Mendelian transcmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens

Summary Insertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity. rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.This paper is dedicated to Prof. Georg Melchers on the occasion of his 75th birthday, in recognition and gratitude for his relentless and enthousiastic pioneering efforts in the field of experimental plant genetics and cell biology     

The lysopinedehydrogenase gene used as a marker for the selection of octopine crown gall cells

Plant cells transformed into octopine-synthesizing tumour cells by the bacterium Agrobacterium tumefaciens survive when cultured in the presence of homo-arginine (HA), whereas both normal plant cells and nopaline producing plant tumour cells do not. Survival of octopine crown gall cells is due to the activity of the enzyme lysopinedehydrogenase (LpDH) in these cells, which converts toxic homo-arginine into non-toxic homo-octopine. The selective toxicity of homo-arginine for normal cells can be applied for the enrichment of octopine Ti plasmid transformed plant cells vs normal plant cells in mixed cultures.     

Methylglyoxal destroys <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> crown gall tumours in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> without any adverse effect on the host plant

Methylglyoxal (MG) is a highly reactive α-oxoaldehyde, demonstrating anticancer effect on plant neoplastic tumours. In in vivo studies it was observed that MG destroyed crown gall tumours in Nicotiana tabacum produced by Agrobacterium tumefaciens, without any adverse effect on the host. The efficacy of MG in comparison to other anticancer drugs viz. cisplatin and ellagic acid in the treatment of crown gall was investigated. A slight degeneration of galls was noted in plants treated with cisplatin and ellagic acid but the plants died subsequently. With MG however, crown galls were completely cured and the plants completed their usual life cycle by flowering and producing seeds. MG inhibited the respiration of crown gall calluses suggesting that energy depletion resulted in tumour destruction.     

Isozyme gene expression in potato tumors incited by Agrobacterium

Summary Two plant tumors (crown galls and hairy roots) were experimentally provoked on potato cv. Désirée by oncogenic strains of Agrobacterium tumefaciens and A. rhizogenes. A marked shift in the expression of some organ-specific genes occurred in crown galls derived from the central zone of tubers: two novel isozyme genes (Est-B and Pox-E) were expressed, two others (Est-C and Pox-F) were suppressed and the remaining ones were maintained in the original state. When the starting tissue was the stem segment, a smaller shift occurred, namely the activation of Adh-A and the suppression of Pox-F. In all cases, the isozyme profiles characterizing all crown galls, whatever their origin, were identical. Under normal aeration conditions, Adh-A was not expressed in either tumoral or non-tumoral roots. However, under the relative anaerobic conditions of in vitro cultures, a difference existed between both types of roots: Adh-A was expressed in normal but not in tumoral roots. This means that hairy roots can tolerate higher levels of anaerobiosis without giving rise to an anaerobic response. For the remaining isozymes, no alteration occurred in either organized (hairy root) or unorganized (crown gall) tumors, as compared to the corresponding non-tumoral tissues (normal root and callus, respectively).     

Occurrence of Agrobacterium tumefaciens Biovar 3 on Grapevine in Brazil

Occurrence of crown gall on grape was observed in a vineyard in Minas Gerais State, Brazil. Isolations of the pathogen yielded a bacterium which incited galls on grapevine, tomato and bryophillum. The bacterium was confirmed as Agrobacterium tumefaciens and its characterization showed it to be biovar 3. This is the first report on the occurrence of the biovar 3 of A. tumefaciens on grapevine in Brazil.     

Patterns of phenolic compounds in leafy galls of tobacco

The chemical composition of ethanolic and aqueous extracts from leafy galls produced after infection of Nicotiana tabacum L. plants with Rhodococcus fascians was drastically changed compared to uninfected controls. Chlorogenic acid was abundant both in uninfected and infected plants, but caffeic acid and another cinnamoyl analogue were new in leafy galls. The most pronounced product induced in leafy galls was identified as 7-O-methyl-6-O-β-d-glucopyranosyl coumarin (7-methyl esculin). This is the first report of the presence of this coumarin derivative in tobacco. Interestingly, 7-methyl esculin did not accumulate in the presence of avirulent R. fascians strains nor was it found in leafy galls on other plant species. However, it did appear in crown galls induced by Agrobacterium tumefaciens on tobacco plants. Intriguingly, none of the phenolics known to accumulate in Solanaceae under pathogen attack were found in leafy galls. 7-Methyl esculin barely affected growth of R. fascians nor was it catabolized. Microscopical analysis showed that autofluorescent compounds were located mainly in the abundant meristematic regions of the leafy galls. We postulate that 7-methyl esculin might locally influence plant cell division. Received: 6 March 1996 / Accepted: 30 August 1996     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra- Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach × almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

Specificity patterns of Agrobacterium tumefaciens phages

Summary Lysogeny was not detected in 10 strains of A. tumefaciens by plating techniques or ultra-violet induction. Fifteen phages were isolated from raw sewage against 13 cultures of A. tumefaciens and purified by single-plaque selections. No phage lysed all of the strains of A. tumefaciens tested; one phage lysed only a single strain; 2 other phages attacked 7 strains. Ten of the 15 phages lysed no more than 3 strains. Three host strains showed identical phage susceptibilities. No relationship was noted between susceptibility to phage and ability of a strain to incite crown galls.Thirteen phages lysed at least 1 of 4 strains of A. radiobacter, but none attacked single strains of A. rubi or A. pseudotsugae. Eleven phages lysed the one strain of A. rhizogenes used. None of the phages had identical host ranges with respect to all the Agrobacterium spp. tested. Similarly none of 5 selected phages attacked any one of 59 strains of bacteria from 12 different genera including 35 strains of rhizobia. Within the limits of this study the phages used were genus-specific.Published with approval of the Director, Wisconsin Agricultural Experiment Station, Madison, Wisconsin, U.S.A. 53706.     

Resistance to crown gall disease in transgenic grapevine rootstocks containing truncated <Emphasis Type="Italic">virE2</Emphasis> of <Emphasis Type="Italic">Agrobacterium</Emphasis>

A truncated form of the Ti-plasmid virE2 gene from Agrobacterium tumefaciens strains C58 and A6, and A. vitis strain CG450 was transferred and expressed in somatic embryos of grapevine rootstocks 110 Richter (Vitis rupestris × V. berlandieri), 3309 Couderc (V. rupestris × V. riparia) and Teleki 5C (V. berlandieri × V. riparia) via Agrobacterium-mediated transformation to confer resistance to crown gall disease. Transformation was confirmed in 98% of the 322 lines by enzyme-linked immunosorbent assay for the neomycin phosphotransferase II protein and 97% of 295 lines by polymerase chain reaction for the truncated virE2 transgene. Southern blot analysis revealed the insertion of truncated virE2 at one to three loci in a subset of seven transgenic 110 Richter lines. In vitro resistance screening assays based on inoculations of shoot internode sections showed reduced tumorigenicity and very small galls in 23 of 154 transgenic lines. Non-transformed controls had a 100% tumorigenicity rate with very large galls. Disease resistance assay at the whole plant level in the greenhouse revealed seven transgenic lines (3 lines of 110 Richter, 2 lines of 3309 Couderc and 2 lines of Teleki 5C) were resistant to A. tumefaciens strain C58 and A. vitis strains TM4 and CG450 with a substantially reduced percentage of inoculation sites showing gall as compared to controls. No association was found between the level of resistance to crown gall disease and the source Agrobacterium strain of virE2. Taken together, our data showed that resistance to crown gall disease can be achieved by expressing a truncated form of virE2 in grapevines.     

The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis L.

The three-dimensional pattern of phloem and xylem in 10-d-to two-month-old tumors induced by Agrobacterium tumefaciens (C58) and in adjacent Ricinus communis L. stem tissues was studied in thick sections by clearing with lactic acid and by staining with lacmoid. The crown galls contained two types of vascular strands: treelike branched bundles, which developed towards the tumor surface in fast-growing regions, and globular bundles in the slowly developing parts. Both types of vascular bundles contained xylem and phloem and were continuous with the vascular system of the host plant. The tumor bundles were interconnected by a dense net of phloem anastomoses, consisting of sieve tubes but no vessels. These vascular patterns reflect the apparent synthesis sites, concentration gradients and flow pathways of the plant hormones additionally produced in the tumors upon expression of the T-DNA-encoded genes. The A. tumefaciens-induced crown gall affected vascular differentiation in the host stem. In the basipetal direction, the tumor induced more xylem differentiation directly below it, where the crown-gall bundles joined the vascular system of the host. In the centripetal direction, the crown gall caused the development of pathologic xylem characterized by narrow vessels, giant rays and absence of fibers. On the other hand, most probably as a consequence of its gibberellic acid content, the host plant stimulated a local differentiation of regenerative phloem and xylem fibers with unique ramifications, only at the base of the tumor. However, fibers were absent from the main body of the crown gall. The study shows that A. tumefaciens-induced crown galls are characterized by a sophisticated network of vascular tissues in the tumor and are accompanied by a perturbated vessel system in the host. The hormonal mechanisms controlling vascular differentiation in the tumor and neighboring host tissues are discussed. In addition, the gall constriction hypothesis is proposed for explaining the mechanism which gives priority in water supply to the growing gall over the host shoot.We thank Dr. Zs. Koncz (Max-Planck-Institut für Züchtungsforschung, Köln, Germany) for Agrobacterium strains and the Deutsche Forschungsgemeinschaft (SFB 199) for financial support to C.I.U.     

Biochemical and genetic characterisation of Agrobacterium tumefaciens the causal agent of walnut crown gall disease in Iran

In 2009–2010, crown tumours were collected from walnut (Juglans regia L.) trees in northern Iran. Gram-negative, rod shaped and aerobic bacteria with circular, convex and white-coloured colonies on potato dextrose agar plus CaCO3 medium were isolated from galls. In pathogenicity tests, tomato seedlings were inoculated with all strains and tumours started to appear three weeks after inoculation. Strains yielded a 224?bp amplicon from the virD2 gene in PCR. When the 16S rRNA gene sequence of strains was compared by BLASTn with nucleotide sequences from GenBank, it showed 99.6% identity with the 16S rRNA sequence of Agrobacterium tumefaciens ATCC 33970. Based on phenotypic and genotypic properties, the bacterium that causes crown gall of walnut trees was identified as A. tumefaciens.     

Potential of Agrobacterium tumefaciens and Octopine-Utilizing Fluorescent Pseudomonas Strains To Attach to Susceptible Potato Tissues

The binding characteristics of two octopine-catabolizing pseudomonads, Pseudomonas fluorescens B99A and E175D, which were isolated from crown galls, have been examined. The binding of strain B99A to potato disks was very weak, followed a Freundlich isotherm, and was temperature and pH independent. Strain E175D displayed strong attachment and followed a Langmuir isotherm. Despite these fundamental differences in binding characteristics, when each strain was placed in competitive binding assays with either Agrobacterium tumefaciens B6 or A. tumefaciens ATCC 15955, the number of bound pseudomonad cells decreased compared with those obtained in independent trials. Furthermore, the binding of A. tumefaciens cells was increased. In prebinding experiments, in which the potato disks were bound with the pseudomonads before exposure to the agrobacteria, the number of bound pseudomonad cells again decreased. This implies that increased desorption was occurring. In these prebinding studies, the numbers of bound A. tumefaciens ATCC 15955 increased, but the number of bound A. tumefaciens B6 remained the same. The mechanism for this observed synergism on the binding of agrobacterial cells and the depression in bound pseudomonad cells is believed to be alterations in the electrostatic or ionic charges on the plant and bacterial cell surfaces. The synergistic effect on A. tumefaciens undermines the use of these pseudomonads as potential biocontrol agents for crown gall.     

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.     

Induction and in vitro culture of soybean crown gall tumors

Induction of crown galls on 4–6 week old soybean (Glycine max (L.) Merr.) plants cultured in growth chambers was obtained with Agrobacterium tumefaciens strains C58, T37 and ACH5. The crown galls were isolated and cultured in vitro as sterile callus and liquid suspension cultures. Transformation was tested by opine tests and by molecular hybridization of restricted cell DNA with T-DNA fragments. Protoplasts were isolated from suspension cultures. Transformed protoplasts regenerate cell walls, divide and form calli without an exogenous supply of hormones.     

Differential accumulation of proteinase inhibitor I in normal and crown gall tissue of tobacco, tomato, and potato

A proteinase inhibitor (inhibitor I) is induced in crown gall tumors of tobacco (Nicotiana tabacum) initiated through infection with the tumorinducing bacterium, Agrobacterium tumefaciens, strains B6 or CG-14. Uninfected tissues do not contain immunologically detectable quantities of inhibitor I. Inhibitor I synthesis in tobacco crown gall tumors paralleled tumor growth at the average rate of about 4.5 μg of inhibitor I per 200 mg of fresh tissue per day. Infection of variegated tobacco mutant Dp-I with A. tumefaciens strain CG-14 produced tumors with 25% more inhibitor than tumors induced with strain B6. Unlike tobacco, tumors induced by either bacterial strain on potato (Solanum tuberosum) and on tomato (Lycopersicum esculentum) did not accumulate inhibitor I. Consequently, inhibitor I accumulation is modulated by the type of plant host used in spite of familial relatedness (Solanaceae) and the strain of A. tumefaciens used for infection.     

Hybrids between tobacco crown gall cells and normal somatic cells of Atropa belladonna

Protoplast fusion of Nicotiana tabacum (B6S3) crown gall cells and Atropa belladonna leaf mesophyll cells was carried out. Hybrids were selected for their capacity to grow on hormone-free media and to green in light. Shoots incapable of rhizogenesis were regenerated on the same media and grafted onto normal plants of different species. 57 hybrid cell lines differing in their genetic constitution were produced. Analysis of hybrid lines involved the determination of the lysopine dehydrogenase (LpDH) activity and the molecular forms of esterase and amylase, a restriction analysis of chloroplast DNA and a cytogenetic study.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - LpDH lysopine dehydrogenase     

Growth requirements ofArabidopsis thaliana crown galls

Crown galls induced onArabidopsis thaliana plants by octopine or nopaline strains ofAgrobacterium tumefaciens were grownin vitro on different media. Dark growth of all tumor tissues was strictly hormone-dependent. In contrast, hormonal autonomy was observed in the light where crown gall calli readily differentiated into teratomas and (sometimes fertile) plants. Differentiating tissues always grew more vigorously than subtended calli. The growth of transformed calli was stimulated by vitamins and partly inhibited by growth regulators in concentrations used for the maintenance of untransformed calli. Crown gall calli, teratomas and sometimes regenerated plants were shown to express lysopine or nopaline dehydrogenase activities.     

A new amino acid derivative present in crown gall tumor tissue

A new amino acid derivative has been found in primary and secondary sunflower crown gall tissue cultures and in fresh crown gall tumors from sunflower plants wound-inoculated with Agrobacterium tumefaciens B₆. Normal plant tissue does not contain detectable levels of the compound. Radioactive labeling and cochromatography experiments strongly suggest that the natural derivative is identical to synthetic N²-(1-carboxyethyl)-L-histidine (histopine). Crown gall tissue cultures contain 1 μmole of histopine/20 g fresh weight. A. tumefaciens strain B₆, but not strain C₅₈, can utilize natural histopine and incorporate the products into macromolecules.     

Expression of nopaline synthesis in tumorous and regenerant tobacco crown galls

Summary Tissues formed in liquid cultures of tobacco (Nicotiana tabacum cv. Wisconsin 38) crown galls incited byAgrobacterium tumefaciens C58 were of three types: unorganized callus, organized teratoma, and organized normal appearing. These tissues contained 400±12, 410±17, and 614±53 μg nopaline/g fresh weight, respectively. Using [¹⁴C]arginine, methods were developed for measuring in vivo nopaline biosynthetic rates. Tissues were incubated in a low concentration (i.e., 3 μM) of [¹⁴C]arginine to minimize disruption of the internal pool (approximately 140 μM free arginine). Radioactivity in the tissue was assayed and the specific radioactivity of free arginine, the precursor of nopaline, was determined. The linear rate of incorporation of radioactivity into nopaline was used to calculate the following biosynthetic rates (expressed as microgram nopaline per gram fresh weight per 24 h): callus, 14; teratoma, 21; normal appearing, 24. These results show conclusively that normal appearing tissues obtained from crown gall tumors can synthesize nopaline. Abnormal growth and opine biosynthesis, therfore, can be expressed independently.