Expression In Protoplasts Research Articles

Intracellular sorting of the tail-anchored protein cytochrome b5 in plants: a comparative study using different isoforms from rabbit and Arabidopsis

Tail-anchored (TA) proteins are bound to membranes by a hydrophobic sequence located very close to the C-terminus, followed by a short luminal polar region. Their active domains are exposed to the cytosol. TA proteins are synthesized on free cytosolic ribosomes and are found on the surface of every subcellular compartment, where they play various roles. The basic mechanisms of sorting and targeting of TA proteins to the correct membrane are poorly characterized. In mammalian cells, the net charge of the luminal region determines the sorting to the correct target membrane, a positive charge leading to mitochondria and negative or null charge to the endoplasmic reticulum (ER). Here sorting signals of TA proteins were studied in plant cells and compared with those of mammalian proteins, using in vitro translation-translocation and in vivo expression in tobacco protoplasts or leaves. It is shown that rabbit cytochrome b5 (cyt b5) with a negative charge is faithfully sorted to the plant ER, whereas a change to a positive charge leads to chloroplast targeting (instead of to mitochondria as observed in mammalian cells). The subcellular location of two cyt b5 isoforms from Arabidopsis thaliana (At1g26340 and At5g48810, both with positive net charge) was then determined. At5g48810 is targeted to the ER, and At1g26340 to the chloroplast envelope. The results show that the plant ER, unlike the mammalian ER, can accommodate cytochromes with opposite C-terminal net charge, and plant cells have a specific and as yet uncharacterized mechanism to sort TA proteins with the same positive C-terminal charge to different membranes.

EpsinR2 Interacts with Clathrin, Adaptor Protein-3, AtVTI12, and Phosphatidylinositol-3-Phosphate. Implications for EpsinR2 Function in Protein Trafficking in Plant Cells

Members of the epsin family of proteins (epsins) are characterized by the presence of an epsin N-terminal homology (ENTH) domain. Epsins have been implicated in various protein-trafficking pathways in animal and yeast (Saccharomyces cerevisiae) cells. Plant cells also contain multiple epsin-related proteins. In Arabidopsis (Arabidopsis thaliana), EPSIN1 is involved in vacuolar trafficking of soluble proteins. In this study, we investigated the role of Arabidopsis EpsinR2 in protein trafficking in plant cells. EpsinR2 contains a highly conserved ENTH domain but a fairly divergent C-terminal sequence. We found that the N-terminal ENTH domain specifically binds to phosphatidylinositol-3-P in vitro and has a critical role in the targeting of EpsinR2. Upon transient expression in protoplasts, hemagglutinin epitope-tagged EpsinR2 was translocated primarily to a novel cellular compartment, while a minor portion localized to the Golgi complex. Protein-binding experiments showed that EpsinR2 interacts with clathrin, AtVTI12, and the Arabidopsis homologs of adaptor protein-3 delta-adaptin and adaptor protein-2 alpha-adaptin. Localization experiments revealed that hemagglutinin epitope-tagged EpsinR2 colocalizes primarily with delta-adaptin and partially colocalizes with clathrin and AtVTI12. Based on these findings, we propose that EpsinR2 plays an important role in protein trafficking through interactions with delta-adaptin, AtVTI12, clathrin, and phosphatidylinositol-3-P.

Transient Expression in Protoplasts

INTRODUCTIONThe study of transient gene expression provides a useful complement to the study of stably transformed plants. Transient assays offer a quick method of testing the effects of genes, using either phenotypic, molecular, or biochemical readouts. Transient assays based on Agrobacterium-mediated transformation of leaf explants have been described for other plant species, but it is not known how well these assays work in Arabidopsis. Alternatives include transient expression in protoplasts or in regenerating roots. The latter is especially suitable for investigating root-specific gene function.

On the phylogeny, expression and targeting of plant nucleoside diphosphate kinases

The plant nucleoside diphosphate kinase (NDPK, EC 2.7.4.6) gene family consists of three groups whose gene products are found in different subcellular locations. In this study we discuss the evolutionary history, localization and expression of the NDPK genes, addressing the question of functional specialization of the different NDPKs. A phylogenetic analysis revealed that the three NDPK isoforms were present already in the last common ancestor of vascular plants and mosses. Our data also imply that the NDPK3 genes possess a higher degree of conservation than the NDPK1 and NDPK2 genes. The expression levels of the different NDPKs in Arabidopsis thaliana inflorescences, leaves and roots were evaluated using quantitative PCR as well as in silico methods. This analysis showed that NDPK1 is the most highly expressed NDPK gene in all the studied tissues. NDPK3a has the second highest NDPK expression, while NDPK3b is expressed to a very low extent. However, expression of NDPK3b is elevated in inflorescence tissue. In situ hybridization experiments performed on inflorescences showed NDPK3a expression in actively dividing cells. NDPK3b expression was observed during later stages of flower development, specifically in the tapetum, ovules and petals. Additionally, we show that an NDPK3 protein is able to direct the green fluorescent protein to both mitochondria and chloroplasts using transient expression in leaf protoplasts. The dual localisation of NDPK3 was confirmed by Western blot, which also demonstrated that the majority of the NDPK3 protein is found in the mitochondria.

Visualization of PtdIns3P dynamics in living plant cells

SummaryTo investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P‐specific biosensor (YFP–2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY‐2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP–2xFYVE fluorescence strongly co‐localized with the pre‐vacuolar marker AtRABF2b, partially co‐localized with the endosomal tracer FM4‐64, but showed no overlap with the Golgi marker STtmd–CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP–2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY‐2 cells expressing YFP–2xFYVE contained twice as much PtdIns3P as YFP‐transformed cells, but this had no effect on cell‐growth or stress‐induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP–2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP–2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY‐2 cells, YFP–2xFYVE‐labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP–2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.

High GUS expression in protoplasts isolated from immature peach fruits

Abstract A protocol for transient expression analysis was developed using protoplast isolated from immature peach fruits. The uid A gene for β-glucuronidase (GUS) was introduced into the protoplast as a reporter gene to evaluate the protoplast activity. Protoplasts isolated from immature fruits at 28–32 days after full bloom (DAFB) showed high GUS activity under the cauliflower mosaic virus 35S promoter. The highest GUS activity was obtained from the protoplasts isolated at 32 DAFB, but it was difficult to isolate protoplasts showing high GUS activity from 34 DAFB. A comparison of the effect of promoter cassette on gene expression showed that the promoter containing the tobacco mosaic virus Ω sequence enhanced GUS activity by at least 10-fold in the peach protoplasts relative to the 35S promoter. The level of GUS activity under the 35S promoter in the peach protoplasts was 0.47-fold as that obtained from maize protoplasts isolated from young greening leaves, indicating that the GUS activity of immature peach protoplast is sufficient for gene expression analysis. Since stable transformation and evaluation of fruit traits in peach transformants are difficult, this transient expression system could be useful for the characterization of genes expressed in peach and other Rosaceae fruit species.

Ectopic Expression of EFFECTOR OF TRANSCRIPTION Perturbs Gibberellin-Mediated Plant Developmental Processes

The plant hormone gibberellin (GA) is known to modulate various aspects of plant cell differentiation and development. The current model of GA-mediated regulation is based on a de-repressible system and includes specific protein modification and degradation. HRT, a zinc finger protein from barley has been shown to have GA-dependent transcriptional repressing activity on the seed-specific alpha-amylase promoter [Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. J. Biol. Chem. 273: 23313-23320]. Here we report the characterization of a dicot homologue from Brassica napus (BnET) and provide evidence for its role in GA response modulation suggesting that this could be a conserved feature of this gene family. When BnET is ectopically expressed in either Arabidopsis or tobacco the phenotypes include dwarfism due to shorter internodes and late flowering, reduced germination rate, increased anthocyanin content and reduced xylem lignification as a marker for terminal cell differentiation. Transient expression in protoplasts supports the notion that this most likely is due to a transcriptional repression of GA controlled genes. Finally, histological analysis showed that in contrast to other GA deficient mutants the shorter internodes were due to fewer but not smaller cells, suggesting a function of BnET in GA-mediated cell division control.

The Evolution of the Abscisic Acid-response in Land Plants: Comparative Analysis of Group 1 LEA Gene Expression in Moss and Cereals

The moss Physcomitrella patens possesses a single copy of a Group 1 LEA gene, designated PpLEA-1. Sequence analysis of the PpLEA-1 gene reveals the gene to contain a single intron in a position conserved in all members of the Group 1 LEA gene family, but also to contain a premature termination codon within the first exon. Nevertheless, a PpLEA-1 transcript accumulates in moss tissues in response both to the imposition of osmotic stress, and to the plant growth regulator abscisic acid (ABA). This response appears to be mediated at the transcriptional level, and observation of the pattern of gene expression, reported by histochemical staining of plants expressing a PpLea-1::GUS transgene suggests that the promoter responds preferentially to ABA in protonemal filaments, whereas osmotic stress induces gene expression primarily in the gametophores. Quantitative analysis of promoter activity by transient expression in Physcomitrella protoplasts shows the PpLEA-1 promoter to be highly active in response to ABA and osmotic stress. ABA-mediated transgene expression from the PpLea-1 promoter occurs at a level similar to that driven by the highly active promoter of the wheat Group 1 LEA gene, E(m). Site-directed mutagenesis of the PpLEA-1 promoter indicates that ABA-inducibility is mediated via an ACGT-core motif similar to that seen in the ABA response elements of higher plant LEA genes. However, whereas the wheat E(m )promoter is active in moss tissues, the moss promoter is not reciprocally active in cereal cells: no activity, ABA-inducible or otherwise was detected in barley aleurone protoplasts transfected with the PpLEA-1::GUS construct. We propose that ABA activation of gene expression in moss cells represents an ancestral state, with only minimal requirements for promoter recognition, whereas cereal cells require the interaction of additional factors with ABA-responsive promoters.

Involvement of Ethylene in Stress-Induced Expression of the TLC1.1 Retrotransposon from Lycopersicon chilense Dun.

The TLC1 family is one of the four families of long terminal repeat (LTR) retrotransposons identified in the genome of Lycopersicon chilense. Here, we show that this family of retroelements is transcriptionally active and its expression is induced in response to diverse stress conditions such as wounding, protoplast preparation, and high salt concentrations. Several stress-associated signaling molecules, including ethylene, methyl jasmonate, salicylic acid, and 2,4-dichlorophenoxyacetic acid, are capable of inducing TLC1 family expression in vivo. A representative of this family, named TLC1.1, was isolated from a genomic library from L. chilense. Transient expression assays in leaf protoplasts and stably transformed tobacco (Nicotiana tabacum) plants demonstrate that the U3 domain of the 5'-LTR region of this element can drive stress-induced transcriptional activation of the beta-glucuronidase reporter gene. Two 57-bp tandem repeated sequences are found in this region, including an 8-bp motif, ATTTCAAA, previously identified as an ethylene-responsive element box in the promoter region of ethylene-induced genes. Expression analysis of wild-type LTR and single and double ethylene-responsive element box mutants fused to the beta-glucuronidase gene shows that these elements are required for ethylene-responsive gene expression in protoplasts and transgenic plants. We suggest that ethylene-dependent signaling is the main signaling pathway involved in the regulation of the expression of the TLC1.1 element from L. chilense.

Visualization of plastid nucleoids in situ using the PEND-GFP fusion protein.

Plastid DNA is a circular molecule of 120-150 kbp, which is organized into a protein-DNA complex called a nucleoid. Although various plastids other than chloroplasts exist, such as etioplasts, amyloplasts and chromoplasts, it is not easy to observe plastid nucleoids within the cells of many non-green tissues. The PEND (plastid envelope DNA-binding) protein is a DNA-binding protein in the inner envelope membrane of developing chloroplasts, and a DNA-binding domain called cbZIP is present at its N-terminus. We made various PEND-green fluorescent protein (GFP) fusion proteins using the cbZIP domains from various plants, and found that they were localized in the chloroplast nucleoids in transient expression in leaf protoplasts. In stable transformants of Arabidopsis thaliana, PEND-GFP fusion proteins were also localized in the nucleoids of various plastids. We have succeeded in visualizing plastid nucleoids in various intact tissues using this stable transformant. This technique is useful in root, flower and pollen, in which it had been difficult to observe plastid nucleoids. The relative arrangement of nucleoids within a chloroplast was kept unchanged when the chloroplast moved within a cell. During the division of plastid, nucleoids formed a network structure, which made possible equal partition of nucleoids.

Heterodimerization and Endocytosis of Arabidopsis Brassinosteroid Receptors BRI1 and AtSERK3 (BAK1)

In Arabidopsis thaliana brassinosteroid (BR), perception is mediated by two Leu-rich repeat receptor-like kinases, BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE3 [AtSERK3]). Genetic, biochemical, and yeast (Saccharomyces cerevisiae) interaction studies suggested that the BRI1-BAK1 receptor complex initiates BR signaling, but the role of the BAK1 receptor is still not clear. Using transient expression in protoplasts of BRI1 and AtSERK3 fused to cyan and yellow fluorescent green fluorescent protein variants allowed us to localize each receptor independently in vivo. We show that BRI1, but not AtSERK3, homodimerizes in the plasma membrane, whereas BRI1 and AtSERK3 preferentially heterodimerize in the endosomes. Coexpression of BRI1 and AtSERK3 results in a change of the steady state distribution of both receptors because of accelerated endocytosis. Endocytic vesicles contain either BRI1 or AtSERK3 alone or both. We propose that the AtSERK3 protein is involved in changing the equilibrium between plasma membrane-located BRI1 homodimers and endocytosed BRI1-AtSERK3 heterodimers.

Tomato heat stress transcription factor HsfB1 represents a novel type of general transcription coactivator with a histone-like motif interacting with the plant CREB binding protein ortholog HAC1.

In contrast with the class A heat stress transcription factors (HSFs) of plants, a considerable number of HSFs assigned to classes B and C have no evident function as transcription activators on their own. However, in the following article, we provide evidence that tomato (Lycopersicon peruvianum) HsfB1 represents a novel type of coactivator cooperating with class A HSFs (e.g., with tomato HsfA1). Provided the appropriate promoter architecture, the two HSFs assemble into an enhanceosome-like complex, resulting in strong synergistic activation of reporter gene expression. Moreover, HsfB1 also cooperates in a similar manner with other activators, for example, with the ASF1/2 enhancer binding proteins of the 35S promoter of Cauliflower mosaic virus or with yet unidentified activators controlling housekeeping gene expression. By these effects, HsfB1 may help to maintain and/or restore expression of certain viral or housekeeping genes during ongoing heat stress. The coactivator function of HsfB1 depends on a histone-like motif in its C-terminal domain with an indispensable Lys residue in the center (GRGKMMK). This motif is required for recruitment of the plant CREB binding protein (CBP) ortholog HAC1. HsfA1, HsfB1, and HAC1/CBP form ternary complexes in vitro and in vivo with markedly enhanced efficiency in promoter recognition and transcription activation in plant and mammalian (COS7) cells. Using small interfering RNA-mediated knock down of HAC1 expression in Arabidopsis thaliana mesophyll protoplasts, the crucial role for the coactivator function of HsfB1 was confirmed.

Isolation of full-length transcript promoter from the Strawberry vein banding virus (SVBV) and expression analysis by protoplasts transient assays and in transgenic plants

A full-length transcript (FLt) promoter was isolated from a genomic clone of Strawberry vein banding virus (SVBV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family, and its activity was analyzed both in protoplasts and transgenic plants. The 5′–3′-boundaries required for maximal promoter activity were determined by 5′- and 3′-end deletion analysis of the SVBV promoter fused to a β-glucuronidase (GUS) reporter gene. A 371-bp promoter fragment (−352 to +19 from the transcription start site; TSS) was found sufficient for maximal promoter activity in a transient protoplast expression assay, and this was chosen for further analysis. Finer deletion analysis of a 90-bp sequence (coordinates −392 to −302 from TSS) of the SVBV FLt promoter revealed the presence of a negative and a positive regulatory element in this region. In gain-of-function experiment, the fusion of the putative positive regulatory elements with minimal promoter showed very little increment in promoter activity, suggesting that a combinatorial action of various cis-sequences is involved in promoter function. The transcription start site of the full-length transcript promoter was mapped to an A-residue that is located 25-bp downstream of the TATA-box. In protoplast transient expression analysis, the SVBV FLt promoter showed about six-fold higher activity in tobacco compared to maize. A quantitative GUS activity assay showed that in transgenic tobacco plants the average promoter activity was about three-fold higher in roots than in leaves, and this higher activity was due to the accumulation of more GUS specific mRNA in roots. Real-time qRT-PCR analysis and quantitative GUS activity assay showed that the relative strength of the SVBV FLt promoter was greater than the CaMV35S promoter in transgenic tobacco plants. The SVBV FLt promoter is a strong, constitutive promoter and has great application potential in expression of foreign genes in plants.

Isolation of full-length transcript promoter from the Strawberry vein banding virus (SVBV) and expression analysis by protoplasts transient assays and in transgenic plants

A full-length transcript (FLt) promoter was isolated from a genomic clone of Strawberry vein banding virus (SVBV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family, and its activity was analyzed both in protoplasts and transgenic plants. The 5′–3′-boundaries required for maximal promoter activity were determined by 5′- and 3′-end deletion analysis of the SVBV promoter fused to a β-glucuronidase (GUS) reporter gene. A 371-bp promoter fragment (−352 to +19 from the transcription start site; TSS) was found sufficient for maximal promoter activity in a transient protoplast expression assay, and this was chosen for further analysis. Finer deletion analysis of a 90-bp sequence (coordinates −392 to −302 from TSS) of the SVBV FLt promoter revealed the presence of a negative and a positive regulatory element in this region. In gain-of-function experiment, the fusion of the putative positive regulatory elements with minimal promoter showed very little increment in promoter activity, suggesting that a combinatorial action of various cis-sequences is involved in promoter function. The transcription start site of the full-length transcript promoter was mapped to an A-residue that is located 25-bp downstream of the TATA-box. In protoplast transient expression analysis, the SVBV FLt promoter showed about six-fold higher activity in tobacco compared to maize. A quantitative GUS activity assay showed that in transgenic tobacco plants the average promoter activity was about three-fold higher in roots than in leaves, and this higher activity was due to the accumulation of more GUS specific mRNA in roots. Real-time qRT-PCR analysis and quantitative GUS activity assay showed that the relative strength of the SVBV FLt promoter was greater than the CaMV35S promoter in transgenic tobacco plants. The SVBV FLt promoter is a strong, constitutive promoter and has great application potential in expression of foreign genes in plants.

Core promoter structure in the oomycete Phytophthora infestans.

We have investigated the core promoter structure of the oomycete Phytophthora infestans. The transcriptional start sites (TSS) of three previously characterized P. infestans genes, Piexo1, Piexo3, and Piendo1, were determined by primer extension analyses. The TSS regions were homologous to a previously identified 16-nucleotide (nt) core sequence that overlaps the TSS in most oomycete genes. The core promoter regions of Piexo1 and Piendo1 were investigated by using a transient protoplast expression assay and the reporter gene beta-glucuronidase. Mutational analyses of the promoters of Piexo1 and Piendo1 showed that there is a putative core promoter element encompassing the TSS (-2 to +5) that has high sequence and functional homology to a known core promoter element present in other eukaryotes, the initiator element (Inr). Downstream and flanking the Inr is a highly conserved oomycete promoter region (+7 to +15), hereafter referred to as FPR (flanking promoter region), which is also important for promoter function. The importance of the 19-nt core promoter region (Inr and FPR) in Piexo1 and Piendo1 was further investigated through electrophoretic mobility shift assays (EMSA). The EMSA studies showed that (i) both core promoters were able to specifically bind a protein or protein complex in a P. infestans whole-cell protein extract and (ii) the same mutations that reduced binding of the EMSA complex also reduced beta-glucuronidase (GUS) levels in transient expression assays. The consistency of results obtained using two different assays (GUS transient assays [in vivo] and EMSA studies [in vitro]) supports a convergence of inference about the relative importance of specific nucleotides within the 19-nt core promoter region.

Analysis of Gene Expression in Yeast Protoplasts Using DNA Microarrays and Their Application for Efficient Production of Invertase and .ALPHA.-Glucosidase

The global gene expression of cultured Saccharomyces cerevisiae protoplasts was compared with that of cells using DNA microarray. Quantitative and qualitative analyses revealed that after 6 h of cultivation, 416 gene transcript levels (about 7.1% in all) in the cultured protoplasts were different from those in the cells. Various characteristics and functions of the protoplasts were predicted from the analysis of the gene functions. The cultured protoplasts were more sensitive to oxidative stress than the cultured cells. Their cell cycles were arrested at the G1 phase and cell wall synthesis was promoted. Carbohydrate metabolism was activated in cultured protoplasts, while amino acid biosynthesis was inhibited. Furthermore, some genes associated with the secretory pathway of metabolites were activated, leading to active secretion of these metabolites into the broth. As an example of the application of DNA microarray analysis, we developed two novel methods for the production of useful enzymes based on the characteristics of protoplasts. One was the production of invertase based on the activated secretory pathway, while the other was the production of α-glucosidase based on the activated carbohydrate metabolism. The secretion of invertase and α-glucosidase was promoted in cultured protoplasts. The invertase and α-glucosidase productivities in the cultured protoplasts were 657 U and 218 U, respectively. On the other hand, only 227 U of invertase was produced, while α-glucosidase was not detected, in the cultured cells. The fragile protoplasts were immobilized in agarose gel to protect them from hydrodynamic stress. Four repeated-batch cultures with the immobilized protoplasts were performed, leading to the production of 1574 U of invertase and 739 U of α-glucosidase. The same productivities were obtained when this system was scaled up by 10-fold (invertase: 13304 U; α-glucosidase: 7688 U).

Analysis of the Arabidopsis nuclear proteome and its response to cold stress.

The nucleus is the subcellular organelle that contains nearly all the genetic information required for the regulated expression of cellular proteins. In this study, we comprehensively characterized the Arabidopsis nuclear proteome. Nuclear proteins were isolated and analyzed using two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Approximately 500-700 spots were detected in reference 2D gels of nuclear proteins. Proteomic analyses led to the identification of 184 spots corresponding to 158 different proteins implicated in a variety of cellular functions. We additionally analyzed the changes in the nuclear proteome in response to cold stress. Of the 184 identified proteins, 54 were up- or downregulated with a greater than twofold change in response to cold treatment. Among these, six proteins were selected for further characterization. Northern analysis data revealed that gene expression of these proteins was also altered by cold stress. Following transient expression in BY-2 protoplasts, two proteins were detected in both the cytoplasm and the nucleus and four others were detected exclusively in the nucleus, which correlates well with the nuclear localization patterns of the proteomic data. Our study provides an initial insight into the Arabidopsis nuclear proteome and its response to cold stress.

Tomato EF-Ts(mt), a functional mitochondrial translation elongation factor from higher plants.

Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Ts(mt) that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts). Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Ts(mt)-GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Ts(mt) to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein. Escherichia coli recombinant LeEF-Ts(mt) co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E. coli, indicating an interaction with bacterial EF-Tu. Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Ts(mt) fraction. The purified LeEF-Ts(mt) stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu. Furthermore, LeEF-Ts(mt) was capable of catalysing the nucleotide exchange reaction with E. coli EF-Tu. Altogether, these data demonstrate that LeEF-Ts(mt) encodes a functional mitochondrial EF-Ts. LeEF-Ts(mt) represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants.

A novel procedure for the localization of viral RNAs in protoplasts and whole plants.

Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.

An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein.

Potato leafroll polerovirus (PLRV) genomic RNA acts as a polycistronic mRNA for the production of proteins P0, P1, and P2 translated from the 5'-proximal half of the genome. Within the P1 coding region we identified a 5-kDa replication-associated protein 1 (Rap1) essential for viral multiplication. An internal ribosome entry site (IRES) with unusual structure and location was identified that regulates Rap1 translation. Core structural elements for internal ribosome entry include a conserved AUG codon and a downstream GGAGAGAGAGG motif with inverted symmetry. Reporter gene expression in potato protoplasts confirmed the internal ribosome entry function. Unlike known IRES motifs, the PLRV IRES is located completely within the coding region of Rap1 at the center of the PLRV genome.