Activin-induced factors maintain goosecoid transcription through a paired homeodomain binding site.

Previous studies in both Xenopus and zebrafish have shown that goosecoid is one of the first genes to be transcribed at the onset of gastrulation. Goosecoid transcription still initiates when embryos are treated with protein synthesis inhibitors, indicating that it is mediated by preexisting factors and suggesting that goosecoid transcription is immediately downstream of the maternal mesoderm-inducing signal. However, goosecoid transcription continues long after this maternal signal has ceased to be active, indicating that there are mechanisms to maintain activin-induced transcription. Our study has focused on understanding the factors required to maintain this transcription. We have defined an element within the zebrafish goosecoid promoter that is sufficient for activin inducibility in both Xenopus and zebrafish embryos. This element, the goosecoid activin element, interacts with two developmentally regulated proteins from Xenopus embryos. A maternal protein interacts through cleavage stages until the midblastula transition, and a second protein binds from the onset of gastrulation. The second protein is zygotically expressed, and its binding is required for activin inducibility in our assay system. We suggest that the zygotic protein we have identified is a good candidate to be involved in the maintenance of goosecoid transcription. Furthermore, this zygotic protein is likely to contain a paired class homeodomain since a consensus binding site for such proteins is present within the goosecoid activin element and is essential for its function.

Neural induction by the secreted polypeptide noggin.

The Spermann organizer induces neural tissue from dorsal ectoderm and dorsalizes lateral and ventral mesoderm in Xenopus. The secreted factor noggin, which is expressed in the organizer, can mimic the dorsalizing signal of the organizer. Data are presented showing that noggin directly induces neural tissue, that it induces neural tissue in the absence of dorsal mesoderm, and that it acts at the appropriate stage to be an endogenous neural inducing signal. Noggin induces cement glands and anterior brain markers, but not hindbrain or spinal cord markers. Thus, noggin has the expression pattern and activity expected of an endogenous neural inducer.

Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish.

The zebrafish dorsoventral axis can first be distinguished at gastrulation, upon formation of the embryonic shield, the site of the organizer. We have asked whether the shield is specified before gastrulation. First, we show that brief exposure of premidblastula embryos to lithium, which is known to shut down the phospho-inositol signaling pathway, produces excessive shield formation and extreme hyper-dorsal development. Second, we show that the zebrafish goosecoid homeobox gene is activated at or just after the midblastula in a localized domain of cells that subsequently populate the most anterior region of the incipient shield and axial hypoblast, goosecoid expression is elevated and radialized by early lithium treatment, suggesting that goosecoid plays a role in establishing the organizer and shield. Our results demonstrate that the zebrafish dorsal axis is signaled by a pathway initiated in the cleavage-stage embryo. Furthermore, they provide novel insights into anterior morphogenesis.

A nontransformable Triticum monococcum monocotyledonous culture produces the potent Agrobacterium vir-inducing compound ethyl ferulate.

Exudates of dicotyledonous plants contain specific phenolic signal molecules, such as acetosyringone, which serve as potent inducers for the expression of the virulence (vir) regulon of the phytopathogen Agrobacterium tumefaciens. This induction activates the Agrobacterium T-DNA transfer process to initiate the genetic transformation of target plant cells. Wounded and metabolically active plant cells are particularly susceptible to Agrobacterium infection, and these cells specifically produce vir-inducing molecules. Most monocotyledonous, as opposed to dicotyledonous, species are resistant to Agrobacterium transformation. One hypothesis for this resistance is that nonsusceptible monocotyledonous cells fail to produce vir signal molecules and, thus, are not recognized by Agrobacterium as transformation targets. Here we demonstrate that monocotyledonous cells make such molecules, and, furthermore, we purify the inducer produced by a Triticum monococcum suspension culture that is resistant to Agrobacterium infection. This molecule is shown to correspond to ethyl ferulate [C12H14O4; 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid ethyl ester], to be more active for vir induction at low concentrations than acetosyringone, and to be produced in quantities giving significant levels of induction. Thus, at least for the wheat cell line used in this study, monocotyledonous resistance to Agrobacterium transformation must result from a block to a step of the T-DNA transfer process subsequent to vir induction.

Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5' virD gene products.

Agrobacterium tumefaciens transfers its Ti-plasmid T-DNA to plant cells. This process is initiated by plant-induced activation of the Ti-plasmid virulence loci, resulting in the generation of single stranded (ss) cleavages of the Ti-plasmid T-DNA border sequences (border nicks) and ss linear unipolar T-DNA molecules (T-strands). A single T-strand is produced from the two-border T-region of the pGV3850 nopaline plasmid. In this paper the induced molecular events for the complex T-region of the pTiA6 octopine plasmid are analyzed. This T-region carries four T-DNA borders delimiting three T-DNA elements (TR, TC and TL). Induction of pTiA6 generates cleavages independently at its border repeats, and six distinct T-strand species corresponding to TR, TR/TC, TR/TC/TL, TC, TC/TL and TL. These T-strand molecules are linear and correspond to the bottom strand of the pTiA6 T-region. Thus, borders can function for both initiation and termination of T-strand synthesis. We propose that the different pTiA6 T-strands are independently generated, and that the distribution of border nicks within the parental T-region determines which T-strand is produced. To identify genes involved in T-strand production, pTiA6 virulence (vir) and chromosomal virulence (chv) mutant strains were analyzed. VirA and VirG, the vir regulatory loci are required. Furthermore, the two 5' cistrons of virD are required for both border nicks and T-strands, suggesting that these genes encode the border endonuclease, and that T-strand production is dependent on border nicks. That no mutants are defective for T-strands alone suggests that functions encoded outside of vir and chv might mediate some of the later reactions of T-strand synthesis.

Site-Specific Nick in the T-DNA Border Sequence as a Result of Agrobacterium vir Gene Expression.

The T-DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products act in trans to mobilize the T-DNA element from the bacterial Ti plasmid. The T-DNA is bounded by 25-base pair direct repeat sequences, which are the only sequences on the element essential for transfer. Thus, specific reactions must occur at the border sites to generate a transferable T-DNA copy. The T-DNA border sequences were shown in this study to be specifically nicked after vir gene activation. Border nicks were detected on the bottom strand just after the third or fourth base (+/- one or two nucleotides) of the 25-base pair transferpromoting sequence. Naturally occurring and base-substituted derivatives of the 25-base pair sequences are effective substrates for acetosyringone-induced border cleavage, whereas derivatives carrying only the first 15 or last 19 base pairs of the 25-base pair sequence are not. Site-specific border cleavages occur within 12 hours after acetosyringone induction and probably represent an early step in the T-DNA transfer process.

Characterization of the virE operon of the Agrobacterium Ti plasmid pTiA6.

The Agrobacterium tumefaciens Ti plasmid contains at least six transcriptional units (designated vir loci) which are essential for efficient crown gall tumorigenesis. Mutations in one of these loci, virE, result in a sharply attenuated virulence phenotype. In the present communication, we have analyzed the virE operon at the molecular level. This locus contains open reading frames coding for two hydrophilic proteins having molecular weights of approximately 7,000 daltons and 60,500 daltons. Using a maxicell strain of E. coli, we have visualized two proteins encoded by virE which correspond in size to these open reading frames. Analysis of codon usage of virE and seven other vir loci indicates that, in contrast to E. coli, all possible codons for a given amino acid are utilized at approximately the same frequency.

A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci.

The vir region of Agrobacterium tumefaciens spans at least six transcriptional loci required for crown gall tumorigenesis. The transcriptional induction of two of these vir loci in response to cocultivation with tobacco suspension cells was measured by using bacteria containing mutations in each of the six vir loci located on the Ti plasmid. Induction of these vir genes occurred only in bacteria that had functional copies of virA and virG. The nucleic acid sequence of a 1.25-kilobase clone encompassing virG contains one open reading frame capable of coding for a protein of about 30,000 daltons. The amino acid sequence of the predicted virG product is homologous to that of eight bacterial proteins, including that of the ompR gene of Escherichia coli. Most, although not all, of these proteins, like VirG, are positive regulatory elements.

virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens.

The Ti plasmid vir loci of Agrobacterium tumefaciens are transcriptionally activated in response to signal molecules produced by plant cells to initiate the T-DNA transfer process. We show that the pTiA6 vir loci are organized as a single regulon whose induction by plants is controlled by virA and virG. Mutations in virA result in attenuated induction. This locus is constitutively transcribed and noninducible. Mutations in virG eliminate vir induction. This locus is constitutively transcribed, plant-inducible, and self-regulated in a complex fashion, and it produces two distinct and differentially regulated transcripts. virA is proposed to encode a transport protein for the plant signal molecule, and virG a positive regulatory protein that together with the plant molecule activates vir expression.

The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens.

The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3-lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty-four vir::Tn3-lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta-galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non-inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant-inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant-inducible loci (pin) which are non-essential for virulence.

A plant cell factor induces Agrobacterium tumefaciens vir gene expression.

The virulence genes of Agrobacterium are required for this organism to genetically transform plant cells. We show that vir gene expression is specifically induced by a small (<1000 Da) diffusible plant cell metabolite present in limiting quantities in the exudates of a variety of plant cell cultures. Active plant cell metabolism is required for the synthesis of the vir-inducing factor, and the presence of bacteria does not stimulate this production. vir-inducing factor is (i) heat and cold stable; (ii) pH stable, although vir induction with the factor is sensitive above pH 6.0; and (iii) partially hydrophobic. Induction of vir gene expression was assayed by monitoring beta-galactosidase activity in Agrobacterium strains that carry gene fusions between each of the vir loci and the lacZ gene of Escherichia coli. vir-inducing factor (partially purified on a C-18 column) induces both the expression in Agrobacterium of six distinct loci and the production of T-DNA circular molecules, which are thought to be involved in the transformation process. vir-inducing factor potentially represents the signal that Agrobacterium recognizes in nature as a plant cell susceptible to transformation.

A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium.

The construction and use of a Tn3-lac transposon, Tn3-HoHo1, is described. Tn3-HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta-galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3-HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta-galactosidase activity. Tn3-HoHo1 carries a non-functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3-HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3-HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3-HoHo1 generated lac fusions.