Publications

bean yellow dwarf virus repa, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.	it has previously been reported that complementary-sense gene products of wheat dwarf virus (wdv), a geminivirus of the genus mastrevirus that infects monocotyledonous plants, bind to human and maize retinoblastoma (rb) protein. rb proteins control cell-cycle progression by sequestering transcription factors required for entry into s-phase, suggesting that the virus modifies the cellular environment to produce conditions suitable for viral dna replication. using a yeast two-hybrid assay, we have ...	1999	10191192
the specificity of polygalacturonase-inhibiting protein (pgip): a single amino acid substitution in the solvent-exposed beta-strand/beta-turn region of the leucine-rich repeats (lrrs) confers a new recognition capability.	two members of the pgip gene family (pgip-1 and pgip-2) of phaseolus vulgaris l. were expressed separately in nicotiana benthamiana and the ligand specificity of their products was analysed by surface plasmon resonance (spr). polygalacturonase-inhibiting protein-1 (pgip-1) was unable to interact with pg from fusarium moniliforme and interacted with pg from aspergillus niger; pgip-2 interacted with both pgs. only eight amino acid variations distinguish the two proteins: five of them are confined ...	1999	10228150
strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism.	the 2b protein encoded by cucumber mosaic cucumovirus (cmv2b) acts as an important virulence determinant by suppressing post-transcriptional gene silencing (ptgs), a natural plant defence mechanism against viruses. we report here that the tomato aspermy cucumovirus 2b protein (tav2b), when expressed from the unrelated tobacco mosaic tobamovirus (tmv) rna genome, activates strong host resistance responses to tmv in tobacco which are typical of the gene-for-gene disease resistance mechanism. domai ...	1999	10329615
subcellular localization and in vivo identification of the putative movement protein of olive latent virus 2.	the gene encoding the 36.5 kda ('36k') nonstructural protein located on rna3 of olive latent virus 2 (olv-2) was cloned, expressed with the escherichia coli pgex-2t system and the purified protein used to raise a polyclonal antiserum. immunoblot analysis of olv-2-infected nicotiana benthamiana plants showed that the 36k protein accumulated in the early stages of infection and was associated with a subcellular fraction enriched in cytoplasmic membranes. in infected cells there were tubular struct ...	1999	10355755
mutational analyses of the putative calcium binding site and hinge of the turnip crinkle virus coat protein.	the turnip crinkle carmovirus (tcv) coat protein (cp) is folded into r (rna-binding), s (shell), and p (protruding) domains. the s domain is an eight-stranded beta barrel common to the coat protein subunits of most rna viruses. a five-amino-acid hinge connects the s and p domains. in assembled particles, each pair of cp subunits is thought to bind a single calcium ion through interactions with three residues of one subunit and two residues of a neighboring subunit. these five residues comprise t ...	1999	10364487
a promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants.	a 1369 bp dna fragment (sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (scbv) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and nicotiana benthamiana). this promoter was also tested for stable expression in transgenic banana and tobacco plants. these experiments showed that this promoter could drive high-level expression of the beta-glucuro ...	1999	10380808
phosphorylation and/or presence of serine 37 in the movement protein of tomato mosaic tobamovirus is essential for intracellular localization and stability in vivo.	the p30 movement protein (mp) of tomato mosaic tobamovirus (tomv) is synthesized in the early stages of infection and is phosphorylated in vivo. here, we determined that serine 37 and serine 238 in the tomv mp are sites of phosphorylation. mp mutants in which serine was replaced by alanine at positions 37 and 238 (lq37a238a) or at position 37 only (lq37a) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants. by contrast, mutation of serine 238 to alanine did not af ...	1999	10400781
the n-terminal 77 amino acids from tobacco n-acetylglucosaminyltransferase i are sufficient to retain a reporter protein in the golgi apparatus of nicotiana benthamiana cells.	in order to investigate sequences of tobacco n-acetylglucosaminyltransferase i (gnti), involved in targeting to and retention in the plant golgi apparatus the cytoplasmic transmembrane stem (cts) region of the enzyme was cloned in frame with the cdna of the green fluorescent protein (gfp) and subsequently transiently expressed in nicotiana benthamiana plants using a tobacco mosaic virus (tmv) based expression vector. confocal laser scanning microscopy showed small fluorescent vesicular bodies in ...	1999	10403396
restoration of a stem-loop structure required for potato virus x rna accumulation indicates selection for a mismatch and a gnra tetraloop.	the 5' region of potato virus x (pvx) rna contains a stem-loop structure, stem-loop 1 (sl1), that is required for efficient plus-strand rna accumulation. to determine how changes to individual elements in sl1 are accommodated by the virus, we inoculated pvx transcripts containing modifications in the terminal tetraloop (tl), stem c (sc), and stem d (sd) regions onto nicotiana benthamiana plants and analyzed progeny rnas over a series of passages. several progeny rnas isolated from plants inocula ...	1999	10417268
interactions between tombusviruses and satellite rnas of tomato bushy stunt virus: a defect in sat rna b1 replication maps to orf1 of a helper virus.	the biological properties of two recently described satellite rnas of tomato bushy stunt virus (tbsv) were analyzed in natural and experimental hosts. full-length cdna clones were constructed for sat rnas b1(822 nt) and b10 (612 nt) and used in inoculations with satellite-free transcripts of different tombusviruses. in all virus-host combinations tested, tbsv sat rna b10 drastically reduced the accumulation of viral genomic rna and attenuated symptoms. in contrast, sat rna b1 caused a less marke ...	1999	10489347
internal initiation of translation directed by the 5'-untranslated region of the tobamovirus subgenomic rna i(2).	previously we reported that, unlike rna of typical tobamoviruses, the translation of the coat protein (cp) gene of a crucifer-infecting tobamovirus (crtmv) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (ires(cp,148)(cr)). the equivalent 148-nt sequence from tmv u1 rna (u1(cp,148)(sp)) was incapable of promoting internal initiation. in the present work, we have found that the 228-nt region upstream of the mov ...	1999	10544089
the first triple gene block protein of peanut clump virus localizes to the plasmodesmata during virus infection.	the subcellular localization of the first triple gene block protein (tgbp1) of peanut clump pecluvirus (pcv) was studied by subcellular fractionation and immunogold cytochemistry using tgbp1-specific antibodies raised against a fusion protein expressed in and purified from bacteria. in the inoculated and apical leaves of virus-infected nicotiana benthamiana, tgbp1 localized to the cell wall and p30 fractions. electron microscopy of immunogold-decorated ultrathin sections of the infected leaf tis ...	1999	10544148
nonstructural proteins of tobacco rattle virus which have a role in nematode-transmission: expression pattern and interaction with viral coat protein.	rna 2 of tobacco rattle virus isolate ppk20 encodes the viral coat protein (cp) and two nonstructural proteins of 40 kda ('40k protein') and 32.8 kda ('32.8k'). the 40k protein is required for transmission of the virus by the vector nematode paratrichodorus pachydermus whereas the 32.8k protein may be involved in transmission by other vector nematode species. an antiserum was raised against the 40k protein expressed in e. coli and used to study the expression and subcellular localization of this ...	1999	10567661
homology-dependent virus resistance in transgenic plants with the coat protein gene of sweet potato feathery mottle potyvirus: target specificity and transgene methylation.	abstract nicotiana benthamiana plants were transformed with the coat protein (cp) coding sequence and the 3' nontranslated region (ntr) of the severe strain of sweet potato feathery mottle potyvirus (spfmv-s). regenerated lines were screened for virus resistance using recombinant potato virus x (pvx) engineered to contain the sequence homologous to the transgene. out of 19 transgenic lines, 7 showed virus resistance after inoculation by the recombinant pvx. in most of the resistant lines, relati ...	1999	18944751
complementation of coat protein mutants of pepper huasteco geminivirus in transgenic tobacco plants.	abstract the role of the pepper huasteco virus (phv) coat protein (cp) gene during the infection was investigated in three different hosts by using mutations that produced truncated proteins and by complementation assays in transgenic plants. the infectivity analysis revealed that mutants that express truncated cp (cp7 and cp191) behave like the wild-type virus when inoculated onto pepper and nicotiana benthamiana plants in terms of symptom expression and viral dna movement. on the contrary, the ...	1999	18944688
detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus using immuno-capillary zone electrophoresis.	abstract immuno-capillary zone electrophoresis (i-cze) is a technique that combines the specificity afforded by serological assays with the sensitivity, rapidity, and automation in detection provided by capillary zone electrophoresis. cymbidium mosaic potexvirus (cymmv) and odontoglossum ringspot tobamovirus (orsv) were detected in their purified forms as well as in the crude saps of infected nicotiana benthamiana leaves and oncidium orchid flowers. the two orchid virus-antibody complexes were r ...	1999	18944725
effect of c-terminal deletions in the movement protein of cowpea chlorotic mottle virus on cell-to-cell and long-distance movement.	in order to elucidate the function of the c-terminal region of cowpea chlorotic mottle bromovirus (ccmv) movement protein (mp) in cell-to-cell movement, a set of deletions ranging from 10 to 80 amino acids (deltamp10, deltamp20, deltamp33, deltamp43, deltamp60 and deltamp80) was engineered into the mp gene encoded by the biologically active clone c3/deltacp-egfp, a variant of ccmv rna3 that contained wild-type (wt) mp and the enhanced green fluorescent protein (egfp) gene in place of the coat pr ...	1999	10374951
synthesis of infectious transcripts of olive latent virus 1: genes required for rna replication and virus movement.	genomic rna of olive latent virus 1 (olv-1) contains five open reading frames (orfs) encoding proteins of 23, 82, 8, 6 and 30 (cp) kda. a full-length cdna copy of olv-1rna was prepared and cloned in a low-copy-number vector (pmuc-19) downstream of or t7 rna polymerase promoter. transcripts derived from this template, denoted pmuc-olv, were highly infectious when inoculated in local and systemic hosts and infected tissues contained virus-like particles. genes required for replication and virus mo ...	1999	10446644
the helper component-proteinase of sweet potato feathery mottle virus facilitates systemic spread of potato virus x in ipomoea nil.	abstract when ipomoea nil was coinfected with sweet potato feathery mottle virus (spfmv), a member of the genus potyvirus, and potato virus x (pvx) typical symptoms caused by pvx were observed on those by spfmv on the first upper true leaves at 14 days postinoculation (dpi). on the other hand, no pvx-induced symptoms were observed on the first upper true leaves at 14 dpi when plants were infected with pvx alone. in the case of coinfection with pvx and spfmv, pvx rna was detected not only in the ...	2000	18944517
brome mosaic virus, good for an rna virologist's basic needs.	abstract taxonomic relationship: type member of the bromovirus genus, family bromoviridae. a member of the alphavirus-like supergroup of positive-sense single-stranded rna viruses. physical properties: virions are nonenveloped icosahedrals made up of 180 coat protein subunits (fig. 1). the particles are 26 nm in diameter and contain 22% nucleic acid and 78% protein. the bmv genome is composed of three positive-sense, capped rnas: rna1 (3.2 kb), rna2 (2.9 kb), rna3 (2.1 kb) (fig. 2). viral protei ...	2000	20572956
recombination between a 3-kilobase tobacco mosaic virus transgene and a homologous viral construct in the restoration of viral and nonviral genes.	transgenic plants harboring various plant virus sequences have shown resistance to viral infections. an environmental risk associated with the use of these plants is the possibility of forming a novel virus by recombination between challenging viruses and transgenic viral mrna. two experiments were designed using tobacco mosaic virus (tmv) vectors and transgenic nicotiana benthamiana to determine if recombinant viral rna would be detectable. n. benthamiana was transformed with a nontranslatable ...	2000	11043947
evidence for a role of the n terminus and leucine-rich repeat region of the mi gene product in regulation of localized cell death.	the tomato mi gene confers resistance against root-knot nematodes and potato aphids. chimeric constructs of the functional gene, mi-1. 2, with a homolog, mi-1.1, were produced, and their phenotypes were examined in agrobacterium rhizogenes-transformed roots. exchange of the leucine-rich repeat (lrr) region of mi-1.1 into mi-1.2 resulted in the loss of ability to confer nematode resistance, as did substitution of a 6-amino acid sequence from the mi-1.1 lrr into mi-1.2. introduction of the mi-1.2 ...	2000	10948252
native electrophoresis and western blot analysis (neweb): a method for characterization of different forms of potyvirus particles and similar nucleoprotein complexes in extracts of infected plant tissues.	a combination of native electrophoresis and immunodetection (western blot) was used for the characterization of nucleoprotein particles of the potyvirus plum pox virus (ppv). virus particles were electrophoresed directly from plant extracts in agarose or mixed acrylamide-agarose gels under native conditions, blotted on nitrocellulose membranes, and characterized with the aid of a coat protein-specific antibody. using this combined methodology, called neweb (native electrophoresis and western blo ...	2000	10950988
a single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing.	we showed previously that 218 and 110 bp n gene segments of tomato spotted wilt virus (tswv) that were fused to the non-target green fluorescent protein (gfp) gene were able to confer resistance to tswv via post-transcriptional gene silencing (ptgs). n gene segments expressed alone did not confer resistance. apparently, the gfp dna induced ptgs that targetted n gene segments and the incoming homologous tswv for degradation, resulting in a resistant phenotype. these observations suggested that mu ...	2000	10900050
transgenic or plant expression vector-mediated recombination of plum pox virus.	different mutants of an infectious full-length clone (p35ppv-nat) of plum pox virus (ppv) were constructed: three mutants with mutations of the assembly motifs rq and df in the coat protein gene (cp) and two cp chimeras with exchanges in the cp core region of zucchini yellow mosaic virus and potato virus y. the assembly mutants were restricted to single infected cells, whereas the ppv chimeras were able to produce systemic infections in nicotiana benthamiana plants. after passages in different t ...	2000	10906199
rotavirus vp6 expressed by pvx vectors in nicotiana benthamiana coats pvx rods and also assembles into viruslike particles.	the rotavirus major inner capsid protein (vp6) has been expressed in nicotiana benthamiana plants using vectors based on potato virus x (pvx). vp6 was expressed either as a fusion with the pvx coat protein or from an additional subgenomic promoter inserted to enable both vp6 and pvx coat protein to be expressed independently. both approaches yielded vp6, which retained the ability to form trimers. vp6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. express ...	2000	10793003
genetic dissection of tomato bushy stunt virus p19-protein-mediated host-dependent symptom induction and systemic invasion.	the plus-sense single-stranded rna of tomato bushy stunt virus (tbsv) encodes a 19-kda protein, which is translated from a 3' proximal open reading frame (p19) that is entirely nested within the cell-to-cell movement gene (p22). expression of the cytosolic p19-protein induces either a systemic lethal collapse in nicotiana benthamiana and n. clevelandii, or necrotic local lesions on resistant n. tabacum. in spinach, the p19-protein is required at high abundance for efficient systemic invasion. th ...	2000	10612662
virus-like particles assemble in plants and bacteria expressing the coat protein gene of indian peanut clump virus.	cdna copies of the coat protein (cp) gene of indian peanut clump virus (ipcv)-h were introduced into cells of nicotiana benthamiana or escherichia coli by transformation with vectors based on prokii or pet respectively. in both plant and bacterial cells, ipcv cp was expressed and assembled to form virus-like particles (vlp). in plant extracts, the smallest preponderant particle length was about 50 nm. other abundant lengths were about 85 and about 120 nm. the commonest vlp length in bacterial ex ...	2000	10640566
evidence of synergism between african cassava mosaic virus and a new double-recombinant geminivirus infecting cassava in cameroon.	stem cuttings were collected in cameroon from cassava plants displaying cassava mosaic disease (cmd) symptoms. the nature of the viruses present was determined by using the pcr with primers specific for the coat protein (cp) genes of african cassava mosaic virus (acmv) and east african cassava mosaic virus (eacmv). all samples were infected by acmv and eight of the 50 samples were infected by both acmv and an eacmv-like virus. the complete nucleotide sequences of dna-a and -b of representative a ...	2000	10640569
role of satellite rna of an indian isolate of cucumber mosaic virus in inducing lethal necrosis of tobacco plants.	lethal necrosis or systemic stem necrosis followed by death of nicotiana benthamiana, severe leaf deformations of n. tabacum cv. white burley and blister formations on n. tabacum cv. samsun nn symptoms were induced by experimental inoculations of cmv rna preparations containing satellite rna (sat-rna). inoculations of rna preparations without sat-rna did not induce that severe symptoms on these plants, only late mild mosaic was observed. it is suggested that sat-rna of cmv isolate has a certain ...	2000	11116536
generation of subgenomic rna directed by a satellite rna associated with bamboo mosaic potexvirus: analyses of potexvirus subgenomic rna promoter.	satellite rna of bamboo mosaic potexvirus (satbamv), a single-stranded positive-sense rna encoding a nonstructural protein of 20 kda (p20), depends on bamboo mosaic potexvirus (bamv) for replication and encapsidation. a full-length cdna clone of satbamv was used to examine the sequences required for the synthesis of potexvirus subgenomic rnas (sgrnas). subgenomic promoter-like sequences (sgps), 107 nucleotides (nt) upstream from the capsid protein (cp) gene of bamv-v, were inserted upstream of t ...	2000	11044078
bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea.	previously, we reported that ccmv(b3a), a hybrid of bromovirus cowpea chlorotic mottle virus (ccmv) with the 3a cell-to-cell movement protein (mp) gene replaced by that of cowpea-nonadapted bromovirus brome mosaic virus (bmv), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. to determine whether the lack of systemic movement of ccmv(b3a) is due to restriction of local spread at specific leaf tissue interfaces, w ...	2000	11059486
the orf1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants.	hybrids of cymbidium ringspot (cymrsv) and carnation italian ringspot (cirv) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. surprisingly, symptoms of nicotiana benthamiana infected with cymrsv/cirv hybrids were distinctly different. it was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents cymrsv and cirv did. we showed here that hybrid ...	2000	11069981
replication of heterologous combinations of helper and defective rna of citrus tristeza virus.	citrus tristeza virus (ctv) populations are among the more complex of plant rna viruses with unusual mixtures of strains and defective rnas (drnas). citrus plants infected with different ctv isolates contain multiple drna molecules that differ in size and relative abundance within and between isolates. additionally, we found mixtures of heterologous drnas in populations. to examine the replication of ctv drnas, the protoplast system had to be extended to support helper-assisted amplification of ...	2000	10662631
mutations in the coat protein gene of plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of nicotiana benthamiana.	two different motifs in the coat protein (cp) of plum pox virus (ppv) (r(3015)q(3016), d(3059)) were mutated by replacing the respective amino acids with others possessing different chemical properties. the mutated cp genes were introduced into an infectious full-length clone of ppv (p35ppv-nat) to investigate their influence on systemic infection of transgenic wild-type ppv cp-expressing and non-transgenic plants of nicotiana benthamiana. all mutants failed to establish systemic infections in n ...	2000	10675394
tagging potato leafroll virus with the jellyfish green fluorescent protein gene.	a full-length cdna corresponding to the rna genome of potato leafroll virus (plrv) was modified by inserting cdna that encoded the jellyfish green fluorescent protein (gfp) into the p5 gene near its 3' end. nicotiana benthamiana protoplasts electroporated with plasmid dna containing this cdna behind the 35s rna promoter of cauliflower mosaic virus became infected with the recombinant virus (plrv-gfp). up to 5% of transfected protoplasts showed gfp-specific fluorescence. progeny virus particles w ...	2000	10675399
the use of transgenic fruit trees as a resistance strategy for virus epidemics: the plum pox (sharka) model.	sharka or plum pox, caused by plum pox virus (ppv: genus potyvirus; family potyviridae), is the most serious disease of prunus. most cultivated prunus species are highly susceptible and conventional breeding has not produced highly resistant and commercially acceptable varieties. success in developing virus-resistant herbaceous crops through genetic engineering led us to investigate this approach for resistance to ppv. our programme aims to develop a biotechnological approach to ppv control that ...	2000	11137162
virus recovery is induced in brome mosaic virus p2 transgenic plants showing synchronous complementation and rna-2-specific silencing.	nicotiana benthamiana plants expressing brome mosaic virus (bmv) p2 protein complemented replication of rnas1 + 3 but, surprisingly, supported little or no replication of rna-2. despite this, the p2 transgenic plants were able to support systemic migration of rnas-1 and -3. kinetic analyses showed identical degradation rates for rnas-2 and -3, greatly detracting from the concept of an induction of an rna-2-specific degradation system. deletion analysis identified a 200-nucleotide sequence that m ...	2000	10707350
suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus.	post-transcriptional gene silencing (ptgs) is a homology-dependent rna degradation process that may target rna exclusively in the cytoplasm. in plants, ptgs functions as a natural defense mechanism against viruses. we reported previously that the 2b protein encoded by cucumber mosaic cucumovirus (cmv) is a virulence determinant and a suppressor of ptgs initiation in transgenic nicotiana benthamiana. by fusion with the green fluorescent protein, we now show that the cmv 2b protein localizes to th ...	2000	10747034
development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus.	to engineer cucumber mosaic virus (cmv-ix) into a gene vector, genome component rna 3 of the virus was modified and split into two sub-components, rna 3a and rna 3b. in rna 3a, the open reading frame of the movement protein (mp) was replaced by a reporter gene encoding the green fluorescent protein (gfp), to monitor virus replication and movement. in rna 3b, the coat protein (cp) gene was eliminated and a multiple cloning site (mcs) was created for foreign gene insertion. each sub-component alon ...	2000	11205118
clones of cotton leaf curl geminivirus induce symptoms atypical of cotton leaf curl disease.	the causative agent of cotton leaf curl disease has previously been shown to be transmissible by the whitefly bemisia tabaci (gennadius) and a begomovirus (geminiviridae) was shown to be associated with the disease. this virus was provisionally called cotton leaf curl virus (clcuv) although no causal relationship between virus and disease was shown. in the present study full-length clones of clcuv, equivalent to the dna a component of bipartite begomoviruses, were obtained. the clones of clcuv w ...	2000	10766303
virus-induced silencing of a plant cellulose synthase gene.	specific cdna fragments corresponding to putative cellulose synthase genes (cesa) were inserted into potato virus x vectors for functional analysis in nicotiana benthamiana by using virus-induced gene silencing. plants infected with one group of cdnas had much shorter internode lengths, small leaves, and a "dwarf" phenotype. consistent with a loss of cell wall cellulose, abnormally large and in many cases spherical cells ballooned from the undersurfaces of leaves, particularly in regions adjacen ...	2000	10810144
rapid production of the major birch pollen allergen bet v 1 in nicotiana benthamiana plants and its immunological in vitro and in vivo characterization.	type i allergies are immunological disorders that afflict a quarter of the world's population. improved diagnosis of allergic diseases and the formulation of new therapeutic approaches are based on the use of recombinant allergens. we describe here for the first time the application of a rapid plant-based expression system for a plant-derived allergen and its immunological characterization. we expressed our model allergen bet v 1, the major birch pollen allergen, in the tobacco-related species n ...	2000	10877820
tobacco mosaic virus replicase-mediated cross-protection: contributions of rna and protein-derived mechanisms.	specific sequences of the tobacco mosaic virus (tmv) rna-dependent rna-polymerase (rdrp) gene were investigated for their ability to confer cross-protection. nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (pol) domains of the tmv rdrp open reading frame were systemically expressed in nicotiana benthamiana using a potato x virus (pvx) vector [chapman, s., kavanagh, t., and baulcombe, d. c. (1992). plant j., 1, ...	2000	10915597
vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in nicotiana benthamiana.	plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. tobacco mosaic virus (tmv) entry into minor, major and transport veins from non-vascular cells of nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (g ...	2000	10929128
delayed activation of post-transcriptional gene silencing and de novo transgene methylation in plants with the coat protein gene of sweet potato feathery mottle potyvirus.	the relationship between post-transcriptional gene silencing (ptgs) and dna methylation was examined using nicotiana benthamiana transformed with the coat protein gene including the 3' non-translated region of sweet potato feathery mottle potyvirus. line 4.28 showed a delayed activation of the transgene silencing in comparison with the other silenced lines, and showed complete resistance against the recombinant potato virus x engineered to contain the sequence homologous to the transgene when th ...	2000	10936519
analysis of transgenic grapevine (vitis rupestris) and nicotiana benthamiana plants expressing an arabis mosaic virus coat protein gene.	a disarmed lba4404 strain of agrobacterium tumefaciens harboring a binary vector which contained chimeric genes encoding the neomycin phosphotransferase (npt ii) and the coat protein (cp) of arabis mosaic nepovirus (armv) was used in co-cultivation experiments with leaf discs of nicotiana benthamiana and somatic embryos of the grapevine rootstock cultivar vitis rupestris. transgenic n. benthamiana expressing the armv cp gene were regenerated and six independent lines were characterized. enzyme-l ...	2000	10936531
the valine anticodon and valylatability of peanut clump virus rnas are not essential but provide a modest competitive advantage in plants.	the role of valine aminoacylation of the two genomic rnas of peanut clump virus (pcv) was studied by comparing the amplification in vivo of rnas with gac, gdeltac, or cca anticodons in the trna-like structure (tls) present at the 3' end of each viral rna. the pcv rna1 tls of isolate pcv2 possesses a gac anticodon and is capable of highly efficient valylation, whereas the rna2 tls has a gdeltac anticodon that does not support valylation. the presence in rna1 of gdeltac or cca anticodons that conf ...	2000	10954573
the p20 gene product of citrus tristeza virus accumulates in the amorphous inclusion bodies.	citrus tristeza virus (ctv) has 10 3' open reading frames (orfs) of unknown function except for the two coat proteins. the highest produced subgenomic rnas are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. the proteins from three orfs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. the p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. ...	2000	10964768
regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein.	cell-to-cell spread of tobacco mosaic virus (tmv) through plant intercellular connections, the plasmodesmata, is mediated by a specialized viral movement protein (mp). in vivo studies using transgenic tobacco plants showed that mp is phosphorylated at its c-terminus at amino acid residues ser258, thr261 and ser265. when mp phosphorylation was mimicked by negatively charged amino acid substitutions, mp lost its ability to gate plasmodesmata. this effect on mp-plasmodesmata interactions was specif ...	2000	10990451
a viral movement protein prevents spread of the gene silencing signal in nicotiana benthamiana.	in plants, viruses induce an rna-mediated defense that is similar to posttranscriptional gene silencing (ptgs) of transgenes. here we demonstrate with potato virus x (pvx) that ptgs operates as a systemic, sequence-specific defense system. however, in grafting experiments or with movement defective forms of pvx, we could not detect systemic silencing unless the 25 kda viral movement protein (p25) was made nonfunctional. investigation of p25 revealed two branches to the ptgs pathway that converge ...	2000	11051555
bipartite geminivirus host adaptation determined cooperatively by coding and noncoding sequences of the genome.	bipartite geminiviruses are small, plant-infecting viruses with genomes composed of circular, single-stranded dna molecules, designated a and b. although they are closely related genetically, individual bipartite geminiviruses frequently exhibit host-specific adaptation. two such viruses are bean golden mosaic virus (bgmv) and tomato golden mosaic virus (tgmv), which are well adapted to common bean (phaseolus vulgaris) and nicotiana benthamiana, respectively. in previous studies, partial host ad ...	2000	11080490
closterovirus encoded hsp70 homolog and p61 in addition to both coat proteins function in efficient virion assembly.	assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. closteroviridae virions are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. the closteroviridae is the only virus family that encodes a protein with similarity to cellular chaperones, a 70-kda heat-shock protein homolog (hsp70h). ...	2000	11112500
partial purification and characterization of cucumber necrosis virus and tomato bushy stunt virus rna-dependent rna polymerases: similarities and differences in template usage between tombusvirus and carmovirus rna-dependent rna polymerases.	tombusviruses are small, plus-sense, single-stranded rna viruses of plants. rna-dependent rna polymerases (rdrp) of two tombusviruses, tomato bushy stunt virus (tbsv) and cucumber necrosis virus (cnv), have been partially purified from infected nicotiana benthamiana plants. the obtained rdrp complexes are capable of de novo initiation of complementary rna synthesis using either plus- or minus-strand templates derived from tombusvirus defective interfering (di) rnas. in addition to template-sized ...	2000	11040120
aphid transmission studies using helper component proteins of potato virus y expressed from a vector derived from potato virus x.	the genes encoding the helper component (hc) proteins of two strains of potato virus y (pvy) were cloned and the proteins expressed from a vector derived from potato virus x (pvx). the expressed hc contained six n-terminal histidine residues to facilitate purification by metal affinity chromatography. approximately 2-4 microg/g of purified hc was obtained from leaves of nicotiana benthamiana plants systemically infected by recombinant pvx. preparations of the hc protein derived from pvy ordinary ...	2000	10725440
soluble, template-dependent extracts from nicotiana benthamiana plants infected with potato virus x transcribe both plus- and minus-strand rna templates.	we have developed a method to convert membrane-bound replication complexes isolated from nicotiana benthamiana plants infected with potato virus x (pvx) to a soluble, template-dependent system for analysis of rna synthesis. analysis of rna-dependent rna polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of pvx genomic rna and the two predominant subgenomic rnas. the endogenous templates were remov ...	2000	10998342
mechanical transmission of potato leafroll virus.	like typical luteoviruses, potato leafroll virus (plrv) cannot be transmitted mechanically by rubbing plants with solutions containing virus particles. however, plrv was found to be mechanically transmissible from extracts of plants that had been inoculated by viruliferous aphids and then post-inoculated with pea enation mosaic virus-2 (pemv-2). unlike the asymptomatic infections induced by either virus alone, double infections in nicotiana benthamiana induced necrotic symptoms with some line pa ...	2000	11038393
complementation of the movement-deficient mutations in potato virus x: potyvirus coat protein mediates cell-to-cell trafficking of c-terminal truncation but not deletion mutant of potexvirus coat protein.	the cell-to-cell movement of the gus-tagged potato virus x (pvx) coat protein (cp) movement-deficient mutant was restored by potyviral cps of potato virus a (pva) and potato virus y (pvy) in nicotiana benthamiana leaves in transient cobombardment experiments. viral cell-to-cell movement of pvx cp mutant was complemented in nicotiana tabacum cv. sr1 transgenic plants expressing pvy cp: pvx rna and polymerase were detected in the pvx cp mutant-inoculated leaves of transgenic plants. these findings ...	2000	10772977
activities associated with the putative replication initiation protein of coconut foliar decay virus, a tentative member of the genus nanovirus.	the putative replication initiation protein (rep) of coconut foliar decay virus (cfdv) was expressed as a 6x his recombinant protein in e. coli and in recombinant baculovirus. purified 6x his-rep protein was demonstrated to possess sequence non-specific rna- and ssdna-binding activities as well as magnesium-dependent atpase/gtpase activity. the yeast two-hybrid system revealed that cfdv rep could interact with itself. subcellular distribution of the cfdv rep was studied by fractionation of insec ...	2000	11086141
tissue specificity of geminivirus infection is genetically determined.	the types of cells and tissues infected by a virus define its tissue tropism. determinants of tissue tropism in animal-infecting viruses have been extensively investigated, but little is known about plant viruses in this regard. some geminiviruses in the genus begomovirus exhibit phloem limitation and are restricted to cells of the vascular system, whereas others can invade mesophyll tissue. to identify viral genetic determinants of tissue tropism, we established a model system using two begomov ...	2000	11090223
evolutionarily related sindbis-like plant viruses maintain different levels of population diversity in a common host.	the levels of population diversity of three related sindbis-like plant viruses, tobacco mosaic virus (tmv), cucumber mosaic virus (cmv), and cowpea chlorotic mottle virus (ccmv), in infections of a common host, nicotiana benthamiana, established from genetically identical viral rna were examined. despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. cmv had the highest levels of diversity, tmv had an intermediate level of d ...	2000	10708428
rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an rna viral vector.	tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. the rice alpha-amylase gene (os103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a rna viral vector. one to two weeks after inoculation, transfected nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. the 46kda recombinant enzyme was purified, and its structural and biological properties were an ...	2000	10713457
subcellular sorting of small membrane-associated triple gene block proteins: tgbp3-assisted targeting of tgbp2.	we studied subcellular distribution of green fluorescent protein (gfp)-tagged movement proteins encoded by the second and the third genes of poa semilatent hordeivirus (pslv) triple gene block (tgb), 15k tgbp2 and 18k tgbp3. gfp-15k transiently expressed in nicotiana benthamiana leaf epidermal cells was associated with the endomembrane system elements. gfp-18k appeared in the membrane bodies at cell periphery. mutation analysis demonstrated that subcellular targeting of gfp-15k depended on the p ...	2000	10725204
potato mop-top virus rna can move long distance in the absence of coat protein: evidence from resistant, transgenic plants.	transgenic expression of a translatable version of the potato mop-top virus (pmtv) coat protein (cp) gene (encoded by rna 3) in nicotiana benthamiana prevented production of symptoms and infective virus particles. rnas 1 and 2 accumulated in inoculated and systemic leaves but, apart from small amounts of cp transgene rna transcript, no genomic-length rna 3 was found. crude leaf extracts from inoculated plants were not infective. however, when rna extracts from such transgenic plants were inocula ...	2000	10656594
production of antimicrobial defensin in nicotiana benthamiana with a potato virus x vector.	a recombinant plasmid, ptxs.th, was constructed to express the gene-encoding wasabi (wasabia japonica) defensin with the potato virus x (pvx) vector. ptxs.th allows the expression of defensin in the host nicotiana benthamiana, and the defensin protein wt1 can be purified from virus-infected leaves by heat treatment and affinity chromatography. wt1 exhibits strong antifungal activity toward the phytopathogenic fungi magnaporthe grisea (50% inhibitory concentration [ic50] = 5 microg/ml) and botryt ...	2001	11204773
replicase-derived resistance against pea early browning virus in nicotiana benthamiana is an unstable resistance based upon posttranscriptional gene silencing.	virus resistance in nicotiana benthamiana plants containing a translatable pea early browning virus (pebv) 54k sequence from the 201k replicase gene has been reported previously. resistant plants contain multiple transgene copies divided between two loci. analysis of a genetic series containing the two loci in separate homozygous or heterozygous condition suggest that only one of the loci is necessary to induce the resistance. the resistance observed in r2 and r3 generations of lines containing ...	2001	11204783
cell-to-cell movement of potato virus x: the role of p12 and p8 encoded by the second and third open reading frames of the triple gene block.	potato virus x (pvx) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (cp) for cell-to-cell movement. when each of these proteins was co-expressed with a cytosolic green fluorescent protein (gfp) in the epidermal cells of nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed gfp, indicating an ability to alter plasmodesmal permeability. p25, p12, and cp, expressed transi ...	2001	11605955
size constraints for targeting post-transcriptional gene silencing and for rna-directed methylation in nicotiana benthamiana using a potato virus x vector.	using a recombinant potato virus x (pvx) vector, we investigated the relationship between the length of rna sequence identity with a transgene and the ability to promote post-transcriptional gene silencing (ptgs) and transgene methylation. the lower size limit required for targeting reporter transgene mrna de novo using ptgs was 23 nucleotides (nt) of complete identity, a size corresponding to that of small rnas associated with ptgs in plants and rna interference (rnai) in animals. the size and ...	2001	11260498
systemic spread of an rna insect virus in plants expressing plant viral movement protein genes.	flock house virus (fhv), a single-stranded rna insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [selling, b. h., allison, r. f. & kaesberg, p. (1990) proc. natl. acad. sci. usa 87, 434-438]. there was no systemic movement of fhv in plants. we tested the ability of movement proteins (mps) of plant viruses to provide movement functions and cause systemic spread of fhv in plants. we compared ...	2001	11296259
rapid production of recombinant allergens in nicotiana benthamiana and their impact on diagnosis and therapy.	type i allergies are immunological disorders that afflict a quarter of the world's population. recombinant allergens have improved the diagnosis of allergic diseases and allow the formulation of new therapeutic approaches. over 50% of all allergens are of plant origin.	2001	11306923
expression of the oligomerization domain of the replication-associated protein (rep) of tomato leaf curl new delhi virus interferes with dna accumulation of heterologous geminiviruses.	the minimal dna binding domain of the replication-associated protein (rep) of tomato leaf curl new delhi virus was determined by electrophoretic mobility gel shift analysis and co-purification assays. dna binding activity maps to amino acids 1-160 (rep-(1-160)) of the rep protein and overlaps with the protein oligomerization domain. transient expression of rep protein (rep-(1-160)) was found to inhibit homologous viral dna accumulation by 70-86% in tobacco protoplasts and in nicotiana benthamian ...	2001	11342533
the suppressor of transgene rna silencing encoded by cucumber mosaic virus interferes with salicylic acid-mediated virus resistance.	the cucumber mosaic virus (cmv)-encoded 2b protein (cmv2b) is a nuclear protein that suppresses transgene rna silencing in nicotiana benthamiana. cmv2b is an important virulence determinant but nonessential for systemic spread in n. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. here, we report that cmv2b became essential for systemic infections in older n. glutinosa plants or in young seedlings pretreated with salicylic acid (sa). expression of cmv2b from ...	2001	11386367
inducible high-level mrna amplification system by viral replicase in transgenic plants.	we have constructed a new system for inducible high-level expression of mrna for foreign genes in transgenic plants by introducing a glucocorticoid-inducible transcription system into the previously developed "mrna amplification system" where target mrna can be amplified as a subgenomic rna by the replicase of a plant tripartite rna virus, brome mosaic virus (bmv). in the new amplification system, the amplification of mrna is tightly regulated by the expression of a subunit of the bmv replicase. ...	2001	11489185
potyviral helper-component proteinase expressed in transgenic plants enhances titers of potato leaf roll virus but does not alleviate its phloem limitation.	coinfection of nicotiana benthamiana with potato virus a (pva, a potyvirus) and potato leaf-roll virus (plrv, a luteovirus) induces a synergistic interaction manifested by enhanced titers of plrv. the helper component proteinase (hc-pro) of potyviruses is involved in viral vascular movement and suppression of an antiviral defense mechanism in plants. data of our study showed that accumulation of plrv in transgenic n. benthamiana expressing the pva hc-pro was enhanced on average by 4.5-fold, as c ...	2001	11336553
the expression of a single-chain fv antibody fragment in different plant hosts and tissues by using potato virus x as a vector.	some aspects of the expression of a single-chain fv antibody fragment (scfv) driven by the plant viral vector potato virus x (pvx) have been studied by quantitative elisa. after inoculation of the infectious transcript, the vector was stable only for a few passages of sap transmission in the inoculated leaves of nicotiana benthamiana and the reversal to wild type was more pronounced in the systemically invaded leaves. the amount of synthesized scfv varied when different solanaceous hosts were te ...	2001	11388801
role of the 3'-untranslated regions of alfalfa mosaic virus rnas in the formation of a transiently expressed replicase in plants and in the assembly of virions.	alfalfa mosaic virus (amv) rnas 1 and 2 encode the replicase proteins p1 and p2, respectively, whereas rna 3 encodes the movement protein and the coat protein (cp). when rnas 1 and 2 were transiently expressed from a t-dna vector (r12 construct) by agroinfiltration of nicotiana benthamiana, the infiltrated leaves accumulated minus-strand rnas 1 and 2 and relatively small amounts of plus-strand rnas. in addition, rna-dependent rna polymerase (rdrp) activity could be detected in extracts of the in ...	2001	11413311
secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants.	we chose the follicle stimulating hormone (fsh), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive n-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. a tobacco mosaic virus (tmv) based transient expression system was used to express a single-chain (sc) version of bovine fsh in the tobacco related species nicotiana benth ...	2001	11502219
interactions of the tgb1 protein during cell-to-cell movement of barley stripe mosaic virus.	we have recently used a green fluorescent protein (gfp) fusion to the gammab protein of barley stripe mosaic virus (bsmv) to monitor cell-to-cell and systemic virus movement. the gammab protein is involved in expression of the triple gene block (tgb) proteins encoded by rnabeta but is not essential for cell-to-cell movement. the gfp fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant tgb-encoded proteins, b ...	2001	11507216
induction of a hypersensitive response by chimeric helicase sequences of tobamoviruses u1 and ob in n-carrying tobacco.	recently, the helicase domain of the tobacco mosaic virus (tmv)-u1 replicase proteins (designated morehel:u1) was identified as the elicitor of the n gene-mediated hypersensitive response (hr) in tobacco. in this study, we used agroinfiltration to express the equivalent morehel domain of the non-hr-inducing tobamovirus strain tmv-ob. it appeared that this morehel:ob sequence did not elicit a hr in n gene-carrying tobacco. both morehel sequences were divided into eight subdomains, and chimeras of ...	2001	11551073
promoters for pregenomic rna of banana streak badnavirus are active for transgene expression in monocot and dicot plants.	two putative promoters from australian banana streak badnavirus (bsv) isolates were analysed for activity in different plant species. in transient expression systems the my (2105 bp) and cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, nicotiana benthamiana, tipu tree), gymnosperm (pinus radiata) and fern (nephrolepis cordifolia). evaluation of ...	2001	11587511
the partial purified rna-dependent rna polymerases from bamboo mosaic potexvirus and potato virus x infected plants containing the template-dependent activities.	rna-dependent rna polymerases (rdrp) isolated from bamboo mosaic potexvirus (bamv) and potato virus x infected nicotiana benthamiana plants and solubilized with the detergent np-40, generated a full-length genomic and two subgenomic double-stranded rnas of respective viruses in an in vitro rdrp assay containing endogenous rna templates. template-dependent and species-specific rdrp activity could be detected after the removal of endogenous rna templates. the 3' untranslated regions (utr) containi ...	2001	11597747
gene c2 of the monopartite geminivirus tomato yellow leaf curl virus-china encodes a pathogenicity determinant that is localized in the nucleus.	expression of the tomato yellow leaf curl virus-china (tylcv-c) c2 protein and green fluorescent protein (gfp) fused to the c2 protein (c2-gfp) in nicotiana benthamiana from a potato virus x (pvx) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. the localization of gfp fluorescence in plant cells infected with pvx/c2-gfp and in insect cells transfected with baculovirus expressing c2-gfp indicates that the ...	2001	11551077
rna-binding properties of the 63 kda protein encoded by the triple gene block of poa semilatent hordeivirus.	the 63 kda '63k' movement protein encoded by the triple gene block of poa semilatent virus (pslv) comprises the c-terminal ntpase/helicase domain and the n-terminal extension domain, which contains two positively charged sequence motifs, a and b. in this study, the in vitro rna-binding properties of pslv 63k and its mutants were analysed. membrane-immobilized 63k and n-63k (isolated n-terminal extension domain) bound rna at high nacl concentrations. in contrast, c-63k (isolated ntpase/helicase d ...	2001	11562549
calcium-dependent protein kinases play an essential role in a plant defence response.	calcium-dependent protein kinases (cdpks) comprise a large family of serine/threonine kinases in plants and protozoans. we isolated two related cdpk cdnas (ntcdpk2 and ntcdpk3) from nicotiana tabacum. these cdpk transcripts are elevated after race-specific defence elicitation and hypo-osmotic stress. transiently expressed myc-epitope-tagged ntcdpk2 in nicotiana benthamiana and n.tabacum leaves showed a rapid transient interconversion to an activated form after elicitation and hypo-osmotic stress ...	2001	11597999
umbravirus-encoded proteins both stabilize heterologous viral rna and mediate its systemic movement in some plant species.	the proteins encoded by open reading frame 3 (orf3) of the umbraviruses pea enation mosaic virus-2 and tobacco mottle virus, like that of groundnut rosette virus, mediated the movement of viral rna through the phloem of infected nicotiana benthamiana or n. clevelandii plants when they were expressed from chimeric tobacco mosaic virus in place of the coat protein. however, these chimeras did not move systemically in n. tabacum. in lysates of n. benthamiana or n. tabacum protoplasts, the chimeric ...	2001	11601910
evidence for rna-mediated defence effects on the accumulation of potato leafroll virus.	in plants infected with potato leafroll virus (plrv), or other luteoviruses, infection is very largely confined to cells in the vascular system. even in tobacco plants transformed with plrv full-length cdna, in which all mesophyll cells should synthesize infectious plrv rna transcripts, only a minority of the mesophyll cells accumulate detectable amounts of virus. we have explored this phenomenon further by transforming a better plrv host, nicotiana benthamiana, with the same transgene, by super ...	2001	11714988
effects of inactivation of the coat protein and movement genes of tomato bushy stunt virus on early accumulation of genomic and subgenomic rnas.	accumulation of rna of tomato bushy stunt virus (tbsv) was examined within the first few hours after infection of nicotiana benthamiana protoplasts to determine the influence of the coat protein (cp), the movement-associated proteins p22 and p19 and rna sequences at very early stages of replication. the results showed that p19 had no effect on early rna replication, whereas the absence of cp and/or p22 expression delayed rna accumulation only marginally. removal of cp-coding sequences had no add ...	2001	11714989
completion of nucleotide sequence and generation of highly infectious transcripts to cucurbits from full-length cdna clone of kyuri green mottle mosaic virus.	the nucleotide sequence of the genome of the type strain of kyuri green mottle mosaic virus (kgmmv-c1) has been completely determined. the genome structure and sequence of the virus were compared to those of yodo strain of kgmmv (kgmmv-y). the genome of kgmmv-c1 is 6,514 nucleotides long consisting of 5' and 3' nontranslated regions (ntrs) and four open reading frames coding for 131 kda and 189 kda viral replicases, 28 kda movement protein and 17 kda coat protein. the nucleotide and amino acid s ...	2001	11765913
a novel strategy for the expression of foreign genes from plant virus vectors.	potato virus x (pvx)-based vector constructs were generated to investigate the use of an internal ribosome entry site (ires) sequence to direct translation of a viral gene. the 148-nucleotide irescp sequence from a crucifer-infecting strain of tobacco mosaic virus was used to direct expression of the pvx coat protein (cp). the ires was inserted downstream of the gene encoding green fluorescent protein (gfp) and upstream of the pvx cp, in either sense or antisense orientation, such that cp expres ...	2001	11165252
genetic analysis of bipartite geminivirus tissue tropism.	the bipartite geminiviruses bean golden mosaic virus (bgmv), cabbage leaf curl virus (cablcv), and tomato golden mosaic virus (tgmv) exhibit differential tissue tropism in nicotiana benthamiana. in systemically infected leaves, bgmv remains largely confined to vascular-associated cells (phloem-limited), whereas cablcv and tgmv can escape into the surrounding mesophyll. previous work established that tgmv bri, the noncoding region upstream from the br1 open reading frame (orf), is required for me ...	2001	11878900
functional equivalence of late gene promoters in bean golden mosaic virus with those in tomato golden mosaic virus.	in the bipartite geminivirus tomato golden mosaic virus (tgmv), the activity of late gene promoters is up-regulated by the multifunctional viral protein al2. cis-acting sequences required for al2-mediated promoter responses have not been well characterized. however, nucleotide sequence analysis has implicated a motif termed the conserved late element (cle). the cle is present in tgmv and many other begomoviruses, although it is not ubiquitous. here we analysed the regulation of late gene express ...	2001	11172109
coat protein gene-mediated resistance to potato virus a in transgenic plants is suppressed following infection with another potyvirus.	high levels of resistance to potato virus a (pva, genus potyvirus), indicated by absence of detectable infection in inoculated leaves, were attained in nicotiana benthamiana transformed with a construct expressing the pva 5'-untranslated region fused with the coat protein (cp)-encoding sequence. low steady-state levels of the transgene transcripts were detected. resistance was pva-specific and did not protect the plants against infection with potato virus y (pvy, genus potyvirus). consequently, ...	2001	11514739
plants expressing tomato golden mosaic virus al2 or beet curly top virus l2 transgenes show enhanced susceptibility to infection by dna and rna viruses.	the al2 gene of the geminivirus tomato golden mosaic virus (tgmv) encodes a transcriptional activator protein (trap) that is required for efficient expression of the viral coat protein (cp) and br1 gene promoters. in contrast, l2, the positional homolog of al2 in the related beet curly top virus (bctv), is not required for cp expression, raising questions about the functional relationship between the al2 and l2 gene products. in this study, transgenic nicotiana benthamiana and n. tabacum var. sa ...	2001	11414806
use of a vector based on potato virus x in a whole plant assay to demonstrate nuclear targeting of potato spindle tuber viroid.	potato spindle tuber viroid (pstvd) is a covalently closed circular rna molecule of 359 nucleotides that replicates within the nucleus of host cells. to determine how this small, highly structured rna enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (gfp) as the reporter molecule. the coding region of gfp was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato st-ls1 gene. a cdna copy of the ...	2001	11369895
5'-coterminal subgenomic rnas in citrus tristeza virus-infected cells.	three unusual 5' coterminal positive-stranded subgenomic (sg) rnas, two of about 0.8 kb and one of 10 kb (designated lmt1, lmt2, and lamt, respectively), from citrus spp. plants and nicotiana benthamiana protoplasts infected with citrus tristeza virus (ctv) were characterized. the 5' termini of the lmt rnas were mapped by runoff reverse transcription and found to correspond with the 5' terminus of the genomic rna. the lmt 5'-coterminal sgrnas consisted of two modal lengths of 744--746 and 842--8 ...	2001	11336562
the c terminus of brome mosaic virus coat protein controls viral cell-to-cell and long-distance movement.	to investigate the functional domains of the coat protein (cp; 189 amino acids) of brome mosaic virus, a plant rna virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley and nicotiana benthamiana. despite its apparent normal replicative competence and cp production, the c-terminal mutant f184a produced no virions. furthermore, virion-forming c-terminal mutants p178a and d182a failed to move from cell to cell in both plant species, and mutants d181a and v18 ...	2001	11333922
cellular basis of potato spindle tuber viroid systemic movement.	viroids are small, nontranslatable pathogenic rnas that replicate autonomously and traffic systemically in their host plants. we have used in situ hybridization to analyze the trafficking pattern of potato spindle tuber viroid (pstvd) in tomato and nicotiana benthamiana. when pstvd was inoculated onto the stem of a plant, it replicated and trafficked to sink, but not source, leaves. pstvd was absent from shoot apical meristems. in the flowers of infected plants, pstvd was present in the sepals, ...	2001	11145890
the size of encapsidated single-stranded dna determines the multiplicity of african cassava mosaic virus particles.	transgenic nicotiana benthamiana plants harbouring a defective interfering (di) dna of african cassava mosaic virus (acmv) and control plants were inoculated with acmv. virus particles were purified from infected plants, separated in sucrose gradients and fractions were analysed by southern blotting. transgenic plant-derived virus particles taken from the top fractions of sucrose gradients contained di dna, middle fractions contained a mixture of genomic and di dna and bottom fractions contained ...	2001	11172110
ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene pto.	we have investigated the origin of the pto disease resistance (r) gene that was previously identified in the wild tomato species lycopersicon pimpinellifolium and isolated by map-based cloning. pto encodes a serine-threonine protein kinase that specifically recognizes strains of pseudomonas syringae pv. tomato (pst) that express the avirulence gene avrpto. we examined an accession of the distantly related wild species lycopersicon hirsutum var. glabratum that exhibits avrpto-specific resistance ...	2001	11172075
complete nucleotide sequence and host range of south african cassava mosaic virus: further evidence for recombination amongst begomoviruses.	complete nucleotide sequences of the dna-a (2800 nt) and dna-b (2760 nt) components of a novel cassava-infecting begomovirus, south african cassava mosaic virus (sacmv), were determined and compared with various new world and old world begomoviruses. sacmv is most closely related to east african cassava mosaic virus (eacmv) in both its dna-a (85% with eacmv-mh and -mk) and -b (90% with eacmv-ug2-mld and eacmv-ug3-svr) components; however, percentage sequence similarities of less than 90% in the ...	2001	11125158