Plant virus movement proteins.
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Nucleic Acids↗
Biomedical subjects
R N Beachy
Publications and source records attributed to R N Beachy.
Seed-specific repression of GUS activity in tobacco plants by antisense RNA.
beta-Conglycinin, the 7S storage protein of soybean, is expressed only in seeds and is regulated predominantly by gene transcription [5]. We applied an antisense strategy to modify expression of a beta-glucuronidase (uidA or gusA) gene in seeds using a promoter from a beta-conglycinin gene. Transgenic tobacco plants harboring the gusA gene under the control of the CaMV 35S promoter were retransformed with a gene construct comprising the beta-conglycinin promoter fused to the gusA gene in the antisense orientation. Double transformants were regenerated and transformation was confirmed by Southern blot hybridization. Seed-specific repression of GUS activity was observed in lines containing high copy numbers of the antisense gusA transgene. Suppression of GUS activity was correlated with the amounts of (-) sense gusA transcript detected and concomitantly with a decrease in gusA transcript levels. Furthermore, the amount of suppression of GUS activity was greatest during mid to late stages of seed development, when expression of the alpha' promoter is high. These results indicate that suppression of GUS activity is due to expression of the antisense gene.
Antigens, Plant↗
Characterization of the genome of rice tungro bacilliform virus: comparison with Commelina yellow mottle virus and caulimoviruses.
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Amino Acid Sequence↗
Effects of terminal deletion mutations on function of the movement protein of tobacco mosaic virus.
A series of carboxy- and amino-terminal deletion mutations in the movement protein (MP) gene of tobacco mosaic virus (TMV) were ligated into a cloned TMV cDNA deleted for the endogenous MP gene. RNA transcripts were produced in vitro from clones carrying the various mutated MP genes. The effect of the deletion mutations on local and systemic movements of the infection was evaluated. Deletion of 9 or 33 amino acids from the carboxy terminus of the movement protein did not effect cell-to-cell movement as reflected by local lesion formation on Nicotiana tabacum cv. Xanthi NN plants. Deletion of 55 amino acids resulted in impaired MP that supported the formation of local lesions of 1 mm in diameter compared to lesions of 3-5 mm caused by the wild-type MP. Deletion of 74 amino acids (or more) from the carboxy terminus resulted in a protein that could not support virus movement. Modified viruses that contained repeated sequences in the 3' region of the MP gene lost the repeated sequences during replication and reverted to the wild type. This was evidenced by the size of the MP produced and by sequence analysis of reverse-transcribed PCR-amplified products, following infection by the modified virus. MP deleted for as few as 3 amino acids at the amino terminus could not support virus movement thus indicating that the amino-terminal domain is critical for MP activity.
Cell Wall↗
Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.
Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.
Cytoplasm↗
Conserved amino acid sequences among plant proteins sorted to protein bodies and plant vacuoles. Can they play a role in protein sorting?
Amino acid sequence comparisons were made between the soybean alpha subunit of beta-conglycinin and 34 members of different plant protein families targeted to seed protein bodies or vacuoles. A number of short conserved amino acid sequences were identified in seed storage proteins, plant protease inhibitors and lectins, and the probable functions of these sequences are discussed. For proteins of known tertiary structure, these sequences map to the surface of the respective molecules. It is postulated that these regions produce a common secondary structure which could interact with other molecules involved in the sorting process. One of these regions, region A, is similar to the yeast carboxypeptidase Y (CPY) vacuolar targeting signal, and is present in both storage proteins and lectins. Computer modeling based upon the tertiary structure of concanavalin A (ConA) was used to generate models representing the structure of two highly related lectins from Dolichos biflorus, one of which is targeted to protein bodies and the other secreted. A different glycosylation pattern together with amino acid sequences upstream of the identified conserved amino acid sequences are predicted to modulate the presentation of the sorting domains in the lectins and be the determinant in the sorting of these lectins.
Amino Acid Sequence↗
Multiple nuclear factors interact with upstream sequences of differentially regulated beta-conglycinin genes.
The expression of the alpha' and beta subunit genes of beta-conglycinin is differentially regulated during soybean embryo development. Although both are expressed solely in developing seeds during mid to late stages of embryo development, the alpha' subunit is expressed more highly on a per gene basis, and alpha' subunit mRNA begins to accumulate three to five days earlier than beta subunit mRNA. In cultured cotyledons, beta subunit gene(s) respond to changes in methionine or abscisic acid levels, whereas expression of the alpha' subunit gene(s) is unaffected by these changes. To investigate the mechanisms by which these genes are transcriptionally regulated, we examined the interactions of nuclear proteins with upstream sequences from the alpha' and beta subunit genes. Four distinct DNA binding factors were identified in nuclear extracts from developing soybean seeds. These factors are termed Soybean Embryo Factors (SEF) 1 through 4. SEF binding sites are distributed non-uniformly between the alpha' and beta subunit genes, and the amount of protein binding is modulated over the course of embryo development. DNA footprinting revealed the sequences recognized by three of these factors. Factors which behave in a manner similar to that of SEF3 were also identified in nuclear extracts from developing tobacco and sunflower seeds.
Antigens, Plant↗
Transient gene expression in cassava using high-velocity microprojectiles.
The bacterial gene encoding beta-glucuronidase (GUS) was transiently expressed in cassava leaves following the introduction of the gene by microparticle bombardment. The DNA expression vector used to introduce the reporter gene is a pUC 19 derivative and consisted of a CaMV 35S promoter (P35S), the GUS coding region and 7S polyadenylation region. Several other promoters and regulating sequences were tested for efficiency in cassava leaves. Two derivatives of the P35S, one including a partial duplication of the upstream region of the P35S and the other containing a tetramer of the octopine synthase enhancer, were found to be expressed at three times the level of the P35S in cassava leaves. The ubiquitin 1 promoter from Arabidopsis thaliana was expressed at the same level as the P35S. No influence on the level of expression was observed when different 3' ends were used. The biolistic transient gene expression system in cassava leaves allows rapid analysis of gene constructs and can serve as a preliminary screen for chimeric gene function in the construction of transgenic cassava plants.
Cloning, Molecular↗
Altered function of the tobacco mosaic virus movement protein in a hypersensitive host.
The N gene in Nicotiana sp. confers hypersensitive resistance to all strains of tobacco mosaic virus (TMV) and limits the rate of virus spread in infected leaves. To examine the role of the movement protein (MP) of TMV in the hypersensitive reaction (HR), transgenic Nicotiana tabacum cv. Xanthi-nc (genotype NN) plants that express the MP gene were produced and the molecular size exclusion limit of plasmodesmata in leaf mesophyll cells was monitored. At the HR-permissive temperature (24 degrees) movement from cell to cell of fluorescein isothiocyanate-labeled dextran of molecular mass 3.9 kDa was detected while 9.4-kDa molecules failed to move. At the HR-nonpermissive temperature (33 degrees) the 9.4-kDa probe moved readily from cell to cell. In contrast, in transgenic Xanthi (genotype nn) which express the MP gene the 9.4-kDa probe moved from cell to cell at 24 and 33 degrees. These results suggest that the N gene may modify the ability of the MP to alter plasmodesmata molecular exclusion limits, although expression of the TMV-MP gene alone did not induce the HR. Furthermore, when MP(+) Xanthi-nc tobacco lines were inoculated with a TMV that lacked a MP gene the HR was induced, and the concentration of MP in the transgenic lines was correlated with the degree of the HR.
Capsid Proteins↗
Transfer of the movement protein gene between two tobamoviruses: influence on local lesion development.
The effects of transfer of the movement gene between the tobamoviruses tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV) were studied. The movement protein (MP) gene of TMGMV was cloned into an infectious cDNA of TMV to build the recombinant virus V23. V23, like TMV and TMGMV, caused systemic infection in Nicotiana tabacum Xanthi. In N. sylvestris V23 and TMV spread systemically although TMGMV produces necrotic local lesions on this host. V23 and TMV cause systemic infection on tomato plants while TMGMV does not infect tomato. In Xanthi nc plants, V23 produced necrotic local lesions similar in size to those produced by TMGMV. On the other hand in transgenic Xanthi nc tobacco plants that express a gene encoding the MP of TMV the necrotic lesions produced by V23 and TMGMV were similar in size to those produced by TMV. These results indicate that the size of necrotic lesions produced by TMGMV and TMV on Xanthi nc plants is influenced by the MP gene.
Amino Acid Sequence↗
In vivo complementation of infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants.
A full-length cDNA clone of the U1 (common) strain of tobacco mosaic virus (TMV) was constructed, and highly infectious transcripts were produced in vitro using bacteriophage T7 RNA polymerase. Frameshift mutations designed to cause premature termination of translation were introduced into either the 30-kDa movement protein (MP) gene or the coat protein (CP) gene. The MP-frameshift mutant was unable to locally or systemically infect inoculated tobacco plants. However, inoculation of transgenic tobacco plants that expressed a wild-type TMV MP gene resulted in both local and systemic viral infection. The CP-frameshift mutant, although unable to move systemically in nontransformed tobacco, exhibited systemic movement in transgenic plants that expressed a wild-type TMV CP gene. Transgenic tobacco plants that expressed the appropriate wild-type TMV gene were thus able to complement, in trans, mutant viruses lacking a functional MP or CP gene.
Amino Acid Sequence↗
The TMV movement protein: role of the C-terminal 73 amino acids in subcellular localization and function.
The role of the C-terminal one-third of the tobacco mosaic virus (TMV) 30-kDa movement protein (MP) on its subcellular localization and on virus spread was investigated. We have constructed eight cDNAs encoding MPs with variable size deletions from the C-terminal end. Expression of the truncated proteins was verified in recombinant yeast using an antiserum directed to a synthetic peptide corresponding to 21 amino acids near the N-terminal end of the MP. In transgenic tobacco plants, MP from which more than 55 amino acids were deleted no longer accumulated in the cell wall fraction of a cellular extract, where the complete MP accumulates. Dye diffusion studies showed that both unmodified and modified MPs that accumulate in the cell wall fraction are able to alter plasmodesmatal size exclusion limits. Biological function of the modified MPs was tested in the transgenic plants with the TMV thermosensitive mutant Ls1 and a TMV genomic RNA transcript lacking a functional MP. There was a correlation between the cell wall localization of the modified MPs and its ability to potentiate virus spread. The results presented here demonstrate the dispensability of the C-terminal 55 amino acids of the MP in its subcellular localization in tobacco plants and its role in virus movement. Moreover, our results show that a stretch of 19 amino acids (195 to 213) is essential for localization of the MP to the cell wall fraction of plant cells.
Capsid↗
Characterization of the genome of rice tungro bacilliform virus: comparison with Commelina yellow mottle virus and caulimoviruses.
Rice tungro disease is caused by an infection of two different viruses, rice tungro spherical virus (a (+) sense RNA virus) and rice tungro bacilliform virus (RTBV) with a genome of circular double-stranded DNA. The genome of an RTBV isolate from the Philippines was cloned, sequenced, and found to be 8000 bp in length. It contains four open reading frames (ORFs) on a single strand, with ORF 1 having an internal termination codon (TAA). The 5' and 3' ends of a polyadenylated viral RNA transcript, of genome length, were mapped by primer extension and cDNA sequence analysis, respectively. The transcript is terminally redundant by 265-268 nucleotides. Purified virus particles contain two major proteins with molecular masses of 37 and 33 kDa, although only the 37-kDa protein was detected in the infected rice tissues. The N-terminal amino acid sequence of the 33-kDa protein was determined and its coding region was identified on the RTBV genome. The identity of the coat protein gene was further confirmed by expressing a region of the genome in Escherichia coli, the products of which reacted with anti-RTBV antibody. The unusually long ORF 3 of RTBV is predicted to encode a polyprotein of 194.1 kDa that includes: the coat protein(s), viral proteinase, reverse transcriptase, and ribonuclease H. The sections of the polyprotein show varying degrees of similarity to the counterparts of Commelina yellow mottle virus (a member of the proposed badnavirus group) and caulimoviruses. The functions of the other three ORFs are unknown.
Amino Acid Sequence↗
The tobacco mosaic virus 30K movement protein in transgenic tobacco plants is localized to plasmodesmata.
Transgenic tobacco plants expressing a gene encoding the tobacco mosaic virus (TMV) movement protein (30K) were studied using immunocytochemical techniques. The movement protein was shown to be localized within or on most of the plasmodesmata observed in the transformed plant. These results are consistent with the idea that the movement protein interacts with the plasmodesmata to facilitate the cell-to-cell spread of TMV.
Capsid Proteins↗
cDNA cloning and nucleotide sequence of the wheat streak mosaic virus capsid protein gene.
The 3'-terminal region of wheat streak mosaic virus (WSMV) genomic RNA was cloned and a cDNA sequence of 1809 nucleotides upstream of the poly(A) tract was determined. The sequence contains a single open reading frame of 1662 nucleotides and a 3' untranslated region of 147 nucleotides. Translation products from WSMV RNA and WSMV cDNA transcripts were immunoprecipitated by WSMV capsid protein antiserum, indicating that the 3'-terminal region of WSMV RNA encodes the capsid protein. Five potential N-terminal capsid protein protease cleavage sites were identified, which would yield proteins ranging from 31.7K to 46.8K. Alignment of the deduced amino acid sequence of the WSMV capsid protein with those of other potyviruses showed significant, but limited, identity as compared to the alignment of two or more aphid-transmitted potyviruses. Although WSMV has characteristics distinct from potyviruses, because of its particle morphology, translation strategy apparently based on polyprotein processing, the ability to form cytoplasmic cylindrical inclusions and the degree of capsid protein homology with aphid-transmitted potyviruses, it should be considered a member of the potyvirus group.
Amino Acid Sequence↗
Complete sequence of a cDNA of alpha subunit of soybean beta-conglycinin.
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Amino Acid Sequence↗
Manipulation of beta-glucuronidase for use as a reporter in vacuolar targeting studies.
It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, beta-glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of beta-glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked glycosylation sites within the GUS gene was altered by site-directed mutagenesis. The second N-linked glycosylation site was not altered because sequence analysis of nucleotide sequences around the second putative glycosylation site revealed that the published sequence was incorrect, and that no such site existed.
Amino Acid Sequence↗
Tissue-specific expression of the TMV coat protein in transgenic tobacco plants affects the level of coat protein-mediated virus protection.
Transgenic tobacco plants were produced that express a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) under the control of the promoter from a ribulose bisphosphate carboxylase small subunit (rbcS) gene. Plant lines expressing comparable levels of CP from the rbcS and cauliflower mosaic virus 35S promoters were compared for resistance to TMV. In whole plant assays the 35S:CP constructs gave higher resistance than the rbcS:CP constructs. On the other hand, leaf mesophyll protoplasts isolated from both plant lines were equally resistant to infection by TMV. This indicated that the difference in resistance between the lines in the whole plant assay reflects differences at the level of short- and/or long-distance spread of TMV. Therefore, we propose that the difference in tissue-specific expression between the 35S and rbcS promoters accounts for greater resistance in the plant lines that express the 35S:CP chimeric genes.
Blotting, Western↗