructive, insect-transmitted disease of noble grapevine cultivars, such as shiraz and merlot, in south africa. earlier studies revealed that, of the three molecular groups of gva that were detected in local vineyards, variants of group ii are closely associated with expression of this disease, and variants of group iii are commonly present in gva-infected sd-negative plants [goszczynski, d.e., 2007. single-strand conformation polymorphism (sscp), cloning and se ...200818822325
the flagellar sigma factor flia is required for dickeya dadantii virulence.the genome sequence of the enterobacteriaceae phytopathogen dickeya dadantii (formerly erwinia chrysanthemi) revealed homologs of genes required for a complete flagellar secretion system and one flagellin gene. we found that d. dadantii was able to swim and swarm but that ability to swarm was dependent upon both growth media and temperature. mutation of the d. dadantii flia gene was pleiotropic, with the alternate sigma factor required for flagella production and development of disease symptoms ...200818842093
transcriptomic and reverse genetic analyses of branched-chain fatty acid and acyl sugar production in solanum pennellii and nicotiana benthamiana.acyl sugars containing branched-chain fatty acids (bcfas) are exuded by glandular trichomes of many species in solanaceae, having an important defensive role against insects. from isotope-feeding studies, two modes of bcfa elongation have been proposed: (1) fatty acid synthase-mediated two-carbon elongation in the high acyl sugar-producing tomato species solanum pennellii and datura metel; and (2) alpha-keto acid elongation-mediated one-carbon increments in several tobacco (nicotiana) species an ...200818931142
inhibition of long-distance movement of rna silencing signals in nicotiana benthamiana by apple chlorotic leaf spot virus 50 kda movement protein.apple chlorotic leaf spot virus 50 kda movement protein (p50) acts as a suppressor of systemic silencing in nicotiana benthamiana. here, we investigate the mode of action of p50 suppressor. an agroinfiltration assay in gfp-expressing n. benthamiana line16c (gfp-plant) showed that p50 could not prevent the short-distance spread of silencing. in grafting experiments, the systemic silencing was inhibited in gfp-plants (scion) grafted on p50-expressing n. benthamiana (p50-plant; rootstock) when gfp ...200818954886
lebzip2 induced by salt and drought stress and transient overexpression by agrobacterium.the full-length cdna of lebzip2 (lycopersicon esculentum bzip2) encodes a protein of 164 amino acids and contains a n-terminal basic-region leucine zipper domain. analysis of the deduced tomato lebzip2 amino acid sequence revealed that it shares 85% sequence identity with both tobacco bzip and pepper ccbzip. lebzip2 mrna is expressed at a high level exclusively in flowers. presently, lebzip2 was strongly increased also following nacl and mannitol treatments. no significant lebzip2 expression was ...200818959814
replication and encapsidation of recombinant turnip yellow mosaic virus rna.turnip yellow mosaic virus (tymv) is a positive strand rna virus that infects mainly cruciferae plants. in this study, the tymv genome was modified by inserting an extra subgenomic rna promoter and a multiple cloning site. this modified tymv was introduced into nicotiana benthamiana using a agrobacterium-mediated t-dna transfer system (agroinfiltration). when a gene encoding beta-glucuronidase or green fluorescent protein was expressed using this modified tymv as a vector, replication of the rec ...200818959822
highly efficient expression of escherichia coli heat-labile enterotoxin b subunit in plants using potato virus x-based vector.a synthetic gene of the b-subunit of escherichia coli heat-labile toxin, optimized for expression in plants, was designed and synthesized. the recombinant viral vector was constructed on the basis of potato virus x containing the ltb gene instead of the removed triple block of transport genes and the coat protein gene, which provides for ltb expression in plants. the vector is introduced into the plant cells during cell infiltration by agrobacteria incorporating a binary vector, the t-dna region ...200818991556
evaluation of two gene-silencing constructs for resistance to tomato yellow leaf curl viruses in nicotiana benthamiana plants.infiltration of agrobacterium tumefaciens into intact plant leaves of n. benthamiana was used to test the efficiency of two virus-based silencing constructs conferring resistance to the closely related begomoviruses. the constructs contained the most conserved sequences of the coat protein (cp) gene and replication-associated protein (rep) gene of tomato yellow leaf curl sardinia virus (sicily strain) (tylcsv-[sic]). both constructs formed a hairpin structure that enhanced the post-transcription ...200818999888
co-infection by two criniviruses alters accumulation of each virus in a host-specific manner and influences efficiency of virus transmission.tomato chlorosis virus (tocv), and tomato infectious chlorosis virus (ticv), family closteroviridae, genus crinivirus, cause interveinal chlorosis, leaf brittleness, and limited necrotic flecking or bronzing on tomato leaves. both viruses cause a decline in plant vigor and reduce fruit yield, and are emerging as serious production problems for field and greenhouse tomato growers in many parts of the world. the viruses have been found together in tomato, indicating that infection by one criniviru ...200819000010
hairy-root organ cultures for the production of human acetylcholinesterase.human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. the practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.200819105816
distinct rna motifs mediate systemic rna trafficking.systemic rna trafficking is emerging as a new paradigm in gene regulation at the whole plant level. the mechanisms that ensure delivery of all sorts of rnas to their respective cellular destinations remain poorly understood. several lines of research suggest that specific sequence/structural motif(s) within an rna directs its trafficking. the first motif was identified in potato spindle tuber viroid (pstvd), which mediates trafficking from bundle sheath to mesophyll in young tobacco leaves. our ...200819704772
wtse, an avre-family type iii effector protein of pantoea stewartii subsp. stewartii, causes cell death in non-host plants.pantoea stewartii subsp. stewartii (pnss) causes stewart's bacterial wilt of sweet corn and leaf blight of maize. the pathogenicity of pnss depends on synthesis of extracellular polysaccharide and an hrp type iii secretion system. wtse, a type iii secreted effector protein, is essential for the virulence of pnss on corn. it belongs to the avre family of effectors, which includes dspa/e from erwinia amylovora and avre1 from pseudomonas syringae. previously, wtse was shown to cause disease-associa ...200819018993
influence of cytoplasmic heat shock protein 70 on viral infection of nicotiana benthamiana.the accumulation of heat shock protein 70 (hsp70) generally occurs in plants infected with viruses. however, the effect of hsp70 accumulation on plant viral infection and pathogenesis remains elusive. in this study, the expression of six hsp70 genes was found to be induced by the four diverse rna viruses, tobacco mosaic virus, potato virus x (pvx), cucumber mosaic virus and watermelon mosaic virus, in nicotiana benthamiana. heat treatment enhanced the accumulation and systemic infection of these ...200819019009
replication promiscuity of dna-beta satellites associated with monopartite begomoviruses; deletion mutagenesis of the ageratum yellow vein virus dna-beta satellite localizes sequences involved in replication.pseudorecombination studies in nicotiana benthamiana demonstrate that ageratum yellow vein virus (ayvv) and eupatorium yellow vein virus (epyvv) can functionally interact with dna-beta satellites associated with ayvv, epyvv, cotton leaf curl multan virus (clcumv) and honeysuckle yellow vein virus (hyvv). in contrast, clcumv shows some specificity in its ability to interact with distinct satellites and hyvv is able to interact only with its own satellite. using an n. benthamiana leaf disk assay, ...200819008407
influenza virus-like particles produced by transient expression in nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice.a strain-specific vaccine represents the best possible response to the threat of an influenza pandemic. rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. plant-based transient expression is one of the few production systems that can meet the anticipated surge requirement. to assess the capability of plant agroinfiltration to produce an influenza vaccine ...200819076615
transport of tmv movement protein particles associated with the targeting of rna to plasmodesmata.the cell-to-cell movement of tobacco mosaic virus through plasmodesmata (pd) requires virus-encoded movement protein (mp). the mp targets pd through the endoplasmic reticulum (er)/actin network, whereas the intercellular movement of the viral rna genome has been correlated with the association of the mp with mobile, microtubule-proximal particles in cells at the leading front of infection as well as the accumulation of the protein on the microtubule network during later infection stages. to unde ...200819281527
subcellular localization and in vivo interactions of the arabidopsis thaliana ethylene receptor family members.the gaseous phytohormone ethylene regulates many developmental processes and responses to environmental conditions in higher plants. in arabidopsis thaliana, ethylene perception and initiation of signaling are mediated by a family of five receptors which are related to prokaryotic two-component sensor histidine kinases. the transient expression of fluorescence-tagged receptors in tobacco (nicotiana benthamiana) epidermal leaf cells demonstrated that all ethylene receptors are targeted to the er ...200819825542
investigating recombinant protein exudation from roots of transgenic tobacco.it is widely acknowledged that plant-made pharmaceuticals (pmps) offer numerous benefits, including inexpensive production, biological safety and the facility for production at agricultural scale. at the same time, it is important to minimize any potential risk associated with this new technology, including the potential release of bioactive proteins into the environment. to address this issue, we studied transgenic nicotiana benthamiana and nicotiana tabacum plants expressing two recombinant si ...200819081009
solanum lycopersicon mill. and nicotiana benthamiana l. under high light show distinct responses to anti-oxidative stress.two experimentally important species, solanum lycopersicon mill. and nicotiana benthamiana l., were propagated in vitro under low light (50 micromol m(-2) s(-1)) and transferred to hl (200 micromol m(-2) s(-1)) under a protocol previously developed for grapevine and chestnut. compared with photooxidative stress parameters already tested in those species, imaging of hydrogen peroxide and superoxide revealed an accumulation on d2-3 and d6 in s. lycopersicon and d1-2 and d5-7 in n. benthamiana. sod ...200817945381
size reversion of a truncated dnabeta associated with tobacco curly shoot virus.our previous results demonstrated that dnabeta associated with tobacco curly shoot virus (tbcsv) is not necessary for infection but intensifies symptoms in some hosts. to better understand the function of dnabeta in virus infection, a betac1 deleted infectious clone of the tbcsv dnabeta was constructed. agroinoculation showed that the truncated dnabeta (dnadeltac1beta) was trans-replicated by tbcsv in tobacco and petunia hybrida plants. however, pcr and southern blot analysis demonstrated that t ...200817950948
resistance to grapevine leafroll associated virus-2 is conferred by post-transcriptional gene silencing in transgenic nicotiana benthamiana.grapevine leafroll-associated virus-2 (glrav-2) is an important component of the leafroll disease complex in grapevine. we have previously sequenced the glrav-2 genome and identified the coat protein (cp) gene. the objective of this study is to test the concept of pathogen-derived resistance against a closterovirus associated with grapevine leafroll disease. because glrav-2 is capable of infecting nicotiana benthamiana, we decided to test the concept on this herbaceous host. thirty-seven t(0) tr ...200817912600
leaf proteome analysis of transgenic plants expressing antiviral antibodies.the expression of exogenous antibodies in plant is an effective strategy to confer protection against viral infection or to produce molecules with pharmaceutical interest. however, the acceptance of the transgenic technology to obtain self-protecting plants depends on the assessment of their substantial equivalence compared to non-modified crops with an established history of safe use. in fact, the possibility exists that the introduction of transgenes in plants may alter expression of endogenou ...200919099506
production of biologically active human myelocytokines in plants.an effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hg-csf and hgm-csf) in nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. the genes of target proteins were cloned into the viral vector driven by actin promoter of arabidopsis thaliana. the expression vectors were delivered into plant cells by agroinjection. maximal synthesis rate was detected 5 days after injection ...200919916932
molecular mechanisms of generation for nitric oxide and reactive oxygen species, and role of the radical burst in plant immunity.rapid production of nitric oxide (no) and reactive oxygen species (ros) has been implicated in the regulation of innate immunity in plants. a potato calcium-dependent protein kinase (stcdpk5) activates an nadph oxidase strboha to d by direct phosphorylation of n-terminal regions, and heterologous expression of stcdpk5 and strbohs in nicotiana benthamiana results in oxidative burst. the transgenic potato plants that carry a constitutively active stcdpk5 driven by a pathogen-inducible promoter of ...200919830396
a leaf-based assay using interchangeable design principles to rapidly assemble multistep recombinant pathways.the assembly of multistep recombinant pathways in stably transformed plants is a cornerstone of crops producing new products yet can be a laborious and time-consuming process. any heterologous expression platform capable of providing a rapid estimation of the functional assembly of an entire pathway would guide the design of such transgenic traits. in this study, we use a nicotiana benthamiana transient leaf expression system to simultaneously express five genes, from five independent t(dna) bin ...200919843252
myosin xi is required for actin-associated movement of plastid stromules.stromules are highly dynamic stroma-filled tubules extending from the surface of plastids and occasionally interconnecting individual plastids, allowing the movement of complex biological molecules between the interconnected plastids. experiments with inhibitors of cytoskeleton assembly have indicated the involvement of an actin-based system in stromule movement. however, the motor protein associated with the system had not been identified. here, we present direct evidence that myosin xi is invo ...200919995729
a myosin xi tail domain homologous to the yeast myosin vacuole-binding domain interacts with plastids and stromules in nicotiana benthamiana.the actin cytoskeleton plays a role in mobility of many different organelles in plant cells, including chloroplasts, mitochondria, golgi, and peroxisomes. while progress has been made in identifying the myosin motors involved in trafficking of various plant organelles, not all of the cargoes mobilized by different members of the myosin xi family have yet been identified. the involvement of myosins in chloroplast positioning and mitochondrial movement was demonstrated by expression of a virus-ind ...200919995734
immunomodulatory activity of a plant extract containing human papillomavirus 16-e7 protein in human monocyte-derived dendritic cells.this study reports the immunomodulatory activity on human monocyte derived dendritic cells (mddcs) of a vaccine preparation shown to be effective against an hpv16-related tumour in an animal model. the vaccine is composed of extract from nicotiana benthamiana leaves containing hpv16 e7 protein expressed by a potato virus x-derived vector (nbpvx-e7). the effect of the extract was evaluated on mddc differentiation and maturation by monitoring the phenotypic expression of specific markers. the resu ...200920074460
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