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molecular dissection of flock house virus protein b2 reveals that electrostatic interactions between n-terminal domains of b2 monomers are critical for dimerization.flock house virus (fhv) encodes a suppressor protein b2 to overcome antiviral rna silencing during infection. biochemical analyses have shown that a homodimer of b2 binds to double-stranded rna to inhibit dicer-mediated cleavage of dsrna and incorporation of small interfering rnas into the rna-induced silencing complex. in this study, using fhv-nicotiana benthamiana system, we identified that the charged amino acids at the n-terminus of b2 are critical for dimerization. interestingly, b2 mutants ...201222721960
functional expression and characterization of sesquiterpene synthases from artemisia annua l. using transient expression system in nicotiana benthamiana.artemisia annua l. produces a number of sesquiterpene synthases, which catalyze the conversion of farnesyl diphosphate to various sesquiterpenes. the cdnas encoding amorpha-4,11-diene synthase (ads), a key enzyme in the artemisinin biosynthesis, and epi-cedrol synthase (ecs), a complex sesquiterpene cyclization synthase, were cloned into cowpea mosaic virus-based viral vector (peaq-ht) with kozak consensus motif and c-terminal histidine tag. the plasmids were transformed into agrobacterium lba44 ...201222565787
cloned tomato golden mosaic virus back in tomatoes.clones of tomato golden mosaic virus (tgmv), a key model for geminivirus research, have been transmitted back to their original host tomato for the first time. in contrast to the high pathogenicity in other solanaceous species, tgmv induced only very mild symptoms: a few chlorotic spots on the leaf lamina for the common variant (formerly strain cs), and limited vein yellowing for the yellow vein variant (yv). symptoms disappeared over time, though viral dna remained detectable in newly developed ...201222677771
identification of tobacco proteins associated with the stem-loop 1 rnas of potato virus x.potato virus x (pvx) contains five viral proteins as well as cis-acting elements like stem-loop 1 (sl1) rnas at the 5' region. sl1 rnas are involved in pvx rna replication, encapsidation, translation, and cell-to-cell movement. in this study, we performed two-dimensional electrophoresis northwestern blot analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry and identified 24 tobacco proteins that interact with sl1 rnas. interestingly, one-third of the identifi ...201222450689
construction of an infectious cdna clone and gene expression vector of tobacco vein banding mosaic virus (genus potyvirus).tobacco vein banding mosaic virus (tvbmv, genus potyvirus) mainly infects solanaceous plants and is of increasing economic importance in china. here, we report sequence determination of the full-length 5'-untranslated region of tvbmv isolate hn39 and construction of an infectious clone. the resultant clone, ptvbmv, which was stabilized by introducing three introns in the p3 and ci-encoding regions, induced similar disease symptoms and accumulated similar titers of virus in plants of nicotiana be ...201222820405
a new viral vector exploiting rna polymerase i-mediated transcription.we have developed a new viral vector system exploiting rna-polymerase i transcription. the vector is based on the crucifer-infecting tobacco mosaic virus (crtmv) cdna inserted into the rrna transcriptional cassette (promoter and terminator). to visualize reproduction of the vector, the coat protein gene was replaced with the gene encoding green fluorescent protein (gfp) resulting in a pr(rrna)-crtmv-gfp construct. our results showed that agroinjection of nicotiana benthamiana leaves with this ve ...201222813595
a game-theoretic model of interactions between hibiscus latent singapore virus and tobacco mosaic virus.mixed virus infections in plants are common in nature and their interactions affecting host plants would depend mainly on plant species, virus strains, the order of infection and initial amount of inoculum. hence, the prediction of outcome of virus competition in plants is not easy. in this study, we applied evolutionary game theory to model the interactions between hibiscus latent singapore virus (hlsv) and tobacco mosaic virus (tmv) in nicotiana benthamiana under co-infection in a plant host. ...201222623970
plasmodesmata formation and cell-to-cell transport are reduced in decreased size exclusion limit 1 during embryogenesis in arabidopsis.in plants, plasmodesmata (pd) serve as channels for micromolecular and macromolecular cell-to-cell transport. based on structure, pd in immature tissues are classified into two types, simple and branched (x- and y-shaped) or twinned. the maximum size of molecules capable of pd transport defines pd aperture, known as the pd size exclusion limit. here we report an arabidopsis mutation, decreased size exclusion limit1 (dse1), that exhibits reduced cell-to-cell transport of the small (524 da) fluore ...201222411811
reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata.maize and arabidopsis thaliana class 1 reversibly glycosylated polypeptides (c1rgps) are plasmodesmata-associated proteins. previously, over-expression of arabidopsis c1rgp atrgp2 in nicotiana tabacum was shown to reduce intercellular transport of photoassimilate, resulting in stunted, chlorotic plants, and inhibition of local cell-to-cell spread of tobacco mosaic virus (tmv). here, we used virus induced gene silencing to examine the effects of reduced levels of c1rgps in nicotiana benthamiana. ...201222274744
sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species.virus-induced gene silencing (vigs) is a robust technique for identifying the functions of plant genes. tobacco rattle virus (trv)-mediated vigs has been commonly used in many plants. in order to overcome the limitations of existing agroinoculation methods, we report an easy and effective method of agroinoculation for virus-induced gene silencing-sprout vacuum-infiltration (svi). using sprout vacuum-infiltration, we have successfully silenced the expression of phytoene desaturase and mg-protopor ...201222717672
tobacco rattle virus (trv)-based virus-induced gene silencing.one of the most effective forms of plant defense against viruses is posttranscriptional gene silencing (ptgs). this process implies that a virus can trigger the host plant's rna-silencing machinery and ultimately become the target of rna silencing. ptgs is, therefore, an attractive endogenous process that can be exploited to study gene function. one of the most efficient approaches of initiating ptgs is through virus-induced gene silencing (vigs). when a recombinant viral vector (vigs vector), c ...201222678574
lectin-mediated resistance impairs plant virus infection at the cellular level.plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. this study analyzed the resistance of certain arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (plamv). map-based positional cloning revealed that the lectin gene jacalin-type lectin required for p ...201222307853
sgt1 is induced by the potato virus x tgbp3 and enhances virus accumulation in nicotiana benthamiana. 201222461666
construction of effective inverted repeat silencing constructs using sodium bisulfite treatment coupled with strand-specific pcr.rna silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (ptgs). in some cases, this technology is difficult to apply due to the instability of inverted repeat (ir) constructs during cloning and plant transformation. although such constructs have been shown to be stabilized with introns and efficiently induce rna silencing, we found that the pdk intron did not stabilize south african cassava mosaic virus (sacmv) silen ...201222482441
engineering of glucosinolate biosynthesis: candidate gene identification and validation.the diverse biological roles of glucosinolates as plant defense metabolites and anticancer compounds have spurred a strong interest in their biosynthetic pathways. since the completion of the arabidopsis genome, functional genomics approaches have enabled significant progress on the elucidation of glucosinolate biosynthesis, although in planta validation of candidate gene function often is hampered by time-consuming generation of knockout and overexpression lines in arabidopsis. to better exploi ...201222999179
opaque1 encodes a myosin xi motor protein that is required for endoplasmic reticulum motility and protein body formation in maize endosperm.myosins are encoded by multigene families and are involved in many basic biological processes. however, their functions in plants remain poorly understood. here, we report the functional characterization of maize (zea mays) opaque1 (o1), which encodes a myosin xi protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (er) structures and an increased numb ...201222892319
rice mitogen-activated protein kinase interactome analysis using the yeast two-hybrid system.mitogen-activated protein kinase (mapk) cascades support the flow of extracellular signals to intracellular target molecules and ultimately drive a diverse array of physiological functions in cells, tissues, and organisms by interacting with other proteins. yet, our knowledge of the global physical mapk interactome in plants remains largely fragmented. here, we utilized the yeast two-hybrid system and coimmunoprecipitation, pull-down, bimolecular fluorescence complementation, subcellular localiz ...201222786887
cis-acting element (sl1) of potato virus x controls viral movement by interacting with the nbmpb2cb and viral proteins.a number of candidate tobacco proteins that bind to cis-acting elements (sl1 rnas) of potato virus x (pvx) have been identified in previous studies. we further characterized tmv-mp30 binding protein 2c (mpb2c) homologous protein. we isolated nbmpb2cb from nicotiana benthamiana and confirmed the interaction of nbmpb2cb with sl1 rnas in vitro. the mrna level of nbmpb2cb was increased upon infection by pvx and tobacco mosaic virus. the movement of pvx was reduced by overexpression of nbmpb2cb and i ...201222405626
recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway.monoacylglycerol acyltransferases (mgats) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (mag) pathway by acylating mag to form diacylglycerol (dag). typical plant triacylglycerol (tag) biosynthesis routes such as the kennedy pathway do not include an mgat step. rather, dag and tag are synthesised de novo from glycerol-3-phosphate (g-3-p) by a series of three subsequent acylation reactions alth ...201222523576
ectopic expression of capsicum-specific cell wall protein capsicum annuum senescence-delaying 1 (casd1) delays senescence and induces trichome formation in nicotiana benthamiana.secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. in a previous study to understand the roles of secreted proteins, capsicum annuum secreted proteins (cas) were isolated by yeast secretion trap. among the secreted proteins, we further characterized capsicum annuum senescence-delaying 1 (casd1), a gene encoding a novel secreted protein that is present only in the genus capsicum. the deduced casd1 contains multiple repeats of the amino acid s ...201222441673
the rice stripe virus pc4 functions in movement and foliar necrosis expression in nicotiana benthamiana.the rice stripe virus (rsv) pc4 has been determined as the viral movement protein (mp). in this study, the pc4 gene was cloned into a movement-deficient tobacco mosaic virus (tmv). the resulting hybrid tmv-pc4, in addition to spreading cell to cell in nicotiana tabacum, moved systemically and induced foliar necrosis in nicotiana benthamiana, indicating novel functions of the rsv mp. a systematic alanine-scanning mutagenesis study established the region k(122)-d(258) of the pc4 substantially asso ...201222305130
role of rice stripe virus nsvc4 in cell-to-cell movement and symptom development in nicotiana benthamiana.our previous work has demonstrated that the nsvc4 protein of rice stripe virus (rsv) functions as a cell-to-cell movement protein. however, the mechanisms whereby rsv traffics through plasmodesmata (pd) are unknown. here we provide evidence that the nsvc4 moves on the actin filament and endoplasmic reticulum network, but not microtubules, to reach cell wall pd. disruption of cytoskeleton using different inhibitors altered nsvc4 localization to pd, thus impeding rsv infection of nicotiana bentham ...201223233857
a novel sec14 phospholipid transfer protein from nicotiana benthamiana is up-regulated in response to ralstonia solanacearum infection, pathogen associated molecular patterns and effector molecules and involved in plant immunity.to elucidate the molecular mechanisms of plant immune responses, we isolated genes whose expression was regulated by inoculation with ralstonia solanacearum. here, we report the characterization of nicotiana benthamiana belonging to the sec14-gene superfamily designated as nicotiana benthamiana sec14 (nbsec14). nbsec14 rescued growth defects and impaired invertase secretion associated with the yeast sec14p temperature-sensitive mutant, while recombinant nbsec14 protein had phospholipids transfer ...201222542247
safety and immunogenicity of a plant-produced recombinant hemagglutinin-based influenza vaccine (hai-05) derived from a/indonesia/05/2005 (h5n1) influenza virus: a phase 1 randomized, double-blind, placebo-controlled, dose-escalation study in healthy adults.recently, we have reported [1,2] on a subunit influenza vaccine candidate based on the recombinant hemagglutinin protein from the a/indonesia/05/2005 (h5n1) strain of influenza virus, produced it using 'launch vector'-based transient expression technology in nicotiana benthamiana, and demonstrated its immunogenicity in pre-clinical studies. here, we present the results of a first-in-human, phase 1 randomized, double-blind, placebo-controlled study designed to investigate safety, reactogenicity a ...201223202523
possible involvement of maize rop1 in the defence responses of plants to viral infection.the expression of host genes can be altered during the process of viral infection. to investigate the viral infection-induced up-regulated gene expression changes of maize at different time intervals post-inoculation with sugarcane mosaic virus (scmv), a suppression subtractive hybridization cdna library was constructed. a total of 454 cdna clones were identified to be viral infection-induced up-regulated genes. the influence of rop1 on the infection of maize by scmv was investigated. the result ...201222332840
transgenic expression of tobacco mosaic virus capsid and movement proteins modulate plant basal defense and biotic stress responses in nicotiana tabacum.plant viruses cause metabolic and physiological changes associated with symptomatic disease phenotypes. symptoms involve direct and indirect effects, which result in disruption of host physiology. we used transgenic tobacco expressing a variant of tobacco mosaic virus (tmv) coat protein (cp(t42w)) or movement protein (mp), and a hybrid line (mp×cp(t42w)) that coexpresses both proteins, to study the plant response to individual viral proteins. findings employing microarray analysis of mp×cp(t42w) ...201222712510
homologous rxlr effectors from hyaloperonospora arabidopsidis and phytophthora sojae suppress immunity in distantly related plants.diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. oomycete pathogens contain large complements of predicted effector genes defined by an rxlr host cell entry motif. the genome of hyaloperonospora arabidopsidis (hpa, downy mildew of arabidopsis) contains at least 134 candidate rxlr effector genes. only a small subset of these genes is conserved in related oomycetes from the phytophthora genus. here, we describe a comparative functional characteriz ...201222709376
plant erd2-like proteins function as endoplasmic reticulum luminal protein receptors and participate in programmed cell death during innate immunity.the hypersensitive response (hr), a form of programmed cell death (pcd), is a tightly regulated innate immune response in plants that is hypothesized to restrict pathogen growth and disease development. although considerable efforts have been made to understand hr pcd, it remains unknown whether the retrograde pathway from the golgi to the endoplasmic reticulum (er) is involved. here we provide direct genetic evidence that two nicotiana benthamiana homologs, erd2a and erd2b, function as er lumin ...201222595145
plant programmed cell death caused by an autoactive form of prf is suppressed by co-expression of the prf lrr domain.in tomato, the nbarc-lrr resistance (r) protein prf acts in concert with the pto or fen kinase to determine immunity against pseudomonas syringae pv. tomato (pst). prf-mediated defense signaling is initiated by the recognition of two sequence-unrelated pst-secreted effector proteins, avrpto and avrptob, by tomato pto or fen. prf detects these interactions and activates signaling leading to host defense responses including localized programmed cell death (pcd) that is associated with the arrest o ...201222451646
ornithine-delta-aminotransferase and proline dehydrogenase genes play a role in non-host disease resistance by regulating pyrroline-5-carboxylate metabolism-induced hypersensitive response.non-host disease resistance involves the production of hypersensitive response (hr), a programmed cell death (pcd) that occurs at the site of pathogen infection. plant mitochondrial reactive oxygen species (ros) production and red-ox changes play a major role in regulating such cell death. proline catabolism reactions, especially pyrroline-5-carboxylate (p5c) accumulation, are known to produce ros and contribute to cell death. here we studied important genes related to proline synthesis and cata ...201222321246
biological and molecular analysis of the pathogenic variant c3 of potato spindle tuber viroid (pstvd) evolved during adaptation to chamomile (matricaria chamomilla).viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small rnas (vsrnas). we describe a new pstvd variant c3, evolved through sequence adaptation to the host chamomile (matricaria chamomilla) after biolistic inoculation with pstvd-kf440-2, which causes extraordinary strong ('lethal') symptoms. the deletion of a single adenine a in the oligoa stretch of the pathogenicity (p) domain appears characteristic of pstvd-c3. the pathog ...201222944665
viroid rna redirects host dna ligase 1 to act as an rna ligase.viroids are a unique class of noncoding rnas: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. viroids replicate through an rna-to-rna rolling-circle mechanism consisting of transcription of oligomeric viroid rna intermediates, cleavage to unit-length strands, and circularization. though the host rna polymerase ii (redirected to accept rna templates) mediates rna synthesi ...201222869737
abiotic stress responses promote potato virus a infection in nicotiana benthamiana.the effect of abiotic stress responses on potato virus a (pva; genus potyvirus) infection was studied. salt, osmotic and wounding stress all increased pva gene expression in infected nicotiana benthamiana leaves. according to the literature, an early response to these stresses is an elevation in cytosolic ca(2+) concentration. the infiltration of 0.1 m cacl(2) into the infected leaf area enhanced the translation of pva rna, and this ca(2+) -induced effect was more profound than that induced sole ...201222340188
transient expression of hemagglutinin antigen from low pathogenic avian influenza a (h7n7) in nicotiana benthamiana.the influenza a virus is of global concern for the poultry industry, especially the h5 and h7 subtypes as they have the potential to become highly pathogenic for poultry. in this study, the hemagglutinin (ha) of a low pathogenic avian influenza virus of the h7n7 subtype isolated from a swedish mallard anas platyrhynchos was sequenced, characterized and transiently expressed in nicotiana benthamiana. recently, plant expression systems have gained interest as an alternative for the production of v ...201222442675
cloning of the complete infectious cdna of the plum pox virus strain ppv-rec.plum pox virus (ppv) is the causal agent of sharka, considered to be the most detrimental viral disease of prunus spp. worldwide. so far, several ppv strains have been recognized, three of them (ppv-d, ppv-m, and ppv-rec) having shown serious economic impact in the european area. infectious cdna clones of plant rna viruses are excellent tools for functional studies of viral genomes. preparation and use of ppv-d and ppv-m infectious clones have been previously reported. here we describe the const ...201222720703
pvx-potyvirus synergistic infections differentially alter microrna accumulation in nicotiana benthamiana.in comparison to single infections, co-infection of nicotiana benthamiana with potato virus x (pvx) and potato virus y (pvy) or plum pox virus (ppv), resulted in increased systemic symptoms (synergism in pathology). previous studies have shown that virus infections affected the accumulation of various micrornas (mirnas) and mirna target genes. our studies revealed that double infection by pvx and pvy or ppv that produced the most severe symptoms in n. benthamiana altered accumulation of mir156, ...201222387565
the 3'-proximal part of the plum pox virus p1 gene determinates the symptom expression in two herbaceous host plants.three major strains of the plum pox virus (ppv) are the most important in europe: ppv-d, ppv-m, and ppv-rec. by combining the genomes of two different strains of ppv (ppv-d with ppv-rec; ppv-d with ppv-m), 20 inter-strain chimeric infectious clones (cicppv) were constructed. biological properties of cicppv were tested by inoculating them on different herbaceous host species susceptible to ppv. four of the seven species tested, exhibited visible symptoms. in nicotiana benthamiana all cicppv induc ...201222367316
production of non-glycosylated recombinant proteins in nicotiana benthamiana plants by co-expressing bacterial pngase f.application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. as such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. for example, plasmodium parasites may have complex post-translationally modified proteins such as pfs48/45 that do not carry n-linked glycans (exp. para ...201222520228
tomato immune receptor ve1 recognizes effector of multiple fungal pathogens uncovered by genome and rna sequencing.fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. the tomato immune receptor ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi verticillium dahliae and verticillium albo-atrum, but the corresponding verticillium effector remained unknown thus far. by high-throughput population genome sequencing, a single 50-kb sequence stretch was identified that only occ ...201222416119
analysis of the antioxidant response of nicotiana benthamiana to infection with two strains of pepper mild mottle virus.the present study was carried out to investigate the role of reactive oxygen species (ros) metabolism in symptom development and pathogenesis in nicotiana benthamiana plants upon infection with two strains of pepper mild mottle virus, the italian (pmmov-i) and the spanish (pmmov-s) strains. in this host, it has been shown that pmmov-i is less virulent and plants show the capability to recover 21 d after inoculation. analyses of oxidative stress biomarkers, ros-scavenging enzyme activities, and a ...201222915745
citrus psorosis and mirafiori lettuce big-vein ophiovirus coat proteins localize to the cytoplasm and self interact in vivo.citrus psorosis (cpsv) and mirafiori lettuce big-vein virus (milbvv) belong to the family ophioviridae, plant viruses with filamentous nucleocapsids and segmented genomes of negative polarity, causing the worldwide distributed citrus psorosis and lettuce big-vein diseases, respectively. to gain insight into the replication cycle of these viruses, the subcellular localization of the viral coat proteins (cp) was studied. immunoblot analysis of fractionated extracts derived from natural and experim ...201222921760
development of viral vectors based on citrus leaf blotch virus to express foreign proteins or analyze gene function in citrus plants.viral vectors have been used to express foreign proteins in plants or to silence endogenous genes. this methodology could be appropriate for citrus plants that have long juvenile periods and adult plants that are difficult to transform. we developed viral vectors based on citrus leaf blotch virus (clbv) by duplicating a minimum promoter (92 bp) either at the 3' untranslated region (clbv3'pr vector) or at the intergenic region between the movement and coat protein (cp) genes (clbvinpr vector). th ...201222670755
transient expression of human papillomavirus type 16 l2 epitope fused to n- and c-terminus of coat protein of potato virus x in plants.transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. in the present study the epitope derived from hpv-16 l2 minor capsid protein (amino acids 108-120) was expressed from potato virus x (pvx)-based vector pgr106 as n- or c-terminal fusion with the pvx coat protein (pvx cp) in transgenic nicotiana benthamiana pl ...201222357210
biological and molecular characterization of a u.s. isolate of hosta virus x.hosta virus x (hvx) is rapidly becoming a serious pathogen of commercially important hosta plants worldwide. we report here biological and molecular characterization of a u.s. isolate of hvx, hvx-37. hvx-37 infectivity was tested in 21 hosta cultivars over three growth seasons, and three types of responses were defined based upon the ability of the virus to cause local and/or systemic infections. four cultivars resistant to systemic hvx infection were identified. the full-length sequence of the ...201222809286
rcy1-mediated resistance to cucumber mosaic virus is regulated by lrr domain-mediated interaction with cmv(y) following degradation of rcy1.rcy1, which encodes a coiled coil nucleotide-binding site leucine-rich repeat (lrr) class r protein, confers the hypersensitive response (hr) to a yellow strain of cucumber mosaic virus (cmv[y]) in arabidopsis thaliana. nicotiana benthamiana transformed with hemagglutinin (ha) epitope-tagged rcy1 (rcy1-ha) also exhibited a defense response accompanied by hr cell death and induction of defense-related gene expression in response to cmv(y). following transient expression of rcy1-ha by agroinfiltra ...201222852808
the p19.7 rna silencing suppressor from grapevine leafroll-associated virus 3 shows different levels of activity across phylogenetic groups.at least five phylogenetic groups have been reported for grapevine leafroll-associated virus 3 (glrav-3). the p19.7 protein encoded by the glrav-3 was previously identified as an rna silencing suppressor. in this study, five constructs of p19.7 belonging to different groups were compared for their suppressing activity. for each p19.7 variant, the accumulation level of green fluorescent protein mrna and specific sirnas were determined using co-infiltration assays in transgenic 16c nicotiana benth ...201222714284
high-level transient expression of er-targeted human interleukin 6 in nicotiana benthamiana.tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as escherichia coli. we therefore expressed the human glycoprotein interleukin 6 (il6) in two commercial tobacco cultivars (nicotiana tabacum cv. virginia and cv. geudertheimer) as well as the model host n. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticu ...201223152824
delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques.filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. here, three anti-ebola virus mouse/human chimeric mabs (c13c6, h-13f6, and c6d8) were produced in chinese hamster ovary and in whole plant (nicotiana benthamiana) cells. in pilot experiments testing a mixture of the three mabs (mb-003), we found that mb-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. ...201223071322
heterologous expression of viral suppressors of rna silencing complements virulence of the hc-pro mutant of clover yellow vein virus in pea.many plant viruses encode suppressors of rna silencing, including the helper component-proteinase (hc-pro) of potyviruses. our previous studies showed that a d-to-y mutation at amino acid position 193 in hc-pro (hc-pro-d193y) drastically attenuated the virulence of clover yellow vein virus (clyvv) in legume plants. furthermore, rna-silencing suppression (rss) activity of hc-pro-d193y was significantly reduced in nicotiana benthamiana. here, we examine the effect of expression of heterologous sup ...201222398917
the movement protein encoded by gene 3 of rice transitory yellowing virus is associated with virus particles.gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing virus (rtyv), has been postulated to encode a cell-to-cell movement protein (mp). trans-complementation experiments using a movement-defective tomato mosaic virus and the p3 protein of rtyv, encoded by gene 3, facilitated intercellular transport of the mutant virus. in transient-expression experiments with the gfp-fused p3 protein in epidermal leaf cells of nicotiana benthamiana, the p3 protein wa ...201222815270
the p19 protein of grapevine algerian latent virus is a determinant of systemic infection of chenopodium quinoa.a previous study showed that both grapevine algerian latent virus (galv) and tomato bushy stunt virus (tbsv) systemically infect nicotiana benthamiana, but galv causes systemic infection whereas tbsv causes only local lesions in chenopodium quinoa (c. quinoa). we recently isolated galv strain naju (galv-n) from limonium sinense and tbsv strain sacheon (tbsv-s) from tomato. both viruses belong to the genus tombusvirus and have a similar genome organization. to identify determinants of systemic in ...201222342277
bipartite and tripartite cucumber mosaic virus-based vectors for producing the acidothermus cellulolyticus endo-1,4-β-glucanase and other proteins in non-transgenic plants.using plant viruses to produce desirable proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. the objective of this work was the development of advanced cmv-based protein production systems to produce acidothermus cellulolyticus endo-1, 4-β-glucanase (e1) in non-transgenic plants.201222999234
viral nanoparticles for in vivo tumor imaging.the use of nanomaterials has the potential to revolutionize materials science and medicine. currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. viral nanoparticles (vnps) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. plant viruses under investigation in the steinmetz lab include icosahedral particles formed by cowpea mosaic virus (cpmv) and brome mosaic virus (bmv), both of which ar ...201223183850
emergence of distinct brome mosaic virus recombinants is determined by the polarity of the inoculum rna.despite overwhelming interest in the impact exerted by recombination during evolution of rna viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. here, by agroinfiltrating nicotiana benthamiana leaves, we show that brome mosaic virus (bmv) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed rna3 negative strand [(-) strand] and faithfully complete the replication cycle. consequently, we sought to exa ...201222357282
utility of the p19 suppressor of gene-silencing protein for production of therapeutic antibodies in nicotiana expression hosts.to study how the p19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different p19 concentrations; compatibility of p19 with various nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with p19 in an rnai-based glycomodified nicotiana benthamiana ...201222984968
effects of the mutation of selected genes of cotton leaf curl kokhran virus on infectivity, symptoms and the maintenance of cotton leaf curl multan betasatellite.cotton leaf curl kokhran virus (clcukov) is a cotton-infecting monopartite begomovirus (family geminiviridae). the effects of mutation of the coat protein (cp), v2, c2 and c4 genes of clcukov on infectivity and symptoms in nicotiana benthamiana were investigated. each mutation introduced a premature stop codon which would lead to premature termination of translation of the gene. mutation of the cp gene abolished infectivity. however, transient expression of the clcukov cp gene under the control ...201222871297
in planta production of a candidate vaccine against bovine papillomavirus type 1.bovine papillomavirus type 1 (bpv-1) is an economically important virus that induces tumourigenic pathologies in horses and cows. given that the bpv-1 l1 major coat protein can self-assemble into highly immunogenic higher-order structures, we transiently expressed it in nicotiana benthamiana as a prelude to producing a candidate vaccine. it was found that plant codon optimization of l1 gave higher levels of expression than its non-optimized counterpart. following protein extraction, we obtained ...201222718313
two distinct sites are essential for virulent infection and support of variant satellite rna replication in spontaneous beet black scorch virus variants.spontaneous point mutations of virus genomes are important in rna virus evolution and often result in modifications of their biological properties. spontaneous variants of beet black scorch virus (bbsv) and its satellite (sat) rna were generated from cdna clones by serial propagation in chenopodium amaranticolor and nicotiana benthamiana. inoculation with recombinant rnas synthesized in vitro revealed bbsv variants with divergent infectious phenotypes that affected either symptom expression or r ...201222971822
the r-rich motif of beet black scorch virus p7a movement protein is important for the nuclear localization, nucleolar targeting and viral infectivity.beet black scorch virus (bbsv) encodes three movement proteins (p7a, p7b and p5') that facilitate its cell-to-cell movement. an arginine-rich motif of p7a n-terminus was found to determine nuclear and nucleolar localization. amino acids substitution or deletion of the r-rich motif interfered with p7a nuclear and nucleolar localization. bimolecular fluorescence complementation (bifc) assays revealed that p7a protein interacted with nicotiana benthamiana nuclear import factor importin α, suggestin ...201222626884
the cysteine-rich proteins of beet necrotic yellow vein virus and tobacco rattle virus contribute to efficient suppression of silencing in roots.many plant viruses encode proteins that suppress rna silencing, but little is known about the activity of silencing suppressors in roots. this study examined differences in the silencing suppression activity of different viruses in leaves and roots of nicotiana benthamiana plants. infection by tobacco mosaic virus, potato virus y and cucumber mosaic virus but not potato virus x (pvx) resulted in strong silencing suppression activity of a transgene in both leaves and roots, whereas infection by b ...201222647371
beet necrotic yellow vein virus subgenomic rna3 is a cleavage product leading to stable non-coding rna required for long-distance movement.beet necrotic yellow vein virus (bnyvv) is a multipartite rna virus. bnyvv rna3 does not accumulate in non-host transgenic arabidopsis thaliana plants when expressed using a 35s promoter. however, a 3'-derivative species has been detected in transgenic plants and in transient expression assays conducted in nicotiana benthamiana and beta macrocarpa. the 3'-derivative species is similar to the previously reported subgenomic rna3 produced during virus infection. 5' race revealed that the truncated ...201222258860
ssef, a type iii effector protein from the mammalian pathogen salmonella enterica, requires resistance-gene-mediated signalling to activate cell death in the model plant nicotiana benthamiana.type iii effector proteins (t3es) of many gram-negative pathogenic bacteria manipulate highly conserved cellular processes, indicating conservation in virulence mechanisms during the infection of hosts of divergent evolutionary origin. in order to identify conserved effector functions, we used a cross-kingdom approach in which we expressed selected t3es from the mammalian pathogen salmonella enterica in leaves of nicotiana benthamiana and searched for possible virulence or avirulence phenotypes. ...201222471508
agroinoculation of the cloned infectious cdnas of lettuce chlorosis virus results in systemic plant infection and production of whitefly transmissible virions.lettuce chlorosis virus (lcv) is a single stranded, positive strand rna virus that is solely transmitted by specific whitefly vectors (bemisia tabaci biotypes a and b) but not by mechanical leaf-rub inoculation. the roles of viral encoded proteins involved in the infection cycle of lcv have not yet been characterized due to the lack of reverse genetic tools. we present here a report of the successful development of an agrobacterium-mediated inoculation system for the cloned cdna constructs of lc ...201222926259
validation of reference genes for gene expression studies in virus-infected nicotiana benthamiana using quantitative real-time pcr.nicotiana benthamiana is the most widely-used experimental host in plant virology. the recent release of the draft genome sequence for n. benthamiana consolidates its role as a model for plant-pathogen interactions. quantitative real-time pcr (qpcr) is commonly employed for quantitative gene expression analysis. for valid qpcr analysis, accurate normalisation of gene expression against an appropriate internal control is required. yet there has been little systematic investigation of reference ge ...201223029521
c2 from beet curly top virus promotes a cell environment suitable for efficient replication of geminiviruses, providing a novel mechanism of viral synergism.• geminiviruses are plant viruses with circular, single-stranded (ss) dna genomes that infect a wide range of species and cause important losses in agriculture. geminiviruses do not encode their own dna polymerase, and rely on the host cell machinery for their replication. • here, we identify a positive effect of the curtovirus beet curly top virus (bctv) on the begomovirus tomato yellow leaf curl sardinia virus (tylcsv) infection in nicotiana benthamiana plants. • our results show that this pos ...201222404507
characterization of plant virus-encoded gene silencing suppressors.agroinfiltration assay using green fluorescent protein (gfp)-expressing nicotiana benthamiana line 16c is a powerful method for screening of putative plant virus-encoded gene silencing suppressors. this method allows the investigator to know whether the putative viral suppressor inhibits silencing in a cell (local silencing) and/or spreading of silencing throughout a plant (systemic silencing). additionally, grafting experiments using transgenic plants expressing the suppressor and the gfp will ...201222678576
involvement of the chloroplastic isoform of trna ligase in the replication of viroids belonging to the family avsunviroidae.avocado sunblotch viroid, peach latent mosaic viroid, chrysanthemum chlorotic mottle viroid, and eggplant latent viroid (elvd), the four recognized members of the family avsunviroidae, replicate through the symmetric pathway of an rna-to-rna rolling-circle mechanism in chloroplasts of infected cells. viroid oligomeric transcripts of both polarities contain embedded hammerhead ribozymes that, during replication, mediate their self-cleavage to monomeric-length rnas with 5'-hydroxyl and 2',3'-phosp ...201222623792
hsp90 interacts specifically with viral rna and differentially regulates replication initiation of bamboo mosaic virus and associated satellite rna.host factors play crucial roles in the replication of plus-strand rna viruses. in this report, a heat shock protein 90 homologue of nicotiana benthamiana, nbhsp90, was identified in association with partially purified replicase complexes from bamv-infected tissue, and shown to specifically interact with the 3' untranslated region (3' utr) of bamv genomic rna, but not with the 3' utr of bamv-associated satellite rna (satbamv rna) or that of genomic rna of other viruses, such as potato virus x (pv ...201222654666
agrobacterium may delay plant nonhomologous end-joining dna repair via xrcc4 to favor t-dna integration.agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (t-dna) into the plant genome. several plant factors that play a role in agrobacterium attachment to plant cells and transport of t-dna to the nucleus have been identified, but the t-dna integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (nhej)-mediated double-str ...201223064322
transient and stable expression of antibodies in nicotiana species.expression and purification of recombinant proteins produced in plants is emerging as an affordable alternative to using more costly mammalian bioreactors since plants are capable of producing mammalian proteins at high concentrations. there are two general methods of expressing foreign proteins in plants, namely, transient expression and stable transgenic expression. both methods have advantages which serve different purposes. nicotiana benthamiana is primarily used as plant host for transient ...201222907365
simple and robust in vivo and in vitro approach for studying virus assembly.in viruses with positive-sense rna genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. such information is vital in formulating novel methods of curbing virus spread and disease control. virus encapsidation can be studied ...201222410612
population diversity of rice stripe virus-derived sirnas in three different hosts and rnai-based antiviral immunity in laodelphgax striatellus.small rna-mediated gene silencing plays evolutionarily conserved roles in gene regulation and defense against invasive nucleic acids. virus-derived small interfering rnas (vsirnas) are one of the key elements involved in rna silencing-based antiviral activities in plant and insect. vsirnas produced after viruses infecting hosts from a single kingdom (i.e., plant or animal) are well described. in contrast, vsirnas derived from viruses capable of infecting both plants and their insect vectors have ...201223029445
[effects of seven rna silencing suppressors on heterologous expression of green fluorescence protein expression mediated by a plant virus-based system in nicotiana benthamiana].to test the effects of 7 virus-encoded rna silencing suppressors (rsss) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (gfp) in nicotiana benthamiana.201223174571
a melting pot of old world begomoviruses and their satellites infecting a collection of gossypium species in pakistan.clcud in southern asia is caused by a complex of multiple begomoviruses (whitefly transmitted, single-stranded [ss]dna viruses) in association with a specific ssdna satellite; cotton leaf curl multan betasatellite (clcumub). a further single ssdna molecule, for which the collective name alphasatellites has been proposed, is also frequently associated with begomovirus-betasatellite complexes. multan is in the center of the cotton growing area of pakistan and has seen some of the worst problems ca ...201222899988
loss of cellulose synthase-like f6 function affects mixed-linkage glucan deposition, cell wall mechanical properties, and defense responses in vegetative tissues of rice.mixed-linkage glucan (mlg) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. based on its occurrence in plants and chemical characteristics, mlg has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. the cellulose synthase-like f (cslf) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. ...201222388489
synthetic human monoclonal antibodies toward staphylococcal enterotoxin b (seb) protective against toxic shock syndrome.staphylococcal enterotoxin b (seb) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. there are currently no vaccines or immunotherapeutics available against this toxin. using phage display technology, human antigen-binding fragments (fabs) were selected against seb, and proteins were produced in escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. ...201222645125
the gef1 proton-chloride exchanger affects tombusvirus replication via regulation of copper metabolism in yeast.replication of plus-strand rna viruses [(+)rna viruses] is performed by viral replicases, whose function is affected by many cellular factors in infected cells. in this paper, we demonstrate a surprising role for gef1p proton-chloride exchanger in replication of tomato bushy stunt virus (tbsv) model (+)rna virus. a genetic approach revealed that gef1p, which is the only proton-chloride exchanger in saccharomyces cerevisiae, is required for tbsv replication in the yeast model host. we also show t ...201223192874
a physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an rna virus.genome packaging is functionally coupled to replication in rna viruses pathogenic to humans (poliovirus), insects (flock house virus [fhv]), and plants (brome mosaic virus [bmv]). however, the underlying mechanism is not fully understood. we have observed previously that in fhv and bmv, unlike ectopically expressed capsid protein (cp), packaging specificity results from rna encapsidation by cp that has been translated from mrna produced from replicating genomic rna. consequently, we hypothesize ...201222438552
sgt1b is required for hopz3-mediated suppression of the epiphytic growth of pseudomonas syringae on n. benthamiana.type iii secreted effectors shape the potential of bacterial pathogens to cause disease on plants. some effectors affect pathogen growth only in specific niches. for example, hopz3 causes reduced epiphytic growth of pseudomonas syringae strain b728a on nicotiana benthamiana. this raises the question of whether genes important for effector-triggered disease resistance are needed for responses to effectors whose major effect is in the epiphytic niche. we report that sgt1b, a protein known to be im ...201222899059
sieve element occlusion (seo) genes encode structural phloem proteins involved in wound sealing of the phloem.the sieve element occlusion (seo) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the fabaceae. more recently, seo genes discovered in various non-fabaceae plants were proposed to encode the common phloem proteins (p-proteins) that plug sieve plates after wounding. we carried out a comprehensive characterization of two tobacco (nicotiana tabacum) seo genes (ntseo). reporter genes controll ...201222733783
rna viral vectors for improved agrobacterium-mediated transient expression of heterologous proteins in nicotiana benthamiana cell suspensions and hairy roots.plant cell suspensions and hairy root cultures represent scalable protein expression platforms. low protein product titers have thus far limited the application of transient protein expression in these hosts. the objective of this work was to overcome this limitation by harnessing a. tumefaciens to deliver replicating and non-replicating rna viral vectors in plant tissue co-cultures.201222559055
lotus japonicus e3 ligase seven in absentia4 destabilizes the symbiosis receptor-like kinase symrk and negatively regulates rhizobial infection.the lotus japonicus symbiosis receptor-like kinase (symrk) is required for symbiotic signal transduction upon stimulation of root cells by microbial signaling molecules. here, we identified members of the seven in absentia (sina) e3 ubiquitin-ligase family as symrk interactors and confirmed their predicted ubiquitin-ligase activity. in nicotiana benthamiana leaves, symrk-yellow fluorescent protein was localized at the plasma membrane, and interaction with sinas, as determined by bimolecular fluo ...201222534128
the destructive citrus pathogen, 'candidatus liberibacter asiaticus' encodes a functional flagellin characteristic of a pathogen-associated molecular pattern.huanglongbing (hlb) is presently the most devastating citrus disease worldwide. as an intracellular plant pathogen and insect symbiont, the hlb bacterium, 'candidatus liberibacter asiaticus' (las), retains the entire flagellum-encoding gene cluster in its significantly reduced genome. las encodes a flagellin and hook-associated protein (fla) of 452 amino acids that contains a conserved 22 amino acid domain (flg22) at positions 29 to 50 in the n-terminus. the phenotypic alteration in motility of ...201223029520
plant erd2s self-interact and interact with gtpase-activating proteins and adp-ribosylation factor 1.erd2s (er luminal protein receptors)-mediated retrograde transport is one of the most substantial processes to maintain the endoplasmic reticulum (er) homeostasis. it is completed by the recognition of the escaped er luminal proteins, the gathering into cop i vesicle, and the fusion and releasing into the er. erd2s can recognize hdel/kdel motifs at the c-terminal of the escaped er luminal proteins at the golgi to initiate the retrograde transport. however, these mechanisms remain largely unknown ...201222899072
endoplasmic reticulum-quality control chaperones facilitate the biogenesis of cf receptor-like proteins involved in pathogen resistance of tomato.cf proteins are receptor-like proteins (rlps) that mediate resistance of tomato (solanum lycopersicum) to the foliar pathogen cladosporium fulvum. these transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. to identify proteins required for the functionality of these rlps, we performed immunopurification of a functiona ...201222649272
requirement of the cytosolic interaction between pathogenesis-related protein10 and leucine-rich repeat protein1 for cell death and defense signaling in pepper.plants recruit innate immune receptors such as leucine-rich repeat (lrr) proteins to recognize pathogen attack and activate defense genes. here, we identified the pepper (capsicum annuum) pathogenesis-related protein10 (pr10) as a leucine-rich repeat protein1 (lrr1)-interacting partner. bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the specific interaction between lrr1 and pr10 in planta. avirulent xanthomonas campestris pv vesicatoria infection induces pr10 ...201222492811
structure-function analysis of the coiled-coil and leucine-rich repeat domains of the rps5 disease resistance protein.the arabidopsis (arabidopsis thaliana) resistance to pseudomonas syringae5 (rps5) disease resistance protein mediates recognition of the pseudomonas syringae effector protein avrpphb. rps5 belongs to the coiled-coil-nucleotide-binding site-leucine-rich repeat (cc-nbs-lrr) family and is activated by avrpphb-mediated cleavage of the protein kinase pbs1. here, we present a structure-function analysis of the cc and lrr domains of rps5 using transient expression assays in nicotiana benthamiana. we fo ...201222331412
repetitive prime-and-realign mechanism converts short capped rna leaders into longer ones that may be more suitable for elongation during rice stripe virus transcription initiation.cucumber mosaic virus (cmv) rnas were found to serve as cap donors for rice stripe virus (rsv) transcription initiation during their co-infection of nicotiana benthamiana. the 5' end of cmv rnas was cleaved preferentially at residues that had multiple-base complementarity to the 3' end of the rsv template. the length requirement for cmv capped primers to be suitable for elongation varied between 12 and 20 nt, and those of 12-16 nt were optimal for elongation and generated more cmv-rsv chimeric m ...201221918010
infection dynamics in viral spread and interference under the synergism between cucumber mosaic virus and turnip mosaic virus.mixed infection of cucumber mosaic virus (cmv) and turnip mosaic virus (tumv) induced more severe symptoms on nicotiana benthamiana than single infection. to dissect the relationships between spatial infection patterns and the 2b protein (2b) of cmv in single or mixed infections, the cmv vectors expressing enhanced green fluorescent or discosoma sp. red fluorescent proteins (egfp [eg] or dsred2 [ds], respectively were constructed from the same wild-type cmv-y and used for inoculation onto n. ben ...201221916556
in planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein.cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. an increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. here, we developed in planta assay systems to detec ...201222424588
cauliflower mosaic virus protein p6 inhibits signaling responses to salicylic acid and regulates innate immunity.cauliflower mosaic virus (camv) encodes a multifunctional protein p6 that is required for translation of the 35s rna and also acts as a suppressor of rna silencing. here we demonstrate that p6 additionally acts as a pathogenicity effector of an unique and novel type, modifying npr1 (a key regulator of salicylic acid (sa)- and jasmonic acid (ja)-dependent signaling) and inhibiting sa-dependent defence responses we find that that transgene-mediated expression of p6 in arabidopsis and transient exp ...201223071821
the potato mop-top virus tgb2 protein and viral rna associate with chloroplasts and viral infection induces inclusions in the plastids.the potato mop-top virus (pmtv) triple gene block 2 (tgb2) movement proteins fused to monomeric red fluorescent protein (mrfp-tgb2) was expressed under the control of the pmtv subgenomic promoter from a pmtv vector. the subcellular localizations and interactions of mrfp-tgb2 were investigated using confocal imaging [confocal laser-scanning microscope, (clsm)] and biochemical analysis. the results revealed associations with membranes of the endoplasmic reticulum (er), mobile granules, small round ...201223269927
microscopic analysis of severe structural rearrangements of the plant endoplasmic reticulum and golgi caused by overexpression of poa semilatent virus movement protein.cell-to-cell transport of plant viruses is mediated by virus-encoded movement proteins and occurs through plasmodesmata interconnecting neighboring cells in plant tissues. three movement proteins coded by the "triple gene block" (tgb) and named tgbp1, tgbp2 and tgbp3 have distinct functions in viral transport. tgbp1 binds viral genomic rnas to form ribonucleoprotein complexes representing the transport form of viral genome, while tgbp2 and tgbp3 are necessary for intracellular delivery of such c ...201222272174
activation of a plant nucleotide binding-leucine rich repeat disease resistance protein by a modified self protein.nucleotide binding-leucine rich repeat (nb-lrr) proteins function as intracellular receptors for the detection of pathogens in both plants and animals. despite their central role in innate immunity, the molecular mechanisms that govern nb-lrr activation are poorly understood. the arabidopsis nb-lrr protein rps5 detects the presence of the pseudomonas syringae effector protein avrpphb by monitoring the status of the arabidopsis protein kinase pbs1. avrpphb is a cysteine protease that targets pbs1 ...201222372664
a microrna superfamily regulates nucleotide binding site-leucine-rich repeats and other mrnas.analysis of tomato (solanum lycopersicum) small rna data sets revealed the presence of a regulatory cascade affecting disease resistance. the initiators of the cascade are microrna members of an unusually diverse superfamily in which mir482 and mir2118 are prominent members. members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the p-loop motif in the mrna sequences for disease resistance proteins with nucleotide ...201222408077
distinct regions of the pseudomonas syringae coiled-coil effector avrrps4 are required for activation of immunity.gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert host defenses. these effectors can be recognized by plant nucleotide-binding-leucine-rich repeat immune receptors, triggering defense responses that restrict pathogen growth. avrrps4, an effector protein from pseudomonas syringae pv. pisi, triggers rps4-dependent immunity in resistant accessions of arabidopsis. to better understand the molecular basis of avrrps4-triggered immunity, we determined the ...201222988101
integration of bioinformatics and synthetic promoters leads to the discovery of novel elicitor-responsive cis-regulatory sequences in arabidopsis.a combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. with arabidopsis (arabidopsis thaliana) microarray data annotated to the pathoplant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. using the binding site estimation suite of tools, best, 407 conse ...201222744985
engineering mammalian mucin-type o-glycosylation in plants.mucin-type o-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. this post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where o-glycans are attached to proteins, and which structures are formed, difficult. because plants are devoid of galnac-type o-glycosylation, we have assessed requirements for establishing human galnac o-gl ...201222334671
visual tracking of plant virus infection and movement using a reporter myb transcription factor that activates anthocyanin biosynthesis.insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. their numerous advantages make fluorescent proteins the markers of choice in most studies. however, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. t ...201222238422
Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system.Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5mg recombinant hGL per gram of infiltrated leaf tissue were obtained. Proteins expressed from two hGL constructs, wild type (wt-hGL) and with a Histidine tag at its C terminal (hGL-His), were purified from leaf tissue using Immobilized Lectin Affinity chromatography and Immobilized Metal Affinity chromatography. Both variants were gly ...201221945702
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