Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.
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ABSTRACT:
SUBMITTER: Rolloos M
PROVIDER: S-EPMC4648448 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:The soil bacterium Agrobacterium tumefaciens is a preferred gene vector not only for plants but also for fungi. Agrobacterium delivers a small set of virulence proteins into host cells concomitantly with transferred DNA (T-DNA) to support the transformation process. Here, we find that expression of one of these proteins, called VirD5, in yeast host cells causes replication stress and DNA damage. This can result in both genomic rearrangements and local mutations, especially small deletions. Delivery of VirD5 during cocultivation with Agrobacterium led to mutations in the yeast genome that were unlinked to the integration of T-DNA. This load of mutations can be prevented by using a virD5 mutant for genome engineering, but this leads to a lower transformation frequency.
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Project description:The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.
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Project description:BackgroundPlant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.ResultsA robust, simple and reproducible barley transformation protocol has been developed that yields average transformation efficiencies of 25%. This protocol is based on the infection of immature barley embryos with Agrobacterium strain AGL1, carrying vectors from the pBract series that contain the hpt gene (conferring hygromycin resistance) as a selectable marker. Results of large scale experiments utilising the luc (firefly luciferase) gene as a reporter are described. The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of the luc gene.ConclusionThis protocol demonstrates significant improvements in both efficiency and ease of use over existing barley transformation methods. This opens up opportunities for the development of functional genomics resources in barley.
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Project description:Agrobacterium tumefaciens is a plant pathogenic bacterium that causes neoplastic growths, called 'crown gall', via the transfer and integration of transferred DNA (T-DNA) from the bacterium into the plant genome. We characterized an acetosyringone (AS)-induced tumour-inducing (Ti) plasmid gene, tzs (trans-zeatin synthesizing), that is responsible for the synthesis of the plant hormone cytokinin in nopaline-type A. tumefaciens strains. The loss of Tzs protein expression and trans-zeatin secretions by the tzs frameshift (tzs-fs) mutant is associated with reduced tumorigenesis efficiency on white radish stems and reduced transformation efficiencies on Arabidopsis roots. Complementation of the tzs-fs mutant with a wild-type tzs gene restored wild-type levels of trans-zeatin secretions and transformation efficiencies. Exogenous application of cytokinin during infection increased the transient transformation efficiency of Arabidopsis roots infected by strains lacking Tzs, which suggests that the lower transformation efficiency resulted from the lack of Agrobacterium-produced cytokinin. Interestingly, although the tzs-fs mutant displayed reduced tumorigenesis efficiency on several tested plants, the loss of Tzs enhanced tumorigenesis efficiencies on green pepper and cowpea. These data strongly suggest that Tzs, by synthesizing trans-zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens.
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Project description:Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is getting more attention recently. Several candidate genes involved in cannabinoid biosynthesis have been elucidated using omics analysis. However, the gene function was not fully validated due to few reports of stable transformation for Cannabis tissues. In this study, we firstly report the successful generation of gene-edited plants using an Agrobacterium-mediated transformation method in C. sativa. DMG278 achieved the highest shoot induction rate, which was selected as the model strain for transformation. By overexpressing the cannabis developmental regulator chimera in the embryo hypocotyls of immature grains, the shoot regeneration efficiency was substantially increased. We used CRISPR/Cas9 technology to edit the phytoene desaturase gene and finally generated four edited cannabis seedlings with albino phenotype. Moreover, we propagated the transgenic plants and validated the stable integration of T-DNA in cannabis genome.
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Project description:Agrobacterium infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301-bar was used for transformation. The two-stage selection was carried out using 4 and 250 mg l-1 BASTA® to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6 min and vacuum infiltrated for 3 min in Agrobacterium suspension, and co-cultivated on MS medium supplemented with 150 µM acetosyringone for 3 days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.
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Project description:Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone "Nanlin895" (Populus deltoides×P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.
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Project description:High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide (glufosinate) painting, and QuickStix Kit for Liberty Link (bar) were used to verify the positive transgenic plants, and absolute quantification PCR confirmed the exogenous gene existed as one to three copies in the soybean genome. This study provides an improved protocol for Agrobacterium-mediated transformation in soybean and a useful reference to improve the transformation efficiency in other plant species.
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Project description:Sorghum (Sorghum bicolor L.) ranks as the fifth most widely planted cereal in the world and is used for food as well as a biomass plant for ethanol production. Use of the TX430 non-tannin sorghum variety has enhanced Agrobacterium-mediated sorghum transformation. These protocols could not be applied, however, to other tannin producing sorghum varieties such as the BTx623 model cultivar for sorghum with full genome information of sorghum. Here we report an improved protocol for Agrobacterium-mediated genetic transformation of tannin-producing sorghum variety BTx623. We successfully developed modification of root regeneration condition for generation of transgenic plant of BTx623. We inoculated immature embryos with Agrobacterium tumefaciens strain EHA105 harboring pMDC32-35S-GFP to generate transgenic plants. In the root regeneration step, we found that regeneration from transformed calli was affected by tannin. For root regeneration, shoots that appeared were not transferred to agar plate, but instead transferred to vermiculite in a plastic pod. Direct planting of regenerated shoots into vermiculite prevented the toxic effect of tannin. Root regeneration efficiency from calli emerged shoots in vermiculite was 78.57%. Presence of sGFP transgene in the genome of transgenic plants was confirmed by PCR and sGFP expression was confirmed in transgenic plants. This improved protocol of Agrobacterium-mediated transformation for tannin-producing sorghum BTx623 could be a useful tool for functional genomics using this plant.
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Project description:Oomycetes are a group of filamentous microorganisms that include some of the biggest threats to food security and natural ecosystems. However, much of the molecular basis of the pathogenesis and the development in these organisms remains to be learned, largely due to shortage of efficient genetic manipulation methods. In this study, we developed modified transformation methods for two important oomycete species, Phytophthora infestans and Plasmopara viticola, that bring destructive damage in agricultural production. As part of the study, we established an improved Agrobacterium-mediated transformation (AMT) method by prokaryotic expression in Agrobacterium tumefaciens of AtVIP1 (VirE2-interacting protein 1), an Arabidopsis bZIP gene required for AMT but absent in oomycetes genomes. Using the new method, we achieved an increment in transformation efficiency in two P. infestans strains. We further obtained a positive GFP transformant of P. viticola using the modified AMT method. By combining this method with the CRISPR/Cas12a genome editing system, we successfully performed targeted mutagenesis and generated loss-of-function mutations in two P. infestans genes. We edited a MADS-box transcription factor-encoding gene and found that a homozygous mutation in MADS-box results in poor sporulation and significantly reduced virulence. Meanwhile, a single-copy avirulence effector-encoding gene Avr8 in P. infestans was targeted and the edited transformants were virulent on potato carrying the cognate resistance gene R8, suggesting that loss of Avr8 led to successful evasion of the host immune response by the pathogen. In summary, this study reports on a modified genetic transformation and genome editing system, providing a potential tool for accelerating molecular genetic studies not only in oomycetes, but also other microorganisms.
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