Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The translocated virulence protein VirD5 causes DNA damage and mutation during Agrobacterium-mediated transformation of yeast.

ABSTRACT: 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.

SUBMITTER: Zhang X

PROVIDER: S-EPMC9668295 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

The translocated virulence protein VirD5 causes DNA damage and mutation during Agrobacterium-mediated transformation of yeast.

Zhang Xiaorong X Hooykaas Marjolein J G MJG van Heusden G Paul GP Hooykaas Paul J J PJJ

Science advances 20221116 46

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 de ...[more]

PMID: 36383666

Similar Datasets

Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.

Project description:Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.

| S-EPMC4648448 | biostudies-other

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.

| S-EPMC4575723 | biostudies-literature

F-Box Gene D5RF Is Regulated by Agrobacterium Virulence Protein VirD5 and Essential for Agrobacterium-Mediated Plant Transformation.

Project description:We previously reported that the Agrobacterium virulence protein VirD5 possesses transcriptional activation activity, binds to a specific DNA element D5RE, and is required for Agrobacterium-mediated stable transformation, but not for transient transformation. However, direct evidence for a role of VirD5 in plant transcriptional regulation has been lacking. In this study, we found that the Arabidopsis gene D5RF (coding for VirD5 response F-box protein, At3G49480) is regulated by VirD5. D5RF has two alternative transcripts of 930 bp and 1594 bp that encode F-box proteins of 309 and 449 amino acids, designated as D5RF.1 and D5RF.2, respectively. D5RF.2 has a N-terminal extension of 140 amino acids compared to D5RF.1, and both of them are located in the plant cell nucleus. The promoter of the D5RF.1 contains two D5RE elements and can be activated by VirD5. The expression of D5RF is downregulated when the host plant is infected with virD5 deleted Agrobacterium. Similar to VirD5, D5RF also affects the stable but not transient transformation efficiency of Agrobacterium. Some pathogen-responsive genes are downregulated in the d5rf mutant. In conclusion, this study further confirmed Agrobacterium VirD5 as the plant transcription activator and identified Arabidopsis thalianaD5RF.1 as the first target gene of VirD5 in regulation.

| S-EPMC7555846 | biostudies-literature

High-throughput Agrobacterium-mediated barley transformation.

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.

| S-EPMC2562381 | biostudies-literature

Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation.

Project description:A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.

| S-EPMC6640349 | biostudies-literature

Agrobacterium-produced and exogenous cytokinin-modulated Agrobacterium-mediated plant transformation.

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.

| S-EPMC6640272 | biostudies-literature

An efficient Agrobacterium-mediated transformation system for poplar.

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.

| S-EPMC4100180 | biostudies-literature

Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.

Project description:The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ0). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.

| S-EPMC5349227 | biostudies-literature

Bacterial transposons are co-transferred with T-DNA to rice chromosomes during Agrobacterium-mediated transformation.

Project description:Agrobacterium tumefaciens is widely utilized for delivering a foreign gene into a plant's genome. We found the bacterial transposon Tn5393 in transgenic rice plants. Analysis of the flanking sequences of the transferred-DNA (T-DNA) identified that a portion of the Tn5393 sequence was present immediately next to the end of the T-DNA. Because this transposon was present in A. tumefaciens strain LBA4404, but not in EHA105 and GV3101, our findings indicated that Tn5393 was transferred from LBA4404 into the rice genome during the transformation process. We also noted that another bacterial transposon, Tn5563, is present in transgenic plants. Analyses of 331 transgenic lines revealed that 26.0% carried Tn5393 and 2.1% contained Tn5563. In most of the lines, an intact transposon was integrated into the T-DNA and transferred to the rice chromosome. More than one copy of T-DNA was introduced into the plants, often at a single locus. This resulted in T-DNA repeats of normal and transposon-carrying TDNA that generated deletions of a portion of the T-DNA, joining the T-DNA end to the bacterial transposon. Based on these data, we suggest that one should carefully select the appropriate Agrobacterium strain to avoid undesirable transformation of such sequences.

| S-EPMC3887757 | biostudies-literature

Agrobacterium virulence factors induce the expression of host DNA repair-related genes without promoting major genomic damage.

Project description:This study aimed to investigate whether the plant DNA damage levels and DNA damage response (DDR) are regulated during Agrobacterium infection and potentially manipulated by Agrobacterium to facilitate T-DNA integration. We investigated the plant genomic response to Agrobacterium infection by measuring gamma H2AX levels, which reflect the levels of double-strand DNA breaks (DSBs), and by characterizing transcription of three major DNA repair marker genes NAC82, KU70, and AGO2. These experiments revealed that, globally, Agrobacterium infection did not result in a major increase in DSB content in the host genome. The transcription of the DNA damage repair genes, on the other hand, was elevated upon the wild-type Agrobacterium infection. This transcriptional outcome was largely negated by a mutation in the bacterial virB5 gene which encodes the virulence (Vir) protein B5, a minor component of Agrobacterium pilus necessary for the translocation of Vir effector proteins into the host cell, suggesting that the transcriptional activation of the cellular DNA damage repair machinery requires the transport into the host cell of the Agrobacterium effectors, i.e., the VirD2, VirD5, VirE2, VirE3, and VirF proteins. Most likely, a combination of several of these Vir effectors is required to activate the host DNA repair as their individual loss- or gain-of-function mutants did not significantly affect this process.

| S-EPMC11487168 | biostudies-literature