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suppression of plant defense responses by extracellular metabolites from pseudomonas syringae pv. tabaci in nicotiana benthamiana.pseudomonas syringae pv. tabaci (pstab) is the causal agent of wildfire disease in tobacco plants. several pathovars of pseudomonas syringae produce a phytotoxic extracellular metabolite called coronatine (cor). cor has been shown to suppress plant defense responses. interestingly, pstab does not produce cor but still actively suppresses early plant defense responses. it is not clear if pstab produces any extracellular metabolites that actively suppress early defense during bacterial pathogenesi ...201323597256
a nuclear localization for avr2 from fusarium oxysporum is required to activate the tomato resistance protein i-2.plant pathogens secrete effector proteins to promote host colonization. during infection of tomato xylem vessels, fusarium oxysporum f. sp. lycopersici (fol) secretes the avr2 effector protein. besides being a virulence factor, avr2 is recognized intracellularly by the tomato i-2 resistance protein, resulting in the induction of host defenses. here, we show that avr2 is highly expressed in root- and xylem-colonizing hyphae three days post inoculation of roots. co-expression of i-2 with avr2 dele ...201323596453
raba members act in distinct steps of subcellular trafficking of the flagellin sensing2 receptor.cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (pm) and ensuing signal transduction. intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. subcellular localization of arabidopsis thaliana flagellin sensing2 (fls2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. however, our understanding about th ...201323532067
theobroxide treatment inhibits wild fire disease occurrence in nicotiana benthamiana by the overexpression of defense-related genes.theobroxide, a novel compound isolated from a fungus lasiodiplodia theobromae, stimulates potato tuber formation and induces flowering of morning glory by initiating the jasmonic acid synthesis pathway. to elucidate the effect of theobroxide on pathogen resistance in plants, nicotiana benthamiana plants treated with theobroxide were immediately infiltrated with pseudomonas syringae pv. tabaci. exogenous application of theobroxide inhibited development of lesion symptoms, and growth of the bacter ...201325288936
coronatine inhibits stomatal closure and delays hypersensitive response cell death induced by nonhost bacterial pathogens.pseudomonas syringae is the most widespread bacterial pathogen in plants. several strains of p. syringae produce a phytotoxin, coronatine (cor), which acts as a jasmonic acid mimic and inhibits plant defense responses and contributes to disease symptom development. in this study, we found that cor inhibits early defense responses during nonhost disease resistance. stomatal closure induced by a nonhost pathogen, p. syringae pv. tabaci, was disrupted by cor in tomato epidermal peels. in addition, ...201323638370
pseudomonas syringae pv. tomato dc3000 cmal (pspto4723), a duf1330 family member, is needed to produce l-allo-isoleucine, a precursor for the phytotoxin coronatine.pseudomonas syringae pv. tomato dc3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with dc3000 pathogenesis. the dc3000 pspto4723 (cmal) gene is located in a genomic region encoding type iii effectors; however, it promotes chlorosis in the model plant nicotiana benthamiana in a manner independent of type iii secretion. coronatine is produced by the ligation of two moieties, coronafacic acid (cfa) and coronamic acid (cma), which are produced by biosynt ...201323144243
use of recombinant tobacco mosaic virus to achieve rna interference in plants against the citrus mealybug, planococcus citri (hemiptera: pseudococcidae).the citrus mealybug, planococcus citri, is an important plant pest with a very broad plant host range. p. citri is a phloem feeder and loss of plant vigor and stunting are characteristic symptoms induced on a range of host plants, but p. citri also reduces fruit quality and causes fruit drop leading to significant yield reductions. better strategies for managing this pest are greatly needed. rna interference (rnai) is an emerging tool for functional genomics studies and is being investigated as ...201324040013
expression and biological activity of the cystine knot bioinsecticide pa1b (pea albumin 1 subunit b).the pa1b (pea albumin 1, subunit b) peptide is an entomotoxin extract from legume seeds with lethal activity on several insect pests, such as mosquitoes, some aphids and cereal weevils. this 37 amino-acid cysteine-rich peptide has been, until now, obtained by biochemical purification or chemical synthesis. in this paper, we present our results for the transient production of the peptide in nicotiana benthamiana by agro-infiltration, with a yield of about 35 µg/g of fresh leaves and maximum produ ...201324349099
molecular evidence for the occurrence of abutilon mosaic virus, a new world begomovirus in india.during an investigation in the year 2010, on the weed reservoir of begomovirus, abutilon pictum showing bright yellow mosaic symptoms was observed in udhagamandalam, tamil nadu, india. the complete bipartite genome of a begomovirus was cloned and sequenced which revealed association of abutilon mosaic virus (abmv). nicotiana benthamiana plants inoculated biolistically with the concatemers generated through rolling circle amplification of the cloned dnas were asymptomatic; however three out of ni ...201324426288
a plant-produced pfs25 vlp malaria vaccine candidate induces persistent transmission blocking antibodies against plasmodium falciparum in immunized mice.malaria transmission blocking vaccines (tbvs) are considered an effective means to control and eventually eliminate malaria. the pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for tbv. it has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (vlps). we engineered ...201324260245
mutations in the capsid protein of brome mosaic virus affecting encapsidation eliminate vesicle induction in planta: implications for virus cell-to-cell spread.positive-strand rna viruses are known to rearrange the endomembrane network to make it more conducive for replication, maturation, or egress. our previous transmission electron microscopic (tem) analysis showed that ectopic expression of wild-type (wt) capsid protein (cp) of brome mosaic virus (bmv) has an intrinsic property of modifying the endoplasmic reticulum (er) to induce vesicles similar to those present in wt bmv infection. in this study, we evaluated the functional significance of cp-me ...201323741003
perception of the novel mamp emax from different xanthomonas species requires the arabidopsis receptor-like protein remax and the receptor kinase sobir.as part of their innate immune system plants carry a number of pattern recognition receptors (prrs) that can detect a broad range of microbe-associated molecular patterns (mamps). in a recently published article (1) we described a novel, proteinaceous mamp termed emax (enigmatic mamp of xanthomonas) that derives from xanthomonas and gets recognized by the receptor-like protein remax (receptor of emax) of arabidopsis thaliana. remax has no ortholog in nicotiana benthamiana and this species does n ...201324384530
biological activity of the tzs gene of nopaline agrobacterium tumefaciens gv3101 in plant regeneration and genetic transformation.agrobacterium tumefaciens has been widely used in plant genetic transformation. hormone-encoding genes residing in the t-dna region have been removed, resulting in disarmed agrobacterium strains that are used in various transformation experiments. nopaline agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. we investigated the activity and function of the tzs gene of a nop ...201324088018
construction of a sonchus yellow net virus minireplicon: a step toward reverse genetic analysis of plant negative-strand rna viruses.reverse genetic analyses of negative-strand rna (nsr) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant nsr viruses. to develop a reverse genetic approach for analysis of plant nsr viruses, we have engineered sonchus yellow net nucleorhabdovirus (synv) minireplicon (mr) reporter cassettes for agrobacterium tumefaciens expression in nicotiana benthamiana leaves. fluorescent reporter ...201323885070
a genetic system for citrus tristeza virus using the non-natural host nicotiana benthamiana: an update.in nature citrus tristeza virus (ctv), genus closterovirus, infects only the phloem cells of species of citrus and related genera. finding that the ctv t36 strain replicated in nicotiana benthamiana (nb) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with rna transcribed from a full-genome cdna clone, a laborious and uncertain system requiring several months for each experiment. we developed a more efficient system based o ...201323847598
a rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta.custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand dna breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dua ...201323625357
agroinoculation of a full-length cdna clone of cotton leafroll dwarf virus (clrdv) results in systemic infection in cotton and the model plant nicotiana benthamiana.cotton blue disease is the most important viral disease of cotton in the southern part of south america. its etiological agent, cotton leafroll dwarf virus (clrdv), is specifically transmitted to host plants by the aphid vector (aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. this limitation has held back the study of this virus and the disease it causes. in this study, a full-length cdna of clrdv was constructed and expressed in vivo un ...201323623981
identification of host proteins modulated by the virulence factor ac2 of tomato chlorotic mottle virus in nicotiana benthamiana.tomato, one of the most important crops cultivated worldwide, has been severely affected by begomoviruses such as the tomato chlorotic mottle virus (tocmov). virulence factor ac2 is considered crucial for a successful virus-plant interaction and is known to act as a transcriptional activator and in some begomoviruses to function as an rna silencing suppressor factor. however, the exact functions of the ac2 protein of the begomovirus tocmov are not yet established. the aim of the present study wa ...201323533094
establishment of an agroinoculation system for broad bean wilt virus 2.we determined the complete nucleotide sequence of a broad bean wilt virus 2 (bbwv-2) isolate from gentian in japan. the full-length rna1 and rna2 sequences, excluding poly(a) tails, were 5955 and 3600 nucleotides long, respectively. analysis indicated that, in contrast to other bbwv-2 isolates, the 5' end of both rna1 and rna2 starts with a guu sequence. we successfully inoculated nicotiana benthamiana with bbwv-2 by infiltrating a mixed suspension of two agrobacterium tumefaciens clones carryin ...201323404460
agroinoculation of beet necrotic yellow vein virus cdna clones results in plant systemic infection and efficient polymyxa betae transmission.agroinoculation is a quick and easy method for the infection of plants with viruses. this method involves the infiltration of tissue with a suspension of agrobacterium tumefaciens carrying binary plasmids harbouring full-length cdna copies of viral genome components. when transferred into host cells, transcription of the cdna produces rna copies of the viral genome that initiate infection. we produced full-length cdna corresponding to beet necrotic yellow vein virus (bnyvv) rnas and derived repl ...201323384276
tsw gene-based resistance is triggered by a functional rna silencing suppressor protein of the tomato spotted wilt virus.as a result of contradictory reports, the avirulence (avr) determinant that triggers tsw gene-based resistance in capsicum annuum against the tomato spotted wilt virus (tswv) is still unresolved. here, the n and nss genes of resistance-inducing (ri) and resistance-breaking (rb) isolates were cloned and transiently expressed in resistant capsicum plants to determine the identity of the avr protein. it was shown that the nss(ri) protein triggered a hypersensitive response (hr) in tsw-containing ca ...201323360130
in planta expression or delivery of potato aphid macrosiphum euphorbiae effectors me10 and me23 enhances aphid fecundity.the interactions between aphids and their host plants seem to be analogous to those of plant-microbial pathogens. unlike microbial pathogen effectors, little is known about aphid effectors and their ability to interfere with host immunity. to date, only three functional aphid effectors have been reported. to identify potato aphid (macrosiphum euphorbiae) effectors, we developed a salivary gland transcriptome using illumina technology. we generated 85 million illumina reads from salivary glands a ...201323194342
the production and characterization of a new active lipase from acremonium alcalophilum using a plant bioreactor.microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. as such, they present the most abundant source for discovery of new enzymes. acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. its optimal conditions for growth are 30°c and ph 9.0-9.2. the genome sequence of acremonium alcalophilum has revealed a large number of genes encoding biom ...201323915965
disruption of sphingolipid biosynthesis in nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to alternaria alternata f. sp. lycopersici.sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. in plants, recent evidence suggests that their metabolic precursors, the long-chain bases (lcbs) act as bioactive molecules in the immune response. interestingly, the virulence of two unrelated necrotrophic fungi, fusarium verticillioides and alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of s ...201322990908
insights into alternanthera mosaic virus tgb3 functions: interactions with nicotiana benthamiana psbo correlate with chloroplast vesiculation and veinal necrosis caused by tgb3 over-expression.alternanthera mosaic virus (altmv) triple gene block 3 (tgb3) protein is involved in viral movement. altmv tgb3 subcellular localization was previously shown to be distinct from that of potato virus x (pvx) tgb3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculation were observed in nicotiana benthamiana when altmv tgb3 was over-expressed from pvx. plants with over-expressed tgb3 showed more lethal damage under dark conditions than under light. yeast-two-hybri ...201323386854
deletions within the 3' non-translated region of alfalfa mosaic virus rna4 do not affect replication but significantly reduce long-distance movement of chimeric tobacco mosaic virus.alfalfa mosaic virus (almv) rnas 1 and 2 with deletions in their 3' non‑translated regions (ntrs) have been previously shown to be encapsidated into virions by coat protein (cp) expressed from rna3, indicating that the 3' ntrs of rnas 1 and 2 are not required for virion assembly. here, we constructed various mutants by deleting sequences within the 3' ntr of almv subgenomic (sg) rna4 (same as of rna3) and examined the effect of these deletions on replication and translation of chimeric tobacco m ...201323867804
molecular identification of ageratum enation virus, betasatellite and alphasatellite molecules isolated from yellow vein diseased amaranthus cruentus in india.natural occurrence of yellow vein disease on amaranthus cruentus was observed at lucknow, india in the year 2008. the causal virus was successfully transmitted through whiteflies (bemisia tabaci) from diseased a. cruentus to healthy seedlings of a. cruentus and other test species which indicated begomovirus infection. the begomovirus dna-a, betasatellite, and alphasatellite components associated with yellow vein disease were amplified by rolling circle amplification using ø-29 dna polymerase fro ...201323963765
dynamic localization of two tobamovirus orf6 proteins involves distinct organellar compartments.orf6 is a small gene that overlaps the movement and coat protein genes of subgroup 1a tobamoviruses. the orf6 protein of tomato mosaic virus (tomv) strain l (l-orf6), interacts in vitro with eukaryotic elongation factor 1α, and mutation of the orf6 gene of tobacco mosaic virus (tmv) strain u1 (u1-orf6) reduces the pathogenicity in vivo of tmv, whereas expression of this gene from two other viruses, tobacco rattle virus (trv) and potato virus x (pvx), increases their pathogenicity. in this work, ...201323052393
a putative rab-gtpase activation protein from nicotiana benthamiana is important for bamboo mosaic virus intercellular movement.the cdna-amplified fragment length polymorphism technique was applied to isolate the differentially expressed genes during bamboo mosaic virus (bamv) infection on nicotiana benthamiana plants. one of the upregulated genes was cloned and predicted to contain a tbc domain designated as nbrabgap1 (rab gtpase activation protein 1). no significant difference was observed in bamv accumulation in the nbrabgap1-knockdown and the control protoplasts. however, bamv accumulation was 50% and 2% in the inocu ...201324210126
chloroplast phosphoglycerate kinase is involved in the targeting of bamboo mosaic virus to chloroplasts in nicotiana benthamiana plants.the bamboo mosaic virus (bamv) is a positive-sense, single-stranded rna virus. previously, we identified that the chloroplast phosphoglycerate kinase (chl-pgk) from nicotiana benthamiana is one of the viral rna binding proteins involved in the bamv infection cycle. because chl-pgk is transported to the chloroplast, we hypothesized that chl-pgk might be involved in viral rna localization in the chloroplasts. to test this hypothesis, we constructed two green fluorescent protein (gfp)-fused misloca ...201324154620
maintaining the structural integrity of the bamboo mosaic virus 3' untranslated region is necessary for retaining the catalytic constant for minus-strand rna synthesis.bamboo mosaic virus (bamv) and the potato virus x (pvx) are members of the genus potexvirus and have a single-stranded positive-sense rna genome. the 3'-untranslated region (utr) of the bamv rna genome was mapped structurally into abc (a cloverleaf-like), d (a stem-loop), and e (pseudoknot) domains. the bamv replicase complex that was isolated from the infected plants was able to recognize the 3' utr of pvx rna to initiate minus-strand rna synthesis in vitro.201323800142
characterization of the polyadenylation activity in a replicase complex from bamboo mosaic virus-infected nicotiana benthamiana plants.bamboo mosaic virus (bamv) has a positive-sense single-stranded rna genome with a 5' cap and a 3' poly(a) tail. to characterize polyadenylation activity in the bamv replicase complex, we performed the in vitro polyadenylation with various bamv templates. we conducted a polyadenylation activity assay for bamv rna by using a partially purified bamv replicase complex. the results showed that approximately 200 adenylates at the 3' end of the rna were generated on the endogenous rna templates. specif ...201323768785
the glutathione transferase of nicotiana benthamiana nbgstu4 plays a role in regulating the early replication of bamboo mosaic virus.bamboo mosaic virus (bamv) is a single-stranded positive-sense rna virus. one of the plant glutathione s-transferase (gst) genes, nbgstu4, responds as an upregulated gene in nicotiana benthamiana post bamv infection. in order to identify the role of nbgstu4 in bamv infection, the expression of nbgstu4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in n. benthamiana in bamv inoculation. the results show a significant decrease in bamv rna accumulation ...201323701112
transgenic resistance to bamboo mosaic virus by expression of interfering satellite rna.plant genetic engineering has broadened the options for plant virus resistance and is mostly based on pathogen-derived resistance. previously, we have shown that interfering satellite rna (satrna) of bamboo mosaic virus (satbamv) greatly reduces bamboo mosaic virus (bamv) accumulation and bamv-induced symptoms in co-inoculated plants. here, we generated a nonviral source of virus-resistant transgenic nicotiana benthamiana and arabidopsis thaliana by introducing interfering satbamv. asymptomatic ...201323675895
ser/thr kinase-like protein of nicotiana benthamiana is involved in the cell-to-cell movement of bamboo mosaic virus.to investigate the plant genes affected by bamboo mosaic virus (bamv) infection, we applied a cdna-amplified fragment length polymorphism technique to screen genes with differential expression. a serine/threonine kinase-like (nbstkl) gene of nicotiana benthamiana is upregulated after bamv infection. nbstkl contains the homologous domain of ser/thr kinase. knocking down the expression of nbstkl by virus-induced gene silencing reduced the accumulation of bamv in the inoculated leaves but not in th ...201323646157
a plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with bacillus anthracis ames spores.the potential use of bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (pa), the principal virulence factor of b. anthracis. among the emerging technologies for the production of these vaccine antigens is our launc ...201323324615
expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides.psittacine beak and feather disease, caused by beak and feather disease virus (bfdv), is a threat to endangered psittacine species. there is currently no vaccine against bfdv, which necessitates the development of safe and affordable vaccine candidates. a subunit vaccine based on bfdv capsid protein (cp), the major antigenic determinant, expressed in the inexpensive and highly scalable plant expression system could satisfy these requirements. full-length cp and a truncated cp (δn40 cp) were tran ...201323578703
enhanced transgene expression in sugarcane by co-expression of virus-encoded rna silencing suppressors.post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. five plant viral suppressors of rna silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the enhanced yellow fluorescent protein (eyfp) or the β-glucuronidase (gus) reporter gene in sugarcane, a major sugar and biomass producing polyploid. functionality of these suppressors was first verified in n ...201323799071
actin cytoskeleton and golgi involvement in barley stripe mosaic virus movement and cell wall localization of triple gene block proteins.barley stripe mosaic virus (bsmv) induces massive actin filament thickening at the infection front of infected nicotiana benthamiana leaves. to determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the bsmv triple gene block (tgb) proteins were coexpressed in cells with the actin marker dsred: talin. tgb ectopic expression experiments revealed that tgb3 is a major elicitor of filament thickening, that tgb2 resulted in formation of intermediate dsred:talin filaments ...201325288925
the benyvirus rna silencing suppressor is essential for long-distance movement, requires both zinc-finger and nols basic residues but not a nucleolar localization for its silencing-suppression activity.the rna silencing-suppression properties of beet necrotic yellow vein virus (bnyvv) and beet soil-borne mosaic virus (bsbmv) cysteine-rich p14 proteins have been investigated. suppression of rna silencing activities were made evident using viral infection of silenced nicotiana benthamiana 16c, n. benthamiana agroinfiltrated with green fluorescent protein (gfp), and gf-fg hairpin triggers supplemented with viral suppressor of rna silencing (vsr) constructs or using complementation of a silencing- ...201323013437
intergeneric recombination between a new, spinach-infecting curtovirus and a new geminivirus belonging to the genus becurtovirus: first new world exemplar.a novel curtovirus, spinach severe curly top virus (ssctv), was associated with symptomatic spinach plants collected from a commercial field in south-central arizona during 2009. in addition, a second viral molecule of about 2.9 kb from the same spinach plants was amplified, cloned and sequenced. the latter isolate, herein named spinach curly top arizona virus (sctav), was found to share 77 % pairwise sequence identity with beet curly top iran virus (bctiv), a leafhopper-transmitted geminivirus ...201323708296
beet yellows virus replicase and replicative compartments: parallels with other rna viruses.in eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. virus-encoded subunits of the replication complex mediate its interaction with membranes. as replication platforms, rna viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (er), golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. closterovirus infections are accompanied by formation of multivesicular complexes from cell ...201323508802
botrytis cinerea mutants deficient in d-galacturonic acid catabolism have a perturbed virulence on nicotiana benthamiana and arabidopsis, but not on tomato.d-galacturonic acid is the most abundant monosaccharide component of pectic polysaccharides that comprise a significant part of most plant cell walls. therefore, it is potentially an important nutritional factor for botrytis cinerea when it grows in and through plant cell walls. the d-galacturonic acid catabolic pathway in b. cinerea consists of three catalytic steps converting d-galacturonic acid to pyruvate and l-glyceraldehyde, involving two nonhomologous galacturonate reductase genes (bcgar1 ...201322937823
a method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. they therefore have great potential for the production of heteromultimeric protein complexes. this work describes the efficient plant-based production and assembly of bluetongue virus-like particles (vlps), requiring the simultaneous expression of four distinct proteins in varying amounts. such particles have the potential to serve as a safe and ...201323647743
arabidopsis 3-ketoacyl-coenzyme a synthase9 is involved in the synthesis of tetracosanoic acids as precursors of cuticular waxes, suberins, sphingolipids, and phospholipids.very-long-chain fatty acids (vlcfas) with chain lengths from 20 to 34 carbons are involved in diverse biological functions such as membrane constituents, a surface barrier, and seed storage compounds. the first step in vlcfa biosynthesis is the condensation of two carbons to an acyl-coenzyme a, which is catalyzed by 3-ketoacyl-coenzyme a synthase (kcs). in this study, amino acid sequence homology and the messenger rna expression patterns of 21 arabidopsis (arabidopsis thaliana) kcss were compare ...201323585652
enhancement of rna-directed dna methylation of a transgene by simultaneously downregulating a ros1 ortholog using a virus vector in nicotiana benthamiana.cytosine methylation can be induced by double-stranded rnas through the rna-directed dna methylation (rddm) pathway. a dna glycosylase repressor of silencing 1 (ros1) participates in dna demethylation in arabidopsis and may possibly counteract rddm. here, we isolated an ortholog of ros1 (nbros1) from nicotiana benthamiana and examined the antagonistic activity of nbros1 against virus-induced rddm by simultaneously inducing rddm and nbros1 knockdown using a vector based on cucumber mosaic virus. ...201323565118
construction of an infectious cdna clone of radish mosaic virus, a crucifer-infecting comovirus.radish mosaic virus (ramv) is a crucifer-infecting comovirus that has been detected worldwide. here, we report the successful construction of a full-length infectious cdna clone of ramv. the full-length cdna clones corresponding to rna1 and rna2 of a japanese isolate of ramv were cloned into the pbluescript plasmid or the binary vector pcambia1301 downstream of the cauliflower mosaic virus 35s promoter. mechanical inoculation or agroinoculation of nicotiana benthamiana with these vectors resulte ...201323447094
construction and biological activities of the first infectious cdna clones of the genus foveavirus.grapevine rupestris stem pitting-associated virus (grspav, genus foveavirus, family betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and syrah decline. little is known about the biology and pathological properties of grspav. in this work, we engineered a full-length infectious cdna clone for grspav and a gfp-tagged variant, both under the transcriptional control of cauliflower mosaic virus 3 ...201323099206
analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps.mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. the sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide r ...201323439009
virus-induced gene silencing using artificial mirnas in nicotiana benthamiana.virus-induced gene silencing using artificial micrornas (mir vigs) is a newly developed technique for plant reverse genetic studies. traditional virus-induced gene silencing (vigs) assays introduce a large gene fragment, which is expressed and then converted into small rnas by the endogenous sirna-based gene silencing machinery of the plant host. by contrast, mir vigs uses well-designed mirnas to induce rna-mediated silencing of the target gene. using a single artificial mirna can provide greate ...201323386298
tombusvirus y-shaped translational enhancer forms a complex with eif4f and can be functionally replaced by heterologous translational enhancers.certain plus-strand rna plant viruses that are uncapped and nonpolyadenylated rely on rna elements in their 3' untranslated region, termed 3'-cap-independent translational enhancers (3'cites), for efficient translation of their proteins. here, we have investigated the properties of the y-shaped class of 3'cite present in the tombusvirus carnation italian ringspot virus (cirv). while some types of 3'cite have been found to function through recruitment of translation initiation factors to the vira ...201323192876
citrus leaf blotch virus invades meristematic regions in nicotiana benthamiana and citrus.to invade systemically host plants, viruses need to replicate in the infected cells, spread to neighbouring cells through plasmodesmata and move to distal parts of the plant via sieve tubes to start new infection foci. to monitor the infection of nicotiana benthamiana plants by citrus leaf blotch virus (clbv), leaves were agroinoculated with an infectious cdna clone of the clbv genomic rna expressing green fluorescent protein (gfp) under the transcriptional control of a duplicate promoter of the ...201323560714
characterization of the complete genome of a novel citrivirus infecting actinidia chinensis.a ssrna virus from kiwifruit (actinidia spp.) was identified as a member of the family betaflexiviridae. it was mechanically transmitted to the herbaceous indicators nicotiana benthamiana, n. clevelandii, n. glutinosa and n. occidentalis. the complete genome was comprised of three orfs and a 3'poly (a) tail. phylogenetic analysis of the entire genome indicated it was a novel member of the genus citrivirus (family betaflexiviridae). the complete nucleotide sequence differed from that of citrus le ...201323494225
root-specific role for nicotiana benthamiana rdr6 in the inhibition of chinese wheat mosaic virus accumulation at higher temperatures.some viruses only infect plants at cool temperatures but the molecular mechanism underlying this low-temperature dependence remains unclear. chinese wheat mosaic virus (cwmv, genus furovirus) was able to infect wheat and nicotiana benthamiana plants at 16 but not at 24°c. when cwmv-infected plants were transferred to 24°c for 2 weeks, the newly emerged leaves and roots became virus free. co-infection with potato virus y rescued cwmv accumulation in n. benthamiana plants after a temperature shift ...201323777430
ophioviruses cpsv and milbvv movement protein is encoded in rna 2 and interacts with the coat protein.citrus psorosis virus (cpsv) and mirafiori lettuce big-vein virus (milbvv), members of the ophioviridae family, have segmented negative-sense single-stranded rna genomes. to date no reports have described how ophioviruses spread within host plants and/or the proteins involved in this process. here we show that the 54k protein of cpsv is encoded by rna 2 and describe its subcellular distribution. upon transient expression in nicotiana benthamiana epidermal cells the 54k protein, and also its 54k ...201323602594
citrus tristeza virus p23: determinants for nucleolar localization and their influence on suppression of rna silencing and pathogenesis.citrus tristeza virus (ctv) encodes a singular protein (p23, 209 amino acids) with multiple functions, including rna silencing suppression (rss). confocal laser-scanning microscopy of green fluorescent protein (gfp)-p23 agroexpressed in nicotiana benthamiana revealed its accumulation in the nucleolus, cajal bodies, and plasmodesmata. to dissect the nucleolar localization signal (nols) typically associated with basic motifs, seven truncated and 10 point-mutated versions of p23 were assayed. delet ...201323387469
self-interaction of the cucumber mosaic virus 2b protein plays a vital role in the suppression of rna silencing and the induction of viral symptoms.the cucumber mosaic virus (cmv) 2b protein is an rna silencing suppressor protein that can also play direct and indirect roles in symptom induction. previous work has shown that a hybrid virus, frad35(2b) -cmv (renamed here as cmv-frad2b-pro), generated by replacement of the 2b gene of strain fny-cmv with that from rad35-cmv, displays markedly lower pathogenicity than fny-cmv on nicotiana species. however, the replacement of proline with leucine at position 55 of the 2b protein of cmv-frad2b-pro ...201323782515
influence of host chloroplast proteins on tobacco mosaic virus accumulation and intercellular movement.tobacco mosaic virus (tmv) forms dense cytoplasmic bodies containing replication-associated proteins (virus replication complexes [vrcs]) upon infection. to identify host proteins that interact with individual viral components of vrcs or vrcs in toto, we isolated viral replicase- and vrc-enriched fractions from tmv-infected nicotiana tabacum plants. two host proteins in enriched fractions, atp-synthase γ-subunit (atpc) and rubisco activase (rca) were identified by matrix-assisted laser-desorptio ...201323096159
long-distance transport of gibberellic acid insensitive mrna in nicotiana benthamiana.the gibberellic acid (ga) signal is governed by the gai (gibberellic acid insensitive) repressor, which is characterized by a highly conserved n-terminal della domain. deletion of the della domain results in constitutive suppression of ga signaling. as the gai transcript is transportable in phloem elements, a δ-della gai (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mrna from the stock. however, little is known about the characteristics of a scion on a g ...201324144190
gene-for-gene relationship in the host-pathogen system malus × robusta 5-erwinia amylovora.fire blight is a destructive bacterial disease caused by erwinia amylovora affecting plants in the family rosaceae, including apple. host resistance to fire blight is present mainly in accessions of malus spp. and is thought to be quantitative in this pathosystem. in this study we analyzed the importance of the e. amylovora effector avrrpt2(ea) , a homolog of pseudomonas syringae avrrpt2, for resistance of malus × robusta 5 (mr5). the deletion mutant e. amylovora ea1189δavrrpt2(ea) was able to o ...201323301854
targeting of envelope domain iii protein of denv type 2 to dec-205 receptor elicits neutralizing antibodies in mice.dengue virus (denv) is the causal agent of severe disease and, in some cases, mortality in humans, but no licensed vaccines against dengue are available. an effective vaccine against dengue requires long-term humoral and cellular immune responses. several viral proteins have been the subjects of intense research, especially the envelope (e) protein, aimed at developing a vaccine. domain iii of the envelope protein (ediii) has been identified as a potential candidate because it is involved in bin ...201323499580
high-level diterpene production by transient expression in nicotiana benthamiana.characterization of plant terpene synthases is typically done by production of recombinant enzymes in escherichia coli. this is often difficult due to solubility and codon usage issues. furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. we report here an optimized agrobacterium-mediated transient expression assay in nicotiana benthamiana for plant diterpene synthase ...201324330621
characterization of two geraniol synthases from valeriana officinalis and lippia dulcis: similar activity but difference in subcellular localization.two geraniol synthases (ges), from valeriana officinalis (voges) and lippia dulcis (ldges), were isolated and were shown to have geraniol biosynthetic activity with km values of 32 µm and 51 µm for gpp, respectively, upon expression in escherichia coli. the in planta enzymatic activity and sub-cellular localization of voges and ldges were characterized in stable transformed tobacco and using transient expression in nicotiana benthamiana. transgenic tobacco expressing voges or ldges accumulate ge ...201324060453
molecular characterization of nbeh1 and nbeh2, two epoxide hydrolases from nicotiana benthamiana.plant epoxide hydrolases (eh) form two major clades, named eh1 and eh2. to gain a better understanding of the biochemical roles of the two classes, nbeh1.1 and nbeh2.1 were isolated from nicotiana benthamiana and steh from potato and heterologously expressed in escherichia coli. the purified recombinant proteins were assayed with a variety of substrates. nbeh1.1 only accepted some aromatic epoxides, and displayed the highest enzyme activity towards phenyl glycidyl ether. in contrast, nbeh2.1 dis ...201323562372
sce1, the sumo-conjugating enzyme in plants that interacts with nib, the rna-dependent rna polymerase of turnip mosaic virus, is required for viral infection.sumoylation, which is catalyzed by small ubiquitin-like modifier (sumo) enzymes, is a transient, reversible posttranslational protein modification that regulates diverse cellular processes. potyviruses, the largest group of known plant viruses, comprise many agriculturally important viruses, such as turnip mosaic virus (tumv). the potyviral genome encodes 11 mature proteins. to investigate if sumoylation plays a role in potyvirus infection, a yeast two-hybrid screen was performed to examine poss ...201323365455
production of a chimeric allergen derived from the major allergen group 1 of house dust mite species in nicotiana benthamiana.plants are widely accepted as a general platform for the large-scale production of recombinant proteins, which has been demonstrated by the successful expression of various exogenous proteins. using plants as a bioreactor for mass production of target proteins for vaccines is thought to show the most potential. this study explores whether a chimeric allergen r8, derived from the major allergen group 1 of house dust mites species (dermatophagoides farinae and dermatophagoides pteronyssinus), is e ...201323354320
specificity of ocimum basilicum geraniol synthase modified by its expression in different heterologous systems.numerous aromatic plant species produce high levels of monoterpenols, using geranyl diphosphate (gpp) as a precursor. sweet basil (ocimum basilicum) geraniol synthase (ges) was used to evaluate the monoterpenol profiles arising from heterologous expressions in various plant models. grapevine (vitis vinifera) calli were transformed using agrobacterium tumefasciens and the plants were regenerated. thale cress (arabidopsis thaliana) was transformed using the floral dip method. tobacco (nicotiana be ...201323108028
the novel grcep12 peptide from the plant-parasitic nematode globodera rostochiensis suppresses flg22-mediated pti.the potato cyst nematode globodera rostochiensis is a biotrophic pathogen that secretes effector proteins into host root cells to promote successful plant parasitism. in addition to the role in generating within root tissue the feeding cells essential for nematode development, (1) nematode secreted effectors are becoming recognized as suppressors of plant immunity. (2)(-) (4) recently we reported that the effector ubiquitin carboxyl extension protein (grubcep12) from g. rostochiensis is processe ...201323803745
a ubiquitin carboxyl extension protein secreted from a plant-parasitic nematode globodera rostochiensis is cleaved in planta to promote plant parasitism.nematode effector proteins originating from esophageal gland cells play central roles in suppressing plant defenses and in formation of the plant feeding cells that are required for growth and development of cyst nematodes. a gene (grubcep12) encoding a unique ubiquitin carboxyl extension protein (ubcep) that consists of a signal peptide for secretion, a mono-ubiquitin domain, and a 12 amino acid carboxyl extension protein (cep12) domain was cloned from the potato cyst nematode globodera rostoch ...201323346875
a strain-specific segment of the rna-dependent rna polymerase of grapevine fanleaf virus determines symptoms in nicotiana species.factors involved in symptom expression of viruses from the genus nepovirus in the family secoviridae such as grapevine fanleaf virus (gflv) are poorly characterized. to identify symptom determinants encoded by gflv, infectious cdna clones of rna1 and rna2 of strain ghu were developed and used alongside existing infectious cdna clones of strain f13 in a reverse genetics approach. in vitro transcripts of homologous combinations of rna1 and rna2 induced systemic infection in nicotiana benthamiana a ...201324088345
a new virus-induced gene silencing vector based on euphorbia mosaic virus-yucatan peninsula for npr1 silencing in nicotiana benthamiana and capsicum annuum var. anaheim.virus-induced gene silencing is based on the sequence-specific degradation of rna. here, a gene silencing vector derived from eumv-yp, named peumv-yp:δav1, was used to silence chli and npr1 genes in nicotiana benthamiana. the silencing of the chli transcripts was efficient in the stems, petioles and leaves as reflected in tissue bleaching and reduced transcript levels. the silencing was stable, reaching the flowers and fruits, and was observed throughout the life cycle of the plants. additionall ...201323546940
the triple gene block movement proteins of a grape virus in the genus foveavirus confer limited cell-to-cell spread of a mutant potato virus x.grapevine rupestris stem pitting-associated virus (grspav) is a member of the genus foveavirus in the family betaflexiviridae. the genome of grspav encodes five proteins, among which are three movement proteins designated the triple gene block (tgb) proteins. the tgb proteins of grspav are highly similar to their counterparts in potato virus x (pvx), as reflected in size, modular structure, conservation of critical amino acid sequence motifs, as well as similar cellular localization. based on th ...201323543158
immunogenicity and protective efficacy of candidate universal influenza a nanovaccines produced in plants by tobacco mosaic virus-based vectors.a new approach for super-expression of the influenza virus epitope m2e in plants has been developed on the basis of a recombinant tobacco mosaic virus (tmv, strain u1) genome designed for agrobacterium-mediated delivery into the plant cell nucleus. the tmv coat protein (cp) served as a carrier and three versions of the m2e sequence were inserted into the surface loop between amino acid residues 155 and 156. cysteine residues in the heterologous peptide were thought likely to impede efficient ass ...201323394564
a plant-produced h1n1 trimeric hemagglutinin protects mice from a lethal influenza virus challenge.the increased worldwide awareness of seasonal and pandemic influenza, including pandemic h1n1 virus, has stimulated interest in the development of economic platforms for rapid, large-scale production of safe and effective subunit vaccines. in recent years, plants have demonstrated their utility as such a platform and have been used to produce vaccine antigens against various infectious diseases. previously, we have produced in our transient plant expression system a recombinant monomeric hemaggl ...201323296194
biological and biochemical characterization of hiv-1 gag/dgp41 virus-like particles expressed in nicotiana benthamiana.the transmembrane hiv-1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of cd4⁺ cells. gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. we hypothesized that hiv enveloped virus-like particles (vlps) consisting of gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplas ...201323506331
immunogenic assessment of plant-produced human papillomavirus type 16 l1/l2 chimaeras.cervical cancer is caused by infection with human papillomaviruses (hpv) and is a global concern, particularly in developing countries, which have ~80% of the burden. hpv l1 virus-like particle (vlp) type-restricted vaccines prevent new infections and associated disease. however, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. thus, there is an urgent need for cheap second-gen ...201323924054
transient expression of vp2 in nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus.infectious bursal disease virus (ibdv) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. this disease causes important economic losses in the poultry industry worldwide. the vp2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. in this context, the aim of this study was to investigate vp2 expression and immunogenicity using an experimental plant-based vaccine against ibdv. we determined tha ...201323583894
mungbean yellow mosaic virus (mymv) ac4 suppresses post-transcriptional gene silencing and an ac4 hairpin rna gene reduces mymv dna accumulation in transgenic tobacco.mungbean yellow mosaic virus (mymv) is a legume-infecting geminivirus that causes yellow mosaic disease in blackgram, mungbean, soybean, frenchbean and mothbean. ac4/c4, which is nested completely within the rep gene, is less conserved among geminiviruses. much less is known about its role in viral pathogenesis other than its known role in the suppression of host-mediated gene silencing. transient expression of mymv ac4 by agroinfiltration suppressed post-transcriptional gene silencing in nicoti ...201323417222
identification of a movement protein of mirafiori lettuce big-vein ophiovirus.mirafiori lettuce big-vein virus (milbvv) is a member of the genus ophiovirus, which is a segmented negative-stranded rna virus. in microprojectile bombardment experiments to identify a movement protein (mp) gene of ophioviruses that can trans-complement intercellular movement of an mp-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (tomv) derivative expressing the gfp was co-bombarded with plasmids containing one of three genes from milbvv rnas 1, ...201323364193
development of apple latent spherical virus-based vaccines against three tospoviruses.apple latent spherical virus (alsv) is characterized by its relatively broad host range, latency in most host plants, and ability to induce gene silencing in host plants. herein, we focus on the above characteristic of alsv and describe our development of alsv vector vaccines against three tospoviruses, namely, impatiens necrotic spot virus (insv), iris yellow spot virus (iysv), and tomato spotted wilt virus (tswv). dna fragments of 201 nt of three tospovirus s-rnas (silencing suppressor (nss) a ...201323850843
expression and assembly of norwalk virus-like particles in plants using a viral rna silencing suppressor gene.binary vector-based transient expression of heterologous proteins in plants is a very attractive strategy due to the short time required for proceeding from planning to expression. however, this expression system is limited by comparatively lower yields due to strong post-transcriptional gene silencing (ptgs) in the host plants. the aim of this study was to optimize a procedure for expression of norovirus virus-like particles (vlps) in plants using a binary vector with co-expression of a ptgs su ...201323925532
how can plant virus satellite rnas alter the effects of plant virus infection? a study of the changes in the nicotiana benthamiana proteome after infection by peanut stunt virus in the presence or absence of its satellite rna.peanut stunt virus (psv), which belongs to the cucumovirus genus, is a pathogen of legumes. certain psv strains associated with a satellite rna (satrna) modify the symptoms of infected plants and interfere with plant metabolism. we used psv-p genomic transcripts (gts) with and without psv-p satrna and a comparative proteomic 2d-dige/ms study to assess their effects on nicotiana benthamiana infection. when the proteomes of the psv-p genomic transcripts-infected (no satrna present) and mock-inocul ...201323580405
the endophytic lifestyle of escherichia coli o157:h7: quantification and internal localization in roots.the foodborne pathogen escherichia coli o157:h7 is increasingly associated with fresh produce (fruit and vegetables). bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. however, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. we have determined the extent of internalization of e. coli o157:h7 in live spinach and lettuce plants and ...201323506361
consequences of flagellin export through the type iii secretion system of pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant nicotiana benthamiana.bacterial flagellin is perceived as a microbe (or pathogen)-associated molecular pattern (mamp or pamp) by the extracellular pattern recognition receptors, fls2 and tlr5, of plants and mammals respectively. flagellin accidently translocated into mammalian cells by pathogen type iii secretion systems (t3sss) is recognized by nucleotide-binding leucine-rich repeat receptor nlrc4 as a pattern of pathogenesis and induces a death-associated immune response. the non-pathogen pseudomonas fluorescens pf ...201323107228
plant-based production of recombinant plasmodium surface protein pf38 and evaluation of its potential as a vaccine candidate.pf38 is a surface protein of the malarial parasite plasmodium falciparum. in this study, we produced and purified recombinant pf38 and a fusion protein composed of red fluorescent protein and pf38 (rfp-pf38) using a transient expression system in the plant nicotiana benthamiana. to our knowledge, this is the first description of the production of recombinant pf38. to verify the quality of the recombinant pf38, plasma from semi-immune african donors was used to confirm specific binding to pf38. e ...201324278216
effects and effectiveness of two rnai constructs for resistance to pepper golden mosaic virus in nicotiana benthamiana plants.tochlpv and pepgmv are begomoviruses that have adapted to a wide host range and are able to cause major diseases in agronomic crops. we analyzed the efficacy of induced resistance to pepgmv in nicotiana benthamiana plants with two constructs: one construct with homologous sequences derived from pepgmv, and the other construct with heterologous sequences derived from tochlpv. plants protected with the heterologous construct showed an efficacy to decrease the severity of symptoms of 45%, while pla ...201324287597
plum pox virus accumulates mutations in different genome parts during a long-term maintenance in prunus host plants and passage in nicotiana benthamiana.plum pox virus (ppv) isolates of the strain ppv-m prevalently infect peaches under natural conditions in middle europe. comparison of complete genome sequences obtained from subisolates of a ppv-m isolate maintained experimentally over a 6-year period in different prunus host species and passaged in nicotiana benthamiana was performed with the aim to highlight the mutations potentially connected with the virus-host adaptation. the results showed that the lowest number of non-silent mutations was ...201324020764
o-glcnac modification of the coat protein of the potyvirus plum pox virus enhances viral infection.o-glcnacylation is a dynamic protein modification which has been studied mainly in metazoans. we reported previously that an arabidopsis thaliana o-glcnac transferase modifies at least two threonine residues of the plum pox virus (ppv) capsid protein (cp). now, six additional residues were shown to be involved in o-glcnac modification of ppv cp. cp o-glcnacylation was abolished in the ppv cp7-t/a mutant, in which seven threonines were mutated. ppv cp7-t/a infected nicotiana clevelandii, nicotian ...201323639873
scion on a stock producing sirnas of potato spindle tuber viroid (pstvd) attenuates accumulation of the viroid.plants can attenuate the replication of plant viruses and viroids by rna silencing induced by virus and viroid infection. in higher plants, silencing signals such as small interfering rnas (sirnas) produced by rna silencing can be transported systemically through phloem, so it is anticipated that antiviral sirna signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. to test whether this is indeed the case, we prepared transgenic tobacco ...201323469061
dicer-like 4 but not dicer-like 2 may have a positive effect on potato spindle tuber viroid accumulation in nicotiana benthamiana. 201323100483
improved silencing suppression and enhanced heterologous protein expression are achieved using an engineered viral helper component proteinase.rna silencing limits transient expression of heterologous proteins in plants. co-expression of viral silencing suppressor proteins can increase and prolong protein expression, but highly efficient silencing suppressors may stress plant tissue and be detrimental to protein yields. little is known whether silencing suppression could be improved without harm to plant tissues. this study reports development of enhanced silencing suppressors by engineering the helper component proteinase (hcpro) of p ...201323933077
interaction of the microtubule-associated host protein hip2 with viral helper component proteinase is important in infection with potato virus a.microtubules (mt) outline and maintain the overall shape of cells and can reorganize cellular membranes to serve as sites of rna virus replication. here, we provide data on involvement of an mt-associated protein in infection of plants with a potyvirus, potato virus a (pva), representing the largest family of plant-infecting rna viruses. our results showed that helper-component proteinase (hcpro)-interacting protein 2 (hip2) of potato (solanum tuberosum) is an mt-associated protein similar to ar ...201323489059
rice yellow stunt rhabdovirus protein 6 suppresses systemic rna silencing by blocking rdr6-mediated secondary sirna synthesis.the p6 protein of rice yellow stunt rhabdovirus (rysv) is a virion structural protein that can be phosphorylated in vitro. however its exact function remains elusive. we found that p6 enhanced the virulence of potato virus x (pvx) in nicotiana benthamiana and n. tabacum plants, suggesting that it might function as a suppressor of rna silencing. we examined the mechanism of p6-mediated silencing suppression by transiently expressing p6 in both n. benthamiana leaves and rice protoplasts. our resul ...201323634838
differential requirements for tombusvirus coat protein and p19 in plants following leaf versus root inoculation.traditional virus inoculation of plants involves mechanical rubbing of leaves, whereas in nature viruses like tomato bushy stunt virus (tbsv) are often infected via the roots. a method was adapted to compare leaf versus root inoculation of nicotiana benthamiana and tomato with transcripts of wild-type tbsv (wttbsv), a capsid (tcp) replacement construct expressing gfp (t-gfp), or mutants not expressing the silencing suppressor p19 (tbsvδp19). in leaves, t-gfp remained restricted to the cells imme ...201323490050
molecular characterization of the interaction between the n-terminal region of potato virus x (pvx) coat protein (cp) and nicotiana benthamiana pvx cp-interacting protein, nbpcip1.using yeast two-hybrid assays and a nicotiana benthamiana cdna library, we previously identified an n. benthamiana protein, nbpcip1, that interacts with potato virus x (pvx) coat protein (cp). we also previously determined that nbpcip1 enhances pvx replication in plants. to determine the domains and/or amino acid residues required for pvx cp and nbpcip1 interaction, here we used yeast two-hybrid and β-galactosidase filter assays to test the effects of deletion and site-directed mutations on the ...201323463175
tgbp3 triggers the unfolded protein response and skp1-dependent programmed cell death.the potato virus x (pvx) triple gene block protein 3 (tgbp3), an 8-kda membrane binding protein, aids virus movement and induces the unfolded protein response (upr) during pvx infection. tgbp3 was expressed from the tobacco mosaic virus (tmv) genome (tmv-p3), and we noted the up-regulation of skp1 and several endoplasmic reticulum (er)-resident chaperones, including the er luminal binding protein (bip), protein disulphide isomerase (pdi), calreticulin (crt) and calmodulin (cam). local lesions we ...201323458484
production of a de-novo designed antimicrobial peptide in nicotiana benthamiana.antimicrobial peptides are important defense compounds of higher organisms that can be used as therapeutic agents against bacterial and/or viral infections. we designed several antimicrobial peptides containing hydrophobic and positively charged clusters that are active against plant and human pathogens. especially peptide sp1-1 is highly active with a mic value of 0.1 μg/ml against xanthomonas vesicatoria, pseudomonas corrugata and pseudomonas syringae pv syringae. however, for commercial appli ...201323242916
a translationally controlled tumor protein negatively regulates the hypersensitive response in nicotiana benthamiana.we have been isolating and characterizing ralstonia solanacearum-responsive genes (rsrgs) in nicotiana plants. in this study we focused on rsrg308, which we renamed nbtctp (n. benthamiana translationally controlled tumor protein) because it encodes a polypeptide showing similarity to translationally controlled tumor proteins. induction of the hypersensitive response (hr) was accelerated in nbtctp-silenced n. benthamiana plants challenged with r. solanacearum 8107 (rs8107). the rs8107 population ...201323788648
in planta effector competition assays detect hyaloperonospora arabidopsidis effectors that contribute to virulence and localize to different plant subcellular compartments.the genome of the pathogenic oomycete hyaloperonospora arabidopsidis is predicted to encode at least 134 high-confidence effectors (harxl) carrying the rxlr motif implicated in their translocation into plant cells. however, only four avirulence genes (atr1, atr13, atr5, and atr39) have been isolated. this indicates that identification of harxl effectors based on avirulence is low throughput. we aimed at rapidly identifying h. arabidopsidis effectors that contribute to virulence by developing met ...201323734779
the phytophthora parasitica rxlr effector penetration-specific effector 1 favours arabidopsis thaliana infection by interfering with auxin physiology.pathogenic oomycetes have evolved rxlr effectors to thwart plant defense mechanisms and invade host tissues. we analysed the function of one of these effectors (penetration-specific effector 1 (pse1)) whose transcript is transiently accumulated during penetration of host roots by the oomycete phytophthora parasitica. expression of pse1 protein in tobacco (nicotiana tabacum and nicotiana benthamiana) leaves and in arabidopsis thaliana plants was used to assess the role of this effector in plant p ...201323594295
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