Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. (57/230)

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.  (+info)

Dissection of cauliflower mosaic virus transactivator/viroplasmin reveals distinct essential functions in basic virus replication. (58/230)

Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is an essential multifunctional viral protein. Dissection of Tav by deletion mutagenesis revealed that the central region is essential for CaMV replication in single cells but that the N- and C-terminal parts are not. Strains with mutations in the central region were defective in the translational transactivator function and could be complemented by coexpressing Gag (capsid protein precursor) and Pol (polyprotein with protease, reverse transcriptase, and RNase H activity) from separate monocistronic plasmids. In contrast, total omission of Tav was only partially complemented by Gag and Pol overexpression from separate plasmids. These results indicate that CaMV basic replication requires both Tav-activated polycistronic translation and some posttranslational function(s) of Tav that is not affected by the deletions in the central region of Tav.  (+info)

Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea. (59/230)

Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.  (+info)

A phaseolin domain involved directly in trimer assembly is a determinant for binding by the chaperone BiP. (60/230)

The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two alpha-helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.  (+info)

Characterization of Cestrum yellow leaf curling virus: a new member of the family Caulimoviridae. (61/230)

Cestrum yellow leaf curling virus (CmYLCV) has been characterized as the aetiological agent of the Cestrum parqui mosaic disease. The virus genome was cloned and the clone was proven to be infectious to C. parqui. The presence of typical viroplasms in virus-infected plant tissue and the information obtained from the complete genomic sequence confirmed CmYLCV as a member of the Caulimoviridae family. All characteristic domains conserved in plant pararetroviruses were found in CmYLCV. Its genome is 8253 bp long and contains seven open reading frames (ORFs). Phylogenetic analysis of the relationships with other members of the Caulimoviridae revealed that CmYLCV is closely related to the Soybean chlorotic mottle virus (SbCMV)-like genus and particularly to SbCMV. However, in contrast to the other members of this genus, the primer-binding site is located in the intercistronic region following ORF Ib rather than within this ORF, and an ORF corresponding to ORF VII is missing.  (+info)

Salicylic acid and the hypersensitive response initiate distinct signal transduction pathways in tobacco that converge on the as-1-like element of the PR-1a promoter. (62/230)

Tobacco pathogenesis-related protein 1a (PR-1a) is induced in plants during the hypersensitive response (HR) after exposure of plants to salicylic acid (SA) and by developmental cues. Gene activation by these diverse stimuli is mediated via an as-1-like element in the PR-1a upstream region. To further analyze the significance of this cis-acting sequence, an authentic as-1 element from the cauliflower mosaic virus 35S RNA promoter was inserted into the PR-1a promoter in place of the as-1-like motif. Reporter gene analysis in transgenic tobacco plants demonstrated that as-1 can functionally replace the as-1-like element in the PR-1a promoter in response to all stimuli. However, reporter gene induction from the as-1 carrying promoter was enhanced in response to SA compared to the wild-type promoter, and the ratio of reporter gene activities in SA treated leaf tissue to tissue exhibiting the HR increased with the as-1 promoter construct. Our findings support a model where PR-1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR-1a as-1-like element. Analysis of PR-1 proteins in plants expressing salicylate hydroxylase yielded additional evidence that an HR dependent pathway leads to high level PR-1 gene induction in tobacco.  (+info)

Low frequency of T-DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences. (63/230)

A powerful system to create gain-of-function mutants in plants is activation tagging using T-DNA based vehicles to introduce transcriptional enhancer sequences. Large Arabidopsis populations of individual plants carrying a quadruple cauliflower mosaic virus (CaMV) 35S enhancer are frequently used for mutant screenings, however the frequency of morphological mutants remains very low. To clarify this low frequency we analyzed a subset of lines generated by this method. The correlation between the number of T-DNA insertion sites, the methylation status of the 35S enhancer sequence and 35S enhancer activity was determined. All plants containing more than a single T-DNA insertion showed methylation of the 35S enhancer and revealed a dramatic decrease in 35S enhancer activity. The results support the notion that in a large proportion of the T-DNA based activation tagged lines the 35S transcriptional enhancer is silenced due to methylation, which is induced by multiple T-DNA integrations.  (+info)

An extracellular aspartic protease functions in Arabidopsis disease resistance signaling. (64/230)

We have used activation tagging with T-DNA carrying cauliflower mosaic virus 35S enhancers to investigate the complex signaling networks underlying disease resistance in Arabidopsis. From a screen of approximately 5000 lines, we identified constitutive disease resistance (CDR1) encoding an apoplastic aspartic protease, the overexpression of which causes dwarfing and resistance to virulent Pseudomonas syringae. These phenotypes reflect salicylic-acid-dependent activation of micro-oxidative bursts and various defense-related genes. Antisense CDR1 plants were compromised for resistance to avirulent P. syringae and more susceptible to virulent strains than wild type. CDR1 accumulates in intercellular fluid in response to pathogen attacks. Induction of CDR1 generates a small mobile signal, and CDR1 action is blocked by the protease inhibitor pepstatin and by mutations in the protease active sites. We propose that CDR1 mediates a peptide signal system involved in the activation of inducible resistance mechanisms.  (+info)