In recent years, several tools have become available for improved gene-targeting (GT) in plants. DNA breaks at specific sites activate local DNA repair and recombination, including recombination with ...ectopic sequences leading to GT. Large-scale transformation with the repair template can be avoided by pre-insertion of the repair template in the genome and liberation by sequence-specific nucleases (in planta GT procedure). Here, we tested whether release of the repair template was required for GT. Plants were transformed with constructs encoding a CRISPR/Cas nuclease with a recognition site in the endogenous PPO gene and a repair template harboring a 5' truncated PPO gene with two amino acid substitutions rendering the enzyme insensitive to the herbicide butafenacil. Selection resulted in so-called true GT events, repaired via homologous recombination at both ends of the gene and transmitted to the next generation. As the template was surrounded by geminiviral LIR sequences, we also tested whether replication of the template could be induced by crossing-in an integrated geminivirus REP gene. However, we could not find evidence for repair template replication by REP and we obtained similar numbers of GT events in these plants. Thus, GT is possible without any further processing of the pre-inserted repair template.
Agrobacterium tumefaciens is the causative agent of crown gall disease and is widely used as a vector to create transgenic plants. Under laboratory conditions, the yeast Saccharomyces cerevisiae and ...other yeasts and fungi can also be transformed, and Agrobacterium‐mediated transformation (AMT) is now considered the method of choice for genetic transformation of many fungi. Unlike plants, in S. cerevisiae, T‐DNA is integrated preferentially by homologous recombination and integration by non‐homologous recombination is very inefficient. Here we report that upon deletion of ADA2, encoding a component of the ADA and SAGA transcriptional adaptor/histone acetyltransferase complexes, the efficiency of AMT significantly increased regardless of whether integration of T‐DNA was mediated by homologous or non‐homologous recombination. This correlates with an increase in double‐strand DNA breaks, the putative entry sites for T‐DNA, in the genome of the ada2Δ deletion mutant, as visualized by the number of Rad52‐GFP foci. Our observations may be useful to enhance the transformation of species that are difficult to transform.
Significance and Impact of the Study: In this study, we have shown that deletion of ADA2, encoding a component of the ADA and SAGA transcriptional adaptor/histone acetyltransferase complexes, from the yeast Saccharomyces cerevisiae resulted in an increased efficiency of Agrobacterium‐mediated transformation. This increased efficiency occurred irrespective of whether T‐DNA integrates by homologous or non‐homologous recombination. The effect on T‐DNA integration by non‐homologous recombination is of special importance as this process is very inefficient in S. cerevisiae. This result may open ways to improve transformation protocols for fungi and yeasts that are difficult to transform.
Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be ...subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.
Agrobacterium tumefaciens transfers part of its Ti plasmid, the T-DNA, to plant cells during tumorigenesis. It is routinely used for the genetic modification of a wide range of plant species. We ...report that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungus. We transformed both protoplasts and conidia with frequencies that were improved up to 600-fold as compared with conventional techniques for transformation of A. awamori protoplasts. The majority of the A. awamori transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. The T-DNA integrated into the A. awamori genome in a manner similar to that described for plants. We also transformed a variety of other filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus, demonstrating that transformation using A. tumefaciens is generally applicable to filamentous fungi.
We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter). The protein encoded by the ZAT (Zn transporter of Arabidopsis thaliana) gene has 398 ...amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants.
The bases of crown gall tumorigenesis Zhu, J; Oger, P M; Schrammeijer, B ...
Journal of bacteriology,
07/2000, Letnik:
182, Številka:
14
Journal Article
Recenzirano
Odprti dostop
Transfer of Ti plasmids into certain nonpathogenic bacteria converts them into tumorigenic pathogens. Most of the genes of the Ti plasmid play direct or indirect roles in some aspect of tumorigenesis ...or tumor colonization.
The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease. In addition, several Virulence proteins must somehow be ...transported to fulfill a function in planta. Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells. Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA.
Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome. Here, we ...show that when this Ti DNA shares homology with the A. awamori genome, integration can also occur by homologous recombination. On the basis of this finding, we have developed an efficient method for constructing recombinant mold strains free from bacterial DNA by A. tumefaciens-mediated transformation. Multiple copies of a gene can be integrated rapidly at a predetermined locus in the genome, yielding transformants free of bacterial antibiotic resistance genes or other foreign DNA. Recombinant A. awamori strains were constructed containing up to nine copies of a Fusarium solani pisi cutinase expression cassette integrated in tandem at the pyrG locus. This allowed us to study how mRNA and protein levels are affected by gene copy number, without the influence of chromosomal environmental effects. Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level. This transformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.
Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T‐DNA), derived from its tumour‐inducing (Ti) plasmid, into plant cells at infection ...sites. Besides these natural hosts, Agrobacterium can deliver the T‐DNA also to monocotyledonous plants, yeasts and fungi. The T‐DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T‐DNA recipient to demonstrate that the non‐homologous end‐joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T‐DNA into the host genome. We discovered a minor pathway for T‐DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T‐DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements.
Type I IFNs play critical roles in orchestrating the antiviral defense by inducing direct antiviral activities and shaping the adaptive immune response. Viruses have evolved numerous strategies to ...specifically interfere with IFN production or its downstream mediators, thereby allowing successful infection of the host to occur. The prototypic human gammaherpesvirus EBV, which is associated with infectious mononucleosis and malignant tumors, harbors many immune-evasion proteins that manipulate the adaptive and innate immune systems. In addition to proteins, the virus encodes >40 mature microRNAs for which the functions remain largely unknown. In this article, we identify EBV-encoded miR-BART16 as a novel viral immune-evasion factor that interferes with the type I IFN signaling pathway. miR-BART16 directly targets CREB-binding protein, a key transcriptional coactivator in IFN signaling, thereby inducing CREB-binding protein downregulation in EBV-transformed B cells and gastric carcinoma cells. miR-BART16 abrogates the production of IFN-stimulated genes in response to IFN-α stimulation and it inhibits the antiproliferative effect of IFN-α on latently infected BL cells. By obstructing the type I IFN-induced antiviral response, miR-BART16 provides a means to facilitate the establishment of latent EBV infection and enhance viral replication.
PDF
