Characterization of the virE locus of Agrobacterium tumefaciens plasmid pTiC58.

The virE locus that is responsible for the efficiency of infection by Agrobacterium tumefaciens (T. Hirooka and C. Kado, J. Bacteriol. 168:237-243, 1986) is located next to the right boundary of the virulence (Vir) region of the nopaline plasmid pTiC58. This locus is very similar to the virE locus of octopine type Ti plasmids on the basis of nucleotide and amino acid sequence comparisons as well as genetic complementation analyses. The nucleotide sequence of virE revealed three open reading frames, arranged as an operon, with a potential coding capacity for proteins of 9, 7.1, and 63.5 kilodaltons. The promoter region of virE was analyzed by using gene fusions to promoterless cat and lux genes. Two different promoters were detected, one which operates in A. tumefaciens and one which operates in Escherichia coli. virE is transcribed from left to right toward the T region. In A. tumefaciens, the expression of virE was induced by acetosyringone and required the presence of pTiC58.     

Mutagenesis of the Agrobacterium VirE2 Single-Stranded DNA-Binding Protein Identifies Regions Required for Self-Association and Interaction with VirE1 and a Permissive Site for Hybrid Protein Construction

The VirE2 single-stranded DNA-binding protein (SSB) of Agrobacterium tumefaciens is required for delivery of T-DNA to the nuclei of susceptible plant cells. By yeast two-hybrid and immunoprecipitation analyses, VirE2 was shown to self-associate and to interact with VirE1. VirE2 mutants with small deletions or insertions of a 31-residue oligopeptide (i31) at the N or C terminus or with an i31 peptide insertion at Leu236 retained the capacity to form homomultimers. By contrast, VirE2 mutants with modifications outside a central region located between residues 320 and 390 retained the capacity to interact with VirE1. These findings suggest the tertiary structure of VirE2 is important for homomultimer formation whereas a central domain mediates formation of a complex with VirE1. The capacity of VirE2 mutants to interact with full-length VirE2 in the yeast Saccharomyces cerevisiae correlated with the abundance of the mutant proteins in A. tumefaciens, suggesting that VirE2 is stabilized by homomultimerization in the bacterium. We further characterized the promoter and N- and C-terminal sequence requirements for synthesis of functional VirE2. A PvirB::virE2 construct yielded functional VirE2 protein as defined by complementation of a virE2 null mutation. By contrast, PvirE or Plac promoter constructs yielded functional VirE2 only if virE1 was coexpressed with virE2. Deletion of 10 or 9 residues from the N or C terminus of VirE2, respectively, or addition of heterologous peptides or proteins to either terminus resulted in a loss of protein function. However, an i31 peptide insertion at Tyr39 had no effect on protein function as defined by the capacity of the mutant protein to (i) interact with native VirE2, (ii) interact with VirE1, (iii) accumulate at abundant levels in A. tumefaciens, and (iv) restore wild-type virulence to a virE2 null mutant. We propose that Tyr39 of VirE2 corresponds to a permissive site for insertion of heterologous peptides or proteins of interest for delivery across kingdom boundaries.     

Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full-length VirD2, VirC2, or VirE2 leads to insertion of aberrant T-DNA structures

Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.     

Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not require VirE1

Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells. The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system. VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell. We analyzed whether the VirE1 chaperone is also essential for transport recognition of VirE2 by the VirB/D4 encoded type IV secretion system. In addition, we assayed whether translocation of VirF and VirE3, which also forms part of the virE operon, is affected by the absence of VirE1. We employed the earlier developed CRAFT (Cre recombinase Reporter Assay For Translocation) assay to detect transfer of Cre::Vir fusion proteins from A. tumefaciens into plants, monitored by stable reconstitution of a kanamycin resistance marker, and into yeast, screened by loss of the URA3 gene. We show that the C-terminal 50 amino acids of VirE2 and VirE3 are sufficient to mediate Cre translocation into host cells, confirming earlier indications of a C-terminal transport signal. This transfer was independent of the presence or absence of VirE1. Besides, the translocation efficiency of VirF is not altered in a virE1 mutant. The results unambiguously show that the VirE1 chaperone is not essential for the recognition of the VirE2 transport signal by the transport system and the subsequent translocation across the bacterial envelope into host cells.     

Control of lysogeny and immunity of Bacillus subtilis temperate bacteriophage SP beta by its d gene.

The d gene from the Bacillus subtilis temperate bacteriophage SP beta was isolated. When introduced into an SP beta-sensitive strain of B. subtilis, the cloned d gene directed the synthesis of a 22-kilodalton protein and conferred on the host immunity to SP beta phage. A frameshift mutation, designated d2, was introduced into the cloned d gene, and it was subsequently crossed back into the SP beta phage genome. The resulting SP beta phage grew lytically and formed clear plaques on sensitive bacteria. Although the cloned d gene confers immunity to the host, we could not detect complementation of the d gene by mixed infection with SP beta d2 and various SP beta c mutants. The nucleotide sequence of the 1,033-base-pair PstI-to-EcoRI fragment containing the d gene was determined; it includes an open reading frame that could potentially encode a protein of 227 amino acids. The gene was mapped within the PstI H fragment on the phage genome, which positions the d gene about 25 kilobases from the right end of the phage genome. It is transcribed from right to left.     

The carboxy-terminus of VirE2 from Agrobacterium tumefaciens is required for its transport to host cells by the virB-encoded type IV transport system

Agrobacterium tumefaciens transfers DNA from the resident 'tumour-inducing' (Ti) plasmid into plant cells, where it can be stably integrated into the plant genome, ultimately resulting in crown gall tumour formation. The mobilized DNA molecule is a single-stranded intermediate with VirD2 covalently bound to its 5' end. Successful transport of the transferred DNA (T-DNA) and integration of the DNA into the genome requires that additional proteins be transported to the plant as well, including the single-stranded (ss)DNA-binding protein, VirE2. The transport of these two different substrates occurs as a result of the activities of a type IV secretion system encoded by the virB operon. Although the substrates have been identified, the mechanism of their transport remains unknown. In the experiments described here, a region in one of these substrates, VirE2, necessary for transport is identified. The addition of a C-terminal FLAG epitope tag to VirE2, or the deletion of its C-terminal 18 amino acids, renders it non-functional in A. tumefaciens. However, transgenic plants expressing either of these virE2 genes respond to virE2 mutants of A. tumefaciens by forming wild-type tumours. These results indicate that this region of VirE2 is necessary for the protein to be transported into the plant cells, but is not necessary for its function within the plant. Additionally, these studies demonstrate that mutant forms of VirE2 lacking this region do not disrupt the activities of the VirB transporter and support the hypothesis that VirE2 and the VirD2 T-strand are transported independently, even when they co-exist in the same cell.     

Agrobacterium rhizogenes GALLS protein substitutes for Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2

Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2.     

Expression of the Agrobacterium tumefaciens chvB virulence region in Azospirillum spp.

Inner membranes of Azospirillum brasilense incubated with UDP-glucose were unable to synthesize beta-(1-2) glucan and lacked the 235-kilodalton intermediate protein known to be involved in the synthesis of beta-(1-2) glucan in Agrobacterium tumefaciens and Rhizobium meliloti. Inner membranes of A. brasilense strains carrying a cosmid containing the chromosomal virulence genes chvA and chvB of Agrobacterium tumefaciens formed beta-(1-2) glucan in vitro and synthesized the 235-kilodalton intermediate protein. No DNA homology to the chvB region was found in different wild-type strains of A. brasilense, but the introduction of a cosmid containing the Agrobacterium tumefaciens chvA and chvB regions yielded strains in which DNA hybridization with the chvB region was detected, provided that the strains were grown under an antibiotic selective pressure.     

Resistance to crown gall disease in transgenic grapevine rootstocks containing truncated <Emphasis Type="Italic">virE2</Emphasis> of <Emphasis Type="Italic">Agrobacterium</Emphasis>

A truncated form of the Ti-plasmid virE2 gene from Agrobacterium tumefaciens strains C58 and A6, and A. vitis strain CG450 was transferred and expressed in somatic embryos of grapevine rootstocks 110 Richter (Vitis rupestris × V. berlandieri), 3309 Couderc (V. rupestris × V. riparia) and Teleki 5C (V. berlandieri × V. riparia) via Agrobacterium-mediated transformation to confer resistance to crown gall disease. Transformation was confirmed in 98% of the 322 lines by enzyme-linked immunosorbent assay for the neomycin phosphotransferase II protein and 97% of 295 lines by polymerase chain reaction for the truncated virE2 transgene. Southern blot analysis revealed the insertion of truncated virE2 at one to three loci in a subset of seven transgenic 110 Richter lines. In vitro resistance screening assays based on inoculations of shoot internode sections showed reduced tumorigenicity and very small galls in 23 of 154 transgenic lines. Non-transformed controls had a 100% tumorigenicity rate with very large galls. Disease resistance assay at the whole plant level in the greenhouse revealed seven transgenic lines (3 lines of 110 Richter, 2 lines of 3309 Couderc and 2 lines of Teleki 5C) were resistant to A. tumefaciens strain C58 and A. vitis strains TM4 and CG450 with a substantially reduced percentage of inoculation sites showing gall as compared to controls. No association was found between the level of resistance to crown gall disease and the source Agrobacterium strain of virE2. Taken together, our data showed that resistance to crown gall disease can be achieved by expressing a truncated form of virE2 in grapevines.     

The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA.

Agrobacterium tumefaciens transfers T-DNA into the plant genome by a process mediated by Ti plasmid-encoded vir genes. Cleavage at T-DNA border sequences by the VirD endonuclease generates linear, single-stranded T-DNA molecules. In the work described in this report, we used electrophoretic mobility shift assays to show that the purified virE2 gene product binds to single-stranded DNA. VirE2 protein associates with T-DNA as shown by immunoprecipitation studies with VirE2-specific antiserum. The VirE2 protein was detected primarily in the cytoplasm, but also in the inner and outer membrane and periplasmic fractions. Virulence of a virE2 mutant was restored by mixed infection with strains carrying an intact vir region, but not with virA, virB, virD, virE, or virG mutants or chvA, chvB, or exoC mutants. We propose that the VirE2 protein is involved in the processing of T-DNA and in T-strand protection during transfer to the plant cell.     

Sequence and structure of the Drosophila melanogaster ovarian tumor gene and generation of an antibody specific for the ovarian tumor protein.

Sequencing cDNA and genomic DNA from the ovarian tumor gene revealed a gene with seven introns spanning 4.5 kilobases. The proline-rich, hydrophilic otu protein is novel. An antibody prepared to a beta-gal-otu fusion protein recognized a 110-kilodalton ovarian protein which was altered in the ovaries of otu gene mutants.     

Major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein.

The spike glycoprotein (S) of coronavirus, the major target for virus-neutralizing antibodies, is assumed to mediate the attachment of virions to the host cell. A 26-kilodalton fragment proteolytically cleaved from transmissible gastroenteritis virus (TGEV) S protein was previously shown to bear two adjacent antigenic sites, A and B, both defined by high-titer neutralizing antibodies. Recombinant baculoviruses expressing C-terminal truncations of the 26-kilodalton region were used to localize functionally important determinants in the S protein primary structure. Two overlapping 223- and 150-amino-acid-long products with serine 506 as a common N terminus expressed all of the site A and B epitopes and induced virus-binding antibodies. Coexpression of one of these truncated protein S derivatives with aminopeptidase N (APN), a cell surface molecule acting as a receptor for TGEV, led to the formation of a complex which could be immunoprecipitated by anti-S antibodies. These data provide evidence that major neutralization-mediating and receptor-binding determinants reside together within a domain of the S protein which behaves like an independent module. In spite of their ability to prevent S-APN interaction, the neutralizing antibodies appeared to recognize a preformed complex, thus indicating that antibody- and receptor-binding determinants should be essentially distinct. Together these findings bring new insight into the molecular mechanism of TGEV neutralization.     

Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains.

Certain virulence region operons of the Agrobacterium tumefaciens Ti plasmid promoted conjugative Ti plasmid transfer. Mutations in the vir region of pTiC58 inhibited conjugative plasmid transfer between A. tumefaciens strains. Mutations in virA, virG, 5' virB, and virE had the greatest effect on plasmid transfer, and mutations in virC had no effect. Transfer inhibition in vir mutants occurred in the presence or absence of acetosyringone.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.