Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance.

KEY MESSAGE: Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance. ABSTRACT: Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. 'Richter 110' rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58.

Identification of putative transcription factor binding sites in rodent selenoprotein W promoter.

To understand transcriptional regulation of the selenoprotein W (SeW) gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter and antibodies, we observed that specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as to the metal response element (MRE). Although competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore, the SeW gene may be a target for Sp1 whose binding to various regulatory sequences of the SeW promoter may activate or repress the transcription of SeW. The MRE, GRE, AP-1 and LF-A1 sites were also tested but no evidence was obtained for specific binding as indicated by lack of competition with unlabeled probes.

Selenoprotein W gene regulation by selenium in L8 cells.

The effects of selenium on selenoprotein W gene expression were examined in cultured L8 rat skeletal muscle cells. Selenoprotein W contains selenium as selenocysteine in the primary protein structure and levels of this selenoprotein are affected by selenium. Northern blots indicated that there were no significant changes (P < 0.05) in selenoprotein W mRNA levels during cell proliferation and differentiation. Reduction of selenium concentration in the medium decreased the selenoprotein W mRNA levels. Nuclear run-on experiments with isolated L8 nuclei showed the same rate of selenoprotein W mRNA synthesis in cells cultured in either low selenium or selenium supplemented medium, suggesting that the transcription rate of the selenoprotein W gene is independent of selenium. Measurement of the selenoprotein W mRNA half-life in myoblasts treated with the transcription inhibitor, alpha-amanitin, showed that selenoprotein W mRNA levels decreased over time with an estimated half-life of 57 h for cells grown in low selenium medium. Selenium treatment increased the selenoprotein W mRNA half-life 2-fold. These data suggest that selenium stabilizes selenoprotein W mRNA but has no effect on transcription.

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58.

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.

The Agrobacterium tumefaciens chaperone-like protein, VirE1, interacts with VirE2 at domains required for single-stranded DNA binding and cooperative interaction.

Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.

Purification, characterization, and glutathione binding to selenoprotein W from monkey muscle.

Selenoprotein W was purified from monkey skeletal muscle to investigate its binding of glutathione. The purification was accomplished by concentration of the cytosol with an Amicon cell, gel filtration using Sephadex G-50, cation-exchange chromatography with CM-Sephadex, and reverse-phase high-pressure liquid chromatography using a C-18 Vydac column. Selenoprotein W was monitored during purification by slot blots. These steps resulted in an electrophoretically pure selenoprotein W preparation that was estimated by gel filtration to have molecular weight of about 10 kDa. N-terminal amino acid sequencing was used to confirm that the pure proteins were selenoprotein W. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) revealed that the proteins existed in three masses of 9635 +/- 7, 9371 +/- 11, and 9330 +/- 5 Da. The theoretical mass of the protein predicted from the cDNA sequence is 9330 Da. The 9635-Da form of the protein was shown to contain bound glutathione (306 Da), which could be released by reduction with dithiothreitol at 50 degreesC. The form with a mass of 9371 Da is assumed to result from binding of an unidentified 41-Da moiety to the 9330-Da form of the protein. MALDI peptide mapping with endoproteinase Glu-C suggested that glutathione is bound to the 36th amino acid (cysteine) of selenoprotein W.

The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor sigmaS and the stress response in Pseudomonas fluorescens Pf-5.

Three global regulators are known to control antibiotic production by Pseudomonas fluorescens. A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production. A mutation in rpoS, which encodes the stationary-phase sigma factor sigmaS, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P. fluorescens to survive exposure to oxidative stress. The gacA gene of P. fluorescens Pf-5 was isolated, and the influence of gacS and gacA on rpoS transcription, sigmaS levels, and oxidative stress response of Pf-5 was determined. We selected a gacA mutant of Pf-5 that contained a single nucleotide substitution within a predicted alpha-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, sigmaS content in gacS and gacA mutants of Pf-5 was less than 20% of the wild-type level. Transcription of rpoS, assessed with an rpoS-lacZ transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase. Mutations in gacS and gacA compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress. The results of this study provide evidence for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in P. fluorescens.

Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration.

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an omega mutation. In addition, a mutant VirD2 protein lacking the omega domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the omega and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the omega domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

Conserved features of selenocysteine insertion sequence (SECIS) elements in selenoprotein W cDNAs from five species.

SECIS elements form stem-loop structures in the 3' untranslated regions (UTR) of eukaryotic mRNAs that encode selenoproteins. These elements direct incorporation of selenocysteine at UGA codons, provided the SECIS element lies a sufficient distance from the UGA. The cDNAs encoding skeletal muscle selenoprotein W from human, rhesus monkey, sheep, rat, and mouse contained highly similar SECIS elements that retained important features common to all known SECIS elements. Comparative analysis of these SECIS elements showed that in some regions both predicted secondary structure and nucleotide sequences were conserved, in other areas secondary structure was maintained using different primary sequence, and in still other portions, base pairing was not conserved. The rodent and sheep selenoprotein W mRNAs used UGA as a stop codon and as a selenocysteine codon. Thus, UGA specified both selenocysteine incorporation and termination in a single mRNA. The selenoprotein W SECIS elements contained an additional highly conserved base-paired stem that may prevent inappropriate selenocysteine incorporation at the UGA stop codons.

Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2.

VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells.

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.

Rat skeletal muscle selenoprotein W: cDNA clone and mRNA modulation by dietary selenium.

Rat skeletal muscle selenoprotein W cDNA was isolated and sequenced. The isolation strategy involved design of degenerate PCR primers from reverse translation of a partial peptide sequence. A reverse transcription-coupled PCR product from rat muscle mRNA was used to screen a muscle cDNA library prepared from selenium-supplemented rats. The cDNA sequence confirmed the known protein primary sequence, including a selenocysteine residue encoded by TGA, and identified residues needed to complete the protein sequence. RNA folding algorithms predict a stem-loop structure in the 3' untranslated region of the selenoprotein W mRNA that resembles selenocysteine insertion sequence (SE-CIS) elements identified in other selenocysteine coding cDNAs. Dietary regulation of selenoprotein W mRNA was examined in rat muscle. Dietary selenium at 0.1 ppm as selenite increased muscle mRNA 4-fold relative to a selenium-deficient diet. Higher dietary selenium produced no further increase in mRNA levels.

Universal PCR primers for detection of phytopathogenic Agrobacterium strains.

Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants.

A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation.

The T-DNA portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events generate linear single-stranded VirD2-bound DNA molecules that include the entire T-DNA (T-strands). VirD2 protein contains a nuclear localization signal (NLS) near the C terminus and may direct bound T-strands to plant nuclei. We constructed mutations in virD2 and showed that the NLS was important for tumorigenesis, although T-strand production occurred normally in its absence. A tobacco etch virus NLS, substituted for the VirD2 NLS, restored tumor-inducing activity. Amino acids (the omega sequence) at the C terminus of VirD2, outside the NLS and the endonuclease domain, contributed significantly to tumorigenesis, suggesting that VirD2 may serve a third important function in T-DNA transfer.

Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism.

During crown gall tumorigenesis, part of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid, the T-DNA, integrates into plant DNA. Direct repeats define the left and right ends of the T-DNA, but tumorigenesis requires only the right-hand repeat. Virulence (vir) genes act in trans to mobilize the T-DNA into plant cells. Transfer of T-DNA begins when the VirD endonuclease cleaves within the right-hand border repeat. Although the T-DNA right-border repeat promotes T-DNA transmission best in its normal orientation, an inverted right border exhibits reduced but significant activity. Two models may account for this diminished tumorigenesis. The right border may function bidirectionally, with strong activity only in its wild-type orientation, or it may promote T-DNA transfer in a unidirectional manner such that, with an inverted right border, transfer proceeds around the entire Ti plasmid before reaching the T-DNA. To determine whether a substantial portion of the Ti plasmid is transferred to plant cells, as predicted by the unidirectional-transfer hypothesis, we examined T-DNAs in tumors induced by strains containing a Ti plasmid with a right border inverted with respect to the T-DNA oncogenes. These tumors contained extremely long T-DNAs corresponding to most or all of the Ti plasmid. To test whether the right border can function bidirectionally, we inserted T-DNAs with either a properly oriented or an inverted right border into a specific site in the A. tumefaciens chromosome. A border situated to transfer the oncogenes first directed T-DNA transfer even from the bacterial chromosome, whereas a border in the opposite (inverted) orientation did not transfer the oncogenes to plant cells. Our results indicate that the right-border repeat functions in a unidirectional manner.

Stimulation of Agrobacterium tumefaciens T-DNA transfer by overdrive depends on a flanking sequence but not on helical position with respect to the border repeat.

T-DNA transfer by Agrobacterium tumefaciens depends on the right border repeat of the T-DNA and is greatly stimulated by overdrive, an adjacent sequence. We report that the function of overdrive does not depend on helical position with respect to the border repeat. A synthetic 24-bp overdrive and a 12-bp region containing a fully conserved 8-bp core overdrive sequence stimulated virulence equally, but full function required additional bases to the left of the 24-bp sequence.

Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers the T-DNA portion of its Ti plasmid to the nuclear genome of plant cells. Upon cocultivation of A. tumefaciens A348 with regenerating tobacco leaf protoplasts, six distinct single-stranded T-DNA molecules (T strands) were generated in addition to double-stranded T-DNA border cleavages which we have previously reported (K. Veluthambi, R.K. Jayaswal, and S.B. Gelvin, Proc. Natl. Acad. Sci. USA 84:1881-1885, 1987). The T region of an octopine-type Ti plasmid has four border repeats delimiting three T-DNA regions defined as T left (TL), T center (TC), and T right (TR). The six T strands generated upon induction corresponded to the TL, TC, TR, TL + TC, TC + TR, and TL + TC + TR regions, suggesting that the initiation and termination of T-strand synthesis can occur at each of the four borders. Most TL + TC + TR T-strand molecules corresponded to the top T-DNA strand, whereas the other five T strands corresponded to the bottom T-DNA strand. Generation of T strands required the virA, virG, and virD operons. Extra copies of vir genes, harbored on cosmids within derivatives of A. tumefaciens A348, enhanced production of T strands. The presence of right and left border repeats in their native orientation is important for the generation of full-length T strands. When a right border repeat was placed in the opposite orientation, single-stranded T-DNA molecules that corresponded to the top strand were generated. Deletion of overdrive, a sequence that flanks right border repeats and functions as a T-DNA transmission enhancer, reduced the level of T-strand generation. Induction of A. tumefaciens cells by regenerating tobacco protoplasts increased the copy number of the Ti plasmid relative to the bacterial chromosome.

Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid.

During crown gall tumorigenesis a specific segment of the Agrobacterium tumefaciens tumour-inducing (Ti) plasmid, the T-DNA, integrates into plant nuclear DNA. Similar 23-bp direct repeats at each end of the T region signal T-DNA borders, and T-DNA transmission (transfer and integration) requires the right-hand direct repeat. A chemically synthesized right border repeat in its wild-type orientation promotes T-DNA transmission at a low frequency; Ti plasmid sequences which normally flank the right repeat greatly stimulate the process. To identify flanking sequences required for full right border activity, we tested the activity of a border repeat surrounded by different amounts of normal flanking sequences. Efficient T-DNA transmission required a conserved sequence (5' TAAPuTPy-CTGTPuT-TGTTTGTTTG 3') which lies to the right of the two known right border repeats. In either orientation, a synthetic oligonucleotide containing this conserved sequence greatly stimulated the activity of a right border repeat, and a deletion removing 15 bp from the right end of this sequence destroyed it stimulatory effect. Thus, wild-type T-DNA transmission required both the 23-bp right border repeat and a conserved flanking sequence which we call overdrive.