production, characterization and use of monoclonal antibodies to grapevine virus a.four stable hybridoma cell lines secreting monoclonal antibodies to grapevine closterovirus a (gva) were obtained by fusing spleen cells of immunized balb/c mice with mouse myeloma cell line sp2/0-ag 14. in elisa all mabs reacted with virus in leaf extracts from nicotiana benthamiana, glass-house-, field-, or in vitro-grown grapevines, or with cortical shavings from mature grape canes. in iem tests, only one of the mabs (pa3.f5) decorated virus particles on the entire surface. this mab was likel ...19921280942
biologically active cymbidium ringspot virus satellite rna in transgenic plants suppresses accumulation of di rna.a full-length dna copy of cymbidium ringspot virus (cyrsv) satellite rna was cloned downstream of the bacteriophage t7 rna polymerase promoter. in vitro transcripts were biologically active in plants when coinoculated with the helper virus or its rna. although the transcripts contained 7 or 29 extra nucleotides at the 3' end, the proper 3' terminus was restored in the satrna progeny. full-length cdna clones of cyrsv satrna under the control of the cauliflower mosaic virus 35s promoter and termin ...19921374981
dna forms of the geminivirus african cassava mosaic virus consistent with a rolling circle mechanism of replication.we have analysed dna from african cassava mosaic virus (acmv)-infected nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected acmv-specific dnas by blot-hybridisation. acmv dna forms including the previously characterised single-stranded, open-circular, linear and supercoiled dnas along with five previously uncharacterised heterogeneous dnas (h1-h5) were resolved. the heterogeneous dnas were characterised by their chromatographic properties on bnd-cellulose and their a ...19912041773
point mutations in the turnip crinkle virus capsid protein affect the symptoms expressed by nicotiana an effort to determine the biological function(s) of the capsid protein protruding domains unique to the plant carmo- and tombusviruses, we constructed turnip crinkle virus (tcv) mutants in which tandem, in-frame translation terminators replaced the first two codons of the five-amino acid hinge between the shell and the protruding domains of the tcv capsid protein. one of the mutants replicated in inoculated leaves and protoplasts without detectable accumulation of capsid protein. the mutant ...19912053278
defective-interfering rnas and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts.tomato bushy stunt virus (tbsv) genomic rna and one of its defective interfering (di) rnas were inoculated in various combinations to protoplasts of nicotiana benthamiana. ethidium bromide staining of electrophoretically separated rnas from infected protoplasts, incorporation of [3h]uridine into tbsv and di rnas, and northern hybridization at different times after inoculation clearly demonstrated reduced accumulation of genomic rna in the presence of di rna. accumulation of genomic rna was very ...19902345965
tomato bushy stunt virus spread is regulated by two nested genes that function in cell-to-cell movement and host-dependent systemic invasion.we have investigated the importance of two small nested genes (p19 and p22) located near the 3' end of the genome of tomato bushy stunt virus (tbsv) for infectivity in several hosts. our results show that both genes are dispensable for replication and transcription and that the p19 gene encodes a soluble protein, whereas the p22 gene specifies a membrane-associated protein. assays using tbsv derivatives that have the beta-glucuronidase gene substituted for the capsid protein gene demonstrate tha ...19957491767
intergeneric transfer and functional expression of the tomato disease resistance gene pto.plant disease resistance loci have been used successfully in breeding programs to transfer traits from resistant germplasm to susceptible plant cultivars. the molecular cloning of plant disease resistance genes now permits the transfer of such traits across species boundaries by genetic transformation of recipient hosts. the tomato disease resistance gene pto confers resistance to strains of the bacterial pathogen pseudomonas syringae pv tomato expressing the avirulence gene avrpto. transformati ...19957580250
nicotiana benthamiana plants transformed with the 54-kda region of the pepper mild mottle tobamovirus replicase gene exhibit two types of resistance responses against viral infection.nicotiana benthamiana plants transformed with the 54-kda region of the pepper mild mottle tobamovirus (pmmv) replicase gene were generated and six independently transformed plant lines were analyzed for resistance to pmmv. two different resistance responses were obtained. some of the transgenic plants from only two lines showed a preestablished, complete, and highly resistant phenotype since no viral symptoms were observed, although a low level of virus replication occurred. the remaining plants ...19957645209
characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene.transgenic nicotiana benthamiana plants expressing the replicase gene of cymbidium ringspot tombusvirus (cymrsv) were tested for the biological activity of the transgene. protoplasts from one resistant and three susceptible transgenic lines were transfected with in vitro synthesized defective interfering (di) rna, a small subviral molecule which requires only the virus replicase for replication. only protoplasts of susceptible transgenic lines were able to sustain the replication of di rna, wher ...19957676638
complementation of african cassava mosaic virus ac2 gene function in a mixed bipartite geminivirus infection.we have previously demonstrated that african cassava mosaic virus (acmv) dnas a and b efficiently complement the systemic spread of tomato golden mosaic virus (tgmv) dna a when co-agroinoculated onto nicotiana benthamiana. here, we show that a mixture of an acmv dna a ac2 mutant and dna b that is normally unable to systemically infect n. benthamiana can do so at low frequency when co-agroinoculated with tgmv dna a. analysis of viral dna showed that the ac2 mutation was retained during infection. ...19957561766
detection of an unidentified potyvirus from roystonea regia palm using the polymerase chain reaction and degenerate, potyvirus specific, primers and potential problems arising from the amplification of host plant dna sequences.degenerate potyvirus-specific primers were used in the pcr to amplify cdna representing a 335 nucleotide region of the coat protein gene in rna purified from an uncharacterised potyvirus isolated from roystonea regia palms in queensland, australia. the rna was also detected by pcr in total nucleic acid extracts from infected nicotiana benthamiana, n. clevelandii and vicia faba. the method also successfully detected pea seed-borne mosaic virus in pisum sativum. in addition the procedure amplified ...19947714044
indian cassava mosaic virus: ultrastructure of infected cells.ultrastructural examination of leaf tissue of nicotiana benthamiana infected with indian cassava mosaic virus (icmv) revealed abnormalities in phloem and, occasionally, xylem cells. nuclei contained granular inclusion bodies which seemed to be largely composed of virus-like particles and were shown by immunogold labelling (igl) to be rich in icmv coat protein. later in infection, hollow spheres made up of fibrillar material were produced. virus coat protein was not a necessary component of these ...19892794977
size reversion of african cassava mosaic virus coat protein gene deletion mutants during infection of nicotiana benthamiana.mutants of african cassava mosaic virus containing extensive deletions across the coat protein gene that remove up to one-third of the genomic component have been constructed and shown to be infectious when mechanically inoculated onto nicotiana benthamiana by leaf abrasion. using n. tabacum protoplasts we demonstrate that mutant pclv.cp delta 11, containing a 712 bp deletion, is competent for replication in its deleted form. however, systemic spread of pclv. cp delta 11 and other deletion mutan ...19892732690
functional expression of the leftward open reading frames of the a component of tomato golden mosaic virus in transgenic tobacco plants.the genome of the geminivirus tomato golden mosaic virus (tgmv) consists of two circular dna molecules designated as components a and b. we have constructed nicotiana benthamiana plants that are transgenic for the three overlapping open reading frames, al1, al2, and al3, from the left side of tgmv a. in the transgenic plants, the al open reading frames are under the control of the cauliflower mosaic virus (camv) 35s promoter. in tgmv infectivity assays, seven of 10 transgenic lines complemented ...19892562559
geminivirus coat protein gene replacement alters insect specificity.chimeric clones have been constructed in which the coat protein encoded by dna a of the bipartite genome of the geminivirus african cassava mosaic virus (acmv) has been replaced by that of beet curly top virus (bctv). constructs containing the coding region inserted in either orientation were infectious when co-inoculated with acmv dna b onto nicotiana benthamiana, producing symptoms typical of acmv infection. the onset of symptom production was delayed relative to plants inoculated with parenta ...19902353465
selection for wild type size derivatives of tomato golden mosaic virus during systemic infection.a chimeric tomato golden mosaic virus (tgmv) a component dna, which results from replacement of the coding region of the viral coat protein gene (cp) with the larger bacterial beta-glucuronidase coding sequence (gus), can replicate in agroinoculated leaf discs but is unstable in systemically infected plants (1). we have made similar replacements of the tgmv cp gene with the gus coding sequence in both the sense and antisense orientations. both derivatives replicated in leaf discs inoculated via ...19902336387
genomic rna of an insect virus directs synthesis of infectious virions in plants.newly synthesized virions of flock house virus (fhv), an insect nodavirus, were detected in plant cells inoculated with fhv rna. fhv was found in whole plants of barley (hordeum vulgare), cowpea (vigna sinensis), chenopodium (chenopodium hybridum), tobacco (nicotiana tabacum), and nicotiana benthamiana and in protoplasts derived from barley leaves. virions produced in plants contained newly synthesized rna as well as newly synthesized capsid protein. these results show that the intracellular env ...19902296598
systemic movement of an rna plant virus determined by a point substitution in a 5' leader sequence.the ability of viruses to move through infected plants is an important determinant of host range and pathogenicity. we have investigated the genetic basis for the inability of the type strain of barley stripe mosaic hordeivirus to undergo long-range systemic movement in the tobacco nicotiana benthamiana. we show that, in this model system, a short open reading frame in the 5' leader of the smallest viral genomic rna prevents long-range vascular movement. as predicted by the ribosome scanning mod ...19902247462
a spontaneous mutation in the movement protein gene of brome mosaic virus modulates symptom phenotype in nicotiana benthamiana.brome mosaic virus (bmv) is a positive-strand rna virus with a multipartite genome that causes symptomless infection in nicotiana benthamiana. we have isolated and characterized a strain of bmv that produced uniform vein chlorosis in systemically infected n. benthamiana. analysis of pseudorecombinants constructed by exchanging rna 1 and 2 and rna 3 components between wild-type (non-symptom-inducing) and vein chlorosis-inducing strains of bmv indicated that the genetic determinant for the inducti ...19957884924
disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block.white clover mosaic virus strain o (wclmv-o), species of the potexvirus genus, contains a set of three partially overlapping genes (the triple gene block) that encodes nonvirion proteins of 26 kda, 13 kda, and 7 kda. these proteins are necessary for cell-to-cell movement in plants but not for replication. the wclmv-o 13-kda gene was mutated (to 13*) in a region of the gene that is conserved in all viruses known to possess triple-gene-block proteins. all 10 13* transgenic lines of nicotiana benth ...19947937946
mutagenesis of the ac3 open reading frame of african cassava mosaic virus dna a reduces dna b replication and ameliorates disease symptoms.small insertions were made independently at each of four unique restriction sites on african cassava mosaic virus (acmv) dna a to disrupt the three overlapping complementary-sense open reading frames (orfs) herein designated ac1, ac2 and ac3. the dna a mutants were assayed for their infectivity by agroinoculation of monomeric constructs to nicotiana benthamiana plants containing chromosomal insertions of acmv dna b. disruption of the ac3 orf alone resulted in a delay and amelioration of disease ...19912045787
african cassava mosaic virus di dna interferes with the replication of both genomic components.natural infections of the geminivirus african cassava mosaic virus (acmv) are known to be associated with low levels of defective interfering (di) dnas. recently it has been demonstrated that extrachromosomal copies of the di dna, mobilized and amplified from an integrated di dna dimer, can ameliorate acmv symptoms in transformed nicotiana benthamiana, providing a possible means for the control of cassava mosaic disease. to further understand the molecular basis of the interference phenomenon, w ...19911853559
systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.tobacco mosaic virus (tmv) produces large quantities of rna and protein on infection of plant cells. this and other features, attributable to its autonomous replication, make tmv an attractive candidate for expression of foreign sequences in plants. however, previous attempts to construct expression vectors based on plant rna viruses, such as tmv, have been unsuccessful in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. a hybrid viral rna (tb2) was ...19911651497
complete nucleotide sequence of the geminivirus tomato yellow leaf curl virus, thailand isolate.the complete genome of a thailand isolate of the geminivirus tomato yellow leaf curl virus (tylcv-th) has been cloned and the nucleotide sequence determined. the genome consists of two dnas each slightly greater than 2700 nucleotides in length and designated a-dna and b-dna. the a-dna contains six open reading frames (orfs) capable of encoding proteins with m(r)s greater than 10k; two orfs were located on the b-dna. the predicted orfs were found at positions on the genome similar to those identi ...19948126446
evidence that orf 1 and 2 are the only virus-encoded replicase genes of cymbidium ringspot tombusvirus.the 33-kda and its 92-kda readthrough protein genes (open reading frames (orfs) 1 and 2) of cymbidium ringspot tombusvirus (cyrsv) were introduced into nicotiana benthamiana plants. protoplasts derived from transgenic plants expressing the first two orfs were inoculated with in vitro transcripts of defective interfering (di) rna. the efficient replication of input di rna, which is incapable of autonomous replication, confirmed the biological activity of the expressed viral replicase. in addition ...19948178483
plants transformed with a region of the 201-kilodalton replicase gene from pea early browning virus rna1 are resistant to virus infection.the 3' proximal portion of the gene encoding the 201-kda putative replicase protein from the tobravirus pea early browning virus (pebv) can potentially be expressed separately as a 54-kda protein. nicotiana benthamiana plants transformed with the open reading frame (orf) encoding the 54-kda protein, designated 54k orf, were resistant to infection by purified pebv at inoculum doses of up to 1 mg/ml, the highest concentration tested. however, resistance was abolished by the introduction into the 5 ...19921631064
characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants.cymbidium mosaic virus (cymv) is the most prevalent virus infecting orchids. here, we report the isolation of partial cdna clones encoding the genomic rna of cymv. like most of the polyadenylated monopartite positive-strand rna viruses, the open reading frame (orf) coding for the viral coat protein (cp) is located at the 3' end. the orf predicts a polypeptide chain of 220 amino acids with a molecular weight of 23,600. sequence comparison of this orf to the cp sequences of potato virus x(pvx) and ...19921600145
complementation and recombination between mutants of complementary sense genes of dna a of abutilon mosaic virus.mutations have been inserted into the complementary sense orfs of dna a of abutilon mosaic virus (abmv). mutation in both orf ac1 and orf ac2/3 resulted in clones incapable of infecting nicotiana benthamiana following agroinoculation. mutation in orf ac1 but not in orf ac2/3 prevented dna replication in leaf discs. thus orf ac1 is essential for abmv dna replication whereas orf ac2/3 is probably involved in some aspect of the spread of the virus through the plant. coagroinoculation of both mutant ...19938212592
expression of maize adh1 intron mutants in tobacco vivo and in vitro gene transfer experiments have suggested that the elements mediating intron recognition differ in mammalian, yeast and plant nuclei. differences in the sequence dependencies, which also exist between dicotyledonous and monocotyledonous nuclei, have prevented some monocot introns from being spliced in dicot nuclei. to locate elements which modulate efficient recognition of introns in dicot nuclei, the maize adh1 gene has been expressed in full-length and single intron constru ...19938220449
sweet potato feathery mottle potyvirus (c1 isolate) virion and rna purification.a procedure for the purification of a peruvian isolate (c1) of sweet potato feathery mottle potyvirus (spfmv) and infective rna has been developed. the use of hepes [n-(2-hydroxyethyl)piperazine-n'-2-ethanesulfonic acid] buffer containing urea and sodium edta as a base for tissue extraction and virus suspension enabled good yields of virus (35-50 mg/100 g) to be obtained from nicotiana benthamiana l. domin. a short rna isolation procedure yielded infectious rna, from which ds cdna of nearly geno ...19938227274
3' splice site selection in dicot plant nuclei is position contrast to mammalian and yeast systems, the mechanism for intron recognition and splice site selection in plant pre-mrnas is poorly understood. splice site sequences and putative branchpoint sequences are loosely conserved in plant introns compared with other eukaryotes. perhaps to compensate for these variations, plant introns are significantly richer in adenosine and uridine residues than are their adjacent exons. to define elements critical for 3' splice site selection in dicotyledonous p ...19938336697
rapid production of full-length, infectious geminivirus clones by abutting primer pcr (abp-pcr).the application of the polymerase chain reaction (pcr) method of dna amplification for the isolation of full-length, infectious clones of geminiviruses is described. non-overlapping, abutting 20-mer oligonucleotide primers were used to produce a linear product from the circular geminivirus genomic template. clones of african cassava mosaic virus (acmv) dna a, obtained by this method, were infectious following mechanical inoculation (in the presence of acmv dna b) onto nicotiana benthamiana. norm ...19938360316
infectious in vivo transcripts of a plum pox potyvirus full-length cdna clone containing the cauliflower mosaic virus 35s rna promoter.a full-length cdna clone of an aphid non-transmissible isolate of plum pox potyvirus (ppv) was rendered biologically active when placed under the control of the cauliflower mosaic virus 35s rna promoter and the nopaline synthase polyadenylation signal. the cdna was constructed so that the exact 5' end of the ppv rna was present at the transcription initiation site. inoculation of plasmid dna onto nicotiana benthamiana led to systemic infection, whereas local lesions were produced in chenopodium ...19921545225
specificity of bipartite geminivirus movement proteins.pseudorecombinants produced by exchanging genome components (dnas a and b) of the geminiviruses african cassava mosaic virus (acmv) and indian cassava mosaic virus (icmv), acmv, and tomato golden mosaic virus (tgmv), and tgmv and abutilon mosaic virus (abmv) are not infectious in their common host nicotiana benthamiana. in each case, dna a was unable to trans-replicate the heterogenomic dna b component in a n. benthamiana leaf disc assay. the non-viability of the pseudorecombinants has been expl ...19938372440
a single missense mutation in the br1 movement protein alters the host range of the squash leaf curl geminivirus.the bipartite geminiviruses causing squash leaf curl disease, sqlcv-e and sqlcv-r, have a broad host range that includes nicotiana benthamiana, pumpkin, squash, and beans. we have characterized a naturally occurring mutant of the sqlcv-r b component (brtd1) which has lost the ability to infect n. benthamiana, but still retains full infectivity for pumpkin and squash. the mutation responsible for this host range alteration was localized to the coding region of br1, a gene essential for viral move ...19938372442
effective resistance to potyvirus infection conferred by expression of antisense rna in transgenic plants.approximately 660 nt including the carboxy-terminal portion of the bean yellow mosaic potyvirus (bymv) coat protein gene, complete 3' noncoding sequence and a short poly(a) tract were introduced to produce antisense rna in transgenic nicotiana benthamiana. original (r0) transformants were selfed, and homozygous second generation (r2) populations challenged with infected sap, purified virus, or viral rna. one transgenic line of 10 examined was extremely resistant to infection by mechanical inocul ...19957579613
mutagenesis of the bc1 and bv1 genes of african cassava mosaic virus identifies conserved amino acids that are essential for spread.the products of three open reading frames encoded by the bipartite geminiviruses have been implicated in viral spread: ac2, bv1 and bc1. alignment of the dna b encoded gene products, bv1 and bc1, from african cassava mosaic virus (acmv) with six other bipartite geminiviruses showed several highly conserved regions. specific amino acids were selected for mutagenic studies to ascertain the tolerance of the virus to change and to identify the regions within these two proteins required for normal fu ...19957730816
purification of tomato yellow leaf curl geminivirus.attempts were made to find a good purification procedure for tomato yellow leaf curl virus (tylcv), a dangerous and continuously spreading whitefly-transmitted germinivirus, up to now only partially purified. electron microscopy, serology and spectrophotometry were used to evaluate different procedures. the scheme finally adopted was the following: collect leaves and stems from nicotiana benthamiana graft-infected 45-60 days previously (5-10 g/plant); homogenize with 0.5 m phosphate buffer ph 6 ...19957553359
identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus x this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (tbsv) on disease symptoms in systemically infected plants and in local lesion hosts. the contribution of individual genes was determined by bioassays with an infectious clone of wild-type tbsv, with p19/p22 mutant derivatives, and by expression of individual tbsv genes from a heterologous potato virus x (pvx) vector. our results showed that tbsv genes could be expressed at high levels from the ...19957549478
dna and rna polymerase activities of nuclei and hypotonic extracts of nuclei isolated from tomato golden mosaic virus infected tobacco leaves.nuclei and hypotonically leached extracts of nuclei prepared from tomato golden mosaic virus (tgmv)-infected nicotiana benthamiana leaves have been used in in vitro dna and rna polymerisation reactions. the synthesis of virus-specific dna was resistant to aphidicolin, sensitive to n-ethylmaleimide and dideoxy ttp, and stimulated by kc1 and atp. variably virion (+) and complementary (-) strand dna of both the a and b genomic components were synthesised. virus-specific rna was synthesised in react ...19854069999
resistance to cymbidium ringspot tombusvirus infection in transgenic nicotiana benthamiana plants expressing the virus coat protein gene.transgenic nicotiana benthamiana plants expressing the coat protein gene of cymbidium ringspot virus (cyrsv) were tested for resistance against infection with cyrsv. transgenic plants showed resistance to infection only when the purified virions concentration in the inoculum was as low as 0.05 micrograms/ml. no protection was observed in transgenic plants inoculated with virion concentrations of 0.5 and 5.0 micrograms/ml or when the inoculum was in vitro synthesized genomic rna.19938448365
defective viral dna ameliorates symptoms of geminivirus infection in transgenic plants.nicotiana benthamiana was transformed with a single copy of a tandem repeat of subgenomic dna b isolated from plants infected with a kenyan isolate of the bipartite geminivirus african cassava mosaic virus. symptoms in transformed plants were less severe than in nontransformed controls when challenged with virus or cloned dna of kenyan or nigerian isolates. symptom amelioration was associated with the mobilization and amplification of the subgenomic dna, producing a comparable reduction in the a ...19902385593
systemic movement and symptom production following agroinoculation with a single dna of tomato yellow leaf curl geminivirus (thailand).two different dna species were cloned from purified tomato yellow leaf curl geminivirus particles after annealing a specific primer to virus dna and generating a second strand; both were approximately 2.8 kbp in length. one clone contains sequences which hybridize to the coat protein gene of tomato golden mosaic virus and most likely represents the a dna of tomato yellow leaf curl virus (thailand). the other clone may represent the b dna of this geminivirus. both clones contain short sequences w ...19902219706
low level of genetic drift in foreign sequences replicating in an rna virus in plants.the accumulation of mutations was measured in foreign sequences constituting a portion of a hybrid virus derived from the 6.4-kb (+) rna virus, tobacco mosaic tobamovirus (tmv). neither of the two foreign sequences tested (dihydrofolate reductase and neomycin phosphotransferase ii) are functionally required by the virus, so they should be free of selective pressures and should be a true measure of viral sequence drift in whole plants. four hybrid virus populations, two of each foreign sequence, ...19938517013
mutational analysis of complementary-sense genes of african cassava mosaic virus dna a.we have investigated the ability of african cassava mosaic virus dna a mutants, containing disrupted complementary-sense genes, to infect nicotiana benthamiana and to replicate in nicotiana tabacum protoplasts. three overlapping open reading frames (orfs) with the capacity to encode proteins with an mr greater than 10k (ac1, ac2 and ac3) are highly conserved between geminiviruses that infect dicotyledonous plants and one (ac4) is less well conserved. of these, only ac1 is a prerequisite for dna ...19912033385
replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral dna.agrobacterium-mediated inoculation of nicotiana benthamiana plants with ti plasmids containing tandem genome repeats derived from different strains of the gemini-virus beet curly top virus (bctv) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants. when two putative plus-strand origins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant ge ...19911896448
inhibition of african cassava mosaic virus systemic infection by a movement protein from the related geminivirus tomato golden mosaic virus.plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. we have analyzed the effect of tomato golden mosaic virus (tgmv) genes bl1 and br1, which are thought to be involved in the process of virus movement, on the infectivity of african cassava mosaic virus (acmv) in nicotiana benthamiana. recombinant genomes were constructed by replacing the acmv coat protein coding sequence with those of either bl1 or br1. replicati ...19921546454
rna-primed complementary-sense dna synthesis of the geminivirus african cassava mosaic virus.the plant dna virus african cassava mosaic virus (acmv) is believed to replicate by a rolling circle mechanism. to investigate complementary-sense dna (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense dna (h3 dna) from infected nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. the presence of an rna moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide ...19921475192
resistance to geminivirus infection by virus-induced expression of dianthin in transgenic plants.ribosome-inactivating proteins (rips) are naturally occurring plant toxins that exhibit antiviral activity against a diverse range of plant and animal viruses. here, the action of dianthin, a potent rip isolated from dianthus caryophyllus, has been exploited to engineer resistance to a plant dna virus, african cassava mosaic virus (acmv), in transgenic nicotiana benthamiana. to achieve this, dianthin has been expressed from the acmv virion-sense promoter that is transactivated by the product of ...19968659104
bromovirus movement protein genes play a crucial role in host specificity.monocot-adapted brome mosaic virus (bmv) and dicot-adapted cowpea chlorotic mottle virus (ccmv) are closely related bromoviruses with tripartite rna genomes. although rnas 1 and 2 together are sufficient for rna replication in protoplasts, systemic infection also requires rna3, which encodes the coat protein and the nonstructural 3a movement protein. we have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by rna3 as well as by rna1 and ...19937682628
pseudorecombination between infectious cloned dna components of tomato mottle and bean dwarf mosaic geminiviruses.a newly described whitefly-transmitted geminivirus infecting tomato plants in florida induces yellow mottling symptoms on leaves, and stunted and distorted growth. the dna-a and dna-b components were cloned from extracts of field-infected tomato tissue; excised monomers or uncut tandem dimers of these clones were infectious when co-inoculated on to nicotiana benthamiana by rub-inoculation. tomato plants inoculated directly with the dna-a and dna-b dimers, or indirectly by sap or graft transmissi ...19938423448
genetic analysis of beet curly top virus: evidence for three virion sense genes involved in movement and regulation of single- and double-stranded dna levels.the monopartite dna genome of beet curly top geminivirus (bctv, strain logan) contains four leftward, complementary sense open reading frames (orfs) designated l1, l2, l3, and l4 and three rightward, virion sense orfs designated r1, r2, and r3 (r1 encodes the coat protein). the r3 orf has not been reported previously in the bctv genome, and evidence for three functional virion sense genes on one genome component has not been presented before for any geminivirus. we investigated the functions of ...19938460493
the 5'-terminal region of a tombusvirus genome determines the origin of multivesicular bodies.multivesicular bodies (mvb) are membranous cytoplasmic inclusions that are invariably associated with tombusvirus infections regardless of the virus species, the host, or the tissue examined. mvb are virus-induced structures since they are absent from tissues of healthy plants and are always present both in infected plants and protoplasts. mvb derive from peroxisomes in cells infected by a number of tombusviruses including cymbidium ringspot virus (cymrsv) and from mitochondria in cells infected ...19968760449
immunity to potato mop-top virus in nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus.nicotiana benthamiana stem tissue was transformed with agrobacterium tumefaciens harboring a binary vector containing the potato mop-top virus (pmtv) coat protein (cp) gene. pmtv cp was expressed in large amounts in some of the primary transformants. the five transgenic lines which produced the most cp were selected for resistance testing. flowers on transformed plants were allowed to self-fertilize. transgenic seedlings selected from the t1 seed were mechanically inoculated with two strains of ...19957756694
resistance to tospoviruses in nicotiana benthamiana transformed with the n gene of tomato spotted wilt virus: correlation between transgene expression and protection in primary transformants.nicotiana benthamiana was transformed with the nucleoprotein (n) gene of an italian isolate of tomato spotted wilt virus (tswv). forty-five t1 primary transformant lines were analyzed for the expression of n protein and for resistance to tswv and three other tospoviruses: impatiens necrotic spot virus (insv), groundnut bud necrosis virus (gbnv), and groundnut ringspot virus (grsv). thirteen of these lines were further characterized. resistance to all tswv isolates tested was found in two lines. ...19957772806
tomato leaf curl geminivirus from india has a bipartite genome and coat protein is not essential for infectivity.genomes of two isolates of tomato leaf curl geminivirus from india (tolcv-india) have been sequenced. tolcv-india contains a and b components, both of which are required for systemic movement and symptom development. the two isolates have 94% sequence identify but one isolate gave mild symptoms in nicotiana benthamiana and tomato. the genome organization of tolcv-india is similar to other whitefly-transmitted geminiviruses (wtgs) with bipartite genomes. however, it contains an additional orf, av ...19957844539
both bipartite geminivirus movement proteins define viral host range, but only bl1 determines viral pathogenicity.bipartite geminiviruses such as squash leaf curl virus (sqlcv) encode two movement proteins (mps), br1 and bl1, that are essential for virus movement and systemic infection of host plants. br1 has been implicated in the host range properties of the virus, and bl1 in viral pathogenic properties. to more precisely examine the roles of each mp, we have introduced missense and deletion mutations into the coding sequence of both br1 and bl1, and examined the effects of these mutations on viral infect ...19957871727
lethal mutations within the conserved stem-loop of african cassava mosaic virus dna are rapidly corrected by genomic recombination.the nonanucleotide motif taatattac occurs in the intergenic region of all geminiviruses that have been examined to date. the motif is invariably located within the loop of a potential stem-loop structure that has been implicated in viral dna replication. to investigate the contribution of these sequences to virus proliferation, african cassava mosaic virus (acmv) dna b mutants have been screened for their ability to infect nicotiana benthamiana when co-inoculated with dna a. mutants in which the ...19947964630
detection and possible functions of african cassava mosaic virus dna b gene products.polyclonal antisera raised against synthetic oligopeptides have been used to detect the dna b gene products bv1 and bc1 of the geminivirus african cassava mosaic virus following sds-page fractionation of nicotiana benthamiana extracts. bv1 antiserum detected a soluble protein of 29 kda, consistent with the size predicted from sequence data. bc1 antiserum detected proteins of 37, 39, and 42 kda in addition to variable, less abundant species, all of which are larger than the predicted size of 34 k ...19938517019
immunodetection of the 33 k/92 k polymerase proteins in cymbidium ringspot virus-infected and in transgenic plant tissue antiserum was raised against the 33 k protein encoded by the 5' proximal gene of cymbidium ringspot tombusvirus rna. this antiserum reacts specifically with the 33 k and 92 k proteins, which constitute the viral replicase, in cyrsv-infected nicotiana benthamiana plants and in transgenic plants transformed with the full-length replicase gene. in inoculated leaves of infected plants, synthesis of the 33 k/92 k proteins stops ten days after inoculation, whereas in newly produced systemically inf ...19947980003
expression of single-chain antibody fragments (scfv) specific for beet necrotic yellow vein virus coat protein or 25 kda protein in escherichia coli and nicotiana benthamiana.the coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mabs) to beet necrotic yellow vein virus (bnyvv) coat protein (cp) or the 25 kda nonstructural protein (p25) were cloned into the pcock vector and expressed as single-chain antibody fragments (scfv) in escherichia coli. for expression in higher plants the scfv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase b (pelb) or the phytohemagglutinin (pha) ...19968980548
movement of a barley stripe mosaic virus chimera with a tobacco mosaic virus movement protein.the tobacco mosaic virus (tmv) 30k movement protein (mp) gene was inserted into a full-length cdna clone of barley stripe mosaic virus (bsmv) rna beta replacing the triple gene block (tgb). the resulting recombinant nd-mpt genome, consisting of infectious wt transcripts of bsmv rnas alpha and gamma, together with the hybrid rna beta transcript, was inoculated onto test plants to study the functional compatibility between the bsmv tgb-adapted genetic system and the tobamovirus transport gene. nd- ...19968610434
movement of tomato yellow leaf curl geminivirus (tylcv): involvement of the protein encoded by orf c4.tomato yellow leaf curl virus (tylcv) is a whitefly-transmitted geminivirus with a monopartite genome. we have investigated the ability of a tylcv dna mutant containing a disrupted orf c4 to infect nicotiana benthamiana and tomato plants. the mutant retained the capability of autonomous replication in protoplast-derived cells of tomato and was able to infect n. benthamiana plants systemically although dna levels were reduced and symptom development was attenuated. however, when tomato plants wer ...19948091687
high similarity among the tomato yellow leaf curl virus isolates from the west mediterranean basin: the nucleotide sequence of an infectious clone from isolate of tomato yellow leaf curl geminivirus, from the first epidemic outbreaks that occurred in murcia, spain (tylcv-m) in 1992, was cloned and its nucleotide sequence was determined. the circular single stranded dna consisted of 2777 nucleotides. the genome organization resembled that of other tylcv sequenced so far; regulatory signal sequences for bidirectional transcription and for polyadenylation of the transcripts were localized in the sequence. infectivity of the cloned dna was demon ...19948198442
transgenic tobacco plants expressing the geminivirus bl1 protein exhibit symptoms of viral disease.bipartite geminiviruses, such as squash leaf curl virus (sqlcv), encode two movement proteins (mps), br1 and bl1, that are essential for viral movement in and subsequent infection of the host plant. to elucidate the biochemical functions of these mps and define their respective contributions to viral infection, we have generated transgenic nicotiana benthamiana plants expressing sqlcv br1 and bl1. transgenic plants expressing br1 or a truncated bl1 were phenotypically indistinguishable from wild ...19938364356
an acidic class iii chitinase in sugar beet: induction by cercospora beticola, characterization, and expression in transgenic tobacco acidic chitinase (se) was found to accumulate in leaves of sugar beet (beta vulgaris) during infection with cercospora beticola. two isoforms, se1 and se2, with mw of 29 kda and pi of approximately 3.0 were purified to homogeneity. se2 is an endochitinase that also exhibits exochitinase activity, i.e., it is capable of hydrolyzing chito-oligosaccharides, including chitobiose, into n-acetyl-glucosamine. partial amino acid sequence data for se2 were used to obtain a cdna clone by polymerase cha ...19938400378
the cdna sequence and infectious transcripts of peanut stripe virus.a full-length cdna clone of the blotch isolate of the peanut stripe potyvirus (pstv) rna genome was constructed downstream from the bacteriophage sp6 rna polymerase promoter. the full-length pstv cdna clone (pstvsf9) was sequenced and compared to the previously published sequence of pstv-b. in vitro-synthesized pstv transcripts capped with m7gpppg were infectious in nicotiana benthamiana plants, and the progeny virus was aphid transmissible. to confirm the origin of infection, a mutant pstv (pst ...19968666291
molecular cloning, in vitro expression and characterization of a plant squalene synthetase cdna.squalene synthetase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, ec catalyzes the first committed step for sterol biosynthesis and is thought to play an important role in the regulation of isoprenoid biosynthesis in eukaryotes. using degenerate oligonucleotides based on a conserved region found in yeast and human squalene synthetase genes, a cdna was cloned from the plant nicotiana benthamiana. the cloned cdna contained an open reading frame of 1234 bp encoding a pol ...19968704125
the tobamovirus capsid protein functions as a host-specific determinant of long-distance movement.the tobamoviruses, tobacco mosaic virus (tmv) and odontoglossum ringspot virus (orsv), differ in the range of plant species that each can systemically infect. both viruses systemically infect nicotiana benthamiana similarly, but differ in the ability to systemically infect tobacco (n. tabacum). orsv is confined to the inoculated leaves of n. tabacum, whereas tmv causes a rapid systemic infection. genetic chimeras were created to identify viral genes involved in specific interactions in long-dist ...19938438560
defective interfering rna-mediated resistance against cymbidium ringspot tombusvirus in transgenic plants.defective interfering (di) rna of cymbidium ringspot tombusvirus was cloned downstream from the bacteriophage t7 rna polymerase promoter. in vitro synthesized rna was biologically active when coinoculated with parental genomic rna onto nicotiana benthamiana plants and prevented the occurrence of apical necrosis. n. benthamiana plants were transformed with the di rna sequences in both the positive and negative orientations relative to the cauliflower mosaic virus 35s promoter. integration of di r ...19938438573
host effects and sequences essential for accumulation of defective interfering rnas of cucumber necrosis and tomato bushy stunt tombusviruses.passage of cucumber necrosis virus (cnv) containing defective interfering (di) rnas through cucumber plants decreased the accumulation of di rnas to undetectable levels. subsequent passages in two nicotiana species (nicotiana benthamiana or n. clevelandii) resulted in the appearance of di rna species that were larger than the di rnas observed during exclusive serial passages of cnv through the nicotiana species. sequence analysis of cloned cdnas corresponding to the two di rna populations indica ...19957793079
replication of wild-type and mutant clones of satellite tobacco mosaic virus in nicotiana benthamiana protoplasts.rna transcribed from cloned satellite tobacco mosaic virus (stmv) cdna replicated in nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (tmgmv) genomic rna, but degraded when inoculated alone. stmv genomic rna extracted from wild-type virions replicated in protoplasts when co-inoculated with tmgmv, tobacco mosaic virus (tmv) or tomato mosaic virus (tomv). transcripts from clones of two stmv coat protein (cp) mutants accumulated to the same level as wild-typ ...19979191918
dna b facilitates, but is not essential for, the spread of abutilon mosaic virus in agroinoculated nicotiana benthamiana.full-length dimers of both dna a and dna b of abutilon mosaic virus have been constructed. these constructs are not infectious when inoculated into nicotiana benthamiana as dna; however, an infection could be obtained using the method of agroinoculation. symptom induction required both dna molecules but agroinoculation with dna a alone resulted in virus spread in the majority of plants. mutations were made in both of the open reading frames of dna b. coagroinoculation of wild-type dna a and muta ...19938503185
analysis of african cassava mosaic virus recombinants suggests strand nicking occurs within the conserved nonanucleotide motif during the initiation of rolling circle dna replication.intact clones containing partial repeats of the genomic components of african cassava mosaic (acmv dnas a and b) are infectious when mechanically coinoculated onto nicotiana benthamiana. monomeric genomic components may be generated either by homologous recombination or, when two copies of the origin of replication (ori) are present, by a modified rolling circle replication mechanism in which nascent single-stranded dna is resolved by the introduction of nicks at both oris. dna b partial repeats ...19957831830
virus and host-specific adaptations in the bl1 and br1 genes of bipartite geminiviruses.the host range of individual geminiviruses may be quite narrow, and closely related viruses can exhibit distinct host adaptations. two such bipartite geminiviruses are bean golden mosaic virus (gbmv) and tomato golden mosaic virus (tgmv). in both, the bl1 and br1 genes are required for the spread of virus infection in plants. we have investigated the contributions of bl1 and br1 to host-specific phenotypes of bgmv and tgmv by constructing hybrid viruses in which these coding regions were exchang ...19958553533
heterologous encapsidation of recombinant pea early browning virus.the coat protein gene of pea early browning virus (pebv) was replaced with that of another tobravirus, tobacco rattle virus (trv strain ppk20). the recombinant virus multiplied efficiently in the systemic host nicotiana benthamiana and, on the local lesion host phaseolus vulgaris, produced symptoms typical of pebv rather than trv showing that viral coat protein is not a determinant for lesion morphology. both viral rnas were encapsidated by trv coat protein although the shorter particles (encaps ...19948207406
destabilization of potato spindle tuber viroid by mutations in the left terminal loop.infectivity studies with highly infectious rna inocula generated by ribozyme cleavage were used to compare the biological properties of three apparently nonviable mutants of potato spindle tuber viroid (pstvd). one of these mutants (pstvd-p) contains three nucleotide substitutions in the left terminal loop, and mechanical inoculation of tomato seedlings with rna transcripts at levels equivalent to 10(3)-10(5) times the id50 for pstvd-intermediate failed to result in systemic infection. viable pr ...19979191908
polygalacturonase-inhibiting proteins (pgips) with different specificities are expressed in phaseolus vulgaris.the pgip-1 gene of phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (pgip), pgip-1 (p. toubart, a. desiderio, g. salvi, f. cervone, l. daroda, g. de lorenzo, c. bergmann, a. g. darvill, and p. albersheim, plant j. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35s promoter in tomato plants via agrobacterium tumefaciens-mediated transformation. transgenic tomato plants with different expression levels of pgip-1 were used in infection experiments w ...19979304859
expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector.a cdna fragment encompassing the aequorea victoria green fluorescent protein-encoding gene (gfp) was introduced into a genomic cdna clone of tobacco mosaic virus (tmv). infectious rna transcripts produced in vitro were used to inoculate tobacco (nicotiana benthamiana) leaves. after 1-2 days, bright green fluorescent areas could be visualized upon illumination with a long-wave ultraviolet (uv) light source. the virus was capable of infecting and expressing gfp in plant tissues both locally (in th ...19968707059
characterization of cucumber mosaic virus. iv. movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus.cdna clones of cucumber mosaic virus (cmv) rna 3 were modified to express the jellyfish green fluorescent protein (gfp) in place of the 3a movement protein (mp) or coat protein (cp), as fusions to the n (gfp-3a) or c (3a-gfp) terminus of the mp or from a separate open reading frame as part of tricistronic rnas 3. cmv rna transcripts containing the individual modified rnas 3 were unable to infect either nicotiana tabacum or nicotiana benthamiana systemically. infection, as measured by confocal mi ...19979356336
molecular analysis of the pothos latent virus genome.pothos latent virus (polv) is an isometric virus with a positive-sense, single-stranded rna genome of 4415 nt. the genome contains five open reading frames (orf), coding for five proteins with approximate molecular masses of 25, 84, 40, 27 and 14 kda, respectively. in vitro synthesized polv rna was infectious to nicotiana benthamiana plants and protoplasts, but could not support replication of the defective interfering (di) and satellite rnas associated with cymbidium ringspot tombusvirus. no di ...19979191911
complete nucleotide sequence of tobacco necrosis virus strain dh and genes required for rna replication and virus movement.the complete genome sequence of tobacco necrosis virus strain d (hungarian isolate, tnv-dh) was determined. the genome (3762 nt) has an organization identical to that reported for tnv-d. highly infectious synthetic transcripts from a full-length tnv-dh cdna clone were prepared, the first infectious necrovirus transcript reported. this clone was used for reverse genetic studies to map the viral genes required for replication and movement. protoplast inoculation with delta 22 and delta 82 mutants ...19979191913
increased pathogenicity in a pseudorecombinant bipartite geminivirus correlates with intermolecular recombination.most whitefly-transmitted geminiviruses possess bipartite dna genomes, and this feature may facilitate viral evolution through pseudorecombination and/or recombination. to test this hypothesis, the dna-a and dna-b components of the geminiviruses bean dwarf mosaic virus (bdmv) and tomato mottle virus (tomov) were exchanged, and the resultant pseudorecombinants were serially passaged through plants. both pseudorecombinants were infectious in nicotiana benthamiana but induced attenuated symptoms an ...19968764054
in vitro transcripts from cloned cdnas of the lettuce infectious yellows closterovirus bipartite genomic rnas are competent for replication in nicotiana benthamiana protoplasts.full-length cloned cdnas of lettuce infectious yellows closterovirus (liyv) rnas 1 and 2 were constructed and fused to the bacteriophage t3 rna polymerase promoter. to assess rna replication, nicotiana benthamiana protoplasts were inoculated with liyv virion rnas and liyv cdna-derived in vitro transcripts. analysis of protoplasts inoculated with liyv virion rnas or capped (m7gpppg) in vitro transcripts from liyv rna 1 and 2 cdnas showed accumulation of liyv genomic and putative subgenomic rnas ( ...19968806497
a dna clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene.a full-length dna clone encoding the genome of odontoglossum ringspot tobamovirus (orsv) was synthesized and placed adjacent to a bacteriophage t7 rna polymerase promoter. capped-rna transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of nicotiana benthamiana and oncidium gower ramsey. a representative clone, designated pot2, caused a disease phenotype identical to that produced by parental viral rna. elisa, western blot analysis, northern blot hybrid ...19989505974
nla and nlb of peanut stripe potyvirus are present in the nucleus of infected cells, but do not form inclusions.we investigated, by immunological and gene-fusion methods, whether the failure of peanut stripe potyvirus (pstv)-encoded nuclear inclusion proteins a (nla) and b (nlb) to form nuclear inclusions is due to the lack of their in vivo accumulation or the inability of one or both proteins to be transported into the nucleus nla domains (nla-vpg and nla-proteinase), full-length nlb, and full-length cylindrical inclusion (ci) protein of pstv were cloned, expressed in escherichia coli, and used for antis ...19968874498
the satellite rnas associated with the groundnut rosette disease complex and pea enation mosaic virus: sequence similarities and ability of each other's helper virus to support their replication.pea enation mosaic virus (pemv) and the causal agents of groundnut rosette disease are diverse examples of disease complexes involving two rna species, one of which is related to the genomes of luteoviruses and the other to those of umbraviruses. in both complexes, these viral rna components may be supplemented with satellite rnas that are dependent on the umbravirus component for replication and systemic movement, and on the luteovirus component for encapsidation and vector transmission. sequen ...19968922480
genetic analysis of the monopartite tomato yellow leaf curl geminivirus: roles of v1, v2, and c2 orfs in viral pathogenesis.tomato yellow leaf curl virus (tylcv) is a whitefly-transmitted geminivirus with a monopartite genome. we have investigated the functions of the v1, v2, and c2 orfs by mutational analysis. we analyzed the ability of tylcv mutants containing disrupted orfs v1, v2, or c2 to replicate, spread, and cause symptoms in nicotiana benthamiana and tomato plants. all the mutants retained the capability of autonomous replication in protoplast-derived cells of tomato and leaf discs of n. benthamiana, althoug ...19979123819
host and viral factors determine the dispensability of coat protein for bipartite geminivirus systemic movement.geminiviruses have unique, twinned icosahedral particles which encapsidate circular single-stranded dna. their genomes are composed of either one or two dna segments. monopartite geminiviruses absolutely require a functional coat protein (cp) for infectivity, whereas bipartite geminivirus cp null mutants can infect plants systemically. however, we show here that a cp mutant of the bipartite tomato golden mosaic virus (tgmv), which can infect nicotiana benthamiana systemically, is confined to the ...19968615033
molecular studies on bromovirus capsid protein. iii. analysis of cell-to-cell movement competence of coat protein defective variants of cowpea chlorotic mottle determine whether the role of coat protein (cp) in cell-to-cell movement of dicot-adapted cowpea chlorotic mottle bromovirus (ccmv) is distinct from that of monocot-adapted brome mosaic bromovirus (bmv), two reporter genes, beta-glucuronidase (gus) and enhanced green fluorescent protein (egfp), were substituted for the cp in a biologically active clone of ccmv rna3 (c3). primary leaves of nicotiana benthamiana, chenopodium quinoa, and cowpea were co-inoculated with wild-type (wt) ccmv rna 1 a ...19979191853
susceptibility of peach gf 305 seedlings and selected herbaceous plants to plum pox virus isolates from western slovakia.the susceptibility of peach gf 305 seedlings and herbaceous plants to five plum pox virus (ppv) isolates from orchards of western slovakia was investigated. ppv was isolated from diseased plum, apricot and peach trees, and transmitted by chip-budding to peach gf 305. the herbaceous plants were infected by mechanical inoculation. the transmission was analysed by symptomatology and double sandwich enzyme-linked immunosorbent assay (das-elisa). infected peaches developed leaf distortion, tissue cle ...19979607094
rapid, high-level expression of biologically active alpha-trichosanthin in transfected plants by an rna viral vector.alpha-trichosanthin, a eukaryotic ribosome-inactivating protein from trichosanthes kirilowii, inhibits the replication of the human immunodeficiency virus (hiv) in vitro. the alpha-trichosanthin gene was placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant rna viral vector. two weeks after inoculation, transfected nicotiana benthamiana plants accumulated alpha-trichosanthin to levels of at least 2% of total soluble protein. the recombinant alpha-trichosanthin ...19938421670
nicotiana benthamiana plants transformed with the plum pox virus helicase gene are resistant to virus infection.nicotiana benthamiana domin. plants were transformed with the cytoplasmic inclusion protein (ci) gene of plum pox potyvirus (ppv) to investigate, whether this non-structural protein would be able to confer resistance. the ci protein is an rna helicase, which contains a conserved nucleotide binding motif (ntbm) and plays an important role in viral replication. two gene constructions were developed for plant transformation. the first contains the original coding sequence of the ci gene under the c ...19989617773
resistance to tomato yellow leaf curl geminivirus in nicotiana benthamiana plants transformed with a truncated viral c1 gene.the c1 gene of tomato yellow leaf curl geminivirus (tylcv) encodes a multifunctional protein (rep) involved in replication. a truncated form of this gene, capable of expressing the n-terminal 210 amino acids (aa) of the rep protein, was cloned under the control of the camv 35s promoter and introduced into nicotiana benthamiana using agrobacterium tumefaciens. the same sequence was also cloned in antisense orientation. when self-pollinated progeny of 19 primary transformants were tested for resis ...19968862407
nontarget dna sequences reduce the transgene length necessary for rna-mediated tospovirus resistance in transgenic plants.rna-mediated virus resistance has recently been shown to be the result of post-transcriptional transgene silencing in transgenic plants. this study was undertaken to characterize the effect of transgene length and nontarget dna sequences on rna-mediated tospovirus resistance in transgenic plants. transgenic nicotiana benthamiana plants were generated to express different regions of the nucleocapsid (n) protein of tomato spotted wilt (tswv) tospovirus. transgenic plants expressing half-gene segme ...19979223349
molecular characterization of a subgroup i geminivirus from a legume in south africa.a south african geminivirus for which we propose the name bean yellow dwarf virus (beydv) has been isolated from french bean (phaseolus vulgaris cv. bonus) showing stunting, chlorosis and leaf curl symptoms. a full-length cloned copy of the viral genome produced characteristic symptoms of the disease when reintroduced into french bean by agroinoculation, and was systemically infectious in nicotiana benthamiana, n. tabacum, lycopersicon esculentum, datura stramonium and arabidopsis thaliana. beyd ...19979267015
molecular cloning and functional expression of cdnas for glycyrrhiza glabra squalene synthase.we have isolated two cdnas (ggsqs1 and ggsqs2) encoding squalene synthase of glycyrrhiza glabra l. by cross-hybridization with that of arabidopsis thaliana squalene synthase under conditions of low stringency. their nucleotide sequences contained an open reading frame for a polypeptide of 413 amino acids (ggsqs1) and 412 amino acids (ggsqs2), respectively. the deduced amino acid sequence of ggsqs1 exhibits 88%, 81%, 78%, 45-44%, and 45-41% identity to those of the ggsqs2, nicotiana benthamiana. ...19968913521
nicotiana benthamiana plants expressing beet necrotic yellow vein virus (bnyvv) coat protein-specific scfv are partially protected against the establishment of the virus in the early stages of infection and its pathogenic effects in the late stages of infection.transgenic plants of nicotiana benthamiana expressing single chain antibody fragments (scfv) specific for the coat protein of beet necrotic yellow vein virus (bnyvv) and non-expressing control plants were inoculated with bnyvv mechanically and by means of the vector polymyxa betae. the scfv were presumably expressed in the endoplasmic reticulum (er). the average time needed for infections to become detectable was longer in the scfv-expressing plants than in the non-expressing control plants. in ...19979672643
tobamovirus cross protection using a potexvirus vector.cross protection is the ability of one virus to prevent or delay infection by a related challenge virus. to examine this phenomena, a potato x potexvirus (pvx) vector (chapman et al., 1992, plant j. 2, 549) was used to systemically express the tobacco mosaic tobamovirus (tmv) coat protein (cp) open reading frame in nicotiana benthamiana. pvx constructs induce mild mosaic symptoms in n. benthamiana, whereas tmv infection results in rapid systemic necrosis and plant death. healthy plants or plants ...19968955042
identification of genes involved in replication and movement of peanut clump virus.the genome of peanut clump pecluvirus (pcv) consists of two messenger rna components which contain, respectively, three and five open reading frames (orfs). inoculation of transcripts from full-length cdna clones derived from the pcv rnas showed that rna-1 is able to replicate in the absence of rna-2 in protoplasts, but both rnas are necessary for plant infection. to investigate the role of different gene products in viral rna replication and movement, transcripts from mutant cdna clones were in ...19989721240
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