Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes.

The T-DNA regions of the root-inducing (Ri) plasmid pRiA4b of Agrobacterium rhizogenes were characterized. Two regions, designated TL-DNA and TR-DNA, were found to be integrated and stably maintained in the plant genome. The TL-DNA spanned a 15- to 20-kilobase region of pRiA4b and was separated from the TR-DNA region by at least 15 kilobases of nonintegrated plasmid DNA. The TR-DNA region also spanned a 15- to 20-kilobase region of pRiA4b and included a region of homology to the tms morphogenic loci of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Eighteen deletions and 95 transposon insertions were generated in the T-DNA regions and tested for alterations in virulence. Insertions into four loci in the TL-DNA affected the morphology of root formation of Kalancho? diagremontiana leaves and stems, but had no visible effects on other host plants. Insertions into two loci (tms-1 and tms-2) in the TR-DNA eliminated virulence symptoms on all plants tested, with the exception of K. diagremontiana stems, where sparse root formation occurred. Complementation experiments with Ri and Ti plasmid T-DNA mutations indicate that the tms genes of the two plasmids serve similar functions and suggest a functional relationship between one or more genes of the TL-DNA and the cytokinin synthesis locus tmr of the Ti plasmid.     

New class of limited-host-range Agrobacterium mega-tumor-inducing plasmids lacking homology to the transferred DNA of a wide-host-range, tumor-inducing plasmid

Biotype 1 and 2 strains of Agrobacterium tumefaciens were isolated from crown gall tumors of Lippia canescens plants growing as ground cover in Arizona. The isolates were agrocin 84 sensitive, did not catabolize octopine, nopaline, agropine, or mannopine, and were limited in their tumorigenic host range. One biotype 2 strain, AB2/73, showed the most limited host range; it incited tumors only on Lippia strains, the cucurbit family of plants, and Nicotiana glauca. Megaplasmids were detected in the isolates by vertical agarose gel electrophoresis. The unusual host range, as well as sensitivity to agrocin 84, were plasmid specified since they were conjugally cotransferred with plasmids from donor strain AB2/73. Correlation of deletions with concomitant loss of virulence and agrocin 84 sensitivity identified the megaplasmid pAtAB2/73d as the virulence element in strain AB2/73. The estimated size of this tumor-inducing plasmid was 500 kilobases. Axenic growth of tumor tissue incited by strains carrying pAtAB2/73d was phytohormone independent. Although the limited-host-range megaplasmid pAtAB2/73d lacked any detectable homology to the phytohormone-biosynthetic genes in wide-host-range transferred DNA (tms-1, tms-2, tmr), it showed homology to the wide-host-range virB, virC, virD, and virG loci. Therefore, pAtAB2/73d represents a new class of tumor-inducing plasmids distinguished by its large size, the absence of determinants for the catabolism of several known opines, the presence of agrocin 84 sensitivity, and its lack of homology to wide-host-range transferred DNA contrasted with its conservation of sequences from the wise-host-range vir region.     

Physical and functional map of an Agrobacterium tumefaciens tumor-inducing plasmid that confers a narrow host range.

Agrobacterium tumefaciens Ag162 induces crown gall disease on an unusually narrow range of host plants. The 231-kilobase Ti plasmid which has been shown to determine host range, was subcloned into the vector pVCK102. By comparing overlaps of cloned insets, maps were constructed for the restriction endonucleases SalI, XhoI, EcoRI, and KpnI. Plasmid incompatibility, octopine catabolism, and at least six virulence genes were localized. Plasmid incompatibility between pTiAg162 and the wide host range plasmid pTiA6 consists of two components: mutual incompatibility and the apparent ability of pTiA6 to block RK2 replication if the pTiAg162 incompatibility locus is linked to the vector pVK102. The octopine catabolism locus maps within the 30 kilobases of DNA separating the two T-DNA regions of pTiAg162. Complementation of avirulent vir mutants of pTiA6 with clones of pTiAg162 DNA did not confer the host range of pTiAg162 but rather restored the wide host range of pTiA6. One potentially important difference between pTiA6 and pTiAg162 is that pTiAg162 T-DNA regions are widely separated.     

DNA from Agrobacterium rhizogenes in transferred to and expressed in axenic hairy root plant tissues

Summary Axenic root tissue cultures were established from primary hairy roots induced on carrot and potato by Agrobacterium rhizogenes strain 15834. cDNA made towards poly-A⁺ RNA isolated from these tissues, hybridized with a limited number of well-defined fragments of the plasmid DNA present in the inciting A. rhizogenes strain. These data therefore demonstrate that at least part of the rootinducing (Ri) plasmid of Agrobacterium rhizogenes is transferred, stably maintained and expressed in hairy-root plant tissues and confirm that hairy roots are a special type of crown gall. The T-DNA in hairy-root cells appears to have several regions which are related in terms of sequence homology and probably also function to the T-DNA in octopine and nopaline crown gall tumours.     

Cloning and nucleotide sequence of the tzs gene from Agrobacterium tumefaciens strain T37.

The trans-zeatin secretion locus (tzs), from the nopaline Ti plasmid of Agrobacterium tumefaciens strain T37, was cloned and the nucleotide sequence determined. This gene is located in the virulence region of pTiT37. The tzs gene is responsible for the secretion of trans-zeatin into bacterial culture medium and in addition has the cytokinin biosynthetic activity, dimethylallylpyrophosphate:AMP dimethylallyltransferase. Sequence analysis showed an open reading frame of 729 nucleotides, capable of encoding a protein of 27,545 daltons. A single new labelled protein of 27,200 daltons was detected in Escherichia coli maxicells expressing the cloned tzs gene. Significant sequence homology was observed between the tzs and the published tmr sequence from pTiT37.     

Characteristics of the nopaline catabolic plasmid in Agrobacterium strains K84 and K1026 used for biological control of crown gall disease

Wild-type Agrobacterium radiobacter strain 84 and its Tra- derivative K1026, used for biological control of crown gall disease, each contain the plasmid pAtK84b. It confers incompatibility to tumor-inducing (Ti) plasmids of pathogenic A. tumefaciens, thus preventing transfer of Ti plasmids into K84 and K1026, and the consequent development of pathogens resistant to the specific antibiotic, agrocin 84 produced by K84 and K1026. pAtK84b also resembles one group of Ti plasmids in its capacity for directing nopaline catabolism. A study of the DNA homology among pAtK84b, pTiC58, and pTiAch5 was carried out. pAtK84b was transferred by conjugation to a plasmidless recipient and, after isolation, was hybridized with Ti plasmid DNA. Areas of DNA homology were located on published maps of pTiC58 and pTiAch5, a restriction enzyme map of pAtK84b was constructed, and areas of homology with DNA of known genetic function were located on the map. Strong and extensive (over 50%) homology was found between pAtK84b and pTiC58 (nopaline catabolic, Noc), but much less between pAtK84b and pTiAch5 (octopine catabolic). There was no detectable homology between pAtK84b and the oncogenic T-DNA and virulence (Vir) regions of either Ti plasmid. The size of pAtK84b was 173 kb and the orientation of regions of identified gene function (Noc, incompatability/origin of replication, and conjugal transfer) on pTiC58 was matched by the locations of homologous areas on pAtK84b. It is concluded that pAtK84b may be a deletion product of a pTiC58-type plasmid which has been disarmed in the oncogenic T-DNA and Vir regions.     

Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids

The abilities of Agrobacterium tumefaciens and A. rhizogenes to transform dicotyle-dons and cause crown gall and hairy root disease are caused by the presence of tumor inducing (Ti) and root inducing (Ri) plasmids. During transformation plasmid T-DNA (transferred DNA) is inserted into the plant genome. The T-region is flanked by 25 bp direct repeats, which are essential for transfer. The T-regions contain oncogenes that are expressed in the plants. Some of these code for enzymes that synthesize auxin or cytokinin. Another type, present in Ri plasmids only, appears to impose a high hormone sensitivity on the infected tissue. The T-DNA also contains genes for enzymes synthesizing opines, which the bacteria catabolize. The T-DNA transfer is initiated by the induction of genes in the virulence (vir) region of the plasmid by phenolic compounds secreted by wounded tissue. The products of the vir -genes and of chromosomal genes mediate transfer of T-DNA to the plant cells. Crown gall disease is caused by production of auxin and cytokinin by the transferred T-DNA. The T-DNA of Ri plasmids codes for at least three genes that each can induce root formation, and that together cause hairy root formation from plant tissue. Current results indicate that the products of these genes induce a potential for increased auxin sensitivity that is expressed when the transformed cells are subjected to a certain level of auxin. After this stage the transformed roots can be grown in culture without exogenous supply of hormones.     

Structural Characterization of the virB Operon on the Hairy-root-inducing Plasmid A4

The hairy-root-inducing plasmid A4 (pRiA4) is capable of conferringtumorigenic symptoms on plants upon infection by its host bacterium,Agrobacterium rhizogenes. The virB operon on pRiA4 has beensequenced and found to be composed of 11 genes, virB1 to virB11,whose products mostly appear to be associated with the cellmembrane. A novel structural characteristic is frequent overlappingsbetween the translation termination and initiation codons ofadjacent genes. This is indicative of fine tuning of relativetranslation frequencies for each VirB protein. These resultssupport the view that VirB multisubunit complexes provide facilitiesfor T-DNA transfer at the bacterial cell membrane. The structuralorganization of the pRiA4 virB operon was essentially identicalto that of the previously reported virB operons of tumorinducingplasmids, pTiC58 and pTiA6, and the corresponding VirB proteinsof the three plasmids were extremely homologous to one another.On the basis of the structural similarity of each VirB protein,the phylogenetic relationship among pRiA4, pTiC58, and pTiA6is discussed.     

Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid

Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids. We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments. Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests. Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments. We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII.     

Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumors.

Methylation of the T-DNA in Agrobacterium tumefaciens and in four octopine-type (A6S/2, E9, 15955/1, 15955/01) and one nopaline-type (HT37#15) crown gall tumors was investigated using the isoschizomeric restriction endonucleases Msp I and Hpa II. T-DNA in the octopine-type Ti-plasmid pTiB6(806) was not methylated at the sequence 5'CCGG3' in Agrobacterium. With two possible exceptions, neither was the T-DNA of the nopaline-type Ti-plasmid pTiT37 methylated in the bacterium. In all tumor lines investigated, at least one copy of the T-DNA was not methylated. DNA methylation was not detected in the lines A6S/2, 15955/1, HT37#15, and the TL region of E9. DNA methylation of some copies of TR in the E9 tumor line, and possibly in the 15955/01 line, was detected. The methylation of some copies of TR in the E9 line may indicate that not all copies of TR are transcribed in this tumor.     

Crown gall disease and hairy root disease : a sledgehammer and a tackhammer

The neoplastic diseases crown gall and hairy root are incited by the phytopathogenic bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively. Although the molecular mechanism of T-DNA transfer to the plant most likely is the same for both species, the physiological basis of tumorigenesis is fundamentally different. Crown gall tumors result from the over-production of the phytohormones auxin and cytokinin specified by A. tumefaciens T-DNA genes. Although the T-DNA of some Riplasmids of A. rhizogenes contains auxin biosynthetic genes, these loci are not always necessary for hairy root formation. Recent experiments suggest that hairy root tumors result from the increased sensitivity of transformed cells to endogenous auxin levels. An understanding of hairy root tumorigenesis will likely result in an increased knowledge of plant developmental processes.     

Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542.

Agrobacterium tumefaciens strains carrying pTiBo542 induce large, fast-appearing tumors and have an unusually wide host range. A clone bank was made from this 250-kilobase plasmid in a wide-host-range vector, and restriction maps were determined for BamHI and SalI. The virulence genes, transferred DNA genes, plasmid incompatibility region, and a region that inhibits growth of certain A. tumefaciens strains were localized. The six virulence genes and two tms genes were highly homologous to the genes of pTiA6, but the tmr gene was not. Mutations in each of the six vir loci of pTiA6 were complemented by clones from the vir region of pTiBo542.     

Genotypic variability of soybean response to agrobacterium strains harboring the ti or ri plasmids

Twenty four diverse cultivars of soybean (Glycine max [L.] Merrill) and three lines of its annual wild progenitor Glycine soja Sieb and Zucc. were tested for their response to Agrobacterium strains harboring either the Ti (tumor-inducing) plasmid (pTi) from Agrobacterium tumefaciens or the Ri (root-inducing) plasmid (pRi) from Agrobacterium rhizogenes following uniform wounding and inoculation. Based upon gall weight at 8 weeks postinfection, three G. max cultivars (Biloxi, Jupiter, and Peking) and one G. soja line, Plant Introduction (PI) 398.693B, were judged highly susceptible to A. tumefaciens strain A348 (pTiA6), ten genotypes moderately susceptible, 11 weakly susceptible, and two nonsusceptible. Of 26 genotypes inoculated with strain R1000 (pRiA4b), only seven responded in a clearly susceptible fashion by forming small, fleshy roots at internodal infection sites. Cotyledons excised from 1- or 3-day old seedlings of Peking and Biloxi cultivars also formed galls when infected in vitro with agrobacteria carrying either the Ti or Ri plasmid. Tumor lines established from cotyledon and stem galls induced by A. tumefaciens A348 (pTiA6) exhibited the T-DNA borne traits of phytohormone-independent growth and octopine synthesis. Additionally, DNA isolated from cultured tumors hybridized with labeled T-DNA probe.     

Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA,and transmission of T-DNA to R1 progeny

Cloned DNA sequences encoding yeast alcohol dehydrogenase and a bacterial neomycin phosphotransferase have been inserted into the T-DNA of Agrobacterium tumefaciens plasmid pTiT37 at the “rooty” locus. Transformation of tobacco stem segments with the engineered bacterial strains produced attenuated crown gall tumors that were capable of regeneration into intact, normal tobacco plants. The yeast gene and entire transferred DNA (T-DNA) were present in the regenerated plants in multiple copies, and nopaline was found in all tissues. The plants were fertile, and seedlings resulting from self-pollination also contained intact and multiple copies of the engineered T-DNA. Expression of nopaline in the germinated seedlings derived from one regenerated plant was variable and did not correlate with the levels of T-DNA present in the seedlings. Preliminary evidence indicates that nopaline in progeny of other similarly engineered plants is more uniform. The disarming of pTiT37 by insertions at the “rooty” locus thus appears to produce a useful gene vector for higher plants.     

我国葡萄根癌农杆菌Ti质粒的特征

以窄宿主葡萄农杆菌Ag162Ti质粒的T-DNA区tmr、tmsl和ocs基因座位以及T_A-DNA和T_B-DNA片段为探针,对12株我国分离的不同生物型、质粒类型和寄主范围的葡萄根癌农杆菌的引质粒转移DNA(T-DNA)进行Southern杂交分析。在9株生物3型octoplne Ti质粒菌株中,与上述探针均同源。其中窄宿主葡萄根癌农杆菌菌株杂交片段彼此较一致。广宿主葡萄根癌农杆菌菌株的杂交片段彼此差异较大。1株无致瘤能力的生物1型菌株与5个探针均不杂交。1株生物3型nopaline Ti质粒菌株及1株诱导冠瘿瘤中只合成精氨酸的菌株,杂交带的变化也大。由此可见葡萄农杆菌在生物进化过程中其转移DNA呈多态性,成为农杆菌中特殊类群。本分析对葡萄根癌农杆菌致病菌株的鉴定亦有帮助。     

Organization and characterization of the virCD genes from Agrobacterium rhizogenes

Summary We have precisely localized virulent (vir) genes of the hairy root-inducing plasmid pRiA4b on the basis of sequence similarity with the tumor-inducing plasmid pTiA6NC, and shown that the overall organizations of vir genes in both plasmids are fairly analogous, although sizes and spacer lengths in some genes differ from each other. Among the vir genes thus mapped, the virC and virD loci were characterized in detail. Transposon insertions in virD led to loss of tumorigenicity on Kalanchoe stems and carrot discs, and one within virC exhibited an attenuated pathogenicity. The avirulent phenotype of the virD2 strain among these mutants was due to the lack of ability to recombine T-DNA border repeats in Agrobacterium cells. The nucleotide sequence of most parts of the virCD loci were similar in both plasmids. The virCD genes of these two plasmids, therefore, seem comparable both functionally and structurally. Phylogeny of pRi and pTi has also been discussed from the sequence data.     

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.     

Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid

Plasmids from two virulent strains of Agrobacterium rhizogenes belonging to biotypes 1 and 2 are compared for DNA homology with the nopaline Ti plasmid from Agrobacterium tumefaciens C58 by means of Southern blot hybridizations. We find that both A. rhizogenes plasmids share strong sequence homology with regions of the Ti plasmid that affect oncogenicity of A. tumefaciens C58. The biotype 1 plasmid shows an additional region of homology at approximately the position of the genes responsible for conjugative transfer of pTiC58. Neither A. rhizogenes plasmid shows any detectable homology with the T-DNA of A. tumefaciens C58. Possible analogies between hairy root and crown gall induction are discussed on the basis of the results presented.     

The virD4 gene is required for virulence while virD3 and orf5 are not required for virulence of Agrobacterium tumefaciens

The virD operon of the resident Ti plasmid of Agrobacterium tumefaciens contains loci involved in T-DNA processing and undefined virulence functions. Nucleotide sequence of the entire virD operon of pTiC58 revealed similarities to the virD operon of the root-inducing plasmid pRiA4b and to that of the octopine-type plasmid pTiA6NC. However, comparative sequence data show that virD of pTiC58 is more akin to that of the pRiA4b than to that of the pTiA6NC. T7f10::virD gene fusions were used to generate polypeptides that confirm the presence of four open reading frames virD1, virD2, virD3, and virD4 within virD which have a coding capacity for proteins of 16.1, 49.5, 72.6, and 73.5 kDa, respectively. virD3 therefore encodes a polypeptide 3.4 times larger (72.6 versus 21.3 kDa) than that encoded by virD3 of octopine Ti plasmids. Non-polar virD4 mutants could not be complemented by a distant homologue, TraG protein of plasmid RP4. An independently regulated fifth ORF (orf5) is located immediately downstream of 3′ end of virD4 and encodes a polypeptide of 97.4 kDa. The expression of orf5 is dependent on its own promoter and is independent of acetosyringone induction in A. tumefaciens. Recently, it has been shown that virD3 of octopine Ri or Ti plasmids is not required for virulence. In this report, we confirm and extend these findings on a nopaline Ti plasmid by using several virD non-polar mutants that were tested for virulence. virD3 and orf5 non-polar mutants showed no effect on tumorigenicity on 14 different plant species, while virD4 mutants lost their tumorigenicity completely on all these test plants. These data suggest that virD3 and orfS are not essential for virulence whereas virD4 is absolutely required on a wide range of host plants.     

Comparison of T-DNA oncogene complements of Agrobacterium tumefaciens tumor-inducing plasmids with limited and wide host ranges.

The T-DNA oncogene complements of the limited-host-range tumor-inducing plasmid pTiAg63 and the wide-host-range plasmid pTiA6 were compared. The resulting data indicate that pTiAg63 has DNA sequences related to most of the genes encoded by the oncogene region, the TL-DNA, of pTiA6 and that these sequences are divided between two T-DNA regions, the TA-DNA, which encoded sequences related to pTiA6 genes 4 (the cytokinin independence gene) and 6a, as well as to a pTiA6 TL-DNA fragment that encoded gene 6b and a portion of gene 3, and the TB-DNA, which encoded sequences related to genes 1 and 2 (the auxin independence genes). Tumor tissues of Nicotiana rustica incited by Agrobacterium tumefaciens harboring either pTiA6 or pTiAg63 grew axenically in vitro on phytohormone-free medium. The morphologies of the tissues, however, differed; whereas those incited with pTiA6 grew as loose, friable, unorganized callus, the tumors incited by pTiAg63 grew as clumps of rootlike structures. Thus, the T-DNA oncogene complements of these plasmids were not equivalent. The results are discussed in relation to the A. tumefaciens host range.