Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens

We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline-type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine-type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane-bound sensor of plant signal molecules.     

The functional organization of the nopaline A. tumefaciens plasmid pTiC58

We have employed the P type plasmid RP4 and the transposons Tn1 and Tn7 to isolate insertion and deletion mutations in the nopaline Ti-plasmid pTiC58. Mutations that inactivate all known Ti phenotypes have been located on the physical map. Most importantly, we have positioned several regions involved in the determination of oncogenicity. They correspond to regions of homology between octopine and nopaline plasmids. One of these regions is part of the T-DNA, the Ti-plasmid DNA present in transformed plant cells. There are also segments of the T-DNA that are not essential for oncogenicity. One of these determines the biosynthesis of nopaline in tumors. The latter regions might allow insertion of foreign DNA that can then be introduced into plant cells.     

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.     

Location of the right boundary of the virulence region on Agrobacterium tumefaciens plasmid pTiC58 and a host-specifying gene next to the boundary.

The right boundary of the virulence (Vir) region of the nopaline plasmid pTiC58 of Agrobacterium tumefaciens was determined by transposon insertion, cartridge emplacement, and deletion mutagenesis. Genetic complementation with mutant and wild-type alleles led to the identification of the virE locus at the right boundary, which was located about 6 kilobases from the left border of the segment of DNA that is transferred into the plant genome. virE is 2.0 kilobases long and encodes at least one protein of 69 kilodaltons. Various mutations in virE resulted in different truncated lengths of the 69-kilodalton protein. As this protein was increasingly truncated from the carboxy terminus, the host range of A. tumefaciens and the frequency of tumor formation diminished concomitantly. Thus, as one of its functions, the 69-kilodalton protein of virE is probably involved in some aspect of the host range specificity of A. tumefaciens and in infection efficiency.     

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.     

Ornithine cyclodeaminase from octopine Ti plasmid Ach5: identification, DNA sequence, enzyme properties, and comparison with gene and enzyme from nopaline Ti plasmid C58.

Octopine and nopaline are two arginine-derived opines synthesized in plant cells transformed with octopine or nopaline plasmids. Utilization in Agrobacterium tumefaciens is mediated by Ti plasmid regions called occ or noc (octopine or nopaline catabolism), and recent experiments showed that noc in pTiC58 codes for a pathway from nopaline to L-proline. The last enzyme is ornithine cyclodeaminase (OCD), an unusual protein converting L-ornithine directly into L-proline. We investigated whether octopine plasmid pTiAch5 also harbors a gene for OCD. The results revealed an ocd gene which is induced by octopine and maps in the occ region. DNA sequence analysis and comparison with the gene from pTiC58 showed that the two genes are related (69% homology in DNA and deduced amino acid sequence), and antiserum against OCD(C58) also reacted with OCD(Ach5). The enzyme activity was characterized, and a comparison with OCD(C58) showed that the properties are similar, but not identical. Differences were detected in the regulation of enzyme activity by L-arginine and L-proline and in the response to varying ratios of NAD+/NADH. It is proposed that this reflects different mechanisms for integration of opine catabolism into general metabolism.     

Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid,pTiC58

The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection. Part of this DNA (the `common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain-specific. Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced. We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM. Gene c codes for a new unknown T-DNA protein. Gene a is homologous to the agrocinopine synthase gene. Genes b, c, d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3. Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes. Our studies show that gene 3 (located on the TR-DNA of octopine strains) is also oncogenic. Although the b–e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora. Shoot formation could be restored by genes c and c in co-infection experiments. Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested.     

Fission yeast Cut2 required for anaphase has two destruction boxes.

The fission yeast Schizosaccharomyces pombe cut2(+) gene is essential for sister chromatid separation. Cut2 protein, which locates in the interphase nucleus and along the metaphase spindle, disappears in anaphase with the same timing as mitotic cyclin destruction. This proteolysis depends on the APC (Anaphase-Promoting Complex)-cyclosome which contains ubiquitin ligase activity. The N-terminus of Cut2 contains two stretches similar to the mitotic cyclin destruction box. We show that both sequences (33RAPLGSTKQ and 52RTVLGGKST) serve as destruction boxes and are required for in vitro polyubiquitination and proteolysis. Cut2 with doubly mutated destruction boxes inhibits anaphase, whereas Cut2 with singly mutated boxes can suppress cut2 mutations. Strong expression of the N-terminal 73 residues containing the destruction boxes leads to the accumulation of endogenous cyclin and Cut2, and arrests cells in metaphase, whereas the same fragment with the mutated boxes does not. Cut2 proteolysis occurs in vitro using Xenopus mitotic extracts in the presence of functional destruction boxes. Furthermore, Cut2 is polyubiquitinated in an in vitro system using HeLa extracts, and this polyubiquitination requires the destruction boxes.     

Gene note. Complete nucleotide sequence of the T-DNA region of the plant tumour-inducing Agrobacterium tumefaciens Ti plasmid pTiC58

The complete nucleotide sequence has been determined of the T-DNA region from the plant tumour-inducing Agrobacterium tumefaciens nopaline Ti plasmid pTiC58. The T-DNA itself consists of 24 782 bp flanked by two direct 25 bp repeats, the border sequences. In addition, 3622 bp located at the left and 1070 bp at the right of the T-DNA borders were sequenced. Twenty-two open reading frames that code for proteins larger than 125 amino acids have been identified.Key words: Agrobacterium tumefaciens, sequence, T-DNA.      

At least two nuclear gene products are specifically required for translation of a single yeast mitochondrial mRNA.

Mitochondrial translation of the oxi2 mRNA, encoding yeast cytochrome c oxidase subunit III (coxIII), has previously been shown to specifically require the mitochondrially located protein product of the nuclear gene PET494. We show here that this specific translational activation involves at least one other newly identified gene termed PET54. Mutations in PET54 cause an absence of the coxIII protein despite the presence of normal levels of its mRNA. pet494 mutations are known to be suppressible by mitochondrial gene rearrangements that replace the normal 5'-untranslated leader of the oxi2 mRNA with the leaders of other mitochondrial mRNAs. In this study we show that pet54, pet494 double mutants are suppressed by the same mitochondrial gene rearrangements, showing that the PET54 product is specifically required, in addition to the PET494 protein, for translation of the oxi2 mRNA. Since, as we show here, PET54 is not an activator of PET494 gene expression, our results suggest that the products of both of these genes may act together to stimulate coxIII translation.     

The Pseudomonas syringae avrRpt2 gene product promotes pathogen virulence from inside plant cells

Several bacterial avr genes have been shown to contribute to virulence on susceptible plants lacking the corresponding resistance (R) gene. The mechanisms by which avr genes promote parasitism and disease, however, are not well understood. We investigated the role of the Pseudomonas syringae pv. tomato avrRpt2 gene in pathogenesis by studying the interaction of P. syringae pv. tomato strain PstDC3000 expressing avrRpt2 with several Arabidopsis thaliana lines lacking the corresponding R gene, RPS2. We found that PstDC3000 expressing avrRpt2 grew to significantly higher levels and often resulted in the formation of more severe disease symptoms in ecotype No-0 plants carrying a mutant RPS2 allele, as well as in two Col-0 mutant lines, cpr5 rps2 and coil rps2, that exhibit enhanced resistance. We also generated transgenic A. thaliana lines expressing avrRpt2 and demonstrated, by using several different assays, that expression of avrRpt2 within the plant also promotes virulence of PstDC3000. Thus, AvrRpt2 appears to promote pathogen virulence from within the plant cell.     

Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance.

There are DnaA protein-binding sites in at least one F origin of replication, and only potentially leaky dnaA(Ts) mutations had ever been used in previous studies indicating that F replication was independent of the dnaA gene product. Here we show that an Escherichia coli dnaA::Tn10 host which does not make a dnaA gene product cannot sustain autonomous or integrated F plasmid maintenance.     

An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into plants

Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ , that is induced by the plant-wound signal molecule, acetosyringone (AS). virJ lies between virA and virB , and is transcribed in the same direction. The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB , required for virulence. virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis. Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains. While virJ is regulated by AS under the control of the virA/virG two-component regulatory system, acvB is not induced by AS. VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction. The strain lacking both acvB and virJ had an impaired ability to transfer T-DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T-DNA transfer from A. tumefaciens to plant cells. acvB is the first chromosomal gene implicated in T-DNA transfer, but whether it functions specifically for this process is not clear. We hypothesize that virJ evolved from acvB , presumably for a more specialized role in tumorigenesis.     

Characterization of the acc operon from the nopaline-type Ti plasmid pTiC58, which encodes utilization of agrocinopines A and B and susceptibility to agrocin 84.

The acc locus from the Ti plasmid pTiC58 confers utilization of and chemotaxis toward agrocinopines A and B (A+B), as well as susceptibility to a highly specific antiagrobacterial antibiotic, agrocin 84. DNA sequence analyses revealed that acc is composed of eight open reading frames, accR and accA through accG. Previous work showed that accR encodes the repressor which regulates this locus, and accA codes for the periplasmic binding protein of the agrocinopine transport system (S. Beck Von Bodman, G. T. Hayman, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 89:643-647, 1992; G. T. Hayman, S. Beck Von Bodman, H. Kim, P. Jiang, and S. K. Farrand, J. Bacteriol. 175:5575-5584, 1993). The predicted proteins from accA through accE, as a group, have homology to proteins that belong to the ABC-type transport system superfamily. The predicted product of accF is related to UgpQ of Escherichia coli, which is a glycerophosphoryl diester phosphodiesterase, and also to agrocinopine synthase coded for by acs located on the T-DNA. The translated product of accG is related to myoinositol 1 (or 4) monophosphatases from various eucaryotes. Analyses of insertion mutations showed that accA through accE are required for transport of both agrocin 84 and agrocinopines A+B, while accF and accG are required for utilization of the opines as the sole source of carbon. Mutations in accF or accG did not abolish transport of agrocin 84, although we observed slower removal of the antibiotic from the medium by the accF mutant compared to the wild type. However, the insertion mutation in accF abolished detectable uptake of agrocinopines A+B. A mutation in accG had no effect on transport of the opines. The accF mutant was not susceptible to agrocin 84 although it took up the antibiotic. This finding suggests that agrocin 84 is activated by AccF after being transported into the bacterial cell.     

An Ustilago maydis gene involved in H2O2 detoxification is required for virulence

The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H(2)O(2) than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H(2)O(2) sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H(2)O(2) treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Deltayap1 mutants. Diamino benzidine staining revealed an accumulation of H(2)O(2) around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H(2)O(2) detoxification system for coping with early plant defense responses.