Agrobacterium tumefaciens ExoR controls acid response genes and impacts exopolysaccharide synthesis, horizontal gene transfer and virulence gene expression.
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ABSTRACT:
ORGANISM(S): Agrobacterium tumefaciens
PROVIDER: GSE59790 | GEO | 2014/07/26
SECONDARY ACCESSION(S): PRJNA256200
REPOSITORIES: GEO
Similar Datasets
Project description:Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the ΔexoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression.
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2014-07-26 | E-GEOD-59790 | biostudies-arrayexpress
Project description:Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.
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Project description:Two novel insertion sequences, IS1312 and IS1313, were found in pTiBo542, the Ti plasmid of Agrobacterium tumefaciens strains Bo542 and A281. Nucleotide sequencing and Southern hybridization revealed that IS1312 and IS1313 are homologous to Rhizobium meliloti ISRm1 and ISRm2, respectively. IS1312, ISRm1, and another Agrobacterium insertion sequence, IS426, belong to the same IS3 family of insertion sequences; however, IS1312 is more closely related to the Rhizobium ISRm1 than it is to the Agrobacterium IS426. The distribution patterns of these insertion elements and their sequence similarities suggest that IS1312 and IS1313 were horizontally transferred from R. meliloti to A. tumefaciens.
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Project description:Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.
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Project description:To elucidate the nature of plant response to infection and transformation by Agrobacterium tumefaciens, we compared the cDNA-amplified fragment length polymorphism (AFLP) pattern of Agrobacterium- and mock-inoculated Ageratum conyzoides plant cell cultures. From 16,000 cDNA fragments analyzed, 251 (1.6%) were differentially regulated (0.5% down-regulated) 48 h after cocultivation with Agrobacterium. From 75 strongly regulated fragments, 56 were already regulated 24 h after cocultivation. Sequence similarities were obtained for 20 of these fragments, and reverse transcription-PCR analysis was carried out with seven to confirm their cDNA-AFLP differential pattern. Their sequence similarities suggest a role for these genes in signal perception, transduction, and plant defense. Reverse transcription-PCR analysis indicated that four genes involved in defense response are regulated in a similar manner by nonpathogenic bacteria, whereas one gene putatively involved in signal transduction appeared to respond more strongly to Agrobacterium. A nodulin-like gene was regulated only by Agrobacterium. These results demonstrate a rapid plant cell response to Agrobacterium infection, which overlaps a general response to bacteria but also has Agrobacterium-specific features.
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Project description:Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.
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Project description:The two-component regulatory system, composed of virA and virG, is indispensable for transcription of virulence genes within Agrobacterium tumefaciens. However, virA and virG are insufficient to activate transcription from virulence gene promoters within Escherichia coli cells, indicating a requirement for additional A. tumefaciens genes. In a search for these additional genes, we have identified the rpoA gene, encoding the alpha subunit of RNA polymerase (RNAP), which confers significant expression of a virB promoter (virBp)::lacZ fusion in E. coli in the presence of an active transcriptional regulator virG gene. We conducted in vitro transcription assays using either reconstituted E. coli RNAP or hybrid RNAP in which the alpha subunit was derived from A. tumefaciens. The two forms of RNAP were equally efficient in transcription from a sigma(70)-dependent E. coli galP1 promoter; however, only the hybrid RNAP was able to transcribe virBp in a virG-dependent manner. In addition, we provide evidence that the alpha subunit from A. tumefaciens, but not from E. coli, is able to interact with the VirG protein. These data suggest that transcription of virulence genes requires specific interaction between VirG and the alpha subunit of A. tumefaciens and that the alpha subunit from E. coli is unable to effectively interact with the VirG protein. This work provides the basis for future studies designed to examine vir gene expression as well as the T-DNA transfer process in E. coli.
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Project description:Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer.
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Project description:Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvE-MmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site.
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