Arginine catabolism: a new function of both octopine and nopaline Ti-plasmids of Agrobacterium.

Summary The oncogenic plasmids of Agrobacterium, the Ti-plasmids, carry genes that enable their bacterial host to catabolize opines. Opines are unusual amino acid derivatives that are only produced in crown gall tumours incited by oncogenic strains of Agrobacterium. The 2 opines, octopine and nopaline, are degraded by Agrobacterium strains carrying the octopine or the nopoline Ti-plasmid, respectively, to arginine and pyruvic acid, and to arginine and -ketoglutaric acid. In this paper it is shown that the Ti-plasmids carry gene(s) involved in the utilisation of arginine as a carbon source. Strains harbouring wild type octopine or nopaline Ti-plasmids in the chromosomal context of strain C58C1 do not grow on arginine as a carbon source. However, they are able to grow on arginine provided that they are induced, or constitutive for opine catabolism. The features of ornithine utilisation are identical. The gene(s) involved in arginine and ornithine utilization in C58C1 (pTi-oct) or C58C1 (pTi-nop) are under the control of the regulator gene that controls octopine or nopaline catabolism. A tentative pathway of octopine utilization is proposed, in which at least two steps are Ti-plasmid coded, and probably belong to the same operon: 1-scission of octopine into arginine and pyruvic acid 2-transformation of an arginine derivative (GSA?) to glutamic acid.Arginine utilization as a carbon source is therefore a new function of the Ti-plasmid. As this function is not inducible by arginine but by opines, it provides a method for selecting regulatory mutants of opine catabolism in the genetic background of strain C58.     

Susceptibility of dry bean (Phaseolus vulgaris L.) to Agrobacterium infection: Transformation of cotyledonary and hypocotyl tissues

A germinating-seed assay was developed to determine the susceptibility of dry bean (Phaseolus vulgaris L.) to infection by Agrobacterium tumefaciens. Seedlings infected one to three days after germination were more susceptible to A. tumefaciens infection than seedlings germinated for five to seven days and the galls that formed on the one to three day seedlings were significantly larger. Nineteen genotypes of dry bean were screened with this assay and all were equally susceptible to nopaline, octopine and agropine biotypes of A. tumefaciens. In addition, cotyledonary nodes and hypocotyls of P. vulgaris were inoculated with disarmed strain A. tumefaciens strain C58Z707 and the avirulent A. rhizogenes strain A4RS (pRiB278b), respectively. Both strains contain the binary plasmid pGA482 which has the neomycin phosphotransferase II (NPT II) gene nested between T-DNA borders. From these infected tissues, callus and root tissues, respectively capable of growing in the presence of kanamycin were obtained. These tissues displayed NPT II activity and integrated copies of the NPT II gene were detected from putative transformed root cultures by genomic blot hybridization.     

Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation

Summary The 200 kb Agrobacterium Ti-plasmid pTiT37 carries a 25 kb segment of T-DNA which it transfers to plant cells during crown-gall tumorigenesis. We have previously engineered into this T-DNA a pBR322-derived cloning vector which enabled us to rescue-clone full length T-DNA from the Ti-plasmid into a 36 kb MINI-Ti plasmid. We report here the deletion of oncogenes from MINI-Ti to produce Micro-Ti containing the nopaline synthase gene and the ampicillin resistance gene and origin of replication of pBR322, flanked by left and right T-DNA borders. Micro-Ti was recloned into the wide host range plasmid pRK290 and transformed into an A. tumefaciens strain carrying a helper plasmid that could supply Virulence (VIR) genes in trans. Using the octopine Ti-plasmid pTiB6-806 as a helper, transformed tobacco cells were obtained which produced both nopaline and octopine. Two cloned cell lines producing both opines were found to be hormone dependent and to produce fertile tobacco plants. We selfed one of these plants and found that the two opine markers segregated in the F1 progeny in a Mendelian fashion. This showed that the T-DNAs were not linked in the transformed plant genome. Southern blot analysis of the genomic DNA from the regenerated plant showed that only part of the (oncogenic) octopine T-DNA was present indicating that it had suffered a deletion in the auxin producing locus (tms region). Presence of the cytokinin autonomy locus presumably accounts for the abnormal rooting behavior of the F1 progeny seedlings containing this T-DNA.Abbreviations NAA Naphtalene acetic acid - IAA Indole-3-acetic acid - BA 6-benzylaminopurine - pCPA para-chlorophenoxyacetic acid Part of this work was presented for her doctoral thesis by A. JdF at the National Institute of Agronomy of Paris-Grignon, January 1983     

Agrobacterium tumefaciens 6bgenes are strain-specific and affect the activity of auxin as well as cytokinin genes

Summary The T-region located 6b gene of Agrobacterium tumefaciens has been found to interfere with cytokinin effects produced by the cotransferred ipt gene. We have compared the biological activity of three different 6b genes: A-6b from Ach5 (octopine, biotype 1), C-6b from C58 (nopaline, biotype 1) and T-6b from Tm4 (octopine, biotype III) by using different biological assays. Each 6b gene was inserted into a disarmed vector and tested on tobacco stems in coinfection experiments with the Ach5 cytokinin (ipt) gene (A-ipt). A-ipt/C-6b coinfections produced tumours with shoots, A-ipt/A-6b coinfections green tumours and A-ipt/T-6b coinfections tumours with a necrotic surface. The tumour phenotypes obtained were independent of the 6b/A-ipt coinfection ratios, indicating that the strain-specific 6b effects result from the activity of a non-diffusible 6b encoded product. Studies with ipt-less Tm4 mutants showed that 6b genes affect other tumour genes besides the ipt gene and pointed to an influence of T-6b on auxin effects resulting from the Tm4 iaa system. T-iaa/T-6b coinfection experiments showed that T-6b did indeed strongly increase tumour formation by the Tm4 iaa genes. The three 6b genes also have effects which do not require other T-region genes. The complex role of the 6b gene in crown gall induction and Agrobacterium host range will be discussed.     

Crown gall transformation of lentil (Lens culinaris Medik.) with virulent strains of Agrobacterium tumefaciens

Summary Four diverse strains of Agrobacterium tumefaciens (C58, Ach5, GV3111, and A281) were capable of inducing tumors at a high frequency on inoculated stems of lentil (Lens culinaris Medik. cultivar Laird) in vivo, and on excised shoot apices in vitro. GV3111 and Ach5 produced the largest and heaviest tumors in vivo, while A281 produced the heaviest tumors in vitro. Tumor formation and opine production are indicative of plant cell transformation and tumors produced appropriate opines: nopaline (C58), octopine (Ach5 and GV3111), and agropine and mannopine (A281). Southern analysis of DNA from a tumor line produced by strain C58 showed that a T-DNA fragment had been transferred into the lentil genome.     

Function of heterologous and pseudo border repeats in T region transfer via the octopine virulence system of Agrobacterium tumefaciens

The successful transfer of the Ti plasmid T region to the plant cell is mediated by its 24 bp border repeats. Processing of the T-region prior to transfer to the plant cell is started at the right border repeat and is stimulated by a transfer enhancer sequence called overdrive. Left and right border repeats differ somewhat in nucleotide sequence; moreover, the repeats of different Ti and Ri plasmids are slightly different. Our data indicate that these differences do not have a significant influence on border activity. However, the overdrive sequence is essential for the efficient transfer of a T region via an octopine transfer system. Our data suggest that an overdrive sequence must also be present next to the right border repeats of the nopaline Ti plasmid and the agropine of octopine and nopaline Ti plasmids express some differences in T-DNA processing activities. of cotopine and nopaline Ti plasmids express some differences in T-DNA processing activities.Furthermore, we demonstrate that certain pseudo border repeats, sequences that resemble the native 24 bp border repeat and naturally occur within the octopine Ti plasmid T-region, are able to mediate T region transfer to the plant cell, albeit with much reduced efficiency as compared to wild-type border repeats.     

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.     

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.     

Octopine- and Nopaline-Inducible Proteins in Agrobacterium tumefaciens Are Also Induced by Arginine

Octopine induced the synthesis of 83, 76, 62, 58, 44, 42, 31, and 22 kDa proteins in Agrobacterium tumefaciens strains harboring the tumor-inducing (Ti) plasmids pTiA6 and pTiAch5. Nopaline induced the synthesis of 83, 76, 62, 58, 56, 44, 42, 31, and 22 kDa proteins in A. tumefaciens strains harboring the Ti plasmids pTiC58 and pTiT37. The molecular masses of proteins induced by octopine and nopaline were very similar. In accordance with the ‘opine concept’, octopine and nopaline were found to induce protein synthesis only in strains harboring the respective Ti plasmids. Arginine, a common catabolic product of octopine and nopaline, induced the synthesis of most of the proteins induced by the two opines. Our results show that only the initial step(s) of octopine and nopaline catabolism are induced by specific opines in the respective strains. The subsequent steps are likely to be regulated by arginine in both strains. Received: 5 January 1996 / Accepted: 21 February 1996     

The interaction of Agrobacterium Ti-plasmid DNA and plant cells

The tumour-inducing plasmids of Agrobacterium tumefaciens (Ti-plasmids) reveal several interesting properties. They are catabolic plasmids, which, instead of rendering Agrobacterium strains capable of catabolizing compounds found in Nature, force a plant to synthesize these catabolites (denoted 'opines'). This situation is obtained by insertion of a segment of the Ti-plasmid (the T-DNA) into the plant nucleus, where T-DNA genes become expressed and intervene in the biosynthesis of these opines. Cells containing the T-DNA behave as neoplasms (crown gall cells). Southern blotting shows that the insertion process responsible for T-DNA transfer probably recognizes special sequences on the T-DNA since the length of the T-DNA segment observed in different, independently isolated tumour lines was found to be similar. For the nopaline Ti-plasmids both left-hand and right-hand borders were found to be constant. For the octopine plasmid the left border was constant and at least two classes of right-hand borders were found. Upon redifferentiation of the transformed plant cells, the T-DNA was found to be conserved in all somatic cells examined. However, small deletions at the border fragments of the T-DNA have been observed. The exact arrangement and copy number of the T-DNA in a nucleus is still under study, but genomic cloning has already revealed that an interspersed tandem arrangement is dominant in nopaline tumours. Clones containing both the right border of one T-DNA and the left border of the neighbouring tandem T-DNA were isolated. In order to identify the different T-plasmid encoded functions an extensive use was made of transposon insertion mutagenesis. When an antibiotic resistance transposon was inserted into the non-essential regions of the T-DNA, a linked transfer to the plant DNA of the transposon together with the T-DNA was observed. This indicates that Ti-plasmids are possible vectors for genetic engineering in plants. A strategy is described for insertion of any cloned DNA segment into the T-DNA.     

Right-border sequences enable the left border of an Agrobacterium tumefaciens nopaline Ti-plasmid to produce single-stranded DNA

The T-region of nopaline-type Ti-plasmids (the portion of the plasmid that is transferred to plant cells) of Agrobacterium tumefaciens is delimited by 23–25 bp direct repeats. They are nicked by the products of the virD locus and the presence of these nicked sites is correlated with the synthesis of single-stranded T-region copies. Despite previous indications to the contrary, we show that the pTiT37 T-region left border is capable of producing single-stranded DNA with high efficiency and that its ability to do so is totally dependent on right border-proximal cis-acting sequences, most probably overdrive, located several kilobases from the border. The absence of overdrive does not affect the single-strand nicking activity of the virD product but only the production of single-stranded copies from the nicked substrate.     

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.     

Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.)

Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.     

Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens

The recognition of the T-DNA left border (LB) repeat is affected by its surrounding sequences. Here, the LB regions were further characterized by molecular analysis of transgenic plants, obtained after Agrobacterium tumefaciens-mediated transformation with T-DNA vectors that had been modified in this LB region. At least the 24-bp LB repeat by itself was insufficient to terminate the T-strand synthesis. Addition of the natural inner and/or outer border regions to at least the LB repeat, even when present at a distance, enhanced the correct recognition of the LB repeat, reducing the number of plants containing vector backbone sequences. In tandem occurrence of both the octopine and nopaline LB regions with their repeats terminated the T-strand synthesis most efficiently at the LB, yielding a reproducibly high number of plants containing only the T-DNA. Furthermore, T-strand synthesis did not terminate efficiently at the right border (RB) repeat, which might indicate that signals in the outer RB region inhibit the termination of T-strand synthesis at the RB repeat.     

Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassica juncea

Summary Brassica napus and Brassica juncea were infected with a number of Agrobacterium tumefaciens strains. Tumourigenesis was very rapid and extremely efficient on B. juncea with all but one of the strains. Tumourigenesis on B. napus varied widely. It was very efficient with the nopaline strains, was reduced with the succinamopine strain A281 and was very weak with the octopine strains. The latter observation was confirmed with six different B. napus rapeseed cultivars. The selectivity was due to differences in the virulence of Ti plasmids with B. napus, rather than the tumourigenicity of the T-DNA or virulence of the chromosomal genes associated with the strains. An exception was strain LBA4404. The virulence of the octopine strains was increased by coinfection with more virulent disarmed strains and by induction with acetosyringone.     

A functional map of the nopaline catabolism genes on the Ti plasmid of Agrobacterium tumefaciens C58

Summary The nopaline catabolism (noc) genes are located in a 14.4 kb region on the pTiC58 plasmid of A. tumefaciens C58. These genes permit the bacterium to grow on nopaline N²-(1,3-dicarboxylpropyl) arginine, a substrate produced in plant tumors initiated by strain C58. The functions of the noc genes include the use of nopaline and L-ornithine as sole carbon and nitrogen sources. Using Tn5 insertional mutants, we have identified and mapped the positions of the genes that are responsible for nopaline catabolism (NopC), ornithine catabolism (OrnC) and nopaline uptake (NopU). A polar relationship was found between these phenotypes, which extended leftward over the noc region to the T-region. The NopC mutants were also deficient in nopaline oxidase, an enzyme that liberates free arginine from nopaline.The noc region also encodes the synthesis of a periplasmic protein, n1 that was induced by nopaline. Tn5 insertional mutations and molecular cloning were used to map the n1 production locus. The recombinant plasmids, pSa4480 and pSa4481, containing the 8.9 kb right-hand end of the noc region, conferred n1 production when introduced into a pTi-free strain of A. tumefaciens. Production of n1 by the strains carrying these plasmids required nopaline induction.We have identified in toto three noc loci: nocB, nocC, and nocA, which confer n1 production, nopaline oxidase production and ornithine catabolism respectively. A model is proposed whereby the noc genes of pTiC58 are contained on a leftward reading operon in the order nocB, nocC, and nocA.     

The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames

Summary Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis (Engstromm et al. 1987) that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.     

Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4

Summary The vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization. The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found. The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids. The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue. We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene. Both sequences show homology to the octopine synthase gene. In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.Abbreviations Ap ampicillin - IS insertion sequence - iaaH indole acetamide hydrolase - iaaM tryptophan monooxygenase - ipt isopentenyl transferase - Km kanamycin - LB Luria broth - m/a mannopine/agropine - o/c octopine/cucumopine - ocs octopine synthase