Isolation and characterization of diverse nopaline type Ti plasmids of Agrobacterium tumefaciens from Japan

Ninety Agrobacterium strains were isolated from naturally appearing crown galls in Japan. They were classified into several groups based on opine type, biovars, tumorigenicity, and indigeneous plasmid profiles. Twenty-nine strains utilized nopaline, but none utilized octopine. Eighteen isolates were tumorigenic, nopaline type strains and thus classified as Agrobacterium tumefaciens. Some strains possessed anomalous traits such as lysine utilization, resistance to agrocin 84, and a lack of motility. Pathogenic strains contained Ti plasmids of either 200 kb or 260 kb, as identified by hybridization to T-DNA of the known Ti plasmid. However, the restriction enzyme cleavage patterns, arising from hybridization to the probe, were different from each other and indicated that nopaline type Ti plasmids possess more diverse T-DNA structures than previously reported. Five of 6 representative strains induced tumors on 6 plant species (tomato, petunia, poplar, kalanköe, apple, and grape). Among these, apple was notable, since only a few strains have been reported to be pathogenic to this plant. On petunia, 4 strains developed large tumors while 2 produced only small tumors. Teratomas were formed on poplar in a strain-dependent manner, but not on tomato. These results suggest that our isolates are wide host range strains, and that host-specificity of these strains is related to diverse T-DNA structures.     

Identification of Different Agrobacterium Strains Isolated from the Same Forest Nursery

Several Agrobacterium strains isolated from the same forest nursery from 1982 to 1988 were compared by serological, biochemical, and DNA-DNA hybridization methods. Similarities among strains belonging to biovar 2 were observed by indirect immunofluorescence, whereas biovar 1 strains showed serological heterogeneity. Electrophoretic analysis of bacterial envelope-associated proteins showed that few bands appeared in the strains belonging to biovar 1, whereas many proteins appeared in the case of biovar 2 strains. Chromosomal DNA was analyzed with six random C58 chromosomal fragments. None of the six probes hybridized to the DNA of the two biovar 2 strains. One of the probes gave the same hybridization pattern with all biovar 1 strains, whereas the other probes yielded different patterns. The vir regions were closely related in the different pathogenic strains. The T-DNA and replication regions were less conserved and showed some variations among the strains.     

Genetic analysis of transfer and stabilization of Agrobacterium DNA in plant cells

In an attempt to elucidate the transfer and integration mechanism of Agrobacterium DNA upon crown gall induction, we translocated a borderless T-DNA to different sites of the C58 Ti plasmid. As a result of the physical linkage of the T-DNA onc genes with other Ti plasmid functions, the concerned strain retained tumor-inducing capacity. However, when the borderless T-DNA is separated on an independent replicon while all other pTi functions are provided in trans, the strain can no longer induce tumors on plants. We provide evidence that the right T-DNA border region harbors one or more in cis active functions essential in the transfer and/or stabilization of the T-DNA into plant cells. The strains used in these experiments allowed us to conclude that some function(s) of the Ti plasmid can induce plant cell proliferations independently of the T-DNA transformation event. The results described here indicate that other Ti plasmid sequences than solely the T-region can be transferred to plant cells.     

Correlative Association between Resident Plasmids and the Host Chromosome in a Diverse Agrobacterium Soil Population

Soil samples collected from a fallow field which had not been cultivated for 5 years harbored a population of Agrobacterium spp. estimated at 3 × 10⁷ CFU/g. Characterization of 72 strains selected from four different isolation media showed the presence of biovar 1 (56%) and bv. 2 (44%) strains. Pathogenicity assays on five different test plants revealed a high proportion (33%) of tumorigenic strains in the resident population. All tumorigenic strains belonged to bv. 1. Differentiation of the strains by restriction fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins, and utilization patterns of 95 carbon substrates (Biolog GN microplate) revealed a diversified bv. 1 population, composed of five distinct chromosomal backgrounds (chr A, C, D, E, and F), and a homogeneous bv. 2 population (chr B). chr A, B, C, and D were detected at similar levels throughout the study site. According to opine metabolism, pathogenicity, and agrocin sensitivity, chr A strains carried a nopaline Ti plasmid (pTi), whereas chr C strains had an octopine pTi. In addition, four of six nontumorigenic bv. 1 strains (two chr D, one chr E, and one chr F) had distinct and unusual opine catabolism patterns. chr B (bv. 2) strains were nonpathogenic and catabolized nopaline. Although agrocin sensitivity is a pTi-borne trait, 14 chr B strains were sensitive to agrocin 84, apparently harboring a defective nopaline pTi similar to pAtK84b. The other two chr B strains were agrocin resistant. The present analysis of chromosomal and plasmid phenotypes suggests that in this Agrobacterium soil population, there is a preferential association between the resident plasmids and their bacterial host.     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.     

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.     

Analysis of core genes supports the reclassification of strains Agrobacterium radiobacter K84 and Agrobacterium tumefaciens AKE10 into the species Rhizobium rhizogenes

Some strains of the former genus Agrobacterium have high biotechnological interest and are currently misclassified. Consequently, in this study, the taxonomic status of the non-pathogenic strain Agrobacterium radiobacter K84, used in biological control, and the tumourigenic strain Agrobacterium tumefaciens AKE10, able to regenerate tobacco transgenic plants, was revised. The phylogenetic analysis of the chromosomal genes rrs, atpD and recA showed that they should be reclassified into Rhizobium rhizogenes. The analysis of virulence genes located in the Ti plasmid (pTi) outside T-DNA showed a common phylogenetic origin among strains AKE10, R. rhizogenes 163C and A. tumefaciens (currently R. radiobacter) C58. However, the genes located inside the T-DNA, mainly the 6b gene, of strain AKE10 were phylogenetically close to those of strain 163C but divergent from those of strain C58. Furthermore, the T-DNA of tumourigenic strains from R. rhizogenes conferred on them the ability to regenerate tumour tissue resembling fasciation in tobacco plants. These results showed the existence of a highly mosaic genetic organization in tumourigenic strains of the genus Rhizobium and provided evidence of the involvement of T-DNA from tumourigenic strains of R. rhizogenes in fasciation of Nicotiana leaves. The data further suggested that pathogenic strains of Rhizobium could be good models to analyse bacterial evolution.     

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K₂TeO₃ was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K₂TeO₃ concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K₂TeO₃-amended medium. The bona fide agrobacterium colonies growing on media amended with K₂TeO₃ were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10³ to 10⁴ agrobacteria · g of dry soil⁻¹ in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.     

Opine synthesis in wild-type plant tissue

Opine production is associated with crown gall tissue, a neoplastic growth caused by infection of dicotyledonous plants with Agrobacterium tumefaciens. Recent publications have claimed that tissues of certain monocotyledonous plants can also be infected by Agrobacterium. Following infection, a part of the Agrobacterium Ti plasmid, T-DNA, is integrated into the chromosome of the infected plant. T-DNA, which codes for opine-synthesizing enzymes, is now used to add foreign genes to plants. A number of laboratories have used opine production in plant tissue, often after arginine feeding or preincubation as evidence for plant transformation by T-DNA vectors. In this report we provide microbiological, chromatographic, spectroscopic and chemical evidence indicating that opines can be formed in normal callus and plant tissue as a result of arginine metabolism. Therefore, researchers studying T-DNA should be aware of the capability of plant tissue to metabolize arginine to opines. Opine production following infection with T-DNA may not always be sufficient evidence to indicate transformation by the Agrobacterium Ti plasmid.     

Tumor Growth Complementation Among Strains of Agrobacterium

The ability of 31 strains of Agrobacterium to initiate the production of a tumor growth factor (TGF) which is associated with crown-gall tumors on primary pinto bean leaves was determined. Extracts from bean leaves inoculated with these bacteria were tested and they showed that 16 of the 19 strains that induced tumors on the leaves also initiated TGF production. The three strains for which no TGF was detected were of low infectivity and included two strains of A. tumefaciens and a strain of A. rhizogenes. Five of the 12 strains that did not induce pinto bean leaf tumors were found to initiate TGF production. Representatives of A. tumefaciens, A. rhizogenes, and A. radiobacter among these 12 strains were present in both categories. Mixed inocula composed of one of the three infectious TGF-negative strains and one of the five nontumorigenic TGF-positive strains resulted in increased growth of tumors induced by the former. These growth changes were not correlated with changes in tumor number. The ability of different strains to show these tumor growth complementation effects corresponded fully with their ability to initiate TGF, as determined by the assay of leaf extracts. The nontumorigenic TGF-positive strains also promoted the growth of tumors initiated by low concentrations of strain B6. These complementation effects were due, therefore, to the same TGF found in extracts of B6 inoculated leaves and of leaves inoculated with most tumorigenic as well as many nontumorigenic strains of Agrobacterium. Heat-inactivated cells of strain B6 failed to initiate sufficient TGF to be detected in extracts, and heat-inactivated cells of several strains failed to show tumor growth complementation, indicating bacterial viability to be one prerequisite for TGF initiation. Heat inactivated cells also inhibited TGF production by viable cells, similar to their ability to inhibit tumor initiation. Consequently, bacteria capable of attaching to the A. tumefaciens infection site may initiate one of four patterns of events: (i) TGF production only, (ii) tumor induction only, (iii) both, or (iv) neither. Suggestive evidence for a second tumor-associated growth factor is presented.     

Non-oncogenic plant vectors for use in the agrobacterium binary system

Summary Agrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.     

Suppression of tumorigenicity by the IncW R plasmid pSa in Agrobacterium tumefaciens

Summary The effect of the IncW R plasmid, pSa, on tumorigenicity and on the expression and maintenance of the Ti plasmid in tumorigenic strains of A. tumefaciens was determined. Plasmid pSa could be transferred into and stably maintained by both octopine-and nopaline-utilizing A. tumefaciens strains. The R plasmid had no effect on Ti plasmid maintenance or on Ti plasmid functions, such as octopine utilization or conjugal bacterial transfer. However, A. tumefaciens strains harboring both the R plasmid and the Ti plasmid in most instances failed to induce tumors on a number of plant species. This effect on tumorigenicity is specific to pSa. When pSa is cured from the A. tumefaciens transconjugants or when their Ti plasmids are genetically transferred to an appropriate recipient, the resultant strains lacking the R plasmid regain tumorigenicity. Restriction endonuclease analysis of plasmid DNA isolated from transconjugants harboring pSa showed no difference in Ti plasmid cleavage patterns when compared to plasmid DNA isolated from the tumorigenic parent strain. These results indicate that pSa does not induce detectable permanent genetic alteration of the Ti plasmid. Rather, it appears that the R plasmid suppresses some Ti plasmid function(s) necessary for tumorigenicity.     

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.     

NewAgrobacterium helper plasmids for gene transfer to plants

We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these disamed Ti plasmids for plant transformation viaAgrobacterium are discussed.     

Isolation of Rhizobium loti Strain-Specific DNA Sequences by Subtraction Hybridization

Mixed-phase (heterogeneous) and single-phase (homogeneous) DNA subtraction-hybridization methods were used to isolate specific DNA probes for closely related Rhizobium loti strains. In the heterogeneous method, DNA from the prospective probe strain was repeatedly hybridized to a mixture of DNA from cross-hybridizing strains (subtracter DNA) which was immobilized on an epoxy-activated cellulose matrix. Probe strain sequences which shared homology with the matrix-bound subtracter DNA hybridized to it, leaving unique probe strain sequences in the mobile phase. In the homogeneous method, probe strain sequences were hybridized in solution to biotinylated, mercurated subtracter DNA. Biotinylated, mercurated subtracer DNA and probe strain sequences hybridized to it were removed by two-step affinity chromatography on streptavidin-agarose and thiol-Sepharose. The specificity of the sequences remaining after subtraction hybridization by both methods was assessed and compared by colony hybridization with R. loti strains. Both methods allowed the rapid isolation of strain-specific DNA fragments which were suitable for use as probes.     

Radiation-sensitive Arabidopsis mutants are proficient for T-DNA transformation

Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure.     

Polygalacturonase Production by Agrobacterium tumefaciens Biovar 3

Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grape. Twenty-two Agrobacterium strains representing biovars 1, 2, and 3 were analyzed for tumorigenicity, presence of a Ti plasmid, ability to cause grape seedling root decay, and pectolytic activity. All of the biovar 3 strains, regardless of their tumorigenicity or presence of a Ti plasmid, caused root decay and were pectolytic, whereas none of the biovar 1 and 2 strains had these capacities. Isoelectrically focused gels that were activity stained with differentially buffered polygalacturonate-agarose overlays revealed that all of the biovar 3 strains produced a single polygalacturonase with a pH optimum of 4.5 and pIs ranging from 4.8 to 5.2. The enzyme was largely extracellular and was produced constitutively in basal medium supplemented with a variety of carbon sources including polygalacturonic acid. Lesions on grape seedling roots inoculated with A. tumefaciens biovar 3 strain CG49 yielded polygalacturonase activity with a pI similar to that of the enzyme produced by the bacterium in culture. These observations support the hypothesis that the polygalacturonase produced by A. tumefaciens biovar 3 has a role in grape root decay.     

Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells

The intact T-region of the B6Ti plasmid of Agrobacterium tumefaciens was stepwise cloned into a site in transposon Tn3. In this way a suitable vehicle (Tn1882) was obtained for translocating the T-region to different replicons, i.e., to other plasmids or the chromosome. The IncP plasmid R772::Tn1882 conferred tumorigenicity on Agrobacterium if the virulence genes were provided in trans in the same cell. This result showed that the T-region present on Tn1882 was transferred efficiently to plant cells. Normal tumor development also occurred if the T-region was placed in the chromosome of A. tumefaciens and an R' plasmid was present carrying virA–E or virA–F. We conclude that the plasmid location of the T-region is not a prerequisite for transfer to the plant cell. The apparently normal delivery of the T-DNA from a bacterial chromosomal location supports a model involving a processing step within Agrobacterium effecting transfer of the T-region as a separate entity.     

Generation of Backbone-Free,Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome

In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five transferred (T)-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector (backbone) into the genome of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events.Since the generation of transgenic plants approximately 25 years ago, Agrobacterium tumefaciens has been widely used for introducing genes into plants for purposes of basic research as well as for generation of commercially used transgenic crops. For plant transformation, the gene of interest is placed between the left and right border repeats of Agrobacterium transferred (T)-DNA (Gelvin, 2003). The T-DNA region harboring the transgene is stably integrated into the plant genome by using an appropriate plant transformation protocol. T-DNA originates from the Agrobacterium tumor-inducing (Ti) plasmid. Because Ti plasmids are large and difficult to manipulate, smaller T-DNA binary vectors are currently predominately used for generation of transgenic plants (de Framond et al., 1983; Lee and Gelvin, 2008).Although Agrobacterium has been used for plant transformation for more than two decades, problems using this bacterium remain. Agrobacterium-mediated transformation generally results in lower transgene copy numbers than do other transformation methods such as particle bombardment or polyethylene glycol-mediated transformation (Kohli et al., 1998; Shou et al., 2004). On the other hand, transformation frequently results in unwanted high copy number T-DNA integration events (Jorgensen et al., 1987; Deroles and Gardner, 1988; Shou et al., 2004; De Buck et al., 2009). Multiple integration events, often coupled with inverted repeat T-DNA integration patterns, may affect the stability of transgene expression by silencing mechanisms (Jorgensen et al., 1996). An additional problem with Agrobacterium-mediated transformation is the propensity for DNA sequences outside the T-DNA region to integrate into the plant genome (Kononov et al., 1997; Wenck et al., 1997; Shou et al., 2004). Integration of such vector backbone sequences can occur with high frequency. For example, Kononov et al. (1997) detected backbone sequences in 75% of tested transgenic tobacco (Nicotiana tabacum) plants, and very often the entire vector backbone is introduced into the plant genome (De Buck et al., 2000). T-DNA vector backbones usually harbor bacterial antibiotic resistance genes that can create governmental regulatory concerns.Here we show that launching T-DNA from the A. tumefaciens chromosome reduces integrated transgene copy number and almost eliminates the presence of T-DNA backbone sequences. We describe several plasmids and bacterial strains to facilitate use of this methodology.