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R N Beachy

Publications and source records attributed to R N Beachy.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cloning of tobacco genes that elicit the hypersensitive response.

We used a functional screening method to isolate genes whose products elicit the hypersensitive response (HR) pathway of defense against plant pathogens. A cDNA library derived from tobacco leaves undergoing the HR was cloned into a tobacco mosaic virus (TMV)-based expression vector. Infectious transcripts were generated and used to inoculate tobacco plants lacking the N resistance gene (genotype Xanthi nn). Approximately 1/1000 of the infectious transcripts produced local lesions, and may thus elicit the HR. The cDNA inserts from 50 lesion-forming clones were recovered by RT-PCR, and 12 unique clones were sequenced. Comparisons with protein databases revealed homologies to (a) ubiquitin, (b) tobacco tumor-related protein, similar to Kunitz-type trypsin inhibitors and (c) ribosomal protein S14. The remaining nine clones revealed no homology to known proteins and are thus considered novel. Five clones were able to induce the expression of PR2, a gene which is specifically activated in the tobacco HR. Northern and western blot analyses of leaves infected by the clone encoding ubiquitin strongly suggest that the infection produced a co-suppression response; the endogenous level of ubiquitin mRNA and protein in infected leaves are ca. 50% less than those found in healthy leaves. This observation supports a previous report on the involvement of the ubiquitin system in the tobacco HR [2], and validates and utility of the functional cloning method.

Base Sequence↗
PubMed1998

Simultaneous accumulation of multiple viral coat proteins from a TEV-NIa based expression vector.

We previously described an expression cassette that relies on the tobacco etch virus (TEV) nuclear inclusion a (NIa) protease and leads to the coordinated accumulation of multiple proteins through self processing of a polyprotein [21]. However, low levels of proteins accumulated when the full-length protease was encoded within the polyprotein [22]. Studies were conducted to evaluate whether the disruption of NIa nuclear localization would affect the levels of proteins produced via the cassette. Modifications comprised either removal of its nuclear localization signals (NLSs), removal of the VPg domain (which includes the NLSs), and fusion to the 6 kDa protein, previously demonstrated to be a viral cytoplasmic anchor [28]. In in vitro translation reactions and in vivo protoplast experiments the modified NIa retained sequence-specific proteolysis. Moreover, the removal of the NLSs correlated with an increase in GUS reporter accumulation. The modified cassette, pPRO10, led to the synthesis of up to three viral coat protein (CPs) in addition to NIa. However, the accumulation of proteins in protoplasts depended upon the position of the CP coding sequence within the cassette as well as on the stability of the protein.

Base Sequence↗
PubMed1998

Functional organization of the cassava vein mosaic virus (CsVMV) promoter.

Cassava vein mosaic virus (CsVMV) is a pararetrovirus that infects cassava plants in Brazil. A promoter fragment isolated from CsVMV, comprising nucleotides -443 to +72, was previously shown to direct strong constitutive gene expression in transgenic plants. Here we report the functional architecture of the CsVMV promoter fragment. A series of promoter deletion mutants were fused to the coding sequence of uidA reporter gene and the chimeric genes were introduced into transgenic tobacco plants. Promoter activity was monitored by histochemical and quantitative assays of beta-glucuronidase activity (GUS). We found that the promoter fragment is made up of different regions that confer distinct tissue-specific expression of the gene. The region encompassing nucleotides -222 to -173 contains cis elements that control promoter expression in green tissues and root tips. Our results indicate that a consensus as1 element and a GATA motif located within this region are essential for promoter expression in those tissues. Expression from the CsVMV promoter in vascular elements is directed by the region encompassing nucleotides -178 to -63. Elements located between nucleotides -149 and -63 are also required to activate promoter expression in green tissues suggesting a combinatorial mode of regulation. Within the latter region, a 43 bp fragment extending from nucleotide -141 to -99 was shown to interact with a protein factor extracted from nuclei of tobacco seedlings. This fragment showed no sequence homology with other pararetrovirus promoters and hence may contain CsVMV-specific regulatory cis elements.

Binding Sites↗
PubMed1998

Expression and inheritance of multiple transgenes in rice plants.

The ability to control integration, inheritance, and expression of multiple transgenes is a prerequisite for manipulating biosynthetic pathways and complex agronomic characteristics in plants. One hundred and twenty-five independent transgenic rice plants were regenerated after cobombarding embryogenic tissues with a mixture of 14 different pUC-based plasmids. Eighty-five percent of the R0 plants contained more than two, and 17% more than nine, of the target genes. Plants containing multiple transgenes displayed normal morphologies and 63% set viable seed. Multigene cotransformation efficiency was correlated with the ratio in which the plasmids were mixed with respect to the selectable marker. All target genes had an equal chance of integration, indicating that the nature of the coding region had no effect on the efficiency of integration. Three plant lines containing 11, 10, and 9 transgenes, respectively, were analyzed for patterns of integration and inheritance until the R3 generation. Integration of multiple transgenes occurred at either one or two genetic loci, with inheritance conforming to a 3:1 Mendelian ratio. Coexpression of four marker genes was investigated until the R2 generation.

Acetyltransferases↗
PubMed1998

Targeting and modification of prokaryotic cell-cell junctions by tobacco mosaic virus cell-to-cell movement protein.

The movement protein (MP) of tobacco mosaic virus (TMV) facilitates the cell-to-cell spread of infection by altering the structure and function of plasmodesmata, the intercellular communication channels in plants. Because the protein was shown to interfere with intercellular communication when expressed in the cyanobacterium Anabaena sp. strain PCC 7120, whether the ability of the protein to target and to modify intercellular communication channels in plants is conserved in this prokaryote was investigated. It was found that the MP localizes to the cell junctions and induces the formation of filamentous structures that traverse the septa. It is proposed that the protein interacts with host components that are similar between plants and Anabaena and that may be evolutionarily related. The observations in Anabaena suggest that the MP modifies plasmodesmata by forming a filamentous aggregate within the pore.

Cell Communication↗
PubMed1998

Domains of the TMV movement protein involved in subcellular localization.

To identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C-terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild-type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9-11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49-51 may be involved in co-alignment of the protein with microtubules. The region around residues 88-101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.

Amino Acid Sequence↗
PubMed1998

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection.

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.

Brefeldin A↗
PubMed1998

Transgenic plants expressing potato virus X ORF2 protein (p24) are resistant to tobacco mosaic virus and Ob tobamoviruses.

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.

Animals↗
PubMed1997

RF2a, a bZIP transcriptional activator of the phloem-specific rice tungro bacilliform virus promoter, functions in vascular development.

Rice tungro bacilliform virus (RTBV) replicates only in phloem cells in infected rice plants and its promoter drives strong phloem-specific reporter gene expression in transgenic rice plants. We isolated a cDNA encoding a basic leucine zipper (bZIP) protein, RF2a, which binds to the Box II cis element that is important for expression from the promoter. RF2a, which stimulates Box II-dependent transcription in a homologous in vitro transcription system, accumulates in nuclei of phloem and certain other cell types in shoots, but is found at only very low levels in roots. Transgenic antisense plants in which RF2a accumulation was suppressed had normal roots but stunted, twisted leaves with small, disorganized vascular bundles, an enlarged sclerenchyma and large air spaces. We propose that the RTBV promoter exploits a host transcription factor that is critical for leaf tissue differentiation and vascular development for its expression.

Amino Acid Sequence↗
PubMed1997

Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV).

Plasmodesmatal gating in epidermal cells of Nicotiana tabacum was examined in expanding infection sites of tobacco mosaic virus (TMV) expressing a fusion between the viral movement protein and the green fluorescent protein (MP-GFP). The infection sites were circular in profile and within 3 days post-inoculation had developed a brightly fluorescent leading edge, giving them a characteristic 'halo' shape. Co-localization of MP-GFP with callose demonstrated that nearly all epidermal cell plasmodesmata were targeted with MP-GFP. The fusion protein was located in the centre of the plasmodesmal pore, between paired callose platelets. Increase in plasmodesmatal size exclusion limit, as determined by the passage of microinjected 10 kDa Texas Red dextran, was restricted predominantly to cells within the fluorescent halo, and was virtually absent from cells in the centre of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labelled with MP-GFP. Injections outside the fluorescent infection site failed to show movement of dextran, while dextran injected into cells at the leading edge moved inwards towards the centre of the lesion but not outwards into cells lacking GFP. Leaf incisions through cells ahead of the infection front halted the advance of the virus, indicating that virus replication was absent in non-fluorescent cells outside the infection site. The data provide the first demonstration that within an expanding infection site plasmodesmatal gating is under temporal control.

Green Fluorescent Proteins↗
PubMed1997

Analysis of the sequence diversity of the P1, HC, P3, NIb and CP genomic regions of several yam mosaic potyvirus isolates: implications for the intraspecies molecular diversity of potyviruses.

Partial sequences from serologically characterized yam mosaic potyvirus (YMV) isolates were determined in conserved (helper-component proteinase, HC; nuclear inclusion b, NIb) and variable (first protein, P1; third protein, P3; and coat protein, CP) regions of the potyviral genome in order to investigate the intraspecies molecular diversity of YMV. Multiple sequence alignments and pairwise comparisons were used to quantify the sequence polymorphism in these regions. Two levels of diversity were observed among YMV isolates: above 90% nucleotide (nt) sequence identities were found between YMV isolates of the same group (intragroup) regardless of the region considered, whereas identities between isolates from different groups (intergroup) were lower and depended upon the protein chosen. For instance, the average intergroup nt sequence identity between YMV isolates was about 65% in the P1 protein and the N terminus of the CP while there was more than 80% nt identity in the HC, P3 and NIb proteins. Thus P3 appeared to be conserved between YMV isolates even though this region was variable between potyvirus species. Similar analysis of the intraspecies molecular diversity of other potyviruses (potato virus Y, zucchini yellow mosaic virus, plum pox virus, pea seed-borne mosaic virus) led to the same results: (i) two levels of intraspecies molecular diversity were found (intragroup and intergroup); (ii) intraspecies molecular diversity differed from interspecies molecular diversity in the P3, P1 and N-terminal regions.

Amino Acid Sequence↗
PubMed1997

Transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide.

An expression cassette based on the highly specific tobacco etch potyvirus (TEV) nuclear inclusion (NIa) proteinase has been developed to produce multiple proteins through the translation of a single self-processing polypeptide. Gene constructs encoding TEV NIa, the tobacco mosaic tobamovirus (TMV) coat protein (CP) and the soybean mosaic potyvirus (SMV) CP were used to develop transgenic tobacco plants. Proper processing of the multifunctional polypeptide was demonstrated, leading to accumulation of separate proteins in planta. Moreover, the viral genes expressed in this way were biologically active and conferred pathogen-derived protection to TMV, TEV and potato potyvirus Y (PVY). Transgenic plants were also derived from gene constructs in which the NIa cleavage site was mutated, resulting in the accumulation of the non-processed polyprotein, as predicted. Although transgenic proteins accumulated in low amounts in all the plant lines analysed, accumulation of the mutant non-processed protein form was greatly increased in plants following infection with TEV, but not TMV, apparently as a consequence of protein stabilization.

Capsid↗
PubMed1997

Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.

Capsid↗
PubMed1996

The role of AV2 ("precoat") and coat protein in viral replication and movement in tomato leaf curl geminivirus.

We analyzed various mutants of tomato leaf curl virus-India to investigate the role of ORFs AV3, AV2, and coat protein (CP) in viral replication, movement, and symptom development. The results of these studies indicate that ORF AV3 does not encode a protein. Plants inoculated with infectious DNA which contained deletions in AV2 developed very mild symptoms and accumulated only low levels of both single-stranded (ss) and double-stranded (ds) viral DNA, whereas inoculated protoplasts accumulated both ss and dsDNA to wild-type levels, showing that AV2 is required for efficient viral movement. However, both plants and protoplasts inoculated with substitution, frameshift, and other similar mutations in AV2 accumulated low levels of viral DNA. The low levels of accumulation of DNA of these mutants were apparently not due to a defect in AV2 synthesis. Mutations in the CP caused a marked decrease in ssDNA accumulation in plants and protoplasts while increasing dsDNA accumulation in protoplasts. Mutations in both AV2 and CP behaved like AV2 mutants in plants and like CP mutants in protoplasts. The results demonstrated that multiple functions provided by AV2, BV1, BC1 are essential for viral movement, and that changes in A-component virion-sense mRNA structure or translation affect viral replication.

Amino Acid Sequence↗
PubMed1996

Cell-to-cell transport of movement-defective cucumber mosaic and tobacco mosaic viruses in transgenic plants expressing heterologous movement protein genes.

To determine if the movement proteins (MPs) of cucumber mosaic cucumovirus (CMV) and tobacco mosaic tobamovirus (TMV) are complementary in function, transgenic plants expressing genes encoding TMV or CMV MP were inoculated with movement-defective mutants of TMV and CMV. Transgenic plants expressing the MP gene of CMV strain S (subgroup II) complemented the cell-to-cell and systemic spread of a movement-defective mutant of CMV strain Fny (subgroup I) but not the local or systemic spread of a movement-defective mutant of TMV. Plants that contained the MP gene from CMV-S were not resistant to wild-type TMV infection. When inoculated with a movement-defective mutant of TMV that produced beta-glucuronidase, transgenic plants with the CMV MP gene supported only subliminal infection. Conversely, immunodetection and in situ localization techniques revealed that transgenic plants accumulating the TMV MP supported cell-to-cell spread, but not systemic transport, of a movement-defective CMV. These studies suggest that the transgenic TMV MP shares some of the functions with the CMV MP required to transport CMV, whereas the transgenic CMV MP is deficient in functions that are needed to mobilize the spread of TMV infection.

Amino Acid Sequence↗
PubMed1996

Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter.

The cassava vein mosaic virus (CVMV) is a double stranded DNA virus which infects cassava plants (Manihot esculenta Crantz) and has been characterized as a plant pararetrovirus belonging to the caulimovirus subgroup. Two DNA fragments, CVP1 of 388 nucleotides from position -368 to +20 and CVP2 of 511 nucleotides from position -443 to +72, were isolated from the viral genome and fused to the uidA reporter gene to test promoter expression. The transcription start site of the viral promoter was determined using RNA isolated from transgenic plants containing the CVMV promoter:uidA fusion gene. Both promoter fragments were able to cause high levels of gene expression in protoplasts isolated from cassava and tobacco cell suspensions. The expression pattern of the CVMV promoters was analyzed in transgenic tobacco and rice plants, and revealed that the GUS staining pattern was similar for each construct and in both plants. The two promoter fragments were active in all plant organs tested and in a variety of cell types, suggesting a near constitutive pattern of expression. In both tobacco and rice plants, GUS activity was highest in vascular elements, in leaf mesophyll cells, and in root tips.

Base Sequence↗