A Genome-Wide Survey of Highly Expressed Non-Coding RNAs and Biological Validation of Selected Candidates in Agrobacterium tumefaciens (original) (raw)
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Novel ways of regulating Ti plasmid functions were investigated by studying small RNAs (sRNAs) that are known to act as posttranscriptional regulators in plant pathogenic bacteria. sRNA-seq analyses of Agrobacterium fabrum C58 allowed us to identify 1,108 small transcripts expressed in several growth conditions that could be sRNAs. A quarter of them were confirmed by bioinformatics or by biological experiments. Antisense RNAs represent 24% of the candidates and they are over-represented on the pTi (with 62% of pTi sRNAs), suggesting differences in the regulatory mechanisms between the essential and accessory replicons. Moreover, a large number of these pTi antisense RNAs are transcribed opposite to those genes involved in virulence. Others are 5′- and 3′-untranslated region RNAs and trans-encoded RNAs. We have validated, by rapid amplification of cDNA ends polymerase chain reaction, the transcription of 14 trans-encoded RNAs, among which RNA1111 is expressed from the pTiC58. Its del...
Scientific Reports
LysR-type transcriptional regulators (LTTRs) are the most common type of transcriptional regulators in prokaryotes and function by altering gene expression in response to environmental stimuli. In the class Alphaproteobacteria, a conserved LTTR named VtlR is critical to the establishment of host-microbe interactions. In the mammalian pathogen Brucella abortus, VtlR is required for full virulence in a mouse model of infection, and VtlR activates the expression of abcR2, which encodes a small regulatory RNA (sRNA). In the plant symbiont Sinorhizobium meliloti, the ortholog of VtlR, named LsrB, is involved in the symbiosis of the bacterium with alfalfa. Agrobacterium tumefaciens is a close relative of both B. abortus and S. meliloti, and this bacterium is the causative agent of crown gall disease in plants. In the present study, we demonstrate that VtlR is involved in the ability of A. tumefaciens to grow appropriately in artificial medium, and an A. tumefaciens vtlR deletion strain is...
The Agrobacterium VirE3 effector protein: a potential plant transcriptional activator
Nucleic Acids Research, 2006
During the infection of plants, Agrobacterium tumefaciens introduces several Virulence proteins including VirE2, VirF, VirD5 and VirE3 into plant cells in addition to the T-DNA. Here, we report that double mutation of virF and virE3 leads to strongly diminished tumor formation on tobacco, tomato and sunflower. The VirE3 protein is translated from a polycistronic mRNA containing the virE1, virE2 and virE3 genes, in Agrobacterium. The VirE3 protein has nuclear localization sequences, which suggests that it is transported into the plant cell nucleus upon translocation. Indeed we show here that VirE3 interacts in vitro with importin-a and that a VirE3-GFP fusion protein is localized in the nucleus. VirE3 also interacts with two other proteins, viz. pCsn5, a component of the COP9 signalosome and pBrp, a plant specific general transcription factor belonging to the TFIIB family. We found that VirE3 is able to induce transcription in yeast when bound to DNA through the GAL4-BD. Our data indicate that the translocated effector protein VirE3 is transported into the nucleus and there it may interact with the transcription factor pBrp to induce the expression of genes needed for tumor development.
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Agrobacterium tumefaciens is a plant pathogen that is widely used in plant transformation. As the process of transgenesis includes the delivery of single-stranded T-DNA molecule, we hypothesized that transformation rate may negatively correlate with the efficiency of the RNA-silencing machinery. Using mutants compromised in either the transcriptional or post-transcriptional gene-silencing pathways, two inhibitors of stable transformation were revealed-AGO2 and NRPD1a. Furthermore, an immunoprecipitation experiment has shown that NRPD1, a subunit of Pol IV, directly interacts with Agrobacterium T-DNA in planta. Using the Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) technique, we demonstrated that the transient down-regulation of the expression of either AGO2 or NRPD1a genes in reproductive organs of Arabidopsis, leads to an increase in transformation rate. We observed a 6.0-and 3.5-fold increase in transformation rate upon transient downregulation of either AGO2 or NRPD1a genes, respectively. This is the first report demonstrating the increase in the plant transformation rate via VIGS-mediated transient down-regulation of the components of epigenetic machinery in reproductive tissue.
Capturing the VirA/VirG TCS of Agrobacterium tumefaciens
Advances in Experimental Medicine and Biology
T wo-component systems (TCS) regulate pathogenic commitment in many interactions and provide an opportunity for unique therapeutic intervention. The VirA/VirG TCS of Agrobacterium tumefaciens mediates inter-kingdom gene transfer in the development of host tumors and sets in motion the events that underlie the great success of this multi-host plant pathogen. Significant proof for the feasibility of interventions has now emerged with the discovery of a natural product that effectively "blinds" the pathogen to the host via inhibition of VirA/VirG signal transduction. Moreover, the emerging studies on the mechanism of signal perception have revealed general sites suitable for intervention of TCS signaling. Given the extensive functional homology, it should now be possible to transfer the models discovered for VirA/VirG broadly to other pathogenic interactions.
Journal of Bacteriology, 1999
The two-component regulatory system, composed of virAand 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 α subunit of RNA polymerase (RNAP), which confers significant expression of avirB promoter (virBp)::lacZ fusion in E. coli in the presence of an active transcriptional regulatorvirG gene. We conducted in vitro transcription assays using either reconstituted E. coli RNAP or hybrid RNAP in which the α subunit was derived from A. tumefaciens. The two forms of RNAP were equally efficient in transcription from a ς70-dependent E. coli galP1 promoter; however, only the hybrid RNAP was able to transcribe virBpin a virG-dependent manner. In addition, we provide evidence tha...
Biochemical and Biophysical Research Communications, 2014
The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5 0 leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTT-ATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5 0 UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5 0 UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5 0 leader region upstream of the gene of interest.
Journal of bacteriology, 1990
The VirD1 and VirD2 proteins encoded by an inducible locus of the virulence (vir) region of the Agrobacterium tumefaciens Ti plasmid are required for site-specific nicking at T-DNA border sites. We have determined the nucleotide sequence of a 3.6-kilobase-pair fragment carrying the virD locus from nopaline Ti plasmid pTiC58. In contrast to the previous report (Hagiya et al., Proc. Natl. Acad. Sci. USA 82:2669-2673, 1985), we found that the first three open reading frames were capable of encoding polypeptides of 16.1, 49.7, and 21.4 kilodaltons. Deletion analysis demonstrated that the N-terminal conserved domain of VirD2 was absolutely essential for its endonuclease activity. When extra copies of the virD1 and virD2 genes were present in an A. tumefaciens strain carrying a Ti plasmid, increased amounts of T-strand and nicked molecules could be detected at early stages of vir induction. Such strains possessed the ability to transform plants with higher efficiency.
Journal of Bacteriology
Certain plant phenolic compounds and monosaccharides induce the transcription of virulence (vir) genes of Agrobacterium tumefaciens through the VirA-VirG two-component regulatory system. The product of the chromosomal virulence gene chvE is homologous to galactose-binding protein of Escherichia coli and is required for vir gene induction by sugars. Adjacent to, but divergent in transcription from, chvE is an open reading frame, now termed gbpR (galactose-binding protein regulator), that is homologous to the LysR family of transcriptional regulators. chvE::lacZ expression was induced by L-arabinose, D-galactose, and D-fucose when