Showing papers on "Cauliflower mosaic virus published in 2001"
01 Jan 2001
TL;DR: It is shown here that overexpression of the cDNA encoding DREB1A in transgenic plants activated the expression of many of these stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing.
Abstract: Plant productivity is greatly affected by environmental stresses such as drought, salt loading and freezing. We reported that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses in Arabidopsis. The transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic Arabidopsis plants activated the expression of many of theses stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress-inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter. As the DRE-related regulatory element is not limited to Arabidopsis the DREB1A cDNA and the rd29A promoter may be useful for improving the stress tolerance of agriculturally important crops by gene transfer.
1,138 citations
TL;DR: These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.
Abstract: A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.
350 citations
TL;DR: Both the levels of DWf4 transcripts and BR phenotypic effects were progressively increased in dwf4, wild-type and AOD4 plants, respectively, suggesting that it will be possible to control plant growth by engineering DWF4 transcription in plants.
Abstract: Plants unable to synthesize or perceive brassinosteroids (BRs) are dwarfs. Arabidopsis dwf4 was shown to be defective in a steroid 22alpha hydroxylase (CYP90B1) step that is the putative rate-limiting step in the BR biosynthetic pathway. To better understand the role of DWF4 in BR biosynthesis, transgenic Arabidopsis plants ectopically overexpressing DWF4 (AOD4) were generated, using the cauliflower mosaic virus 35S promoter, and their phenotypes were characterized. The hypocotyl length of both light- and dark-grown AOD4 seedlings was increased dramatically as compared to wild type. At maturity, inflorescence height increased >35% in AOD4 lines and >14% in tobacco DWF4 overexpressing lines (TOD4), relative to controls. The total number of branches and siliques increased more than twofold in AOD4 plants, leading to a 59% increase in the number of seeds produced. Analysis of endogenous BR levels in dwf4, Ws-2 and AOD4 revealed that dwf4 accumulated the precursors of the 22alpha-hydroxylation steps, whereas overexpression of DWF4 resulted in increased levels of downstream compounds relative to Ws-2, indicative of facilitated metabolic flow through the step. Both the levels of DWF4 transcripts and BR phenotypic effects were progressively increased in dwf4, wild-type and AOD4 plants, respectively. This suggests that it will be possible to control plant growth by engineering DWF4 transcription in plants.
337 citations
TL;DR: Direct genetic evidence is presented that the plant antiapoptotic protein AtBI-1 is biologically active in suppressing the mammalian Bax action in planta.
Abstract: We recently isolated the AtBI-1 (Arabidopsis Bax Inhibitor-1) gene, the expression of which suppressed Bax-induced cell death in yeast. To determine whether the same is true in the plant system, transgenic Arabidopsis plants overexpressing Bax protein under a dexamethasone (DEX)-inducible promoter were generated. On DEX treatment, such transgenic plants exhibited marked cell death at the whole-plant level, cell shrinkage, membranous destruction, and other apoptotic phenotypes. Transgenic Bax plants were retransformed with a vector containing the AtBI-1 gene (tagged with green fluorescent protein) under the control of the cauliflower mosaic virus 35S promoter. Plants expressing both Bax and AtBI-1 were able to maintain growth on DEX-treatment by sustaining intracellular integrity. Thus, we present here direct genetic evidence that the plant antiapoptotic protein AtBI-1 is biologically active in suppressing the mammalian Bax action in planta.
244 citations
TL;DR: A full-length cDNA clone encoding a peroxisomal type ascorbate peroxidase was isolated from barley and introduced into A. thaliana under control of the 35S RNA promoter of the cauliflower mosaic virus, resulting in transgenic plants that were significantly more tolerant to heat stress as compared with the wild-type.
153 citations
TL;DR: All of the transgenic tomato plants expressing ACC deaminase showed some increased tolerance to flooding stress and were less subject to the deleterious effects of root hypoxia on plant growth than were non-transformed plants.
142 citations
TL;DR: Results illustrate that SAS is a potent tool for the manipulation of SA levels in plants and helps to generate plants that are more disease-resistant.
Abstract: Summary Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.
121 citations
TL;DR: In vivo evidence that IDS3 is the “MA synthase” that converts DMA to MA is shown, indicating that the 5′-flanking region of Ids3 works as a strong Fe-deficiency-inducible promoter in rice, as well as in barley.
Abstract: We proposed that an Fe-deficiency-induced gene, Ids3 (Iron deficiency specific clone no. 3), from barley (Hordeum vulgare L.) roots encodes a dioxygenase that catalyzes the hydroxylation step from 2′-deoxymugineic acid (DMA) to mugineic acid (MA). To prove this hypothesis, we introduced the Ids3 gene into rice (Oryza sativa L.), which lacks Ids3 homologues and secretes DMA, but not MA. Transgenic rice plants, carrying either Ids3 cDNA or a barley genomic DNA fragment (20 kb) containing Ids3, were obtained using Agrobacterium-mediated transformation. Ids3 cDNA under the control of the cauliflower mosaic virus 35S promoter was constitutively expressed in both the roots and the leaves of the transgenic rice, regardless of Fe nutrition status. In contrast, in the roots of transformants carrying a barley genomic fragment, transcripts of Ids3 were markedly increased in response to Fe deficiency. Slight expression of Ids3 was also observed in the leaves of the Fe-deficient plants. Western blot analysis confirmed the induction of Ids3 in response to Fe deficiency in the roots of the transformants carrying a genomic fragment. These expression patterns indicate that the 5′-flanking region of Ids3 works as a strong Fe-deficiency-inducible promoter in rice, as well as in barley. Both kinds of transgenic rice secreted MA in addition to DMA under Fe-deficient conditions, but wild-type rice secreted only DMA. This is in vivo evidence that IDS3 is the “MA synthase” that converts DMA to MA.
115 citations
TL;DR: The results suggest that activity of the CsVMV promoter, in contrast to the CaMV 35S promoter, was under developmental regulation in transgenic grape plants as compared with theCaMV35S promoter.
105 citations
TL;DR: The results indicate that co-suppressing endogenous genes can cause dominant phenotypes as expected and it is predicted that this system will be generally useful for identifying genes that yield phenotypes upon over-expression as well.
Abstract: We have developed a system to over-express or co-suppress random cDNAs in Arabidopsis thaliana upon Agrobacterium tumefaciens-mediated transformation We constructed a binary vector containing a novel Arabidopsis cDNA library driven by the cauliflower mosaic virus (CaMV) 35S promoter The vector, 35SpBARN, offers in terra selection with glufosinate ammonium (BASTA) and the ability to identify the cDNA insert using PCR with flanking primers We introduced this overexpression library into Arabidopsis and selected over 30,000 transformants A random sample of 50 T1 plants was analyzed to determine the quality of the cDNA library in planta About 90% of T1 plants in the collection have inserts, the average insert size is ca 11 kb, and ca 43% of these inserts appear to encode full-length proteins T1 plants were screened for visible abnormalities, and one mutant, V5, was chosen for further study This mutant displays a pale green phenotype, and its transgene contains a partial petH cDNA encoding chloroplast ferredoxin-NADP+ reductase (EC 11812) This construct co-suppresses the endogenous petH transcript We recapitulated the mutant phenotype by expressing either the full-length or truncated petH cDNA from the CaMV 35S promoter in wild-type Arabidopsis Our results indicate that co-suppressing endogenous genes can cause dominant phenotypes as expected As we have also used the 35SpBARN vector to successfully over-express other transcripts in planta, we predict that this system will be generally useful for identifying genes that yield phenotypes upon over-expression as well
93 citations
TL;DR: The results suggest that GS1 is regulated at the level of RNA stability and protein turnover, and no increase in GS activity or polypeptide level was detected in the leaves of transgenic plants.
Abstract: Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH4+ with glutanate to yield glutamine. Gene constructs consisting of the cauliflower mosaic virus (CaMV) 35S promoter driving a cytosolic isoform of GS (GS1) gene have been introduced into alfalfa (Medicago sativa). Although transcripts for the transgene were shown to accumulate to high levels in the leaves, they were undetectable in the nodules. However, significant amounts of beta-glucuronidase activity could be detected in nodules of plants containing the CaMV 35S promoter-beta-glucuronidase gene construct, suggesting that the transcript for the GS1 transgene is not stable in the root nodules. Leaves of alfalfa plants with the CaMV 35S promoter-GS1 gene showed high levels of accumulation of the transcript for the transgene when grown under low-nitrogen conditions and showed a significant drop in the level of GS1 transcripts when fed with high levels of NO3-. However, no increase in GS activity or polypeptide level was detected in the leaves of transgenic plants. The results suggest that GS1 is regulated at the level of RNA stability and protein turnover.
TL;DR: It is demonstrated that p23 is involved in symptom development and that it most likely plays a key role in CTV pathogenesis, the first case in which a protein encoded by a woody plant-infecting RNA virus has been identified as being directly involved in pathogenesis in its natural host.
Abstract: Summary
The 23 kDa protein (p23) coded by the 3′-terminal gene of Citrus tristeza virus (CTV), a member of the genus Closterovirus with the largest genome among plant RNA viruses, is an RNA-binding protein that contains a motif rich in cysteine and histidine residues in the core of a putative zinc-finger domain. On this basis, a regulatory role for CTV replication or gene expression has been suggested for p23. To explore whether over-expression of this protein in transgenic plants could affect the normal CTV infection process, transgenic Mexican lime plants were generated carrying the p23 transgene, or a truncated version thereof, under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Constitutive expression of p23 induced phenotypic aberrations that resembled symptoms incited by CTV in non-transgenic lime plants, whereas transgenic plants expressing the p23 truncated version were normal. The onset of CTV-like symptoms in p23-transgenic plants was associated with the expression of p23, and its accumulation level paralleled the intensity of the symptoms. This demonstrates that p23 is involved in symptom development and that it most likely plays a key role in CTV pathogenesis. This is the first case in which a protein encoded by a woody plant-infecting RNA virus has been identified as being directly involved in pathogenesis in its natural host. This finding also delimits a small region of the large CTV genome for the future mapping of specific pathogenic determinants.
TL;DR: Two putative promoters from Australian banana streak badnavirus isolates for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.
Abstract: Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.
TL;DR: The results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.
Abstract: We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection ...
TL;DR: It is shown that the resistance and cell death that comprise the HR elicited by W260 can indeed be uncoupled, and it is found that cell death and resistance segregated independently in the F2 population of a cross between N. edwardsonii and N. clevelandii.
Abstract: Cauliflower mosaic virus strain W260 elicits a hypersensitive response (HR) in leaves of Nicotiana edwardsonii, an interspecific hybrid derived from a cross between N. glutinosa and N. clevelandii. Interestingly, we found that N. glutinosa is resistant to W260, but responds with local chlorotic lesions rather than necrotic lesions. In contrast, N. clevelandii responds to W260 with systemic cell death. The reactions of the progenitors of N. edwardsonii to W260 infection indicated that each contributed a factor toward the development of HR. In this study, we present two lines of evidence to show that the resistance and cell death that comprise the HR elicited by W260 can indeed be uncoupled. First, we showed that the non-necrotic resistance response of N. glutinosa could be converted to HR when these plants were crossed with N. clevelandii. Second, we found that cell death and resistance segregated independently in the F2 population of a cross between N. edwardsonii and N. clevelandii. We concluded that the...
TL;DR: The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants, and a non-infectious MP mutant with two amino acid changes in the N-terminal third of the MP failed to interact withMPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the2- Hybrid system.
Abstract: Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is required for virus movement. The CaMV movement protein (MP) was used in a yeast 2-hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-interacting (MPI) proteins. Three different clones were found encoding proteins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV MP. The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants. A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the 2-hybrid system. MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis. MPI7 is related in sequence, size and hydropathy profile to mammalian proteins (such as rat PRA1) described as a rab acceptor. The gene encoding MPI7 is expressed widely is Arabidopsis plants, and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentrated in punctate spots. In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed. At these sites, fluorescence resonance energy transfer (FRET) between fluorophores was observed indicating an interaction in planta between the CaMV MP and MPI7.
TL;DR: A new system for inducible high-level expression of mRNA for foreign genes in transgenic plants is constructed by introducing a glucocorticoid-inducible transcription system into the previously developed "mRNA amplification system" where target mRNA can be amplified as a subgenomic RNA by the replicase of a plant tripartite RNA virus, Brome mosaic virus.
Abstract: Summary
We have constructed a new system for inducible high-level expression of mRNA for foreign genes in transgenic plants by introducing a glucocorticoid-inducible transcription system into the previously developed ‘mRNA amplification system’ where target mRNA can be amplified as a subgenomic RNA by the replicase of a plant tripartite RNA virus, Brome mosaic virus (BMV). In the new amplification system, the amplification of mRNA is tightly regulated by the expression of a subunit of the BMV replicase. Transgenic Nicotiana benthamiana plants (designated GVG1 × 2FR) were produced that contained cDNA of BMV RNA1 coding a subunit of replicase under the control of a tightly regulated, glucocorticoid-inducible promoter. In addition GVG1 × 2FR plants contain cDNAs of BMV RNA2 coding another subunit of the replicase, and a replicable engineered BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN) gene under the control of the Cauliflower mosaic virus 35S promoter. When transgenic plants were treated with dexamethasone (DEX), a strong synthetic glucocorticoid, induction of replication and amplification of the 35S-driven FCP2IFN and synthesis of subgenomic mRNA for IFN were observed. Accumulation levels of amplified FCP2IFN were over 300 times higher than those of the 35S-driven FCP2IFN in the GVG1 × 2FR plant without the treatment and those of the mRNA for IFN were 30–230 times higher than in the previous, non-inducible mRNA amplification system. Without DEX treatment, no subgenomic mRNA for IFN was detected in the GVG1 × 2FR plant. The advantages and potential uses of this system are also discussed.
TL;DR: These transgenic lines with the CP gene of SPFMV-S can be used for coat protein-mediated resistance to the virus, and are shown to be highly resistant not only to primary but also to secondary infection by SPFMv-S.
Abstract: One of the most-serious diseases of sweet potato [Ipomoea batatas (L.) Lam] is russet crack disease caused by sweet potato feathery mottle virus (SPFMV). We constructed an expression vector carrying the coat protein (CP) and hygromycin phosphotransferase (hpt) genes driven by cauliflower mosaic virus 35 S promoters. Accordingly, we introduced the expression vector into sweet potato variety Chikei 682-11 by the electroporation method. Among 449 calli obtained after antibiotic selection, 19 plants from seven independent calli grew to form adventitious shoots. Three transgenic lines were obtained from independent calli, based on analysis of the CP and hpt genes. The transcription and translation of the CP gene were shown in these transgenic lines by Northern- and Western-blot analyses. To assay the virus resistance of the transgenic lines, each line was vegetatively propagated and then grafted with morning glory (Ipomoea nil) that had been infected with SPFMV-S. A PAS-ELISA assay with polyclonal antiserum of the CP demonstrated that virus accumulation 3 months after grafting with the infected morning glory was suppressed in the transgenic lines as compared with non-transgenic ones. These transgenic lines were shown to be highly resistant not only to primary but also to secondary infection by SPFMV-S. Thus we concluded that the three transgenic lines with the CP gene of SPFMV-S can be used for coat protein-mediated resistance to the virus.
TL;DR: 82 unique cDNA clones of diverse functions are isolated from tomato, including those with a putative role in the oxidative burst, proteolysis, the hypersensitive response, signal transduction, and a number of genes with unknown functions that will assist in the characterization of defense pathways activated during disease resistance.
Abstract: The tomato (Lycopersicon esculentum) resistance gene Pto confers resistance to the bacterial pathogen Pseudomonas syringae pv tomato carrying the avirulent gene avrPto. Overexpressing Pto under the control of the cauliflower mosaic virus 35S promoter constitutively activates defense responses in the absence of pathogen infection and nonspecifically enhances disease resistance. To elucidate the mechanisms underlying this resistance, we isolated cDNAs corresponding to transcripts that accumulated in 35S::Pto plants. By using suppression subtractive hybridization, we isolated 82 unique cDNA clones, most of which corresponded to differentially expressed transcripts. Most of the genes examined were also induced by pathogen inoculation. Sequence analysis showed that a large number of genes encode defense-related proteins, and most had not been previously isolated from tomato. The isolated cDNAs also include those with a putative role in the oxidative burst, proteolysis, the hypersensitive response, signal transduction, and a number of genes with unknown functions. The isolation of these cDNAs of diverse functions will assist in the characterization of defense pathways activated during disease resistance.
TL;DR: The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level ofGNA present in the plants, and that conditions under which the plants were grown affect the levels of G NA expression and subsequent levels of insect resistance.
Abstract: Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.
TL;DR: It is demonstrated that P3 directly interacts with purified viral particles and unassembled coat protein without the need for any other factor and that P2 mediates the association of P2 with purified virus particles.
Abstract: Transmission of cauliflower mosaic virus (CaMV) by aphids requires two viral nonstructural proteins, the open reading frame (ORF) II and ORF III products (P2 and P3). An interaction between a C-terminal domain of P2 and an N-terminal domain of P3 is essential for transmission. Purified particles of CaMV are efficiently transmitted only if aphids, previously fed a P2-containing solution, are allowed to acquire a preincubated mixture of P3 and virions in a second feed, thus suggesting a direct interaction between P3 and coat protein. Herein we demonstrate that P3 directly interacts with purified viral particles and unassembled coat protein without the need for any other factor and that P3 mediates the association of P2 with purified virus particles. The interaction domain of P3 is located in its C-terminal half, downstream of the P3-P2 interaction domain but overlapping a region which binds nucleic acids. Mutagenesis of P3 which interferes with the interaction between P3 and virions is correlated with the loss of transmission by aphids. Taken together, our results demonstrate that P3 plays a crucial role in the formation of the CaMV transmissible complex by serving as a bridge between P2 and virus particles.
TL;DR: The result suggests that cytokinins cannot serve as mobile signals to elicit the release of apical dominance in tissues compromised for enhanced cytokinin synthesis, and the usefulness of the dx-on/tc-off system for experiments addressing gene function was demonstrated.
Abstract: The chimeric transcriptional activator TGV mediates dexamethasone (dx)-inducible and tetracycline (tc)-repressible transgene expression in tobacco (dx-on/tc-off system). The expression profiles of four different synthetic target promoters, comprising multiple TGV binding sites upstream of a core promoter, were characterised using the sensitive luciferase assay. Induction factors of over 1000 were measured in roots and leaves of over 30% of the transgenic plants, irrespective of the promoter used. Promoters PTF and PTax, which carry the –48 to +1 region of the Cauliflower Mosaic Virus 35S promoter, showed higher expression levels in both the uninduced and induced states than PTop10 and PTFM, which harbour several point mutations in this region. Moreover, PTax expressed higher background activities than PTF, indicating that the sequence of the synthetic regulatory region can influence background levels. The usefulness of the dx-on/tc-off system for experiments addressing gene function was demonstrated by using it to control the expression of isopentenyl transferase. This enzyme catalyses the rate-limiting step in cytokinin biosynthesis and causes phenotypic effects even at low expression levels. Only dx-induced transgenic plants displayed phenotypic alterations indicative for increased cytokinin synthesis (e.g. outgrowth of lateral buds). Simultaneous treatment of selected buds with the anti-inducer tc suppressed bud growth. This result suggests that cytokinins cannot serve as mobile signals to elicit the release of apical dominance in tissues compromised for enhanced cytokinin synthesis.
TL;DR: Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants.
Abstract: Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5 0 - untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T0 and T1 plants, and assayed for b-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T1 plants segregated in a 3:1 Mendelian ratio.
TL;DR: The full-length cDNA sequences of two FtsZ2 genes from Arabidopsis thaliana are determined and it is found that the genes encode polypeptides of 478 and 473 amino acids, respectively, and both contain N-terminal extensions beyond what have previously been predicted.
TL;DR: The virion-associated protein (VAP) is conserved among all members of the Caulimoviridaefamily and contains a coiled-coil structure that has been shown to assemble as a tetramer in the case of cauliflower mosaic virus.
Abstract: All plant pararetroviruses belong to the Caulimoviridae family. This family contains six genera of viruses with different biological, serological, and molecular characteristics. Although some important mechanisms of viral replication and host infection are understood, much remains to be discovered about the many functions of the viral proteins. The focus of this study, the virion-associated protein (VAP), is conserved among all members of the group and contains a coiled-coil structure that has been shown to assemble as a tetramer in the case of cauliflower mosaic virus. We have used the yeast two-hybrid system to characterize self-association of the VAPs of four distinct plant pararetroviruses, each belonging to a different genus of Caulimoviridae. Chemical cross-linking confirmed that VAPs assemble into tetramers. Tetramerization is thus a common property of these proteins in plant pararetroviruses. The possible implications of this conserved feature for VAP function are discussed.
TL;DR: The apoplastic protein fractions of sbwAFP expressing tobacco lines exhibited enhanced antifreeze activity as demonstrated by the ability to inhibit ice re-crystallization and increased thermal hysteresis.
TL;DR: Three instability determinants in the termini of the cauliflower mosaic virus capsid protein precursor are described, of which one is still present in the mature capsidprotein p44, which implies that the other two signals may target a novel degradation pathway.
Abstract: Targeted protein degradation plays an important regulatory role in the cell, but only a few protein degradation signals have been characterized in plants. Here we describe three instability determinants in the termini of the cauliflower mosaic virus (CaMV) capsid protein precursor, of which one is still present in the mature capsid protein p44. A modified ubiquitin protein reference technique was used to show that these motifs are still active when fused to a heterologous reporter gene. The N-terminus of p44 contains a degradation motif characterized by proline, glutamate, aspartate, serine and threonine residues (PEST), which can be inactivated by mutation of three glutamic acid residues to alanines. The signals from the precursor do not correspond to known degradation motifs, although they confer high instability on proteins expressed in plant protoplasts. All three instability determinants were also active in mammalian cells. The PEST signal had a significantly higher degradation activity in HeLa cells, whereas the precursor signals were less active. Inhibition studies suggest that only the signal within the N-terminus of the precursor is targeting the proteasome in plants. This implies that the other two signals may target a novel degradation pathway.
TL;DR: Evidence is provided that gene expression in transformed maize plants can be impacted by simply altering the order, orientation or regulatory sequences of adjacent genes.
Abstract: Anther-targeted expression of E. coli DNA (Adenosine-N6-)-Methyltransferase (DAM) in maize was tested as a means to produce male-sterile plants. A high frequency of male-sterile plants with reduced anther size was observed when DAM was regulated by the maize anther-specific promoter 5126 (5126:DAM) and placed upstream of the herbicide resistance gene, pat, regulated by the cauliflower mosaic virus (CaMV) 35S promoter (35S:PAT). In contrast, placement of 5126:DAM upstream of a pat gene regulated by either the maize ubiquitin (UBI:PAT) or rice actin (rACTIN:PAT) promoters resulted in male-fertile plants. Based on these observed differences, DAM-mediated sterility was used as a phenotypic marker to assess the contribution of factors affecting gene expression such as orientation of the transcription units, choice of regulatory sequences mediating expression of adjacent genes, and effects of varying the anther-specific promoter regulating DAM. Constructs that place a portion of the CaMV 35S promoter, including the native AS-1 sequences, between 5126:DAM and UBI:PAT yielded a high frequency of male-sterile plants with reduced anther size. Significant differences in the frequency of male-sterile events and the associated anther size were also observed when the position of 35S:PAT was changed relative to 5126:DAM. These data provide evidence that gene expression in transformed maize plants can be impacted by simply altering the order, orientation or regulatory sequences of adjacent genes.
TL;DR: It is demonstrated that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity, and by using an artificial stem structure that blocks scanning, direct evidence that ribosomes shunt operates during CaMV infection is obtained.
Abstract: Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of reverse transcription of a terminally redundant pregenomic 35S RNA that is also used as a polycistronic mRNA. The leader of 35S RNA is long, highly structured, and contains multiple short ORFs (sORFs), which strongly interfere with the ribosome scanning process. Translation of this RNA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5′-proximal short ORF (sORF A), then skip a large region of the leader containing a putative RNA encapsidation signal and reinitiate translation at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of shunt-dependent expression and the degree of shunt enhancement by a CaMV-encoded translation transactivator (TAV), consequently reduced infectivity of the virus in turnip plants. First- or second-site reversions appeared in the viral progeny. The second-site reversions restored shuntdependent expression to an extent correlating with their relative abundance in the progeny. Mutations that abolished both the basal and TAV-activated components of shunting proved to be lethal. Finally, by using an artificial stem structure that blocks scanning, we obtained direct evidence that ribosome shunt operates during CaMV infection.
TL;DR: It is established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits, and it is shown that P2 can also polymerize as long paracrystalline filaments.
Abstract: The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% a-helical. Most a-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2. Most plant viruses are transmitted from one host to another by insect vectors (23), the majority in a noncirculative manner (25). In this mode of transmission, virus particles are acquired while the vector feeds on infected plants and are specifically retained in the food canal of the mouthparts (stylets). The virus is subsequently released from the stylets and inoculated to a new host when the insect vector feeds on another plant. This process implies that specific interactions occur between virus particles and the cuticle lining of the vector’s stylets. Two distinct molecular strategies mediating these interactions can be distinguished (24). In the capsid strategy, a motif within the virus coat protein directly recognizes a binding site in the vector stylets, whereas in the helper strategy, binding is not direct but is mediated by a so-called helper component (HC), a viral nonstructural protein acting as a reversible molecular bridge between virus and vector (12). The helper strategy is frequently adopted by plant viruses, and although aphid transmission has been extensively studied for the genera Caulimovirus (4) and Potyvirus (26), the biochemical and structural features of HCs in these two virus groups remain largely unknown.