A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border

Summary The DNA sequence from a Rhizobium leguminosarum 300 (RL300) plasmid that contains homology to the T_c-DNA of Agrobacterium tumefaciens is described. The RL300 sequence has 78% homology to a 359 bp sequence in the T_c-DNA of pTi15955. The RL300 homology starts approximately 100 bp from the 24 bp border sequence of the T_L-DNA and ends approximately 3 bp from an IS66 homolog in the T_c-DNA. An unusual feature of the RL300 homology is the presence of 81 bp direct repeats with T_c-DNA homology, separated by 201 bp. One end of each direct repeat has a 12 bp palindrome. Four cloned sequences of RL300 with homology to the T DNA region were hybridized to plasmid lysates of RL300 derivatives to determine the source of each plasmid. The sequenced homolog, originally on pRH228, was isolated from pRL7JI; the other 3 homologs were isolated from the transmissable plasmids pRL7JI (pRH235) and pRL8JI (pRH235 and pRH236).     

Sequence of the iaa and ipt region of different Agrobacterium tumefaciens biotype III octopine strains: reconstruction of octopine Ti plasmid evolution

The TA regions of biotype III octopine/cucumopine (OC) Ti plasmids are closely related to the TL region of the biotype I octopine Ti plasmids pTiAch5 and pTi15955. Sequence analysis shows that the limited and wide host range biotype III OC TA regions are derived from a common ancestor structure which lacked the 6a gene found in the biotype I octopine TL region. The TA region of the wide host range OC Ti plasmids has conserved most of the original TL-like structure. In most wide host range OC isolates the TA-iaaH gene is inactivated by the insertion of an IS866 element. However, the TA region of the wide host range isolate Hm1 carries an intact TA-iaaH gene. This gene encodes a biologically active product, as shown by root induction tests and indole-3-acetic acid measurements.The limited host range OC Ti plasmids pTiAB3 and pTiAg57 have shorter TA regions which are derived from a wide host range TA region. The AB3 type arose by an IS868-mediated, internal TA region deletion which removed the iaa genes and part of the ipt gene and left a copy of IS868 at the position of the deleted fragment. The pTiAB3 iaa/ipt deletion was followed by insertion of a second IS element, IS869, immediately 3 of the ipt gene. pTiAg57 underwent the same iaa-ipt deletion as pTiAB3, but lacks the IS868 and IS869 elements.Analysis of the various TA region structures provides a detailed insight into the evolution of the biotype III OC strains.     

Analysis of octopine left border-directed DNA transfer fromAgrobacterium to plants

We constructed a binary plasmid, pVR30, with a neomycin phosphotransferase II (nptII) plant expression cassette flanked by a pTiA6 left border on its right and a pTiA6 right border on its left. This plasmid was used to study transfer of DNA to plants from a left border in the presence of a right border. Infection of tobacco leaf discs with a wild type octopine strain ofAgrobacterium tumefaciens harbouring the binary plasmid resulted in the generation of kanamycin resistant calli at 18 to 26% frequency. Southern hybridization analysis of DNA isolated from eight transformed lines to different probes indicated that left border could mediate DNA transfer to plants in the presence of a right border in cis. Our results also suggest that transfer events corresponding to transfer of T- centre DNA of octopine Ti plasmid pTiA6 do occur. We have shown the relevance of left border- initiated T- DNA transfer by specifically selecting for such events and have confirmed it by Southern hybridization analysis. We also found that a border could be skipped in a few T- DNA transsfer events. This work was presented at “Plant Molecular Biology and Plant Biotechnology” symposium held in ICGEB, New Delhi during December 14–17, 1994.     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified by the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Ars region in TL-DNA on octopine type Ti plasmids

Summary In the TL-DNA region of the octopine type Ti plasmids, an ars region was assigned as the DNA segment conferring the replicational ability to YIp5 in Saccharomyces cerevisiae. T-DNA:YIp5 hybrid plasmids containing a particular T-DNA region could transform yeast cells at a frequency of 10³–10⁴ transformants per g plasmid DNA and they were rescued in Escherichia coli, although the transformed phenotype was mitotically unstable. The instability was inferred to be caused by segregation of the plasmids due to their low efficiency of replication. The ars region was mapped on the noncoding region between the coding regions corresponding to no. 5 and no. 7 mRNA, and its minimal length determined in this experiment was about 150 bp.Abbreviations Ti plasmid tumor inducing plasmid - T-DNA transferred DNA or tumor DNA - TL-DNA left T-DNA - ars autonomously replicating sequences     

Functional analysis of the Agrobacterium tumefaciens octopine Ti-plasmid left and right T-region border fragments

Summary Border fragments of the octopine Ti-plasmid were tested for their ability to restore tumorigenicity of an avirulent mutant carrying a deleted right border. It was found that neither introduction of left border fragments nor that of small right border fragments at the position of the deletion resulted in a complete restoration of oncogenicity. However, insertion of a larger right border fragment in the deletion mutant gave fully oncogenic strains. In the latter case sequences to the right side of the right border repeat were found to be responsible for a complete restoration of oncogenicity. Also a left border repeat inserted together with this enhancer sequence fully restored the oncogenicity of the deletion mutant. The enhancer-sequence on itself was not able to mediate the transfer of the T-region to the plant cell. Border fragments inserted in inverted orientation in the deletion mutant were able to mediate the transfer of the T-region to the plant cell, but at a reduced frequency.     

Inactivation of a transgene due to transposition of insertion sequence (IS136) of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Agrobacterium strains harbour insertion sequences, which are known to transpose into genomes as well as into Ti plasmids. In this study we report the inactivation of a transgene due to transposition of the A. tumefaciens insertion sequence IS136. The transposition was discovered following transformation of plant tissues, although the fidelity of the binary vector was confirmed following transformation into Agrobacterium. Such transpositions are rare but can occur and it is thus important to check the fidelity of the binary vector at different times of Agrobacterium growth in order to avoid failure in achieving transgene expression.     

Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA

We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy.     

Ti plasmid containing Rhizobium meliloti are non-tumorigenic on plants,despite proper virulence gene induction and T-strand formation

We examined the expression of the vir genes of the Agrobacterium tumefaciens Ti plasmid in Rhizobium meliloti, which remains non-tumorigenic on plants after introduction of a Ti- or Ri-plasmid. Both the levels of virulence (vir) gene expression, induced by the plant phenolic compound acetosyringone, and of subsequent T-strand formation were comparable to what is observed in Agrobacterium. In contrast to the situation in Agrobacterium, though, vir induction in R. meliloti did not require a low pH (5.3) of the induction medium and the optimum temperature for induction in R. meliloti was significantly lower than in Agrobacterium. At 37°C no induction of the vir genes was found both in Agrobacterium and R. meliloti. We postulate that the lack of tumorigenicity of Ti carrying R. meliloti strains is due either to a lack of proper attachment of the bacteria to plant cells, or to an improper assembly of a virB-determined essential structure in the cell wall of R. meliloti.     

Independent integration and seed-transmission of the TR-DNA of the octopine Ti plasmid pTi Ach5 in Nicotiana plumbaginifolia

Summary After co-cultivation of diploid Nicotiana plumbaginifolia protoplasts with an octopine-type Agrobacterium tumefaciens strain (LBA 4013) putative transformants were selected for hormone-independent growth, and were tested for T-DNA markers. The number of transformants expressing only T_L-DNA markers, i.e. phytohormone autotrophy and octopine synthase, was an order of magnitude higher than that of the cell lines which were simultaneously positive for both T_L- and T_R-DNA markers (the latter being mannopine and agropine). In one transformant, line no. 101, only the T_R-DNA markers were found. Not each of the T_L-, or T_R-DNA markers were expressed in each transformant resulting in a variety of phenotypes. It included the unorganized or the shoot-teratoma type of growth combined with the presence or absence of opines; e.g. agropine was absent from some of the transformants containing its precursor, mannopine. 5-Azacytidine did not induce agropine synthesis in these lines. Southern blot analysis showed that the T_R-DNA region coding for agropine synthesis was rearranged or absent in one of these lines. Similar variation in the expression of agropine and mannopine production was observed in transformants obtained with the leucinopine-type strain A281.From line 101 plants could be easily regenerated with the ability to synthesize agropine and mannopine. The segregation in the self-progeny fitted to a 3:1 ratio, indicating that the T_R-DNA was carried by a single chromosome. The Southern blot analysis showed that only opine-positive plants contained T_R-DNA. It also confirmed the absence of the T_L-DNA, demonstrating the independent integration of the T_R-region of the octopine-type Ti plasmid pTi Ach5.     

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.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

Single-stranded DNA Plant Pathogens in Eilat

An international conference on ``Inter- and Intracellular Dynamics of ssDNA Plant Pathogens: Implications for Improving Resistance' was sponsored by the United States-Israel Binational Agricultural Research and Deveoplment Fund (BARD) and organized in Eilat, Israel in November 2005. The topic of this meeting was single-stranded plant pathogens, their inter- as well as intra-cellular dynamics and their implications for improving resistance. Most of the talks concentrated on new and very new findings on principles of virus and bacterium-host interactions, studies that no doubt will lead eventually to the establishment of plants resistant to viral and bacterial infections.     

Insertion sequence (IS) hybridization supports classification of Rhizobium meliloti by phage typing

Sixty-one isolates of Rhizobium meliloti from two field sites which had been previously classified into 15 phage types on the basis of sensitivity to 16 typing phages, were subjected to insertion sequence (IS) hybridization using DNA probes for ISR m 3 and ISR m 5. Isolates from all but one phage type contained ISR m 3 (apparent copy no. 1–11) and all isolates contained ISR m 5 (apparent copy no. 3–11). The isolates were placed into 24 IS classes based on differences in their respective ISR m 3 and ISR m 5 hybridization profiles. At either field site, isolates representing different phage types possessed IS hybridization profiles that differed from each other, while those comprising a specific type had identical or closely related profiles. Isolates from one phage type were unusual since they did not react with any of the typing phages and were shown by IS hybridization to constitute a heterogeneous group. Evidence for spatial effects were provided by isolates from two of six types present at both sites which fell into separate IS classes on the basis of their site of origin. These data have ecological implications and suggest that for a particular site, phage typing may be employed for the rapid assessment of the genetic diversity among field isolates.     

Double infection of tobacco plants by two complementing octopine T-region mutants of Agrobacterium tumefaciens

The molecular basis of complementation by a mixture of two different types of octopine T-region mutants (LBA4060 and LBA4210) was studied. Six randomly chosen cellular clones derived from a tumor obtained after mixed infection were analyzed for their T-DNA content via Southern blot hybridization. The clones appeared to contain T-DNA that originated from each of both mutants, indicating that they developed from doubly infected single cells. Genetic complementation, therefore, might explain at least in part the observed complementation phenomenon. However, complementation as a result of cross-feeding between separately transformed cells could not be excluded. Following protoplast isolation, small aggregates might have formed that developed into the clones analyzed.     

Studies on the structure of cointegrates between octopine and nopaline Ti-plasmids and their tumour-inducing properties

Stable cointegrates between incRh-1 octopine (Ach5) and nopaline (C58) Ti-plasmids, present in ten independently isolated Agrobacterium tumefaciens strains, showed identical restriction endonuclease patterns. Each cointegration event had taken place in the common sequence between the T-regions of both Ti-plasmids. This illustrates a high preference for this region when used in the formation of cointegrates. Four crown gall tissues, obtained after transformation of Nicotiana tabacum cells by one of the mutants, were analysed by using Southern blot analysis for their T-DNA structure. The borders of T-DNA frequently appeared to differ from T-DNA borders previously detected in tumour tissues that had been induced by Agrobacterium strain C58 or Ach5. Therefore, it was concluded that possibly a less stringent mechanism exists for the integration into plant DNA of T-DNA, derived from a composite (octopine/nopaline) T-region than for integration of T-DNA from a normal (octopine or nopaline) T-region.Abbreviations Agr sensitivity to agrocin 84 - Ape phage Apl exclusion - Cb resistance to carbenicillin - Occ octopine catabolism - Ocs octopine synthesis - Noc nopaline catabolism - Nos nopaline synthesis - Rec recombination - Tra transfer - Vir virulence     

Mapping and genetic organization of pTiChry5, a novel Ti plasmid from a highly virulent Agrobacterium tumefaciens strain

Agrobacterium tumefaciens Chry5, a wild-type strain originally isolated from chrysanthemum, is unusually tumorigenic, particularly on soybean. We have mapped the Chry5 Ti plasmid by genomic walking and restriction endonuclease analysis, and have located its virulence, T-DNA, plasmid incompatibility, and l,l-succinamopine utilization loci. Southern analysis has revealed that about 85% of the Chry5 Ti plasmid is highly homologous to another Ti plasmid, pTiBo542. Although all the functions that we have located on pTiChry5 are encoded by pTiBo542-homologous regions, the two Ti plasmids differ in their genetic organization. The overall patterns of restriction sites in the plasmids also differ, with the exception of an approximately 12 kb segment of the virulence region, where the BamHI sites appear to be conserved. Complementation analysis has shown that deletion of a DNA segment which flanks the oncogenic T-DNA results in severe attenuation of virulence. This region also contains a sequence that is repeated in the Chry5 genome outside the Ti plasmid, and that is widely distributed in the Rhizobiaceae.