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[17] Binary ti vectors for plant transformation and promoter analysis
... Preliminary tests have shown that vacuum-infiltration of the Type I AFP of winter flounder into the extracellular spaces of canola, potato and Arabidopsis thaliana leaves resulted in an average of 1.8°C decrease in the spontaneous freezing temperature of treated tissue (Cutler et al., 1989). While Bernard (Duncker, Chen, Davies, & Walker, 1995;Duncker, Hermans, Davies, & Walker, 1996) and Rancourt (Rancourt, Davies, & Walker, 1992;1987) suggested that the Type II and Type III AFPs might be better choices for transfer the antifreeze activity to other organisms than the linear Type I AFP, because of Type I AFP has no tertiary structure and is less stable than the globular AFPs. Identification of hyperactive Type III AFPs could yield utilization in improving cold resistance in other organisms including plants. ...
... The PEBV-based pCAPE vector system is a binary vector including two plasmids, pCAPE1 (assistant plasmid) and pCAPE2-GFP (control plasmid). So assistant vector, control vector and the recombinant vector were transformed into Agro-bacterium tumefaciens strain GV3101 by the liquid nitrogen freeze-thaw method (An, 1987). The transformed agrobacteria were grown overnight at 28°C under kanamycin and rifampicin selection and then positive clones were used to transform into tobacco (Jia et al., 2008). ...
... Preliminary tests have shown that vacuum-infiltration of the Type I AFP of winter flounder into the extracellular spaces of canola, potato and Arabidopsis thaliana leaves resulted in an average of 1.8°C decrease in the spontaneous freezing temperature of treated tissue (Cutler et al., 1989). While Bernard (1995;Duncker, Hermans, Davies, & Walker, 1996) and Rancourt (Rancourt et al., 1992;1987) suggested that the globular Type II and Type III AFPs are better choices for antifreeze Q. Huang, et al. Aquaculture and Fisheries xxx (xxxx) xxx-xxx transfer to other organisms than the linear Type I AFP, because of Type I AFP has no tertiary structure and is less stable than the globular AFPs. ...
Type III antifreeze proteins (AFPIIIs) are small globular proteins that found in some polar fishes function to protect organisms from freezing damage. The transgenic expression of AFPs holds great promise for conferring cold tolerance to commercially important plant and animal species. We found from an Antarctic eelpout (Lycodichthys dearborni) multiple AFPIII genes that encode larger AFPIII isoforms containing up to 12 of the conventional AFPIII domains. Here we cloned AFPIII genes encode for monomer (LD1), dimer (LD2), trimer (LD3) and tetramer (LD4) AFPIII isoforms, and introduced them into tobacco plants. Pot-grown 4-week-old plants were then treated at 4 °C for 30 days, we found LD1, LD2, LD3 and LD4 plants showed relatively lower electrolyte leakage and lower content of malondialdehyde (MDA), but accumulated higher content of proline than control plants, indicating considerably improved membrane integrity under low temperature stress and better cold resistance. Comparing between the transgenic plants, the plants with the tetramer- and trimer-domain encoding AFPIII genes demonstrated higher cold-tolerant levels than those transformed by dimer- and monomer domain AFPIIIs. This study validated that fish AFPIIIs, especially the multidomain proteins may protect cells from non-freezing hypothermic stresses, apart from its well-known function as ice inhibition molecules at freezing temperature.
... In addition to ChromoP, replacements with other genes were also examined (Only the results of one replacement (EPSPS) are provided in Supplementary 3, while other data are not included). These constructs were subsequently introduced into the Agrobacterium tumefaciens strain EHA105, following the protocol outlined by An (1987) (An 1987). The modified strain was then employed for the genetic transformation of the Iranian rice cultivar, Hashemi. ...
... In addition to ChromoP, replacements with other genes were also examined (Only the results of one replacement (EPSPS) are provided in Supplementary 3, while other data are not included). These constructs were subsequently introduced into the Agrobacterium tumefaciens strain EHA105, following the protocol outlined by An (1987) (An 1987). The modified strain was then employed for the genetic transformation of the Iranian rice cultivar, Hashemi. ...
Rice (Oryza sativa L.) is a vital staple food for a significant portion of the global population. Meeting the demand and enhancing productivity in the face of climate change and population growth necessitate innovative strategies, including molecular modification, genetic engineering, and genome editing. This study presents a simplified gene transformation protocol for the rice cultivar Hashemi using 4 to 7-day-old calluses. The method, based on mature rice seeds and gene transformation to a 4 to 7-day-old callus, achieves a maximum 40% efficiency, circumventing complications associated with using immature. Notably, a novel reporter gene, ChromoP (a codon-optimized chromoprotein), is introduced for the first time to facilitate the optimization of gene transformation. The transgenic calli containing ChromoP exhibit a distinctive pinkish-purple coloration, enabling easy visual identification. Furthermore, the study introduces a simple, fast, and cost-effective protocol, named semi-universal inverse PCR (SUN-iPCR), for detecting individual events and determining the insertion site of a transgene. In conclusion, this study successfully outlines a gene transformation protocol for rice, covering the process until the detection of an independent transformant. Furthermore, the reporter gene and SUN-iPCR methodology utilized in this study hold promising potential for optimizing gene transformation in various other plant species.
... 27 using the freezethaw method of. 28 Arabidopsis wild types (Landsberg erecta Ler or Columbia-0 Col-0) or ft mutants (CS56 ft-1, Cs185 ft-3) were transformed by the floral dipping method. 29 Primary transformants (T1 generation) were selected by spraying 120 mg l −1 BASTA © (Glufosinate-ammonium; Bayer, Germany, 150 g l −1 ) three times at 3-7 day-intervals, starting with 7 dayold seedlings grown on soil. ...
The FLOWERING LOCUS T (FT) gene is the essential integrator of flowering regulatory pathways in angiosperms. The paralogs of the FT gene may perform antagonistic functions, as exemplified by BvFT1, that suppresses flowering in Beta vulgaris, unlike the paralogous activator BvFT2. The roles of FT genes in other amaranths were less investigated. Here, we transformed Arabidopsis thaliana with the FLOWERING LOCUS T like (FTL) genes of Chenopodium ficifolium and found that both CfFTL1 and CfFTL2–1 accelerated flowering, despite having been the homologs of the Beta vulgaris floral promoter and suppressor, respectively. The floral promotive effect of CfFTL2–1 was so strong that it caused lethality when overexpressed under the 35S promoter. CfFTL2–1 placed in an inducible cassette accelerated flowering after induction with methoxyphenozide. The spontaneous induction of CfFTL2–1 led to precocious flowering in some primary transformants even without chemical induction. The CqFT2–1 homolog from Chenopodium quinoa had the same impact on viability and flowering as CfFTL2–1 when transferred to A. thaliana. After the FTL gene duplication in Amaranthaceae, the FTL1 copy maintained the role of floral activator. The second copy FTL2 underwent subsequent duplication and functional diversification, which enabled it to control the onset of flowering in amaranths to adapt to variable environments.
... The vector was sequenced in order to verify the correct insertion of the cassette, then it was introduced into A. tumefaciens strain AGL1 [67] for O. basilicum 'FT Italiko' transformation, according to the protocol described by [68]. pDirect22c_sg442-462 was also introduced in A. rhizogenes strain LB9402 to evaluate the efficiency of genome editing in the élite cv of O. basilicum hairy roots system. ...
Abstract: Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately1000 ha of “Genovese-type” basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE)
approach mediated by CRISPR/Cas9 in O. basilicum ‘Italiko’, the élite cultivar used to produce “Pesto Genovese D.O.P”, was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in
ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of ‘FT Italiko’. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.
... 27 using the freezethaw method of. 28 Arabidopsis wild types (Landsberg erecta Ler or Columbia-0 Col-0) or ft mutants (CS56 ft-1, Cs185 ft-3) were transformed by the floral dipping method. 29 Primary transformants (T1 generation) were selected by spraying 120 mg l −1 BASTA © (Glufosinate-ammonium; Bayer, Germany, 150 g l −1 ) three times at 3-7 day-intervals, starting with 7 dayold seedlings grown on soil. ...
The FLOWERING LOCUS T ( FT) gene is the essential integrator of flowering regulatory pathways in angiosperms. The paralogs of the FT gene may perform antagonistic functions, as exemplified by BvFT1 , that suppresses flowering in Beta vulgaris , unlike the paralogous activator BvFT2 . The roles of FT genes in other amaranths were less investigated. Here, we transformed Arabidopsis thaliana with the FLOWERING LOCUS T like ( FTL ) genes of Chenopodium ficifolium and found, that both CfFTL1 and CfFTL2-1 accelerated flowering, despite having been the homologs of the Beta vulgaris floral promoter and suppressor, respectively. The floral promotive effect of CfFTL2-1 was so strong that it caused lethality when overexpressed under the 35S promoter. CfFTL2-1 placed in inducible cassette accelerated flowering after the induction with methoxyphenozide. The spontaneous induction of CfFTL2-1 led to precocious flowering in some primary transformants even without chemical induction. The CqFT2-1 homolog from Chenopodium quinoa had the same impact on viability and flowering as CfFTL2-1 , when transferred to A. thaliana . After the FTL gene duplication in Amaranthaceae, the FTL1 copy maintained the role of floral activator. The second copy FTL2 underwent subsequent duplication and functional diversification, which enabled to control the onset of flowering in amaranths to adapt to variable environments.
... The plasmid vectors with a cassette were transferred into Agrobacterium tumefaciens strain EHA105 (Hood et al. 1993) using the freeze-thaw method of (An 1987). Arabidopsis wild types (Landsberg erecta Ler or Columbia-0 Col-0) or ft mutants (CS56 ft-1, Cs185 ft-3) were transformed by the oral dipping method (Clough and Bent 1998). ...
The FLOWERING LOCUS T ( FT) gene is the essential integrator of flowering regulatory pathways in angiosperms. The paralogs of the FT gene may perform antagonistic functions, as exemplified by BvFT1 , that suppresses flowering in Beta vulgaris , unlike the paralogous activator BvFT2 . The roles of FT genes in other amaranths were less investigated. Here, we transformed Arabidopsis thaliana with the FLOWERING LOCUS T like ( FTL ) genes of Chenopodium and found, that both FTL1 and FTL2-1 accelerated flowering, despite having been the homologs of the Beta vulgaris floral promoter and suppressor, respectively. The floral promotive effect of FTL2-1 was so strong that it caused lethality when overexpressed under the 35S promoter. FTL2-1 placed in inducible cassette accelerated flowering after the induction with methoxyphenozide. The spontaneous induction of FTL2-1 led to precocious flowering in some primary transformants even without chemical induction. After the FTL gene duplication in Amaranthaceae, the FTL1 copy maintained the role of floral activator. The second copy FTL2 underwent subsequent duplication and functional diversification, which enabled to control the onset of flowering in amaranths to adapt to variable environments.
... A potential target sequence (5'-ATGTCATCGTACTTCTCCCA-3') within the first exon of OsWRKY7 was cloned into the CRISPR vector pRGEB32 through the BsaI restriction site. The construct was transformed into the Agrobacterium tumefaciens strain LBA4404 using the freeze-thaw method (An, 1987). Transgenic rice plants were obtained via the Agrobacterium-mediated co-cultivation method (Lee et al., 1999). ...
Sucrose controls various developmental and metabolic processes in plants. It also functions as a signaling molecule in the synthesis of carbohydrates, storage proteins, and anthocyanins, as well as in floral induction and defense response. We found that sucrose preferentially induced OsWRKY7, whereas other sugars (such as mannitol, glucose, fructose, galactose, and maltose) did not have the same effect. A hexokinase inhibitor mannoheptulose did not block the effect of sucrose, which is consequently thought to function directly. MG132 inhibited sucrose induction, suggesting that a repressor upstream of OsWRKY7 is degraded by the 26S proteasome pathway. The 3-kb promoter sequence of OsWRKY7 was preferentially induced by sucrose in the luciferase system. Knockout mutants of OsWRKY7 were more sensitive to the rice blast fungus Magnaporthe oryzae, whereas the overexpression of OsWRKY7 enhanced the resistance, indicating that this gene is a positive regulator in the plant defense against this pathogen. The luciferase activity driven by the OsPR10a promoter was induced by OsWRKY7 and this transcription factor bound to the promoter region of OsPR10a, suggesting that OsWRKY7 directly controls the expression of OsPR10a. We conclude that sucrose promotes the transcript level of OsWRKY7, thereby increasing the expression of OsPR10a for the defense response in rice.
... R. Kim et al., 2009;Pasriga et al., 2019). After chimeric molecules in Escherichia coli were constructed, they were transformed into Agrobacterium tumefaciens LBA4404 using the freezethaw method, as described previously (An, 1987). The agrobacteriummediated cocultivation method was used to obtain transgenic rice plants (S. Lee et al., 1999). ...
Floral transition starts in the leaves, when florigens respond to various environmental and developmental factors. Among several regulatory genes that are preferentially expressed in the inflorescence meristem during floral transition, this study examines the homeobox genes OsZHD1 and OsZHD2 for their roles in regulating this transition. Although single mutations in these genes did not result in visible phenotype changes, double mutations in these genes delayed flowering. Florigen expression was not altered in the double mutants, indicating that the delay was due to a defect in florigen signaling. Morphological analysis of shoot apical meristem at early developmental stage indicated that inflorescence meristem development was significantly delayed in the double mutants. Overexpression of ZHD2 causes early flowering because of downstream signals after the generation of florigens. Expression levels of the auxin biosynthesis genes were reduced in the mutants and the addition of indole-3-acetic acid recovered the defect in the mutants, suggesting that these homeobox genes play a role in auxin biosynthesis. A rice florigen, RFT1, binds to the promoter regions of homeobox genes. These results indicate that florigens stimulate the expression of homeobox genes, enhancing inflorescence development in the shoot apex. This article is protected by copyright. All rights reserved.
... The amplified PCR products were digested with AscI and inserted into the pBA002 binary vector (Meera and Anu, 2020). The recombinant vector was transformed into A. tumefaciens strain GV3101 using the freeze-thaw method (An, 1987). Transformed Agrobacterium cells were resuspended in Arabidopsis transformation medium containing 5% sucrose and 0.05% Silwet L-77 (OSi Specialties, Inc., Danbury, CT, USA). ...
The formation of a hydrophobic cuticle layer on the aerial plant parts was a critical innovation for protection from the terrestrial environment during the evolution of the land plants. However, little is known about the molecular mechanisms underlying cuticle biogenesis in early terrestrial plants. Here, we report an AP2/ERF1 transcriptional activator, PpWIN1, involved in cutin and cuticular wax biosynthesis in Physcomitrium patens and Arabidopsis. The levels of PpWIN1 transcripts were 2.5-fold higher in gametophores than in the protonema and increased by approximately 3- to 4.7-fold in the protonema and gametophores under salt and osmotic stresses. PpWIN1 harboring transcriptional activation activity is localized in the nucleus of tobacco leaf epidermal cells. Δppwin1 knockout mutants displayed a permeable cuticle, increased water loss, and cutin- and wax-deficient phenotypes. In contrast, increased total cutin and wax loads and decreased cuticular transpiration rates were observed in PpWIN1-overexpressing Arabidopsis plants. The transcript levels of CYP86A4, CYP86A7, HTH-like, GPAT4, KCS2, CER1, and CER4 genes involved in cutin or wax biosynthesis were significantly upregulated in PpWIN1-overexpressing Arabidopsis lines, indicating that PpWIN1 acts as a transcriptional activator in cuticle biosynthesis. This study suggests that Arabidopsis WIN1/SHN1 orthologs may be functionally conserved from the early to the vascular land plants.
... The pKCS4P::KCS4-GUS and pKCS4P::KCS4 vectors were introduced into A. tumefaciens GV3101 using the freeze-thaw method (An 1987), and then Arabidopsis (Col-0) was transformed using A. tumefaciens cells harboring the pKCS4P::KCS4-GUS or pKCS4P::KCS4 vector using the vacuum infiltration method described by Bechtold et al. (1993). Harvested seeds were sterilized and germinated on half-strength MS medium supplemented with 25 μg·mL −1 kanamycin. ...
Your article is protected by copyright and all rights are held exclusively by Korean Society of Plant Biologist. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com". Abstract Very long-chain fatty acids (VLCFAs) are essential precursors of membrane lipids, such as phospholipids and sphingolipids, cuticular waxes, suberins, and Brassica seed oils. The first step of VLCFA synthesis is mediated by 3-ketoacyl-CoA synthase (KCS), which catalyzes the condensation of a C2 unit from malonyl-CoA to acyl-CoA. In the present study, we investigated the role of KCS4 in pollen tube and root growth. KCS4 was predominantly expressed in shoot and root apical meristems, leaf veins, mature and germinated pollen grains, and developing embryos. The fluorescent signals of KCS4 fused with enhanced yellow fluorescent protein (KCS4:eYFP) were detected in the endoplasmic reticulum of tobacco epidermis. KCS4 disruption inhibited pollen tube elongation and root growth, whereas KCS4 promoter-driven KCS4 expression rescued the growth-retarded phenotype to wild type (WT) in kcs4 complementation lines. Root growth assay of WT and kcs4 lines treated with metazachlor and mefluidide, which are specific KCS inhibitors, and fatty acid analysis of their roots and seeds revealed that KCS4 is involved in the elongation of longer than C24 VLCFAs, which are essential for root and pollen tube growth.
... The deleted or point-mutated coding regions of KCS9 were generated by the overlap extension PCR cloning method (Bryksin and Matsumural, 2010) and cloned into the pSPYNE-35S and pSPYCE-35S vectors for BiFC or membrane Y2H vectors (MoBiTec). The generated recombinant constructs were transformed into Agrobacterium GV3101 using the freeze-thaw method (An, 1987). For the generation of binary constructs harboring KCS9 mutants, the point-mutated coding regions of KCS9 were amplified with KCS9 YFPN F/KCS9 YFPC R primer sets using MbPY2H_KCS9_D308G, MbPY2H_KCS9_RRR309-311GGG, and MbPY2H_KCS9_D339G constructs as templates (Supplementary Table S1). ...
Fatty acid elongase (FAE), which catalyzes the synthesis of very-long-chain fatty acids (VLCFAs), is a multiprotein complex; however, little is known about its quaternary structure. In this study, BiFC and/or yeast two hybrid (Y2H) assays showed that 1) homo-interactions were observed in β-ketoacyl-CoA synthases (KCS2, KCS9, and KCS6), Eceriferum2-like proteins [CER2 and CER2-Like2 (C2L2)], and FAE complex proteins (KCR1, PAS2, ECR, and PAS1), except for CER2-Like1 (C2L1). 2) Hetero-interactions were observed between KCSs (KCS2, KCS9, and KCS6), between CER2-LIKEs (CER2, C2L2, and C2L1), and between FAE complex proteins (KCR1, PAS2, ECR, and PAS1). 3) PAS1 interacts with FAE complex proteins (KCR1, PAS2, and ECR), but not with KCSs (KCS2, KCS9, and KCS6) and CER2-LIKEs (CER2, C2L2, and C2L1). 4) Asp (308 aa) and three Arg (309 to 311 aa) residues of KCS9 were essential for the homo-interactions of KCS9 and hetero-interactions between KCS9 and PAS2 or ECR. An Asp (339 aa) residue of KCS9 is involved in its homo- and hetero-interactions with ECR. Complementation analysis of Arabidopsis kcs9 mutant by the expression of amino acid-substituted KCS9 mutant genes showed that 5) Two Asp residues (308 and 339 aa) of KCS9 are involved in the synthesis of C24 VLCFAs from C22. This study suggests that protein-protein interaction (PPI) in FAE complexes is important for VLCFA synthesis and provides insight into the quaternary structure of FAE complexes for efficient synthesis of VLCFAs.
... The three constructs (as prepared above) were then separately introduced into the competent cells of Agrobacterium tumefaciens LBA4404 strain using freeze-thaw method (An 1987). The bacterial cells containing pBI: OsFEX promoter were used to infect N. benthamiana leaf discs. ...
Key message
Early induction of OsFEX was insufficient for fluoride adaptation in IR-64. Overexpression ofOsFEX in yeast andNicotiana benthamiana enhanced fluoride tolerance.
Abstract
The present study delineates the regulation of fluoride exporter (FEX) in the fluoride-sensitive rice cultivar, IR-64 and its efficacy in generating high fluoride tolerance in transgenic Nicotiana benthamiana. Gene and protein expression profiling revealed that OsFEX exhibited early induction during fluoride stress in the vegetative and reproductive tissues of IR-64, although the expression was suppressed upon prolonged stress treatment. Analysis of OsFEX promoter in transgenic N. benthamiana, using β-glucuronidase reporter assay confirmed its early inducible nature, since the reporter expression and activity peaked at 12 h of NaF stress, after which it was lowered. OsFEX expression was up regulated in the presence of gibberellic acid (GA) and melatonin, while it was suppressed by abscisic acid (ABA). Complementation of ΔFEX1ΔFEX2 yeast mutants with OsFEX enabled high fluoride tolerance, thus validating the functional efficiency of the transgene. Bioassay of transgenic N. benthamiana lines, expressing OsFEX either under its own promoter or under CaMV35S promoter, established that constitutive overexpression, rather than early induction of OsFEX was essential and crucial for generating fluoride tolerance in the transgenics. Overall, the suppression of OsFEX in the later growth phases of stressed IR-64 due to enhanced ABA conservation and lowered synthesis of GA, as supported by the application of the respective phytohormone biosynthetic inhibitors, such as sodium tungstate and paclobutrazol, accounted for the fluoride-hyperaccumulative nature of the rice cultivar.
... Schematic diagrams of the T-DNA regions of the plasmids Figure 1. The resulting plasmids were transformed into E. coli Top10 (Thermo Fisher Scientific, United States), and after isolation and sequence verification they were eventually transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method (An, 1987). ...
Methods for simple and fast assembly of exchangeable standard DNA parts using Type II S restriction enzymes are becoming more and more popular in plant synthetic and molecular biology. These methods enable routine construction of large and complex multigene DNA structures. Two available frameworks emphasize either high cloning capacity (Modular Cloning, MoClo) or simplicity (GoldenBraid, GB). Here we present a set of novel α-level plasmids compatible with the GB convention that extend the ability of GB to rapidly assemble more complex genetic constructs, while maintaining compatibility with all existing GB parts as well as most MoClo parts and GB modules. With the use of our new plasmids, standard GB parts can be assembled into complex assemblies containing 1, 5, 10 and up to theoretically 50 units in each successive level of infinite loop assembly. Assembled DNA constructs can be also combined with conventional binary GB-assemblies (1, 2, 4, 8… units). We demonstrate the usefulness of our framework on single tube assembly of replicating plant expression constructs based on the geminivirus Bean yellow dwarf virus (BeYDV).
... The SlCYP90B3 RNA interference fragment was recombined to the plant binary vector pBIN19, generating SlCYP90B3-RNAi (Supplementary Table 2). The OE and RNAi vectors were then introduced into Agrobacterium tumefaciens strain LBA4404 (Tiangen, Beijing, China), and tomato transformation was performed as described 49 . Both independent OE (OE-4, OE-5) and RNAi (RNAi-9, RNAi-10) single-insertion lines were subsequently assessed after cultivation for three successive generations. ...
The essential role of ethylene in fruit ripening has been thoroughly studied. However, the involvement of brassinosteroids (BRs) in the regulation of fruit ripening and their relationship with the ethylene pathway are poorly understood. In the current study, we found that BRs were actively synthesized during tomato fruit ripening. We then generated transgenic lines overexpressing or silencing SlCYP90B3 , which encodes a cytochrome P450 monooxygenase that catalyzes the rate-limiting step of BR synthesis. The expression level of SlCYP90B3 was positively related to the contents of bioactive BRs as well as the ripening process in tomato fruit, including enhanced softening and increased soluble sugar and flavor volatile contents. Both carotenoid accumulation and ethylene production were strongly correlated with the expression level of SlCYP90B3 , corroborated by the altered expression of carotenoid biosynthetic genes as well as ethylene pathway genes in transgenic tomato fruits. However, the application of the ethylene perception inhibitor 1-methycyclopropene (1-MCP) abolished the promotion effect of SlCYP90B3 overexpression on carotenoid accumulation. Taken together, these results increase our understanding of the involvement of SlCYP90B3 in bioactive BR biosynthesis as well as fruit ripening in tomato, thus making SlCYP90B3 a target gene for improvement of visual, nutritional and flavor qualities of tomato fruits with no yield penalty.
... The amplified products were digested with the restriction enzymes XbaI/SacI and inserted into the pBI121 vector to form the SlbHLH95-OE vector. These recombinant plasmids were transformed into Agrobacterium tumefaciens (strain LBA4404) by the freeze-thaw method (An, 1987). The WT tomato was used to create SlbHLH95-RNAi and SlbHLH95-OE transgenic lines, and the rin mutant was used to produce SlbHLH95-OE-rin transgenic lines. ...
Ethylene signaling pathways regulate several physiological alterations that occur during tomato fruit ripening, such as changes in colour and flavour. However, the mechanisms underlying the transcriptional regulation of genes in these pathways remain unclear, although the role of RIN has been widely reported. In this report, we describe a bHLH transcription factor SlbHLH95, whose transcripts accumulated abundantly in breaker+4 and breaker+7 fruits compared to rin (ripening inhibitor) and Nr (never ripe). Moreover, the promoter activity of SlbHLH95 was regulated by RIN in vivo. Suppression of SlbHLH95 resulted in reduced sensitivity to ethylene, decreased accumulation of the total carotenoid, lowered GSH content, and inhibited expression of fruit ripening- and glutathione metabolism-related genes. Conversely, up-regulation of SlbHLH95 in wild-type tomato resulted in higher sensitivity to ethylene, increased accumulation of the total carotenoids, and caused slightly premature ripening, and elevated accumulation of GSH, soluble sugar and starch content. Notably, overexpression of SlbHLH95 in rin led to the up-regulated expression of fruit ripening related genes (FUL1, FUL2, SAUR69, ERF4 and CNR) and multiple glutathione metabolism-related genes (GSH1, GSH2, GSTF1 and GSTF5). These results clarified that SlbHLH95 participates in the regulation of fruit ripening, affects ethylene sensitivity and multiple metabolisms targeted by RIN in tomato.
... Schematic diagrams of the T-DNA regions of the plasmids pGB-R-GFP, pGB-R-GFP-P19, pGB-R-DsRed, pGB-R-DsRed-P19, pGB-R-GFP-DsRed and pGB-R-DsRed-GFP are shown in Fig. 1. The resulting plasmids were transformed into Escherichia coli (Top10, Thermo Fischer Scientific), and after isolation and sequence verification they were eventually transformed into Agrobacterium tumefaciens (EHA 105) by the freeze-thaw method (An 1987). ...
Key message:
This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.
... Afterward, to confirm the gene orientation, double restriction digestion was done using BamHI and XbaI. We used the freeze-thaw method to transfer pBI121 carrying the JrPPO1 fragment into competent cells of Agrobacterium tumefaciens strain LBA4404 (An, 1987). ...
Walnut (Juglans regia L.) is economically important for both its wood and nut nutritional value, but it is susceptible to diseases such as walnut bacterial blight, caused by X. arboricola pv. juglandis (Xaj). Walnuts contain many phenolic compounds, providing a good model on which to study polyphenol oxidase (PPO). We inoculated the detached walnut fruits of ‘Ford’, ‘Chandler’, ‘Franquette’, ‘Robert Livermore’ and ‘Payne’ with Xaj (108 cfu/mL) and measured the induction of PPO activity in infected and adjacent to infected sites. PPO activity was induced significantly in areas adjacent to infected sites in all cultivars except ‘Ford’, compared to infected and uninfected sites. ‘Ford’ and ‘Franquette’, presenting the lowest and the highest PPO activity, showed the largest and smallest mean diameter spots in response to Xaj, respectively. Polyacrylamide gel electrophoresis confirmed monophenol oxidase activity of walnut PPO in the assessed tissues. Then, we revealed the anti‐pathogenic potential of walnut PPO through Agrobacterium tumefaciens‐mediated walnut JrPPO1 gene transfer into tobacco (Nicotiana tobacum L.). Two transformed tobacco lines overexpressing JrPPO1 gene were regenerated successfully and challenged with Pseudomonas syringae pv. tabaci. Transgenic line 1 and 2, showed significantly higher PPO activity and lower disease severity to the pathogen compared to control. However, a significant difference in disease severity and PPO activity level was observed between transgenic tobacco line 1 and 2. Our results demonstrate a potential defense‐related role of PPO in transgenic tobacco and its induction in areas adjacent to infection sites in cultivars treated with Xaj.
... The KpnI-XbaI fragment of FcLFY was generated by PCR with primers FcLFY-KpnI-F and FcLFY-XbaI-R (Table S1).The fragment of FcLFY was cloned into the pCAMBIA1302 vector with KpnI and XbaI adapter sites and driven by CaMV 35S promoter (Fig. S3B). The construct was transformed into Agrobacterium tumefaciens strain EHA 105 via the freeze-thaw method (An, 1987) (Fig. S3C) and then transformed into WT and lfy-15 mutant Arabidopsis plant by floral dip transformation N. Ma, et al. Scientia Horticulturae xxx (xxxx) xxxx method (Steven and Andrew, 2008) (Fig. S3D). ...
Fig (Ficus carica L.) is high in fruit yield and nutritious, and easy to form flower buds. Fig tree, differing from many other fruit trees, has unique flower bud differentiation and fruit-bearing characteristics. Its flower bud (inflorescence) differentiation takes place throughout the shoot elongation period, and it occurs with fruits simultaneously. The entire process of flower bud differentiation to fruit ripening can be completed in one growing season. Studying the unique flowering characteristics of fig provides an important theoretical basis for
its cultivation, flowering regulation, fruit production, and molecular breeding. However, the molecular mechanism of flower bud differentiation in fig has not been elucidated, so identifying the relevant genes is essential for understanding the characteristics of fig flower bud differentiation. Here, we isolated a FLORICAULA/LEAFY homologue gene from fig, FcLFY, and identified its function and expression patterns. FcLFY contains a 1377 bp open reading frame and encodes 458 amino acids. The amino acid sequence has a typical LFY/FLO family domain, containing a unique glycine rich region. Moreover, FcLFY expressed the highest in apical bud meristem of fig. In addition, Arabidopsis transgenic lines L23 and L28, which overexpressed FcLFY, bolted 8–9 days and flowered 6 days earlier than WT, while the line C7, which rescued the abnormal flower phenotype of lfy-15 mutant, flowered 8 days earlier than the mutant. Analysis with qRT-PCR further showed that the expressions of flowering promoting genes, including AP1, LFY, CAL and SEP3 were up-regulated while the flowering inhibitory genes FLC and TFL1 were down-regulated in the FcLFY transgenic Arabidopsis. Taken together, FcLFY overexpression
shortened the period of vegetative growth, promoted early flowering, partially complemented the phenotypic defects of lfy15 mutant and increased the expression of floral meristem and flower organ genes in
Arabidopsis, indicating that FcLFY is involved in the flower bud differentiation and plays an important role in apical meristem formation of fig. This study provides useful information for us to better understand the characterization and function of FcLFY in regulating flower initiation, and is helpful for establishing molecular breeding systems in fig.
... The amplified PCR product was digested with Bgllll and BstEll and then cloned in the same restriction enzyme sites of the pCSEN binary vector containing the SEN1 promoter. The generated binary constructs were subsequently transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method (An 1987). ...
Malonyl-ACP is a key building block for the synthesis of fatty acids, which are important components of cell membranes, storage oils, and lipid-signaling molecules. Malonyl CoA-acyl carrier protein (ACP) malonyltransferase (MCAMT) catalyzes the production of malonyl-ACP and CoA from malonyl-CoA and ACP. Here, we report that MCAMT plays a critical role in cell division and has the potential to increase the storage oil content in Arabidopsis. The quantitative RT-PCR and MCAMT promoter:GUS analyses showed that MCAMT is predominantly expressed in shoot and root apical meristems, leaf hydathodes, and developing embryos. The fluorescent signals of MCAMT:eYFP were observed in both chloroplasts and mitochondria of tobacco leaf protoplasts. In particular, the N-terminal region (amino acid residues 1 to 30) of MCAMT was required for mitochondrial targeting. The Arabidopsis mcamt-1 and -2 mutants exhibited an embryo-lethal phenotype because of the arrest of embryo development at the globular stage. The transgenic Arabidopsis expressing antisense MCAMT RNA showed growth retardation caused by the defects in cell division. The overexpression of MCAMT driven by the promoter of the senescence-associated 1 (SEN1) gene, which is predominantly expressed in developing seeds, increased the seed yield and storage oil content of Arabidopsis. Taken together, the plastidial and mitochondrial MCAMT is essential for Arabidopsis cell division and is a novel genetic resource useful for enhancing storage oil content in oilseed crops.
... Subsequently, the fragment was recombined into the destination vector pK7WGF2 [52] using L/R-Clonase. The construct was verified by sequencing and was transferred to the Agrobacterium tumefaciens GV3101 strain using the freeze/ thaw method [53]. Transient transformation of tobacco leaf epidermal cells was performed as described in [54]. ...
Background
Developing tolerant cultivars by incorporating resistant genes is regarded as a potential strategy for controlling Verticillium wilt that causes severe losses in the yield and fiber quality of cotton.
Results
Here, we identified the gene GbHyPRP1 in Gossypium barbadense, which encodes a protein containing both proline-rich repetitive and Pollen Ole e I domains. GbHyPRP1 is located in the cell wall. The transcription of this gene mainly occurs in cotton roots and stems, and is drastically down-regulated upon infection with Verticillium dahliae. Silencing HyPRP1 dramatically enhanced cotton resistance to V. dahliae. Over-expression of HyPRP1 significantly compromised the resistance of transgenic Arabidopsis plants to V. dahliae. The GbHyPRP1 promoter region contained several putative phytohormone-responsive elements, of which SA was associated with gene down-regulation. We compared the mRNA expression patterns of HyPRP1-silenced plants and the control at the global level by RNA-Seq. A total of 1735 unique genes exhibited significant differential expression. Of these, 79 DEGs involved in cell wall biogenesis and 43 DEGs associated with the production of ROS were identified. Further, we observed a dramatic thickening of interfascicular fibers and vessel walls and an increase in lignin in the HyPRP1-silenced cotton plants compared with the control after inoculation with V. dahliae. Additionally, silencing of HyPRP1 markedly enhanced ROS accumulation in the root tips of cotton inoculated with V. dahliae.
Conclusions
Taken together, our results suggest that HyPRP1 performs a role in the negative regulation of cotton resistance to V. dahliae via the thickening of cell walls and ROS accumulation.
Electronic supplementary material
The online version of this article (10.1186/s12870-018-1565-1) contains supplementary material, which is available to authorized users.
... Finally, the 4X ABRE: 35S and 2X ABRC: 35S inducible promoters were subcloned separately in the binary vector pBI121 at HindIII and BamHI site by replacing CaMV 35S constitutive promoter following standard procedures (Sambrook and Russell 2001). The two constructs were separately transferred into the competent cells of Agrobacterium tumefaciens LBA4404 strain (An 1987). Transformation of tobacco leaf discs was done according to RoyChoudhury et al. (2007) to generate T 0 plants, which were allowed to flower and set seeds under normal environmental conditions. ...
In this communication, we report the binding of abscisic acid responsive elements (ABREs) of rice Osem, namely motif A and motif B, with a cognate trans-acting factor present in the nuclear extract of tobacco leaf. The binding
is specific as both the complexes were disrupted with an excess of homologous non-radioactive DNA like motif A or motif B
themselves or with cis-elements of rice Rab16A, motif I (ABRE) and motif IIa (non-ACGT ABRE-like sequences). Four tandem repeats of ABRE from wheat Em (4X ABRE) or two tandem repeats of Em ABRE, plus two copies of coupling element (CE1) from barley HVA22 (2X ABRC), also showed specific complexes, that were competed out by an excess of homologous competitors like motif I, motif
IIa, motif A, motif B, 4X ABRE and 2X ABRC, but not by the unrelated 4X DRE sequence. Elution of the protein from all the
complexes showed a single 26 kDa polypeptide band. Introgression of two of the above synthetic promoters 4X ABRE and 2X ABRC,
each fused with minimal promoter of cauliflower mosaic virus 35S (CaMV 35S), could induce the expression of the reporter gene
β-glucuronidase (gus) in transgenic tobacco in response to high NaCl concentration, dehydration or abscisic acid, but not at the constitutive
level, proving that they can be used as efficient stress-inducible promoters. Our work shows both in vivo and in vitro activity of the promoters from monocot genes in the model dicot plant tobacco.
... The amplified products were digested with XbaI/SacI and linked to the plant binary vector pBI121 at XbaI/SacI restriction sites. Finally, the RNAi vector was transformed into WT tomato and the overexpression vector was transformed into the tomato mutant rin through the freezethaw method, using Agrobacterium tumefaciens strain LBA4404 (An, 1987). Transgenic lines were selected on the basis of kanamycin (50 mg l −1 ) resistance. ...
Fruit ripening represents a process changing flavor and appearance and also a process dramatically increasing fruit softening. Fruit softening and textural variations are mainly resulted from the disrupted cell wall of fruit throughout ripening, whereas, the exact mechanisms and specific modifications of cell wall remain unclear. Plant-specific GRAS proteins play a critical role in development and growth. To date, few GRAS genes have been functionally categorized in tomato. The expression of a novel GRAS gene revealed herein is designated as SlFSR (fruitshelf-liferegulator), specifically increased during fruit ripening, whereas significantly decreased in tomato mutant rin (ripening inhibitor). RNAi repression of SlFSR resulted in reduced expression of multiple cell wall modification-related genes, decreased PG (polygalacturonase), TBG (tomato β-galactosidase), CEL (cellulase) and XYL (β-D-xylosidase) activities, and significantly prolonged fruit shelf-life. Furthermore, overexpression of SlFSR in mutant rin gave rise to up-regulated expression of multiple cell wall modification-related genes, such as PG, TBG4, CEL2, XYL1, PL, PE, MAN1, EXP1 and XTH5, and significantly shortened fruit shelf-life. Our findings make it possible to reveal the genetic mechanisms underlying fruit cell wall metabolisms and suggest that SlFSR gene is another biotechnological targeted control of tomato fruit shelf-life.
... The resultant vector was named 'DEWAX2P::GUS'. The recombinant vectors CaMV35SP::DEWAX2:eYFP and DEWAX2P::GUS were transformed into Agrobacterium tumefaciens GV3101 as described by An (1987). Arabidopsis was transformed via Agrobacteriummediated transformation using the floral-dip method (Zhang et al. 2006). ...
The aerial parts of terrestrial plants are covered with hydrophobic wax layers, which represent the primary barrier between plant cells and the environment and act to protect plants from abiotic and biotic stresses. Although total wax loads are precisely regulated in environmental or organ-specific manner, regulatory mechanisms underlying cuticular wax biosynthesis remain largely unknown. In this study, we characterized the DEWAX2, which encodes an AP2/ERF-type transcription factor and is predominantly expressed in young seedlings, and rosette and cauline leaves. Total wax loads increased by approximately 12 and 16% in rosette and cauline leaves of dewax2, respectively, but were not significantly altered in the stems of dewax2 relative to wild type (WT). The excess wax phenotype of dewax2 leaves was rescued upon expression of DEWAX2 driven by its own promoter. Overexpression of DEWAX2 decreased total wax loads by approximately 15 and 26% in the stems and rosette leaves compared with those of the WT, respectively. DEWAX2:eYFP was localized to the nucleus in Arabidopsis roots and hypocotyls. DEWAX2 possessed transcriptional repression activity in tobacco protoplasts. Transcriptome and quantitative RT-PCR analyses showed that the transcript levels of CER1, ACLA2, LACS1, LACS2, and KCS12 were downregulated in DEWAX2 overexpression lines compared with WT. Transient transcriptional assays showed that DEWAX2 represses the expression of its putative target genes. Quantitative chromatin immunoprecipitation-PCR revealed that DEWAX2 binds directly to the GCC motifs of the LACS1, LACS2, KCS12, and CER1 promoters. These results suggest that DEWAX2-mediated transcriptional repression may contribute to the total wax load in Arabidopsis leaves.
... A 3.6 kb Not I fragment containing the hpRNA cassette was isolated from the plasmid pHANNIBAL(TSV-CP) and sub-cloned into the binary vector pART27 (Gleave 1992). This chimeric gene construct was then inserted into Agrobacterium tumefaciens strain LBA4404 (Jefferson et al. 1987) via triparental mating using pRK2013 as a helper as described by An (1987). Insertion of the chimeric gene construct was confirmed by colony PCR. A. tumefaciens harbouring the plasmid with the hpRNA cassette was used for transformation of sunflower and tobacco. ...
... The constructs were transformed into the Solanum lycopersicum cv. Ailsa Craig plants by Agrobacterium tumefaciens strain LBA4404 [64]. ...
The basic leucine zipper (bZIP) transcription factors have crucial roles in plant stress responses. In this study, the bZIP family gene SlbZIP38 (GenBank accession No: XM004239373) was isolated from a tomato (Solanum lycopersicum cv. Ailsa Craig) mature leaf cDNA library. The DNA sequence of SlbZIP38 encodes a protein of 484 amino acids, including a highly conserved bZIP DNA-binding domain in the C-terminal region. We found that SlbZIP38 was differentially expressed in various organs of the tomato plant and was downregulated by drought, salt stress, and abscisic acid (ABA). However, overexpression of SlbZIP38 significantly decreased drought and salt stress tolerance in tomatoes (Ailsa Craig). The findings that SlbZIP38 overexpression reduced the chlorophyll and free proline content in leaves but increased the malondialdehyde content may explain the reduced drought and salt tolerance observed in these lines. These results suggest that SlbZIP38 is a negative regulator of drought and salt resistance that acts by modulating ABA signaling.
... The PttMYB021, PttNAC058 and PttNAC085 effector constructs were as described previously (Winzell et al., 2010;Ratke et al., 2015). The PttMYB167 effector construct was produced by amplification of the coding sequence from hybrid aspen xylem cDNA using PCR primers PtMYB52-U and PtMYB52-L (Table S1), followed by cloning of the cDNA fragment into pGA581 vector (An, 1987) containing a 35S promoter. The nomenclature of the NAC and MYB transcription factors is adapted from Hu et al. (2010) and Wilkins et al. (2009). ...
Metacaspases (MCs) are cysteine proteases that are implicated in programmed cell death of plants. AtMC9 ( Arabidopsis thaliana Metacaspase9) is a member of the Arabidopsis MC family that controls the rapid autolysis of the xylem vessel elements, but its downstream targets in xylem remain uncharacterized.
PttMC13 and PttMC14 were identified as AtMC9 homologs in hybrid aspen ( Populus tremula × tremuloides ). A proteomic analysis was conducted in xylem tissues of transgenic hybrid aspen trees which carried either an overexpression or an RNA interference construct for PttMC13 and PttMC14 .
The proteomic analysis revealed modulation of levels of both previously known targets of metacaspases, such as Tudor staphylococcal nuclease, heat shock proteins and 14‐3‐3 proteins, as well as novel proteins, such as homologs of the PUTATIVE ASPARTIC PROTEASE3 (PASPA3) and the cysteine protease RD21 by PttMC13 and PttMC14.
We identified here the pathways and processes that are modulated by PttMC13 and PttMC14 in xylem tissues. In particular, the results indicate involvement of PttMC13 and/or PttMC14 in downstream proteolytic processes and cell death of xylem elements. This work provides a valuable reference dataset on xylem‐specific metacaspase functions for future functional and biochemical analyses.
... pBI121-GFP and pBI121-DCAS-GFP were introduced into Agrobacterium tumefaciens LBA4404 by electroporation. BY-2 cells were transformed by A. tumefaciens infection as described (An, 1987) and selected on solidified Murashige and Skoog medium containing kanamycin (100 mg mL 21 ). The suspension cultures of the transgenic cells were established in liquid Murashige and Skoog medium containing kanamycin and subcultured every 7 d. ...
Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from Rhododendron dauricum. We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of R. dauricum. The gene coded for a 533-amino acid polypeptide with moderate homologies to FAD oxidases from other plants. The primary structure contained an N-terminal signal peptide, and conserved amino acid residues to form bicovalent linkage to FAD isoalloxazine ring at His112 and Cys175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris, exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa, whereas DCA synthase catalyzes a novel cyclization reaction of farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at chemical, biochemical and molecular levels. Extracellular localization of DCA was also confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA; the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells.
... This vector contains the herbicide resistance marker gene PAT (Phosphinothricin acetyltransferase), which enables selection of the transgenic plants. These constructs were transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method (An, 1987), which were then used for the transformation of Arabidopsis by the Agrobacterium-mediated floral dip method (Zhang et al., 2006). ...
Triacylglycerol (TAG) is an energy-rich reserve in plant seeds that is composed of glycerol esters with three fatty acids. Since TAG can be used as a feedstock for the production of biofuels and bio-chemicals, producing TAGs in vegetative tissue is an alternative way of meeting the increasing demand for its usage. The WRINKLED1 (WRI1) gene is a well-established key transcriptional regulator involved in the upregulation of fatty acid biosynthesis in developing seeds. WRI1s from Arabidopsis and several other crops have been previously employed for increasing TAGs in seed and vegetative tissues. In the present study, we first identified three functional CsWRI1 genes (CsWRI1A. B, and C) from the Camelina oil crop and tested their ability to induce TAG synthesis in leaves. The amino acid sequences of CsWRI1s exhibited more than 90% identity with those of Arabidopsis WRI1. The transcript levels of the three CsWRI1 genes showed higher expression levels in developing seeds than in vegetative and floral tissues. When the CsWRI1A. B, or C was introduced into Arabidopsis wri1-3 loss-of-function mutant, the fatty acid content was restored to near wild-type levels and percentages of the wrinkled seeds were remarkably reduced in the transgenic lines relative to wri1-3 mutant line. In addition, the fluorescent signals of the enhanced yellow fluorescent protein (eYFP) fused to the CsWRI1 genes were observed in the nuclei of Nicotiana benthamiana leaf epidermal cells. Nile red staining indicated that the transient expression of CsWRI1A. B, or C caused an enhanced accumulation of oil bodies in N. benthamiana leaves. The levels of TAGs was higher by approximately 2.5- to 4.0-fold in N. benthamiana fresh leaves expressing CsWRI1 genes than in the control leaves. These results suggest that the three Camelina WRI1s can be used as key transcriptional regulators to increase fatty acids in biomass.
... From this plasmid, PCR products were amplified with the pPaLAR3A_2 and pPaLAR3B_2 primer sets (Additional file 1). These two PCR products were subsequently cloned into the destination plasmid pCF201 which was adapted from the pGA580 vector used for Agrobacterium transformation [48] by overlap extension PCR. To be able to do so, the destination plasmid was amplified into two separate PCR products. ...
Background
The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato.
Results
The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183–196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3’H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes.
Conclusions
PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-016-0952-8) contains supplementary material, which is available to authorized users.
... The recombinant plasmids pCAPE2-CaM and pCAPE2-CaMm were transformed into the competent cells of Agrobacterium tumefaciens (strain GV3101) by using the liquid nitrogen freeze-thaw method (An 1987). Five-week old N. benthamiana plants were used for A. tumefaciens-mediated gene transformation. ...
Calmodulin (CaM) is a highly conserved calcium sensor protein associated with chilling tolerance in living organisms. It has four EF-hand domains for binding of four Ca²⁺, two of them located in the N-terminus, and the other two in the C-terminus. A notothenioid CaM gene fragment (CaMm), which only codes for N-terminus of CaM (with two EF-hand domains), was introduced into Nicotiana benthamiana. Effects of its overexpression on chilling tolerance in plants were explored. During 4°C or 0°C chilling treatment, both CaMm and CaM transgenic plants showed higher PSII maximum quantum yield, actual quantum yield, and soluble protein content, lower electrolyte leakage and malondialdehyde content than that of the control. The changes in these physiological indices were comparable between the CaMm and CaM transgenic plants during the treatments. These results indicate that the N-terminus of calmodulin is likely the key functional domain involved in the adaptive response to cold stress.
... The Agrobacterium tumefaciens strain LBA4404 was transformed with EcJAZ-pCAMBIA1303 following the standard protocol (Gynheung 1987). Agrobacrterium cells (an absorbance A 600 ≈ 0.5) were pelleted, resuspended with 0.25 M NaCl, again pelleted, resuspended with 20 mM CaCl 2 , mixed with 1 ug of EcJAZ-pCAMBIA1303 DNA, and incubated on ice for 30 min. ...
Jasmonate ZIM domain (JAZ) proteins are key regulators of the jasmonic acid (JA) signaling pathway. Repressors of JAZ remain bound to the Myelocytomatosis 2 (MYC2) or MYC3/MYC4 transcription factors in the absence of JA and negatively regulate transcription of the JA responsive genes. In the presence of JA, JAZ proteins interact with coronative insensitive 1 (COI1), the recognition molecule of E3 ubiquitin ligase SCFCOI1 (COI1 stabilized by Skp, cullin, F-box containing complex), get ubiquitinated and subsequently degraded by the 26S proteasome. However, there is a dearth of knowledge about this gene family in monocot cereals, specifically its role in finger millet is unknown till date. Here we present the isolation and characterization of a novel JAZ family repressor gene from nonsequenced Eleusine coracana (EcJAZ) utilizing available genome information of Oryza, Sorghum, and Setaria. The EcJAZ sequence showed the presence of a conserved ZIM domain, the Jas motif, and N-terminal motif 7 like other Group1 TIFY sequence containing proteins. We observed coronatine (an analog of JA-Ile) dependent and time dependent degradation of recombinant EcJAZ that thereby fulfilled the basic characteristic of the JAZ proteins. We found a proteasome inhibitor N-(phenylmethoxy) carbonyl-L-leucyl-N-[(1R)-1-formyl-3-methylbutyl]-L-leucinamide) (MG132) mediated degradation inhibition of EcJAZ that supported its 26S proteasome mediated degradation. Our study shows the nuclear localization of GFP-EcJAZ by Agrobacterium mediated transient transformation of onion scale epidermal cells. In Eleusine leaves, transcription of EcJAZ increased 4.2-fold by salt stress and 5.5-fold by coronatine application; thus ascertained its inducibility by the abiotic stress as well as by bioactive JA-Ile. Taken together, all these results contribute to our understanding on the JA signaling pathway in Eleusine coracana.
... A 3696-bp NotI fragment containing the hpRNA cassette was isolated from the plasmid pHANNIBAL(TSV-Rep) and sub-cloned into the binary vector pART27. This chimeric gene construct was then introduced into Agrobacterium tumefaciens strain LBA4404 [25] via triparental mating using pRK2013 as a helper as described by An [3]. Cotyledon explants of groundnut (cv. ...
The feasibility of controlling peanut stem necrosis disease caused by Tobacco streak virus (TSV) in groundnut (Arachis hypogaea L.) was explored by expressing double-stranded RNA of the replicase (Rep) gene of TSV in groundnut through genetic engineering. A hairpin (hp) RNAi construct containing 535-bp sense and antisense TSV-Rep sequences flanking a 742-bp spacer sequence (Pdk intron) under the control of the constitutive Cauliflower mosaic virus 35S promoter was made in the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Cotyledon explants of groundnut cultivar TMV-7 were transformed with A. tumefaciens harboring the hpRNA cassette. The presence of the transgene in the transgenic plants was confirmed up to T3 generation by PCR amplification of the 535-bp fragment of TSV-Rep gene. The bioassay results indicated that necrotic lesions were observed on the leaves of the wild-type plants 7–9 days after inoculation with TSV and stem necrosis appeared 16–20 days after inoculation, whereas the transgenic plants did not develop symptoms until harvest. ELISA results indicated that the wild-type plants inoculated with TSV recorded the highest virus concentration as compared to the transgenic lines.
... The plasmid DNA was digested with EcoRI/HindIII, and an approximately 3.3 kb fragment of MYB94 genomic DNA, including its promoter region, was ligated into EcoRI/HindIII-digested pCAMBIA3300. The generated constructs were transformed into an Agrobacterium strain (GV3101) using the freeze-thaw method (An 1987), and myb94 and myb96 mutant plants were transformed with the Agrobacterium cells using the vacuum infiltration method (Clough and Bent 1998). The seeds harvested from transgenic plants were sterilized and selected on 1/2 MS agar medium containing 5 mg ml À1 (w/v) phosphinothricin or 25 mg ml À1 (w/v) kanamycin. ...
Aerial plant surfaces are coated by a cuticular wax layer to protect against environmental stresses, such as desiccation. In this study, we investigated the functional relationship between MYB94 and MYB96 transcription factors involved in cuticular wax biosynthesis. Both MYB94 and MYB96 transcripts were abundantly expressed in the aerial organs of Arabidopsis, and significantly induced at the same or similar time-points under conditions of drought. MYB94 complemented the wax-deficient phenotype of the myb96 loss-of-function mutant under both well-watered and drought stress conditions. The magnitude of decrease in total wax load in the myb94 myb96 double mutant was almost equal to the sum of the reduced wax loads in the individual myb94 and myb96 mutants under both conditions. Leaves of the myb94 myb96 mutant lost water through the cuticle faster than those of myb94 or myb96 plants. Transcript levels of wax biosynthetic genes were decreased in the single mutants, and further reduced in the double mutant, relative to wild-type, under drought and abscisic acid treatment conditions. MYB94 and MYB96 interact with the same regions containing MYB consensus motifs in the promoter regions of wax biosynthetic genes. The data collectively indicate that MYB94 and MYB96 exert an additive effect on cuticular wax biosynthesis, which may represent an efficient adaptive mechanism of response to drought in plants.
Trihelix proteins are plant-specific transcription factors that play crucial roles in plant development and stress responses. However, the involvement of trihelix proteins in fruit ripening and transcriptional regulatory mechanisms remains largely unclear. In this study, we cloned a trihelix gene SlGT31, whose relative expression was significantly induced by the application of exogenous ethylene but repressed by 1-methylcyclopropene (1-MCP). Suppression of SlGT31 resulted in delayed fruit ripening, decreased accumulation of total carotenoids and ethylene content, and inhibition of relative expression of genes related to ethylene and fruit ripening. Conversely, the opposite results were observed in SlGT31-overexpression lines. Yeast one-hybrid and dual-luciferase assays suggested that SlGT31 could bind to the promoters of two key ethylene biosynthesis genes ACO1 and ACS4. These results indicate that SlGT31 may act as a positive modulator during fruit ripening.
Very long-chain fatty acids (VLCFAs) are essential precursors of membrane lipids, such as phospholipids and sphingolipids, cuticular waxes, suberins, and Brassica seed oils. The first step of VLCFA synthesis is mediated by 3-ketoacyl-CoA synthase (KCS), which catalyzes the condensation of a C2 unit from malonyl-CoA to acyl-CoA. In the present study, we investigated the role of KCS4 in pollen tube and root growth. KCS4 was predominantly expressed in shoot and root apical meristems, leaf veins, mature and germinated pollen grains, and developing embryos. The fluorescent signals of KCS4 fused with enhanced yellow fluorescent protein (KCS4:eYFP) were detected in the endoplasmic reticulum of tobacco epidermis. KCS4 disruption inhibited pollen tube elongation and root growth, whereas KCS4 promoter-driven KCS4 expression rescued the growth-retarded phenotype to wild type (WT) in kcs4 complementation lines. Root growth assay of WT and kcs4 lines treated with metazachlor and mefluidide, which are specific KCS inhibitors, and fatty acid analysis of their roots and seeds revealed that KCS4 is involved in the elongation of longer than C24 VLCFAs, which are essential for root and pollen tube growth.
Una herramienta importante en la ingeniería genética de plantas son los vectores o constructos génicos que contienen genes reporteros, estos son utilizados para facilitar la estandarización de protocolos de transformación genética. El objetivo de este estudio fue generar de manera rápida y eficiente una colección base de constructos génicos en Escherichia coli cepa DH5α y Agrobacterium tumefaciens cepas LBA 4404, EHA 105 y C58, empleando 25 vectores binarios que contienen los genes reporteros gfp y gus utilizando las técnicas de choque térmico y electroporación. La confirmación de los transformantes se realizó mediante PCR y cortes con enzimas de restricción (Eco RI y Xho I), permitiendo verificar la presencia de estos 25 vectores dentro de las bacterias empleadas. La creación de esta colección es valiosa, para el adelanto de trabajos de estandarización de protocolos de transformación genética vía Agrobacterium tumefasciens, en especies de interés agronómico.
All higher plants express several different acyl carrier protein (ACP) isoforms in a tissue-specific manner. We provide evidence that expression of mRNA for the most abundant ACP isoform in Arabidopsis leaves (ACP4) is increased severalfold by light, whereas mRNA levels for ACP isoforms 2 and 3 are independent of light. The presence of GATA-like motifs in the upstream region of the Acl1.4 gene (encoding for ACP4) and the similarity in light-mediated induction to ferredoxin-A mRNA suggests a direct role of light in Acl1.4 gene activation. Polyribosomal analysis indicated that light also affects the association of ACP transcripts with polysomes, similarly to mRNAs encoding ferredoxin-A. ACP2, ACP3, and ACP4 mRNA levels were also examined in Arabidopsis cell suspension culture and were found to be differentially controlled by metabolic and/or growth derived signals. Comparison of 5′-untranslated regions (UTRs) of ACP mRNAs of diverse plant species revealed two motifs that have been conserved during evolution, a CTCCGCC box and C-T-rich sequences. Fusions of the 5′-UTR sequences of ACP1 and ACP2 to luciferase and expression in transgenic plants indicated that the ACP1 leader contributes to preferential expression in seeds, whereas the ACP2 5′-UTR favored expression in roots. The deletion of 58 bp containing the conserved motifs of the ACP1 5′-UTR resulted in 10- to 20-fold lower gene expression in leaf and seed tissues of transgenic Arabidopsis plants.
This study was conducted to develop an antibiotics marker-free potato (Solanum tuberosum L., cv. Taedong valley) plant having resistance against two herbicides. Agrobacterium tumefaciens strain EHA105, harboring a binary vector plasmid pCAMBIA3300 containing bar gene under the control of a promoter CaMV35S and linked CP4-EPSPS genes driven by CaMV35S promoter, was used in the current study. The leaf segments of newly bred potato variety (cv. Taedong Valley) was co-cultured with Agrobacterium. Then, the regenerated individual shoots were excised and transferred to potato multiplication medium supplemented with 0.5 mg/L phosphinothricin. The shoots were rooted in MS medium without hormone and obtained putative transgenic plant E3-6. Integration of target genes into the E3-6 plant and their expression was confirmed by PCR, Southern analysis, and ELISA test. The tissue necrosis test on young leaf blade and shikimic acid accumulation test using the tissue of E3-6 plant were conducted to investigate the resistance to glufosinate-ammonium and glyphosate, respectively. The transgenic plants (E3-6) simultaneously showed a high resistance to both herbicides. The same results were surely obtained also in the whole plants foliar-treated with alone or mixture of two herbicides, glufosinate-ammonium and glyphosate.
Jatropha curcas seeds are an excellent biofuel feedstock, but seed yields of Jatropha are limited by its poor flowering and fruiting ability. Thus, identifying genes controlling flowering is critical for genetic improvement of seed yield. We isolated the JcLFY, a Jatropha ortholog of Arabidopsis thaliana LEAFY (LFY), and identified JcLFY function by overexpressing it in Arabidopsis and Jatropha. JcLFY is expressed in Jatropha inflorescence buds, flower buds, and carpels, with highest expression in the early developmental stage of flower buds. JcLFY overexpression induced early flowering, solitary flowers, and terminal flowers in Arabidopsis, and also rescued the delayed flowering phenotype of lfy-15, a LFY loss-of-function Arabidopsis mutant. Microarray and qPCR analysis revealed several flower identity and flower organ development genes were upregulated in JcLFY-overexpressing Arabidopsis. JcLFY overexpression in Jatropha also induced early flowering. Significant changes in inflorescence structure, floral organs, and fruit shape occurred in JcLFY co-suppressed plants in which expression of several flower identity and floral organ development genes were changed. This suggests JcLFY is involved in regulating flower identity, floral organ patterns, and fruit shape, although JcLFY function in Jatropha floral meristem determination is not as strong as that of Arabidopsis.
The gene fatty acid desaturase 2 (FAD2) exists in multiple copies in the Brassica napus genome and encodes an enzyme that catalyzes the conversion of oleic acid to linoleic acid. In the present study, we characterized the regulatory region controlling the expression of an FAD2 gene located on chromosome C5 of Brassica napus and named it BnFAD2-C5. A long intron was found within the 5′-untranslated region (5′-UTR) of the BnFAD2-C5 gene. This intron, compared with an intron-less control, conferred up to a sixfold increase in green fluorescent protein (GFP) expression in transgenic Arabidopsis, thus suggesting that it makes function through intron-mediated enhancement. The sequence containing the promoter and intron was identified to promote high levels of gene expression in genital organs, particularly in seeds, using qRT-PCR and transgenic Arabidopsis. We identified the different promoter regions responsible for the tissue-specific gene expression through a deletion analysis of the BnFAD2-C5 promoter and a β-glucuronidase and GFP reporter system. The results showed that the −1020 to −319 bp region primarily controls BnFAD2-C5 gene expression in the root, whereas the −1020 to −581 bp region controls expression in the stem, the −581 to −319 bp region controls expression in the leaf, and the −1257 to −1020 bp region probably controls expression in the floral parts. The −319 to −1 bp region is also important, conferring high-level transcription in the seeds. The transcription of BnFAD2-C5 could be induced by salicylic acid and jasmonic acid, and the relative response elements were identified in the −1257 to −1020 bp region and −319 to −1 bp region, respectively.
A range of procedures has been developed for transferring specific DNA into plant cells. These include the uptake of DNA by protoplasts (Krens et al. 1982; Potrykus et al. 1985), DNA transfer mediated by Agrobacterium, and more recently the microprojectile (Klein et al. 1987) or shoot gun method, which is of great interest for the transformation of plants whose tissues are recalcitrant to regeneration of whole plants from cells.
Citrus is widely grown throughout the tropical and subtropical regions of the world. However, despite breeding programs that have been vigorously pursued in a number of countries during the last century, most presently grown citrus rootstock and scion cultivars have not arisen through conventional breeding. Instead, they originated as chance seedlings or limb or bud sport mutations that were recognized to be horticulturally superior and vegetatively propagated (Hodgson 1967).
Ginseng (Panax ginseng C.A. Meyer) belongs to the Araliaceae family. In China and Korea, ginseng has been used medicinally for over 1000 years. Old literature recounts that ginseng cultivation in Korea began at least around 400 a.d. and in China around 40 b.c.
The circular plasmid pSRl from Zygosaccharomyces rouxii includes a pair of inverted repeat sequences of 959 bp that contain recombination sites (RS; 58 bp at most) for intramolecular recombination [1], Experiments performed in vitro with this recombination system indicated that the system requires only the R protein, the recombinase, that is encoded by the R gene of pSRl [2]. The pSRl recombination system (R-RS system) is similar, in terms of its recombination mechanism, to the Cre-loxP system derived from bacteriophage Pl [40] and the FLP-FRT system of the 2-µm plasmid of Saccharomyces cerevisiae [7] Use of these site-specific recombination systems in heterologous organisms seems to offer several advantages: recombination takes place only between specific sequences, which are usually several dozen base pairs (bp) in length (high specificity); recombination is catalyzed by a single recombinase protein, and no other protein is required (simple mechanism); and the recombination frequency is remarkably high.
Camelina (Camelina sativa), which belongs to the Brassicaeae family, is an emerging oilseed crop with the potential to expand biodiesel production to arid land. During storage oil synthesis, diacylglycerol acyltrasferase1 (DGAT1) catalyzes the conversion of diacylglycerol (DAG) and free fatty acids to triacylglycerol (TAG). In this study, three DGAT1 genes (CsDGAT1A, CsDGAT1B, and CsDGAT1C) were isolated from developing C. sativa seeds. The deduced amino acid sequences of the three CsDGAT1 genes shared more than 84 % identity with those of DGAT1 genes from Arabidopsis thaliana and Brassica napus. CsDGAT1A, B, and C transcripts were detected in various C. sativa organs, including developing seeds. Fluorescent protein-fused CsDGAT1A, B, and C were localized in the endoplasmic reticulum (ER) of tobacco epidermal cells. When the CsDGAT1A, B, and C genes under the control of the BnNapin promoter were expressed in an Arabidopsis AS11 mutant, which is defective in DGAT1, the amounts and composition of total fatty acids in dry seeds were restored to those of the wild type, indicating the three CsDGAT1 genes to be functionally active. In transgenic C. sativa plants overexpressing CsDGAT1B, the levels of total seed oils were increased by ~24 % compared with non-transgenic lines. Transgenic C. sativa embryos with enhanced seed oil contents harbored larger embryonic cells and a greater number of cells compared with the wild type. Transgenic Camelina plants with increased oil contents can be used as renewable resources for the production of biodiesel and non-petroleum-based biomaterials.
Biotechnology explores the metabolic properties of living organisms for the production of valuable products of a very different structural and organizational level. Plant serves as an important source of primary and secondary metabolites used in pharmacy, biotechnology, and food technology. Plant biotechnology has gained importance in the recent past for augmenting the quality and quantity of agricultural, horticultural, ornamental plants, and in manipulating the plants for improved agronomic performance. Plant tissue culture is the most popular technique of plant biotechnology, which has diverse applications in the various fields. To understand the basic facts related with plant in vitro studies it is worth acknowledging historical principles of plant tissue culture science, which takes its roots from ground-breaking research like discovery of cells followed by the propounding of cell theory. This chapter covers various major historical achievements such as the concept of cellular totipotency, which was inherent in cell theory and was further elaborated by Haberlandt in 1902. This historical account created the scope and development for plant tissue culture science such as research and production of transgenic plants and their products, which could be of use to mankind as food, medicine, and life-saving drugs.
Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.
Expression of the three chlorophyll a/b binding protein (cab) genes of Arabidopsis thaliana was studied in transformed tobacco tissues. For each cab gene, approximately 1000 bp of the promoter region plus a portion of the structural gene was inserted into a promoter-expression vector such that a translational fusion between the cab gene and the promoter-less chloramphenicol acetyltransferase (cat) gene was formed. The constructed molecules were introduced into either cultured tobacco cells or tobacco leaves and the promoter activity was monitored as chloramphenicol acetyltransferase activity. The light-grown tissues exhibited 1.5- to 60-fold greater promoter activity than did dark-grown tissues. Expression of the cab promoters was tissue specific: activities were much stronger in green leaves than other tissues. The cab promoters were almost equally active in transformed calli or shoots derived from leaves. However, in cultured tobacco cells, one promoter was two to three times stronger than the other two. The chimeric gene fusion, cab-cat, segregated in the F1 generation as a dominant Mendelian trait.
Using a promoter expression vector system based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, we have studied the molecular structure of the nopaline synthase (nos) promoter which is active constitutively in transformed plant tissues. The system uses the sensitive and reliable chloramphenicol acetyltransferase (CAT) assay for the analysis of promoter strength in plant cells. Two sets of mutants were generated by sequential deletion of the nos promoter region from both 5 and 3 ends. These promoter fragments were linked to the cat coding sequence within the expression vector. The strength of the mutant promoters was measured in transformed tobacco calli as CAT activity. 3 deletions up to-17 bp did not significantly affect the promoter strength. Further deletions into the TATA box region reduced the promoter strength by about ten-fold. Analysis of the 5 deletion mutants showed that an upstream region is required for the nos promoter activity in addition to the TATA box and CCAAT box regions.
Le crown gall (galle de la couronne) est une tumeur végétale provoquée par Faction spécifique de la bacterieAgrobacterium tumefaciens. Ses plantes notes ne sont en général pas clairement décrites ou sont simplement considérées etre limitées a la classe des dicotylédones. Nous avons examiné des données sur la susceptibilité de 1193 espèces appartenantes à 588 genres et 138 families; 643 sont des plantes nôtes qui appartiennent à 331 genres et 93 families. Notre liste semble être la plus grande source d’information concernant la susceptibilité des plantes à la galle de la couronne. En outre, nous avons essayé d’établir un rapport entre la susceptibilité des plantes au crown gall et les relations taxonomiques connues et/ou proposées (d’après les systèmes taxonomiques d’Engler et de Takhtajan). On ne connaît pas une plante inférieure qui est sensible au crown gall. Soixante pour cent des gymnospermes et des dicotylées étudiées ont développé des tumeurs. II n’y a pas de rapport significatif entre la position taxonomique d’une famille dicotylée et sa sensibilité. Selon la litérature les monocotylées sensibles appartiennent toutes aux ordres desLiliales et desArales. L’opinion générate que les plantes nôtes du crown gall appartiennent toutes à la classe des dicotyledones est donc inexacte.
The effect of a large number of Tn3 insertions in the vir region of the Ti plasmid pTiA6NC on the virulence of Agrobacterium was determined. The Vir- insertions were mapped in three of the five loci that have been defined previously. Merodiploid Rec- strains carrying one insertion mutation on the Ti plasmid and another insertion mutation (or the homologous wild-type region) on a compatible plasmid were constructed and used in complementation tests for virulence in test plants. This analysis has revealed that there are ten units of gene expression, presumably transcription units in the vir region. Mutation in one of these units is confirmed to be dominant while those in all others are recessive. Co-infection of test plants with pairs of insertion mutants did not restore virulence.
Mutants with Tn5 insertions in the vir region of the Agrobacterium tumefaciens TiC58 plasmid are unable to form crown-gall tumors. Complementation tests of these vir region mutants were carried out by constructing merodiploids in a recombination-deficient strain. Each merodiploid possessed a mutant TiC58 plasmid and a recombinant plasmid containing either the homologous wild-type DNA region or the homologous region containing a second Tn5 insertion. The analysis identified six complementation groups. Mutations in one of these complementation groups were not complemented in trans and represent a cis-dominant locus. The mutation in one complementation group showed variation in host range.
We have constructed a set of small vectors based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which allow the transfer of exogenous DNA into plant chromosomes. These vectors contain: (i) a chimeric gene containing the transcriptional control signals from the nopaline synthase gene and the coding sequence for neomycin phosphotransferase; (ii) the ColE1 replicon; (iii) the cos site of bacteriophage lambda; (iv) the border sequences from the ends of the T-DNA region of the Ti plasmid; and (v) a wide host range replicon. Due to the small size of these cosmid vectors, DNA fragments up to 35 kbp can be inserted by an in vitro packaging method in Escherichia coli. The ability of these vectors to be stably replicated in both E. coli and A. tumefaciens allows their subsequent transfer to and maintenance in Agrobacterium without intermediate genetic manipulations. We demonstrate that DNA cloned into these vectors in A. tumefaciens can efficiently transform plants when in trans with a wild-type Ti plasmid which donates the functions necessary for DNA transfer and integration. We also show that only the right border of the T-DNA is necessary for DNA transformation.