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A set of plant expression vectors for transcriptional and translational fusions

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... In restriction sites PacI and XhoI within this polylinker the chalcone synthase promoter pchs_H1 [47] was integrated based of primers 45 and 46 providing PacI and XhoI restriction ends to the promoter sequence (Table S1). The new expression cassette was then completed by integration of the XhoI/AscI fragment from vector pRT-100 [75] providing the CaMV terminator. We named the resulting plant vector pJM14. ...
... NtAGO5 cDNAs was then cloned directly into the chs_H1(600) promoter cassette of binary vector pJM14 (see above). NbTFIIIA-7ZF was first cloned into pRT-100 via unique XhoI and BamHI sites [75] and then re-cloned together with the terminator sequence into pJM14 using XhoI and AscI. Finally, both NtAGO5 and NbTFIIIA-7ZF bearing plant vectors were transformed into A. tumefaciens LBA4404 and used for transient expression assays. ...
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Some viroids-single-stranded, non-coding, circular RNA parasites of plants-are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (−) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming "comets" on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.
... With obtained PCR amplicon, a tobacco petE (plastocyanin) transit peptide (NtPc sig.) was fused for chloroplast targeted expression of the gene. The NtPc sig-UfCyt c 6 cassette was placed in-between CaMV 35S promoter and NOS terminator of pRT 101 vector [23]. Thereafter, the entire cassette containing CaMV 35S-NtPc sig-UfCyt c 6 gene and terminator was digested with PstI and cloned into pCAMBIA1301 vector at PstI site. ...
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Cytochromes are important components of photosynthetic electron transport chain. Here we report on genetic transformation of Cytochrome c6 (UfCyt c6) gene from Ulva fasciata Delile in tobacco for enhanced photosynthesis and growth. UfCyt c6 cDNA had an open reading frame of 330 bp encoding a polypeptide of 109 amino acids with a predicted molecular mass of 11.65 kDa and an isoelectric point of 5.21. UfCyt c6 gene along with a tobacco petE transit peptide sequence under control of CaMV35S promoter was transformed in tobacco through Agrobacterium mediated genetic transformation. Transgenic tobacco grew normal and exhibited enhanced growth as compared to wild type (WT) and vector control (VC) tobacco. Transgenic tobacco had higher contents of photosynthetic pigments and better ratios of photosynthetic pigments. The tobacco expressing UfCyt c6 gene exhibited higher photosynthetic rate and improved water use efficiency. Further activity of the water-splitting complex, photosystem II quantum yield, photochemical quenching, electron transfer rate, and photosynthetic yield were found comparatively higher in transgenic tobacco as compared to WT and VC tobacco. Alternatively basal quantum yield of non-photochemical processes in PSII and non-photochemical quenching were estimated lower in tobacco expressing UfCyt c6 gene. As a result of improved photosynthetic performance the transgenic tobacco had higher contents of sugar and starch, and exhibited comparatively better growth. To the best of our knowledge this is the first report on expression of UfCyt c6 gene from U. fasciata for improved photosynthesis and growth in tobacco.
... HvPIP2;4 was sub cloned into a constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter/CaMV strain Cabb B-D polyadenylation signal cassette, within the intermediate vector pRT101 (Tӧpfer et al., 1987). Using the restriction site HindIII, the CaMV35S promoter-HvPIP2;4-35S terminator cassette was subsequently transferred to T-DNA portion of plant binary vector pCAMBIA 2301 (11.6 kb). ...
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p>In the present study HvPIP2;4 was overexpressed in Arabidopsis thaliana to engineer enhanced salt tolerance. Barley Aquaporin was selected since barley shows fairly good tolerance to drought, salt stress and low temperature compared to many other crops including rice, and it was thought that analysis of barley aquaporin will provide a good insight into the molecular mechanisms involved in transport of water & their efficacy during abiotic stress condition. Arabidopsis line expressing HvPIP2;4 from annual crop plant Hordeum vulgare (Barley) under the control of constitutive promoter was used to analyze the expression of HvPIP2 ; 4 and its efficacy during salt stress when NaCl concentration gradually increased. The pattern of expression of Hv PIP2; 4 were found to be NaCl dose dependent during salt stress. The constitutive expression of HvPIP2;4 enhanced salt stress tolerance in Arabidopsis . HvPIP2;4 played a dominant role in improving plant salt tolerance. It may be very well presumed that overexpression of HvPIP2;4 in crop plant might benefit them by enhancing their salt tolerance capacity. Int J Appl Sci Biotechnol, Vol 4(4): 498-512</p
... The four bar variants were cloned independently, in a modified vector pRT100 (Töpfer et al. 1987) downstream to the Upstream Regulatory Module (URM), 35SsynJ (Kanoria and Burma 2012). These expression cassettes that differ only in the sequences of the bar gene were further mobilized into the binary vector pZP200N (Bhullar et al. 2003) as a HindIII fragment (Fig. 1a). ...
Article
Information of codon usage bias has been used for modifying genes for improved expression in heterologous systems. Codon modifications are carried out using the species-specific codon usage so that they reflect the codon usage pattern of the host, without modifying the amino acid sequence of the encoded protein. In the present study we analyzed the effect of codon optimization on the expression of the bacterial gene bar in tobacco. In order to identify the percentage of optimal codons needed to achieve high levels of protein expression, bar genes with different percentage and placement of optimal codons were analyzed in transgenic tobacco lines. It was observed that there was no gain in bar protein expression when the percentage of optimal codons was increased from 63.9% (as observed in the wild type bar gene) to 93.9%. However, reducing the percentage of optimal codons to 54.0 led to a drop in the levels of the bar protein. Further, in silico analysis was also carried out in ~ 4500 genes present on chromosome 2 of Arabidopsis thaliana to study the distribution of optimal codons. It was observed that majority (88%) of the genes have 30–50% of optimal codons and none of the gene was found to have more than 80% of optimal codons. The present study showed that there may not be a one-to-one correlation between the percentage of optimal codons and the expression levels of the transgene. A certain percentage of optimal codons is probably enough to achieve high levels of transgene expression. Any increase in the percentage of optimal codons beyond this level may not necessarily lead to any further improvement in expression.
... In order to design the expression vector containing a PVX modified sequence and the gb gene silencing suppressor from Poa semilatent virus (PSLV) a gb amplicon was generated using SalI-BgP-forw and SpeI-BgP-rev primers and pPgb plasmid [33] as a template. The PCR product was cut with SalI and SpeI restriction enzymes and cloned into the XhoI-XbaI digested pRT103 plasmid generating the pRT-35S-gb-ter construct [34]. A 35S-gb-ter expression cassette was released from the pRT-35S-gb-ter plasmid by PstI and inserted into NsiI digested pLH-Dbar binary vector resulting in pLH-gb plasmid. ...
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Salmonella Typhimurium is one of the most important zoonotic pathogens worldwide and a major cause of economic losses in the pig production chain. The emergence of multi-drug resistant strains over the past years has led to considerations about an enhanced surveillance of bacterial food contamination. Currently, ELISA is the method of choice for high throughput identification of S. Typhimurium. The sensitivity and specificity of this assay might be improved by application of new diagnostic antibodies. We focused on plant-based expression of candidate diagnostic TM43-E10 antibodies discovered using as antigen the S. Typhimurium OmpD protein. The scFv-TM43-E10 and scFv-Fc-TM43-E10 antibody derivatives have been successfully produced in N. benthamiana using a deconstructed movement-deficient PVX vector supplemented with the γb silencing suppressor from Poa semilatent virus. The plant-made antibodies showed the same antigen-binding specificity as that of the microbial/mammalian cell-produced counterparts and could recognize the OmpD antigen in S. Typhimurium infected plant samples.
... For tobacco transformation, the cDNA of JcWRKY (Agarwal et al., 2014, NCBI acc no KC191643), was PCR amplified using JcWRKYTF and JcWRKYTR primers (Supplementary Table S1) containing KpnI and XbaI restriction sites, respectively, and the digested fragment was cloned in KpnI/XbaI sites of pRT101 vector (Topfer et al., 1987). Thereafter, the entire expression cassette containing 35S: JcWRKY: Poly A was cloned in the pCAMBIA 1301 at the HindIII site and mobilized into the Agrobacterium strain LBA4404. ...
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Plants, being sessile, have developed intricate signaling network to specifically respond to the diverse environmental stress. The plant-specific WRKY TFs form one of the largest TF family and are involved in diverse plant processes, involving growth, development and stress signaling through auto and cross regulation with different genes and TFs. Here, we report the functional characterization of a salicylic acid-inducible JcWRKY TF. The JcWRKY overexpression confers salinity tolerance in transgenic tobacco, as was evident by increased chlorophyll content and seed germination potential. The transgenic plants showed increased soluble sugar, membrane stability, reduced electrolyte leakage and generation of reactive oxygen species (H 2 O 2 and O •−) as compared to the wild type. 2 Furthermore, the low SA treatment along with salinity improved the tolerance potential of the transgenics by maintaining ROS homeostasis and high K + /Na + ratio. The transcript expression of SA biosynthetic gene ICS1 and antioxidative enzymes (CAT and SOD) showed upregulation during stress. Thus, the present study reflects that JcWRKY is working in coordination with SA signaling to orchestrate the different biochemical and molecular pathways to maneuvre salt stress tolerance of the transgenic plants.
... Tomato EST clones cLEC80N18 (GenBank accession BI923467) and cTOC20K10 (GenBank accession BI931548) representing either incomplete or disordered SlMlo1 cDNAs were recombined to obtain a bona fide full-length SlMlo1 cDNA clone. The respective cDNA was shuttled via plasmid pRT101 (harboring Cauliflower mosaic virus 35S promoter and terminator sequences; Töpfer et al. 1987) into binary vector pPZP211 (Gen-Bank accession number U10490; Hajdukiewicz et al. 1994), using appropriate restriction sites. Agrobacterium-mediated transformation of resistant line R26 and subsequent selection of transgenic lines was performed as described (Knapp et al. 1994). ...
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The resistant cherry tomato (Solanum lycopersicum var. cerasiforme) line LC-95, derived from an accession collected in Ecuador, harbors a natural allele (ol-2) that confers broad-spectrum and recessively inherited resistance to powdery mildew (Oidium neolycopersici). As both the genetic and phytopathological characteristics of ol-2–mediated resistance are reminiscent of powdery mildew immunity conferred by loss-of-function mlo alleles in barley and Arabi-dopsis, we initiated a candidate-gene approach to clone Ol-2. A tomato Mlo gene (SlMlo1) with high sequence-related-ness to barley Mlo and Arabidopsis AtMLO2 mapped to the chromosomal region harboring the Ol-2 locus. Comple-mentation experiments using transgenic tomato lines as well as virus-induced gene silencing assays suggested that loss of SlMlo1 function is responsible for powdery mildew resistance conferred by ol-2. In progeny of a cross between a resistant line bearing ol-2 and the susceptible tomato cul-tivar Moneymaker, a 19-bp deletion disrupting the SlMlo1 coding region cosegregated with resistance. This polymor-phism results in a frameshift and, thus, a truncated non-functional SlMlo1 protein. Our findings reveal the second example of a natural mlo mutant that possibly arose post-domestication, suggesting that natural mlo alleles might be evolutionarily short-lived due to fitness costs related to loss of mlo function.
... For analysis of ABA3 transcriptional regulation 1.0 kb upstream of the PtABA3 coding sequence was amplified from Populus trichocarpa genomic DNA (Primer : Table S1) and the resulting putative ptaba3 promoter sequence was cloned into pRT99GUS [37] using restriction enzymes SmaI and EcoRI. Subsequently, the cassette consisting of the aba3 promotor, the β-glucuronidase (GUS) coding sequence was transferred into the binary vector pBIN19 [38] using HindIII restriction sites at the 5' end of the ptaba3 promotor and the 3' end of the terminator. ...
... The OsAnn5 was cloned into pRT100 vector (Töpfer et al., 1987) using KpnI and BamHI restriction enzyme sites for a transcriptional fusion with the CaMV 35S promoter and polyadenylation signal. This expression cassette CaMV35S:OsAnn5:polyA was cloned into pCAMBIA2300 binary vector (CAMBIA, Australia) at the HindIII restriction enzyme site and the recombinant vector was confirmed by digestion and PCR ( Supplementary Fig. 2). ...
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The current study on putative rice annexin OsAnn5 was tried to know its functional role in the abiotic stress tolerance. For this an in silico analysis of its protein sequence and upstream region was carried out. This results in identification of several probable potential sites for post-translational modifications and cis-elements respectively. We have studied the effect of OsAnn5 in the amelioration of abiotic stress tolerance through heterologous expression in transgenic tobacco and E.coli. It is observed that OsAnn5 over expression leads to enhanced tolerance to abiotic stress through efficient scavenging of the ROS and balanced expression of SOD and CAT antioxidant enzymes in both the systems, under stress treatments. Fluorescent signal for transiently expressed EGFP:OsANN5 fusion protein was localized in the peripheral region of the onion epidermal cells under salt stress treatment. Expression analysis of OsAnn5 under ABA synthesis inhibitor, fluridone and salinity stress revealed that OsAnn5 appears to act through an ABA-independent pathway under salt stress and in support to this 35S:OsAnn5 transgenics seedlings exhibited less sensitivity to externally applied ABA.
... The volume of the protoplast suspension was adjusted to 4 × 10 6 cells/mL. Protoplasts were cotransfected with 2 µg of plasmid carrying one of the PR1 promoter::GUS constructs and 6 µg of effector plasmid pRT101 (Töpfer et al., 1987) carrying 35S::AtWRKY50, 35S::AtWRKY51 or 35S::AtWRKY59. As a control, co-transformation of PR1::GUS fusions with the empty expression vector pRT101 was carried out. ...
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Arabidopsis PR1 is a salicylic acid (SA) inducible marker gene for systemic acquired resistance (SAR). However, the regulation of PR1 in plants is poorly understood. In this study, we showed that AtWRKY50 transcription factor binds to two promoter elements of PR1 via its DNA binding domain. Interestingly, the DNA-binding sites for AtWRKY50 deviate significantly from the consensus WRKY binding W-box. The binding sites are located in close proximity to the binding sites for TGA transcription factors. Transactivation experiments in Arabidopsis protoplasts derived from wild type, npr1-1 and tga256 mutant plants indicated that AtWRKY50 alone was able to induce expression of a PR1::β-glucuronidase (GUS) reporter gene, independent of TGAs or NPR1. However, co-expression of TGA2 or TGA5 with AtWRKY50 synergistically enhanced expression to high levels. Yeast-2-hybrid assays and bimolecular fluorescence complementation (BiFC) experiments revealed that AtWRKY50 could interact with TGA2 and TGA5. Using electrophoretic mobility shift assays (EMSA) it was established that AtWRKY50 and TGA2 or TGA5 simultaneously bind to the PR1 promoter. Taken together, these results support a role of AtWRKY50 in SA-induced expression of PR1. Highlights: AtWRKY50 specifically binds to LS10 region of PR1 promoter and interacts with TGAs to synergistically activate PR1 expression.
... Full-length MYB3-HA, LNK1-HA, LNK2-HA, LNK3-HA, and LNK4-HA, or mutatedLNK1D556N-HA, LNK1D614N-HA, LNK1D556N/D614N-HA, and chimeric ENT-LNK3-HA (HA tag, influenza virus hemagglutinin tag) were PCR amplified cloned into effector plasmid pRT101 under the CaMV 35S promoter (Töpfer et al., 1987). The promoter fragments of C4H-fused GUS cloned in reporter plasmid GusXX was described previously (Zhou et al., 2015b). ...
... PCR product was sequenced commercially for the confirmation of the reading frame. The ORF of AdDjSKI was cloned at the NcoI-SmaI site into the plant expression cassette of pRT100 vector [31]. The AdDjSKI expression cassette containing the CaMV-35S promoter and the termination signal was released with PstI digestion and was subsequently cloned in the binary vector pCAMBIA2300 [32] at the PstI restriction site. ...
... forward 5'-GAGGTCGACATGGAAATATTCTC-TTCTGGGGA-3′and reverse 5-GATGGTACCGCAAGCAATATCAAGTAT-CAAACTG-3′. SalI sites in forward primers and KpnI sites in reverse primers are underlined.Coding regions of BBX19, BBX32, and LHY2 were introduced into pRT104-3xHA and pRT104-3xMyc(Fülöp et al., 2005, Töpfer, Matzeit, Gronenborn, Schell, & Steinbiss, 1987 in frame behind the 35S Cauliflower Mosaic Virus promoter and Myc or Humaninfluenza hemagglutinin (HA)-epitope tags. To produce CYCD3 tagged with the MYC epitope, the full coding region of CYCD3 (Potri.014G023000.1) ...
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Trees are carbon dioxide sinks and major producers of terrestrial biomass with distinct seasonal growth patterns. Circadian clocks enable the co-ordination of physiological and biochemical temporal activities, optimally regulating multiple traits including growth. To dissect the clock's role in growth, we analysed Populus tremula × P. tremuloides trees with impaired clock function due to down-regulation of central clock components. late elongated hypocotyl (lhy-10) trees, in which expression of LHY1 and LHY2 is reduced by RNAi, have a short free-running period and show disrupted temporal regulation of gene expression and reduced growth, producing 30-40% less biomass than wild-type trees. Genes important in growth regulation were expressed with an earlier phase in lhy-10 , and CYCLIN D3 expression was misaligned and arrhythmic. Levels of cytokinins were lower in lhy-10 trees, which also showed a change in the time of peak expression of genes associated with cell division and growth. However, auxin levels were not altered in lhy-10 trees and the size of the lignification zone in the stem showed a relative increase. The reduced growth rate and anatomical features of lhy-10 trees were mainly caused by misregulation of cell division, which may have resulted from impaired clock function.
... The SbSI-1 gene was amplified using S. brachiata cDNA with AccuPrime TM Pfx DNA polymerase using forward (5 ′ -TCCGAGCTCATGCCTAATAAACATATCATGG-3 ′ ) and reverse (5 ′ -CGCGGATCCTTAACGGTTCCCTTGTTTC-3 ′ ) primers containing Sac I and BamH 1 sites, respectively. The SbSI-1 amplicon with BamH 1/Sac I sites was cloned into pRT101 vector (Töpfer et al., 1987). Further, the cassette containing the CaMV35S constitutive promoter, SbSI-1 gene and terminator was cloned into the pCAMBIA2301 vector at Pst I site (Supplementary Figure 1C) and subsequently mobilized into Agrobacterium tumefaciens (LBA 4404). ...
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A novel Salicornia brachiata Salt Inducible (SbSI-1) gene was isolated and overexpressed in tobacco for in planta functional validation subjected to drought and salt stress. SbSI-1 is a nuclear protein. The transgenic tobacco overexpressing SbSI-1 gene exhibited better seed germination, growth performances, pigment contents, cell viability, starch accumulation, and tolerance index under drought and salt stress. Overexpression of SbSI-1 gene alleviated the build-up of reactive oxygen species (ROS) and curtailed the ROS-induced oxidative damages thus improved the physiological health of transgenic tobacco under stressed conditions. The higher activities of antioxidant enzymes, lower accumulation of ROS, higher membrane stability, relative water content, and polyphenol contents indicated the better survival of the transgenic tobacco than wild-type (WT) tobacco under stressed conditions. Transgenic tobacco had a higher net photosynthetic rate, PSII operating efficiency, and performance index under drought and salt stress. Higher accumulation of compatible solutes and K+/Na+ ratio in transgenic tobacco than WT showed the better osmotic and redox homeostasis under stressed conditions. The up-regulation of genes encoding antioxidant enzymes (NtSOD, NtAPX, and NtCAT) and transcription factors (NtDREB2 and NtAP2) in transgenic tobacco under stressed conditions showed the role of SbSI-1 in ROS alleviation and involvement of this gene in abiotic stress tolerance. Multivariate data analysis exhibited statistical distinction among growth responses, physiological health, osmotic adjustment, and photosynthetic responses of WT and transgenic tobacco under stressed conditions. The overexpression of SbSI-1 gene curtailed the ROS-induced oxidative damages and maintained the osmotic homeostasis under stress conditions thus improved physiological health and photosynthetic efficiencies of the transgenic tobacco overexpressing SbSI-1 gene.
... To rescue the nfh1-3 mutant phenotype by MtNFH1 re-expression, a similar DNA fragment [containing the 2051-bp the promoter sequence and the coding sequence of MtNFH1 or MtNFH1(D148A)] was inserted into pRT104 (Töpfer et al., 1987) and finally [with the poly(A) tail from pRT104] cloned into the binary vector pCAMBIA1302. ...
Article
Establishment of symbiosis between legumes and nitrogen-fixing rhizobia depends on bacterial Nod factors (NFs) that trigger symbiosis-related NF signaling in host plants. NFs are modified oligosaccharides of chitin with a fatty acid moiety. NFs can be cleaved and inactivated by host enzymes, such as MtNFH1 (MEDICAGO TRUNCATULA NOD FACTOR HYDROLASE 1). In contrast to related chitinases, MtNFH1 hydrolyzes neither chitin nor chitin fragments, indicating a high cleavage preference for NFs. Here, we provide evidence for a role of MtNFH1 in the symbiosis with Sinorhizobium meliloti. Upon rhizobial inoculation, MtNFH1 accumulated at the curled tip of root hairs, in the so-called infection chamber. Mutant analysis revealed that lack of MtNFH1 delayed rhizobial root hair infection, suggesting that excess amounts of NFs negatively affect the initiation of infection threads. MtNFH1 deficiency resulted in nodule hypertrophy and abnormal nodule branching of young nodules. Nodule branching was also stimulated in plants expressing MtNFH1 driven by a tandem CaMV 35S promoter and plants inoculated by a NF-overproducing S. meliloti strain. We suggest that fine-tuning of NF levels by MtNFH1 is necessary for optimal root hair infection as well as for NF-regulated growth of mature nodules.
... To make the effector constructs pRT101-Oshox12 and pRT101-Oshox14, the full length cDNAs of Oshox12 and Oshox14 were cut as EcoRI-BamHI fragments from λFLC-1-B-Oshox12 and λFLC-1-B-Oshox14 respectively, and cloned into pRT101 cut with the same restriction enzymes [49]. ...
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The homeodomain-leucine zipper (HD-Zip) transcription factor family plays vital roles in plant development and morphogenesis as well as responses to biotic and abiotic stresses. In barley, a recessive mutation in Vrs1 (HvHox1) changes two-rowed barley to six-rowed barley, which improves yield considerably. The Vrs1 gene encodes an HD-Zip subfamily I transcription factor. Phylogenetic analysis has shown that the rice HD-Zip I genes Oshox12 and Oshox14 are the closest homologues of Vrs1. Here, we show that Oshox12 and Oshox14 are ubiquitously expressed with higher levels in developing panicles. Trans-activation assays in yeast and rice protoplasts demonstrated that Oshox12 and Oshox14 can bind to a specific DNA sequence, AH1 (CAAT(A/T)ATTG), and activate reporter gene expression. Overexpression of Oshox12 and Oshox14 in rice resulted in reduced panicle length and a dwarf phenotype. In addition, Oshox14 overexpression lines showed a deficiency in panicle exsertion. Our findings suggest that Oshox12 and Oshox14 may be involved in the regulation of panicle development. This study provides a significant advancement in understanding the functions of HD-Zip transcription factors in rice.
... To transiently express S11-GFP in plants and protoplasts, we amplified the S11-GFP fusion DNA from pBS-S11 with a two-step recombinant polymerase chain reaction (PCR), using primers F1&R2, F2&R1 for step 1 and F1&R1 for step 2 (See Table S1 in Supporting Information for the primer sequences). The PCR products were digested with NcoI and KpnI and ligated into pRTL2 vector (Töpfer et al., 1987), and then linearized with the same enzymes to generate pRTL2-S11GFP. Plasmid pRTL2-S11 (1−131 aa) GFP (F1&R3, F3&R1 for step 1 and F1&R1 for step 2) was constructed similarly. ...
Article
RNA silencing is a potent antiviral mechanism in plants and animals. As a counter-defense, many viruses studied to date encode one or more viral suppressors of RNA silencing (VSR). In the latter case, how different VSRs encoded by a virus function in silencing remains to be fully understood. We previously showed that the nonstructural protein Pns10 of a Phytoreovirus, Rice dwarf virus (RDV), functions as a VSR. Here we present evidence that another nonstructural protein, Pns11, also functions as a VSR. While Pns10 was localized in the cytoplasm, Pns11 was localized both in the nucleus and chloroplasts. Pns11 has two bipartite nuclear localization signals (NLSs), which were required for nuclear as well as chloroplastic localization. The NLSs were also required for the silencing activities of Pns11. This is the first report that multiple VSRs encoded by a virus are localized in different subcellular compartments, and that a viral protein can be targeted to both the nucleus and chloroplast. These findings may have broad significance in studying the subcellular targeting of VSRs and other viral proteins in viral-host interactions.
... The hybrid protein constructs consisting of an A. thaliana LYK ectodomain (LYK1 ED , 693-bp fragment of LYK1; LYK4 ED , 813-bp fragment of LYK4; LYK5 ED , 831-bp fragment of LYK5) and an intracellular domain of a L. japonicus Nod factor receptor of (NFR1 ID , 1203-bp fragment of NFR1; NFR5 ID , 1086-bp fragment of NFR5) were obtained by overlap extension PCR. The constructs (namely LYK1 ED -NFR1 ID , LYK4 ED -NFR1 ID , LYK5 ED -NFR1 ID , LYK1 ED -NFR5 ID , LYK4 ED -NFR5 ID , LYK5 ED -NFR5 ID ) were inserted into pRT104 [36] to yield expression cassettes containing a CaMV 35S promoter and a poly-A tail. The chimeric receptor pair constructs were then introduced into the pCAMBIA-NINp vector by using the Hieff Clone ™ Plus Multi One ...
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Background: Plant receptors with lysin motifs (LsyM) recognize microbial signals such as fungal chitin and lipo-chitooligosaccharidic Nod factors of nitrogen-fixing rhizobia. It is generally assumed that ligand-induced dimerization of LysM receptors is an essential step in activation of intracellular kinase domains and downstream signaling. Consequently, genes required for plant defense and establishment of symbiosis are expressed. We recently found that three LysM receptor proteins (namely LYK1, LYK4 and LYK5) of Arabidopsis thaliana form a tripartite receptor complex to perceive chitin. However, constitutive and ligand-induced interactions of LysM receptors generally remain difficult to be characterized. Results: Interactions between ectodomains of LYK1, LYK4 and LYK5 were investigated by a chimeric receptor approach using hairy roots of the legume Lotus japonicus. Synthetic receptor pairs consisting of a LYK ectodomain and the intracellular domain of a L. japonicus Nod factor receptor (NFR1 and NFR5, respectively) were tested for their capacity to activate expression of the symbiotic NIN (nodule inception) gene. The results indicated constitutive (LYK4ED-LYK4ED, LYK4ED-LYK5ED) and chitin-induced interactions (LYK1ED-LYK1ED, LYK1ED-LYK5ED) of the examined ectodomains. Conclusion: We present a method to functionally analyze constitutive and ligand-induced interactions of LysM-type proteins.
... In this study, we have used the vector PGJ357 (Hajdukiewicz et al. 1994) in which the 35S cassette from the cauliflower mosaic virus of the pRT101 plasmid (Töpfer et al. 1987) was inserted (pGJpRT). The Vv-α-gal/SIP gene was cloned into the pGJpRT along with the ampicillin resistance gene for bacterial selection and the kanamycin marker gene for plant selection. ...
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Grapevine is an important fruit crop cultivated worldwide. Previously, we have reported the characterization of a salt stress-inducible gene Vv-α-gal/SIP isolated from the tolerant grapevine cultivar Razegui. In this study, we performed functional studies in both Escherichia coli and tobacco systems to gain more insights in the role of the Vv-α-gal/SIP gene. Our data revealed that the recombinant E. coli cells harboring the pET24b⁺ expression vector with the Vv-α-gal/SIP showed higher tolerance to desiccation and salinity compared to E. coli cells harboring the vector alone. In addition, the transgenic tobacco plants expressing the Vv-α-gal/SIP gene exhibited a higher percentage of seed germination and better growth under salt stress than the wild-type (WT) tobacco seedlings. This stress mitigation might be related to the putative function of this gene, which is thought to be involved in carbohydrate metabolism regulation. Collectively, these results suggest that Vv-α-gal/SIP is potentially a candidate gene for engineering drought and salt tolerance in cultivated plants.
... The open reading frame (ORF) of JcPR-10a cDNA (Agarwal et al., 2013), was PCR amplified using JcPR-10TF and JcPR-10TR primers (Supplementary Table S1) flanked with XhoI and XbaI restriction sites, respectively, and cloned in in XhoI/XbaI sites of pRT101vector (Topfer et al., 1987). Thereafter, expression cassette containing 35S: JcPR-10a: Poly A was cloned in the pCAMBIA 1301 at the HindIII site and mobilized into the Agrobacterium strain LBA4404. ...
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Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle, and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth, and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants.
... Plasmids carrying VBF-promoter-GUS and plasmids expressing VirE3 and/or JAZ8 were cotransformed into Arabidopsis Col-0 leaf protoplasts by polyethylene-glycolmediated transformation as previously described (Schirawski et al., 2000). Cotransformation of pRT101 with GUSXX plasmids was used as control (Töpfer et al., 1987). Protoplasts were incubated at 25 • C for 16 h and harvested by centrifugation. ...
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Agrobacterium tumefaciens can cause crown gall tumors by transferring both an oncogenic piece of DNA (T-DNA) and several effector proteins into a wide range of host plants. For the translocated effector VirE3 multiple functions have been reported. It acts as a transcription factor in the nucleus binding to the Arabidopsis thaliana pBrp TFIIB-like protein to activate the expression of VBF , an F-box protein involved in degradation of the VirE2 and VIP1 proteins, facilitating Agrobacterium -mediated transformation. Also VirE3 has been found at the plasma membrane, where it could interact with VirE2. Here, we identified AtJAZ8 in a yeast two-hybrid screening with VirE3 as a bait and confirmed the interaction by pull-down and bimolecular fluorescence complementation assays. We also found that the deletion of virE3 reduced Agrobacterium virulence in a root tumor assay. Overexpression of virE3 in Arabidopsis enhanced tumorigenesis, whereas overexpression of AtJAZ8 in Arabidopsis significantly decreased the numbers of tumors formed. Further experiments demonstrated that AtJAZ8 inhibited the activity of VirE3 as a plant transcriptional regulator, and overexpression of AtJAZ8 in Arabidopsis activated AtPR1 gene expression while it repressed the expression of AtPDF1.2 . Conversely, overexpression of virE3 in Arabidopsis suppressed the expression of AtPR1 whereas activated the expression of AtPDF1.2 . Our results proposed a novel mechanism of counter defense signaling pathways used by Agrobacterium , suggesting that VirE3 and JAZ8 may antagonistically modulate the salicylic acid/jasmonic acid (SA/JA)-mediated plant defense signaling response during Agrobacterium infection.
... The coding sequence of DsRed1 was PCR-amplified from pX-DR [46] using primers listed in Additional file 1: Table S2. The amplicon was inserted with XhoI and XbaI into pRT104 containing a CaMV 35S promoter and a poly(A) terminator [47]. The expression cassette was then excised with HindIII and cloned into the single HindIII site of pISV2678. ...
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Background Protocols for Agrobacterium rhizogenes -mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus . The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL , nopM , and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1 . A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti . Conclusion We have established a novel protocol for hairy root transformation of L. japonicus . The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.
... Eight genes are directly involved in the seco-iridoid pathway, two genes to boost the precursors' formation necessary to support the target pathway, and two genes involved in downstream alkaloid biosynthesis (Miettinen et al. 2014). Interestingly, these multi-transgenes were delivered using a well-established vector for transcriptional fusion (pRT101) used as a donor (Tö pfer et al. 1987) in combination with Agrobacterium binary vector pCAMBIA1300. These are only a few of the many examples of combinatorial pathway reconstruction in microorganisms and plants. ...
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The evolution of new traits in living organisms occurs via the processes of mutation, recombination, genetic drift, and selection. These processes that have resulted in the immense biological diversity on our planet are also being employed in metabolic engineering to optimize enzymes and pathways, create new-to-nature reactions, and synthesize complex natural products in heterologous systems. In this review, we discuss two evolution-aided strategies for metabolic engineering—directed evolution, which improves upon existing genetic templates using the evolutionary process, and combinatorial pathway reconstruction, which brings together genes evolved in different organisms into a single heterologous host. We discuss the general principles of these strategies, describe the technologies involved and the molecular traits they influence, provide examples of their use, and discuss the roadblocks that need to be addressed for their wider adoption. A better understanding of these strategies can provide an impetus to research on gene function discovery and biochemical evolution, which is foundational for improved metabolic engineering. These evolution-aided approaches thus have a substantial potential for improving our understanding of plant metabolism in general, for enhancing the production of plant metabolites, and in sustainable agriculture.
... Binary plasmids for transient assays were prepared as follow. DNA fragments encompassing a basal CaMV35S promoter without enhancer sequences (Töpfer et al., 1987), modified MGD2 5 0 -UTRs, and the YFP CDS were ordered from GeneCust (http://www.genecust.com/fr) and subcloned into a Gateway donor vector. ...
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Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants.
... PCR products were cloned into pGEM-T Easy vector (Promega). Following sequence verification, ORFs were subcloned into a pRT100 [36] derivative using PCR-introduced 5′ NcoI and 3′ NotI restriction sites to generate in-frame Cterminal triple-myc epitope or GFP fusion constructs. The ACS6 C-terminal domain corresponds to amino acid positions 435 to 495 [20] and was C-terminally fused as a 5′ NcoI -3′ NotI fragment to GFP in pGreenII-0029 vector, driven by a double 35S promoter. ...
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Background Protein kinases are important components of signalling pathways, and kinomes have remarkably expanded in plants. Yet, our knowledge of kinase substrates in plants is scarce, partly because tools to analyse protein phosphorylation dynamically are limited. Here we describe Kinase-Associated Phosphoisoform Assay, a flexible experimental method for directed experiments to study specific kinase-substrate interactions in vivo.The concept is based on the differential phosphoisoform distribution of candidate substrates transiently expressed with or without co-expression of activated kinases. Phosphorylation status of epitope-tagged proteins is subsequently detected by high-resolution capillary isoelectric focusing coupled with nanofluidic immunoassay, which is capable of detecting subtle changes in isoform distribution. ResultsThe concept is validated by showing phosphorylation of the known mitogen-activated protein kinase (MAPK) substrate, ACS6, by MPK6. Next, we demonstrate that two transcription factors, WUS and AP2, both of which are shown to be master regulators of plant development by extensive genetic studies, exist in multiple isoforms in plant cells and are phosphorylated by activated MAPKs. Conclusion As plant development flexibly responds to environmental conditions, phosphorylation of developmental regulators by environmentally-activated kinases may participate in linking external cues to developmental regulation. As a counterpart of advances in unbiased screening methods to identify potential protein kinase substrates, such as phosphoproteomics and computational predictions, our results expand the candidate-based experimental toolkit for kinase research and provide an alternative in vivo approach to existing in vitro methodologies.
... Sequences were compiled in a '.txt' file and uploaded on the GSDS server for obtaining the line diagram of gene structures. The full-length coding sequence of GmPIF4b (Glyma.14G032200.1) was cloned downstream of the 35S promoter in a cloning vector pRT-101 (Töpfer et al., 1987), and resulting construct (35S::GmPIF4b::polyA) was transferred to the binary vector pUQC10255 (from The University of Queensland) for Agrobacterium-mediated (EHA105) transformation of soybean. Soybean was transformed using the protocol detailed in Method 1. Steps of soybean transformation and shoot regeneration from transgenic calli are shown in Figures S1 and S2. ...
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Phytochrome Interacting Factor 4 act as a signalling hub for integrating multiple environmental cues like light and temperature. While the function of PIF4 in the model plant Arabidopsis has been studied, there is limited knowledge regarding the role of PIF4 in agronomically important legume crop soybean. Here, we employed a constitutive over-expression approach to functionally characterise a soybean PIF4 homolog, GmPIF4b in a determinate short-day cultivar, Bragg. Multiple sequence alignment of seven soybean PIF4 homologs (GmPIF4a-g) with Arabidopsis PIF4 revealed the presence of an Active Phytochrome Binding (APB) domain in the N-terminal region of six soybean PIF4 homologs. Cis-elements related to plant hormone biosynthesis, stress response, meristem, and endosperm gene expression were located in the promoter region of soybean PIF4s. Interestingly, transgenic soybean plants carrying 35s::GmPIF4b::polyA construct showed reduced plant height, reduced leaf surface area, decreased branching, early flowering, and faster transition from full bloom flowering stage to full maturity stage without any decline in yield. Further, pod colour of transgenic soybean plants changed to dark brown, whereas wild type plants showed tan or light brown pod colour. Clear hilum was observed in seeds obtained from transgenic plants as opposed to the dark or black hilum of wild type seeds. Transcripts of soybean florigens GmFT2a and GmFT5a were also elevated in transgenic plants. Collectively, our results suggest that GmPIF4b over-expression could affect phenotypes related to plant morphology and reproductive stages in soybean, and can be used as a gene target for soybean improvement programs to ensure future food security.
... We performed endonuclease digestion of the pUC57-SPOP vector with NcoI and Bsp120I (PspOMI) to obtain the insert. The fragment was ligated into the cloning vector pRTRA [19,20], which was previously cut with NcoI and NotI. ...
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Determining the function of proteins remains a key task of modern biology. Classical genetic approaches to knocking out protein function in plants still face limitations, such as the time-consuming nature of generating homozygous transgenic lines or the risk of non-viable loss-of-function phenotypes. We aimed to overcome these limitations by acting downstream of the protein level. Chimeric E3 ligases degrade proteins of interest in mammalian cell lines, Drosophila melanogaster embryos, and transgenic tobacco. We successfully recruited the 26S proteasome pathway to directly degrade a protein of interest located in plant nuclei. This success was achieved via replacement of the interaction domain of the E3 ligase adaptor protein SPOP (Speckle-type POZ adapter protein) with a specific anti-GFP nanobody (VHHGFP4). For proof of concept, the target protein CENH3 of A . thaliana fused to EYFP was subjected to nanobody-guided proteasomal degradation in planta . Our results show the potential of the modified E3-ligase adapter protein VHHGFP4-SPOP in this respect. We were able to point out its capability for nucleus-specific protein degradation in plants.
... The open reading frame (ORF) of JcPR-10a cDNA (Agarwal et al. 2013), was PCR amplified using JcPR-10TF and JcPR-10TR primers (Table S1) flanked with XhoI and XbaI restriction sites, respectively, and cloned in in XhoI/XbaI sites of pRT101vector (Topfer et al. 1987). Thereafter, expression cassette containing 35S: JcPR-10a: Poly A was cloned in the pCAMBIA 1301 at the HindIII site and mobilized into the Agrobacterium strain LBA4404. ...
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Plants in ecosystems are simultaneously exposed to abiotic and biotic stresses, which restrict plant growth and development. The complex responses to these stresses are largely regulated by plant hormones, which in turn, orchestrate the different biochemical and molecular pathways to manoeuvre stress tolerance. The PR-10 protein family is reported to be involved in defence regulation, stress response and plant growth and development. The JcPR-10a overexpression resulted in increased number of shoot buds in tobacco (Nicotiana tabacum), which could be due to high cytokinin to auxin ratio in the transgenics. The docking analysis shows the binding of three BAP molecules at the active sites of JcPR-10a protein. JcPR-10a transgenics showed enhanced salt tolerance, as was evident by increased germination rate, shoot and root length, relative water content, proline, soluble sugar and amino acid content under salinity. Interestingly, the transgenics also showed enhanced endogenous cytokinin level as compared to WT, which, further increased with salinity. Exposure of gradual salinity resulted in increased stomatal conductance, water use efficiency, photosynthesis rate and reduced transpiration rate. Furthermore, the transgenics also showed enhanced resistance against Macrophomina fungus. Thus, JcPR-10a might be working in co-ordination with cytokinin signalling in mitigating the stress induced damage by regulating different stress signalling pathways, leading to enhanced stress tolerance
... A05521.1, [32] for a transcriptional fusion with the CaMV 35S promoter and polyadenylation signal. The expression cassettes were further cloned in to the plant binary vector pCAMBIA 2300 (http://www.cambia.org) ...
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Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.
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The ubiquitous small heat shock proteins (sHSPs) are well-documented to act in vitro as molecular chaperones to prevent irreversible aggregation of heat sensitive proteins. However, in vivo activities of sHSPs remain unclear. To investigate the two most abundant classes of plant cytosolic sHSPs (CI and CII), RNAi and over-expression lines were created in Arabidopsis thaliana and shown to have reduced or enhanced tolerance, respectively, to extreme heat stress. Affinity purification of CI and CII sHSPs from heat-stressed seedlings recovered translation factors eEF1B (α, β, γ subunits) and eIF4A (three isoforms), although association with CI sHSPs was stronger, and additional proteins involved in translation were recovered with CI sHSPs. eEF1B subunits became partially insoluble during heat stress, and, in the CI and CII RNAi lines, showed reduced recovery to the soluble cell fraction after heat stress, which was also dependent on HSP101. Furthermore, after heat stress, CI sHSPs showed increased retention in the insoluble fraction in the CII RNAi line and vice versa. Immunolocalization revealed both CI and CII sHSPs were present in cytosolic foci, some of which colocalized with HSP101 and eEF1Bγ and β. Thus, CI and CII sHSPs have both unique and overlapping functions and act either directly or indirectly to protect specific translation factors in cytosolic stress granules.
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Little is known about the molecular mechanism of the R2R3-MYB transcriptional repressors involved in plant phenylpropanoid metabolism. Here, we describe one R2R3-type MYB repressor, FtMYB11 from Fagopyrum tataricum. It contains the SID-like motif GGDFNFDL and it is regulated by both the importin protein 'Sensitive to ABA and Drought 2' (SAD2) and the jasmonates signalling cascade repressor JAZ protein. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that FtMYB11 interacts with SAD2 and FtJAZ1. Protoplast transactivation assays demonstrated that FtMYB11 acts synergistically with FtSAD2 or FtJAZ1 and directly represses its target genes via the MYB-core element AATAGTT. Changing the Asp122 residue to Asn in the SID-like motif results in cytoplasmic localization of FtMYB11 because of loss of interaction with SAD2, while changing the Asp126 residue to Asn results in the loss of interaction with FtJAZ1. Overexpression of FtMYB11or FtMYB11(D126N) in F. tataricum hairy roots resulted in reduced accumulation of rutin, while overexpression of FtMYB11(D122N) in hairy roots did not lead to such a change. The results indicate that FtMYB11 acts as a regulator via interacting with FtSAD2 or FtJAZ1 to repress phenylpropanoid biosynthesis, and this repression depends on two conserved Asp residues of its SID-like motif.
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Type III protein secretion systems of nitrogen-fixing rhizobia deliver effector proteins into leguminous host cells to promote or inhibit the nodule symbiosis. However, mechanisms underlying effector-triggered inhibition of nodulation remain largely unknown. Nodulation outer protein T (NopT) of Sinorhizobium sp. NGR234 is an effector protease related to the Pseudomonas effector AvrPphB (Avirulence protein Pseudomonas phaseolicola B). Here, we constructed NGR234 mutants producing different NopT variants and found that protease activity of NopT negatively affects nodulation of smooth crotalaria (Crotalaria pallida). NopT variants lacking residues required for autocleavage and subsequent lipidation showed reduced symbiotic effects and were not targeted to the plasma membrane. We further noticed that Sinorhizobium fredii strains possess a mutated nopT gene. S. fredii USDA257 expressing nopT of NGR234 induced considerably fewer nodules in soybean (Glycine max) cv. Nenfeng 15 but not in other cultivars. Effector perception was further examined in NopT-expressing leaves of Arabidopsis (Arabidopsis thaliana) and found to be dependent on the target protein AtPBS1 (Arabidopsis AvrPphB susceptible 1) and the associated resistance protein AtRPS5 (Arabidopsis Resistance to Pseudomonas Syringae 5). Experiments with Nicotiana benthamiana plants indicated that the soybean homolog GmPBS1-1 associated with AtRPS5 can perceive NopT. Further analysis showed that NopT cleaves AtPBS1 and GmPBS1-1 and thus can activate these target proteins. Insertion of a DKM motif at the cleavage site of GmPBS1-1 resulted in increased proteolysis. Nodulation tests with soybeans expressing an autoactive GmPBS1-1 variant indicated that activation of a GmPBS1-1-mediated resistance pathway impairs nodule formation in cv. Nenfeng 15. Our findings suggest that legumes face an evolutionary dilemma of either developing effector-triggered immunity against pathogenic bacteria or establishing symbiosis with suboptimally adapted rhizobia producing pathogen-like effectors.
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Manipulation of a single abiotic stress-related gene could improve plant performance under abiotic stress conditions. To simultaneously increase plant tolerance to multiple stresses, it is usually required to overexpress two (or more) genes in transgenic plants. The common strategy is to assemble two or more expression cassettes, where each gene has its own promoter and terminator, within the same T-DNA. Does the arrangement of the two expression cassettes affect expression of the two transgenes? Can we use the Drosophila gypsy insulator sequence to increase the expression of the two transgenes? Answers to these questions would contribute to design better transformation vectors to maximize the effects of multi-gene transformation. Two Arabidopsis genes, PP2A-C5 and AVP1, and the gypsy insulator sequence were used to construct six transformation vectors with or without the gypsy insulator bracketing the two expression cassettes: uni-directional transcription, divergent transcription, and convergent transcription. Total RNAs were isolated for reverse transcription- quantitative real-time polymerase chain reaction (RT-qPCR) assays and a thorough statistical analysis was conducted for the RT-qPCR data. The results showed that the gypsy insulator does promote the expression of two transgenes in transgenic plants. Besides, the plants containing the divergent transcription cassettes tend to have more correlated expression of both genes.
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Jasmonates (JAs) are plant hormones that induce many secondary metabolites accumulation, such as rutin in buckwheat, via regulation of JAs-responsive transcription factors (TFs). Here, we report on the identification of a clade of JAs-responsive subgroup 4 MYB TFs, FtMYB13, FtMYB14, FtMYB15 and FtMYB16, directly repressing rutin biosynthesis in Fagopyrum tataricum. Immunoblot analysis showed that FtMYB13, FtMYB14 and FtMYB15 could be degraded via 26S proteasome in COI1-dependent JA signaling pathway, and this degradation is due to the SID motif in their C-terminus. Yeast two hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC) assays revealed that FtMYB13, FtMYB14 and FtMYB15 interact with the importin protein ‘Sensitive to ABA and Drought 2’ (FtSAD2) in stem and inflorescence. Furthermore, the key repressor of JAs signaling FtJAZ1 specifically interacts with FtMYB13. Point mutation analysis showed that the conserved Asp residue of SID domain contributes to mediate the protein-protein interaction. Protoplasts transient activation assays demonstrated that FtMYB13, FtMYB14 and FtMYB15 directly repress phenylalanine ammonia lyase (FtPAL) gene expression and FtSAD2 and FtJAZ1 significantly promote the repressing activity of FtMYBs. These findings may ultimately be promising for further engineering of plant secondary metabolisms.
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Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering. Electronic supplementary material The online version of this article (10.1007/s11240-018-1398-5) contains supplementary material, which is available to authorized users.
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Hydroxycinnamic acid amides (HCAAs) are a class of antimicrobial metabolites involved in plant defense against necrotrophic pathogens, including Alternaria brassicicola and Botrytis cinerea. The agmatine coumaroyl transferase (AtACT) is the key enzyme to catalyze the last reaction in the biosynthesis of HCAAs, including p‐coumaroylagmatine (CouAgm) and feruloylagmatine in Arabidopsis thaliana. However, it is currently unknown about the regulatory mechanism of the AtACT gene expression. Yeast one‐hybrid screening using the AtACT promoter as bait isolated the key positive regulator ORA59 that is involved in jasmonic acid/ethylene (JA/ET) mediated plant defense responses. AtACT gene expression and HCAAs biosynthesis were synergistically induced by a combination of JA/ET. In the AtACT promoter, two GCC boxes function equivalently for the trans‐activation by ORA59 in Arabidopsis protoplasts, and mutation of either GCC box abolished the AtACT mRNA accumulation in transgenic plants. Site‐directed mutation analysis demonstrated that the specific Leu residue at position 228 of the ORA59 EDLL motif mainly contributed to its transcriptional activity on AtACT gene expression. Importantly, MEDIATOR25 (MED25) and ORA59 homodimer are also required for ORA59‐dependent activation of the AtACT gene. These results suggest that ORA59 and two functionally equivalent GCC‐boxes form the regulatory module together with MED25 that enables the AtACT gene expression and the HCAAs biosynthesis to respond to simultaneous activation of the JA/ET signaling pathways. This article is protected by copyright. All rights reserved.
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Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, we expressed PEPBs in a heterologous context to determine their ectopic activity. We found that heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, we identified deregulated genes encoding proteases that may contribute to proteome stability at equilibrium. Our results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes we observed.
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Crown roots (CRs) are essential components of the rice root system. Several genes involved in CR initiation or development have been indentified but our knowledge about how they organize to form a gene regulatory network (GRN) is still limited. To characterize the regulatory cascades acting during CR formation, we used a systems biology approach to infer the gene regulatory network controlling CR formation downstream of CROWN ROOTLESS1 (CRL1), coding for an ASL (asymmetric leaves‐2‐like)/LBD (LOB domain) transcription factor necessary for CR initiation. A time‐series transcriptomic dataset was generated after synchronised induction of CR formation by dexamethasone‐mediated expression of CRL1 expression in a crl1 mutant background. This time‐series revealed three different genome expression phases during the early steps of CR formation and was further exploited to infer a GRN using a dedicated algorithm. The predicted GRN was confronted to experimental data and 72% of inferred links were validated. Interestingly, this network revealed a regulatory cascade linking CRL1 to other genes involved in CR initiation, in root meristem specification and maintenance such as QUIESCENT‐CENTER SPECIFIC HOMEOBOX and in auxin signalisation. This predicted regulatory cascade was validated in vivo using transient activation assays. Thus, the CRL1‐dependant GRN reflects major gene regulation events at play during CR formation and constitutes a valuable source of discovery to better understand this developmental process. This article is protected by copyright. All rights reserved.
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Jasmonates (JAs) are plant hormones that regulate the biosynthesis of many secondary metabolites, such as hydroxycinnamic acid amides (HCAAs), through JA-responsive transcription factors (TFs). HCAAs are renowned for their role in plant defense against pathogens. The multidrug and toxic compound extrusion (MATE) transporter DETOXIFICATION18 (DTX18) has been shown to mediate the extracellular accumulation of HCAAs p-coumaroylagmatine (CouAgm) at the plant surface for defense response. However, little is known about the regulatory mechanism of DTX18 gene expression by TFs. Yeast one-hybrid screening using the DTX18 promoter as bait isolated the key positive regulator Redox Responsive Transcription Factor 1 (RRTF1), which is a member of the AP2/ERF family of proteins. RRTF1 is a JA-responsive factor that is required for the transcription of the DTX18 gene, and it thus promotes CouAgm secretion at the plant surface. As a result, overexpression of RRTF1 caused increased resistance against the fungus Botrytis cinerea, whereas rrtf1 mutant plants were more susceptible. Using yeast two-hybrid screening, we identified the BTB/POZ-MATH (BPM) protein BPM1 as an interacting partner of RRTF1. The BPM family of proteins act as substrate adaptors of CUL3-based E3 ubiquitin ligases, and we found that only BPM1 and BPM3 were able to interact with RRTF1. In addition, we demonstrated that RRTF1 was subjected to degradation through the 26S proteasome pathway and that JA stabilized RRTF1. Knockout of BPM1 and BPM3 in bpm1/3 double mutants enhanced RRTF1 accumulation and DTX18 gene expression, thus increasing resistance to the fungus B. cinerea. Our results provide a better understanding of the fine-tuned regulation of JA-induced TFs in HCAA accumulation.
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Main conclusion Inducible lineage analysis and cell ablation via conditional toxin expression in cells expressing the DORNRÖSCHEN-LIKE transcription factor represent an effective and complementary adjunct to conventional methods of functional gene analysis. Abstract Classical methods of functional gene analysis via mutational and expression studies possess inherent limitations, and therefore, the function of a large proportion of transcription factors remains unknown. We have employed two complementary, indirect methods to obtain functional information for the AP2/ERF transcription factor DORNRÖSCHEN-LIKE (DRNL), which is dynamically expressed in flowers and marks lateral organ founder cells. An inducible, two-component Cre–Lox system was used to express beta-glucuronidase GUS in cells expressing DRNL , to perform a sector analysis that reveals lineages of cells that transiently expressed DRNL throughout plant development. In a complementary approach, an inducible system was used to ablate cells expressing DRNL using diphtheria toxin A chain, to visualise the phenotypic consequences. These complementary analyses demonstrate that DRNL functionally marks founder cells of leaves and floral organs. Clonal sectors also included the vasculature of the leaves and petals, implicating a previously unidentified role for DRNL in provasculature development, which was confirmed in cotyledons by closer analysis of drnl mutants. Our findings demonstrate that inducible gene-specific lineage analysis and cell ablation via conditional toxin expression represent an effective and informative adjunct to conventional methods of functional gene analysis.
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We hereby report in planta function characterization of a novel galactosyl transferase-like (SbGalT) gene from Salicornia brachiata for enhanced abiotic stress tolerance. The SbGalT gene had an open reading frame of 1563 bp. The ectopic expression of SbGalT gene in tobacco improved the seed germination, seedling growth, biomass accumulation and potassium/sodium ratio under salt and osmotic stress. The SbGalT over-expression delayed stress-induced senescence, pigment breakdown and ionic cytotoxicity in tobacco. Higher contents of organic solutes and potassium under stress maintained the osmotic homeostasis and relative water content in tobacco. Higher activity of antioxidant enzymes under stress in transgenic tobacco curtailed accumulation of reactive oxygen species (ROS) and maintained the membrane integrity. The chlorophyll a fluorescence transient indicated no influences of imposed strengths of stress on basal state of photosystem (PS) I in transgenic tobacco over-expressing the SbGalT gene. Due to better membrane integrity the transgenic tobacco exhibited improved photosynthesis, stomatal conductance, intercellular CO2, transpiration, maximum quantum yield and operating efficiency of PSII, electron transport, photochemical and non-photochemical quenching. In agreement with photosynthesis, physiological health, tolerance index and growth data, transgenic tobacco accumulated higher contents of sugar, starch, amino acid, polyphenol and proline under stress conditions. The multivariate data analysis exhibited significant statistical distinctions among osmotic adjustment, physiological health and growth, and photosynthetic responses in control and SbGalT transgenic tobacco under stress conditions. The results strongly indicated novel SbGalT gene as a potential candidate for developing smart agriculture.
Article
The plants endomembrane system of the cellular compartments with its complex membrane trafficking network facilitates transport of macromolecules. The endomembrane dynamics is essential for maintaining basic and specific cellular functions including adaptation to the extracellular environment. The plant vacuole serves as a reservoir for nutrients and toxic metabolites and performs detoxification processes to maintain cellular homeostasis. The overexpression of AlRab7, a vesicle trafficking gene from Aeluropus lagopoides, improved germination and growth and reduced ionic and oxidative stress in transgenics. Moreover, the root and shoot of transgenic tobacco showed differential accumulation of phytohormone ABA and IAA with different ionic stresses. The improved growth (root and shoot length) can be co‐related with higher IAA accumulation with NaCl stress. The low Na+/K+ ratio with different NaCl stress treatments indicate better ion homeostasis in transgenics. Furthermore, the increased stomatal density and higher number of open stomata on both leaf surfaces in transgenics during NaCl stress suggest better gaseous exchange/functioning of guard cells. The maintained or increased SOD, CAT, APX, POX and GR antioxidative enzyme activities suggest that an extensive ROS scavenging system was triggered to detoxify cellular ROS, which remained at low levels in transgenics during the different stress treatments. Our results suggest that, the AlRab7 transgenic tobacco ameliorates ionic stress by facilitating differential and selective ion transport at vacuolar membrane regulating hormone signalling, ROS homeostasis, stomatal development and movement.
Article
An earlier investigation showed that annexin OsANN5 of rice was expressed to high levels in various stress treatments. In the present study, we have investigated the molecular mechanism underlying stress responses of OsANN5 through heterologous expression in transgenic tobacco and E. coli. E. coli cells expressing OsANN5 showed improved growth under high salt and heat stress treatments. Transgenic tobacco plants overexpressing OsANN5 exhibited tolerance to salinity, drought and osmotic stress during seed germination and post-germinative stages. The transgenic plants showed increased accumulation of proline, reduced chlorosis and membrane damage as compared to the wild type plants. Higher expression of OsANN5 also resulted in efficient scavenging of reactive oxygen species through the modulation of anti-oxidant enzymes, superoxide dismutase and catalase under stress treatments. OsANN5 exhibited calcium binding activity and under salt stress, relocated from the peripheral regions to cytosol. This suggests that OsANN5 has multiple functions in rice growth and stress tolerance. Promoter analysis of OsANN5 revealed the presence of several cis-acting elements that are well known to be involved in hormonal and stress response regulation. The expression patterns of OsANN5 in the presence of ABA synthesis inhibitor, fluoridone and salinity stress revealed that upregulation of OsANN5 transcripts is regulated by both ABA-dependent and independent pathways. This indicates OsANN5 may play a role in cross-talk between ABA-dependent and independent pathways during the abiotic stress response.
Article
Brassica juncea (mustard) is a major oilseed crop of India grown in around five to six million hectares of land under low moisture availability. In around two million hectares of rain-fed area under mustard cultivation in the states of Rajasthan, Haryana, Madhya Pradesh, and Uttar Pradesh, the crop is severely affected by the root parasite Orobanche aegyptiaca. Glyphosate, a broad-spectrum systemic herbicide has been reported to control O. aegyptiaca besides the general weed flora. We report glyphosate-resistant transgenic lines in B. juncea variety Varuna using three different genes—cp4 epsps, gox, and gat. The cp4 epsps gene encodes a protein that is insensitive to glyphosate; the gox and gat genes encode proteins that detoxify the herbicide. Transgenes were expressed either under a CaMV 35S promoter or a predominantly meristem-specific promoter; two 5′ UTRs—SYN J and PHOTO were used to enhance the transgene expression. Nine different constructs were made and used to develop 327 transgenic lines. Single copy transgenic lines were identified and tested for resistance to glyphosate by germinating seeds on different concentrations of the herbicide. Glyphosate at 50 mg/L was lethal for the seedlings of untransformed Varuna; seedlings of many transgenic lines grew normally at the highest tested concentration of 500 mg/L. Transgenic lines containing the cp4 epsps gene along with a double mutant als gene could provide long-term control of O. aegyptiaca in the highly affected areas.
Article
Cyanogenic glucosides (CNglcs) play an important role in plant defense response, however, the regulation mechanism of CNglcs synthesis by external environment and endogenous hormones is largely unclear. In this study, we found that jasmonates (JAs) promoted the synthesis of CNglcs by activating the expression of CNglcs biosynthesis genes in Lotus japonicus. Several differentially expressed bHLH family genes related to the synthesis of cyanogenic glucosides were identified by RNA-seq. The LjbHLH7 can directly activate the expression of CYP79D3 gene, the first step of CNglcs synthesis, by binding to the G-box sequence of its promoter. Transgenic plants overexpressing LjbHLH7 exhibited higher relative CNglcs content and enhanced insect resistance than the wild type. Furthermore, the transcriptional activity of LjbHLH7 was suppressed by the interaction with L. japonicus JASMONATE-ZIM DOMAIN protein LjJAZ4. Based on these results, we proposed that LjbHLH7 acts as an activator and LjJAZ4 acts as a repressor of JAs-induced regulation of cyanogenic glucosides biosynthesis in L. japonicus.
Article
Comparative results of the studied effectiveness of two new promoters, pro-SmAMP1 and pro- SmAMP2, from chickweed (Stellaria media L.) in various types of cultivated plants with transient expression and in stable transformants are given. The effectiveness of the promoters was evaluated through the expression of the reporter uidA gene by measuring the activity of its GUS protein product. It was found that the deletion variant (442 bp) of the pro-SmAMP1 promoter was significantly stronger in plants of Nicotiana benthamiana (Domin) with transient expression than the deletion variant (455 bp) of the pro-SmAMP2 promoter. The effectiveness of these short deletion variants of both promoters under transient expression in the plants of rapeseed (Brassica napus L.) and sugar beet (Beta vulgaris L.) was comparable with that of the viral CaMV35S promoter. The functionality of the pro-SmAMP2 promoter in the calluses of common flax plants (Linum usitatissimum L.) was shown. In the homozygous lines of transgenic tobacco plants (Nicotiana tabacum L.), all deletion variants of the pro-SmAMP1 promoter and the shortest version of pro-SmAMP2 were twice as strong as the CaMV35S viral promoter. The effectiveness of short variants of both promoters from the chickweed in controlling the gene encoding neomycin phosphotransferase II in the transgenic plants of tobacco and arabidopsis (Arabidopsis thaliana L.) growing on media supplemented with recommended concentrations of kanamycin are not inferior to the duplicated 2хCaMV35S viral promoter. The obtained experimental data show that short deletion variants of pro-SmAMP1 (442 bp) and pro-SmAMP2 (455 bp) promoters may be recommended as strong constitutive promoters for use in the biotechnology of crop plants.
Article
Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M 13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.
Article
We have determined the complete primary structure (8031 base pairs) of an infectious clone of cauliflower mosaic virus strain CM1841. The sequence was obtained using the strategy of cloning shotgun restriction fragments in the sequencing vector M13mp7. Comparison of the CM1841 sequence with that published for another CaMV strain (Strasbourg) reveals 4.4% changes, mostly nucleotide substitutions with a few small insertions and deletions. The six open reading frames in the sequence of the Strasbourg isolate are also present in CM1841.
Article
5′-Noncoding sequences have been tabulated for 211 messenger R'JAs from higher eukaryotic cells. The 5′-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions −1, −2, −4 and −5, just upstream from the initiator codon. The sequence (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the −1 to −5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.
Article
Four RNA transcripts encoded by cauliflower mosaic virus DNA have been detected in the polyadenylated RNA from virus-infected turnip leaves. Two of these transcripts, the major 35S and the 8S species, have the same 5' termini, at nucleotide 7435. A viral DNA fragment encompassing this region directs transcription initiation at this point in vitro. The 5' terminus of the 19S transcript is at nucleotide 5764, and a corresponding viral DNA fragment also directs transcription initiation in vitro. The major 35S RNA is a complete transcript of the circular viral genome, and is 3'-coterminal with 19S RNA at nucleotide 7615. The 8S RNA has its 3' extremity at delta 1, the single-stranded interruption in the transcribed strand of virion DNA. A minor 35S RNA has also been detected that has its 5' and 3' termini at delta 1.
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