Bioassay and attributes of a growth factor associated with crown gall tumors

An improved bioassay is described for a factor that promotes tumor growth which was first obtained from extracts of pinto bean leaves with crown gall tumors. Sixteen primary pinto bean leaves per sample are inoculated with sufficient Agrobacterium tumefaciens to initiate about 5 to 10 tumors per leaf and treated with tumor growth factor at day 3 after inoculation. The diameters of 30 to 48 round tumors (no more than 3 randomly selected per leaf) are measured per test sample at day 6. Mean tumor diameter increased linearly with the logarithm of the concentration of tumor growth factor applied. The tumor growth factor was separated by column chromatography from an ultraviolet light-absorbing compound previously reported to be associated with fractions having maximal tumor growth factor activity. Partly purified tumor growth factor showed no activity in a cytokinin bioassay or an auxin bioassay, and negligible activity in gibberellin bioassays. Representatives of these three classes of growth factors did not promote tumor growth. Extracts from crown gall tumors on primary pinto bean leaves, primary castor bean leaves, Bryophyllum leaves, carrot root slices, and tobacco stems showed tumor growth factor activity, whereas extracts from healthy control tissues did not. Extracts from actively growing parts of healthy pinto beans, Bryophyllum, and tobacco, however, showed tumor growth factor activity. Tumor growth factor is proposed to be a normal plant growth factor associated with rapidly growing tissues. Its synthesis may be activated in nongrowing tissues by infection with Agrobacterium sp.     

Response of various cucurbits to infection by plasmid-harboring strains of agrobacterium

Tumor formation in cucurbit cultivars resulting from infection by various strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes is environmentally affected. In all instances, tumors could be induced on excised cotyledons while inoculating attached cotyledons or stems resulted in no tumor formation. In addition, buttercup squash (Cucurbita maxima Duch. buttercup) was most susceptible to tumor formation, while butterbush squash (Cucurbita maxima Duch. butterbush) failed to form tumors when inoculated with any of the strains of Agrobacterium. Other tested cucurbit cultivars showed intermediate susceptibility to tumor induction by the various Agrobacterium strains.     

Effect of gibberellic Acid on crown gall tumor induction in aging primary pinto bean leaves

Gibberellic acid was tested for its effect on tumor induction by Agrobacterium tumefaciens in primary pinto bean (Phaseolus vulgaris) leaves in various stages of development. The hormone was found to promote tumor induction in partially aged leaves but did not effect tumor induction in very young leaves or in fully matured leaves. It is suggested that the natural loss of susceptibility to tumor induction in maturing pinto bean leaves is associated with a concomitant loss of endogenous gibberellins and/or a sensitivity to gibberellins.     

Extraction, Assay and Partial Purification of a Factor from Tumorous Leaves that Promotes Crown-gall Tumor Growth

A tumor growth factor was extracted from primary pinto bean leaves {Phaseolus vulgaris L. cv. “Pinto”) having crown-gall tumors. Application of these extracts to primary pinto bean leaves at day 3 after inoculation with Agrobacterium tumefaciens (Smith and Town.) Conn resulted in an increase in the diameter of tumors on these leaves after 24 hours that was 25–60 percent greater than that of the controls. No growth promoting activity was detected in comparable extracts from control leaves or from cultures of the bacterium. The tumor growth factor did not affect tumor number when applied in this fashion. The active extracts were fractionated on Sephadex (1–15 columns and an active component obtained which eluted in a volume suggesting a molecular weight in the range of 100–200. A spectrum of the material in this fraction is shown and some of the characteristics of this material are described. Reasons are presented for considering this growth factor to be identical with at least one of the growth factors previously hypothesized to account for the greater tumor growth observed as tumor number is increased in this system.     

Identification of a crown-gall tumor growth factor as GABA

A crown-gall tumor growth factor (TGF-II) isolated from bean leaves inoculated with Agrobacterium tumefaciens strain 13333 is shown to be γ-aminobutyric acid (GABA). This identification is based on the comparative behavior of purified TGF-II and authentic GABA with respect to elution from preparative ion exchange and molecular sieve columns, ninhydrin reaction, TLC, co-chromatography on an automated amino acid analyzer, MS analysis and biological activity. GABA is detected by bioassay only in bean leaves infected with the bacterium and is in growth limiting supply when only a few tumors are present per leaf. GABA promotes tumor growth when as little as 1 ng is applied per leaf.     

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots byAgrobacterium tumefaciens andAgrobacterium rhizogenes

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls or hairj⁷ roots) inKalanchoe plants and carrot root slices. The MiniSa plasmid caused only a slight decrease of the frequency and size of tumor growths induced. The mechanism of suppression of virulence by the Sa plasmid inAgrobacterium tumefaciens andAgrobacterium rhizogenes seems to be similar.     

Plasmid Content and Tumor Initiation Complementation by Agrobacterium tumefaciens IIBNV6

Avirulent strains IIBNV6 and NT1, derived from virulent strains of Agrobacterium tumefaciens, were tested for their ability to enhance tumor initiation (complement) on coinoculation with tumorigenic strains. Strain NT1, cured of the Agrobacterium virulence plasmid, failed to complement when inoculated with its virulent parental strain or with other virulent strains. Strain IIBNV6, however, complemented with all virulent strains tested. Attachment to host wound sites by both strain IIBNV6 and the virulent strain was essential for this effect. Inoculation of the tumorigenic strain at different times on leaves previously inoculated with IIBNV6 showed that the capacity to complement is lost during the period between 4 and 8 h after IIBNV6 inoculation. The rate of tumor appearance obtained with an inoculum containing IIBNV6 and a virulent auxotrophic strain was characteristic of the appearance rate obtained with prototrophic bacteria. Evidence is summarized which suggests that strain IIBNV6 can induce tumors when supplied with a substance produced or induced by a virulent bacterium at a separate site. A deoxyribonucleic acid plasmid about 40% the size of the Agrobacterium virulence plasmid was obtained from strain IIBNV6. We propose that this plasmid accounts for the ability of strain IIBNV6 to complement and that it contains part of the genetic information necessary for tumor initiation.     

QUANTITATIVE MEASUREMENTS OF INTERACTIONS BETWEEN CROWN-GALL TUMORS AND THE PINTO BEAN HOST

Interactions between crown-gall tumors on the primary pinto bean leaf and the pinto bean seedling (Phaseolus vulgaris L. ‘Pinto‘) were estimated by quantitative measurements of tumor initiation and growth as affected by certain modifications of the host. Effects of the tumors on the host were estimated by measurements of host growth and correlation responses. The presence of crown-gall tumors was found to reduce the growth of the leaf in area but to nearly double the weight of the leaf 9 days after inoculation with Agrobacterium tumefaciens (Smith and Town.) Conn, strain B6. The presence of tumors on only one of the two primary leaves resulted in a decrease in the weight of the leaf without tumors, showing the tumors to be effective mobilization centers. Tumors also delayed the abscission of petiole explants and delayed the growth of the epicotyl bud, both reminiscent of auxin effects. The excision of the cotyledons, the epicotyl bud, or one of the pair of primary leaves at the time of inoculation increased the number of tumors initiated per leaf. Removing the epicotyl bud or one of the primary leaves, or placing a cytokinin on one of the leaves, altered leaf growth but failed to alter tumor growth, indicating that tumor growth is not affected by the changes responsible for the compensatory growth effects induced by these treatments. Tumor growth was shown to be generally correlated with leaf growth from day 2 through 8 after inoculation, suggesting that the factors normally limiting leaf growth in a determinate type leaf are also active in limiting tumor growth. The changes in the plant cell responsible for the enhanced rate of growth seen in crown-gall tumor cells, therefore, appear to occur in regulatory systems other than those normally limiting leaf growth. The regulatory systems that are affected may be identical with those activated in compensatory host growth effects.     

THE QUANTITATIVE DETERMINATION OF THE INFECTIVITY OF AGROBACTERIUM TUMEFACIENS

A bioassay relating number of Agrobacterium tumefaciens cells in the inoculum quantitatively to the number of crown-gall tumors initiated on primary pinto bean leaves is described. Variability in estimation of infectious titers by this assay is similar to that observed in comparable plant virus assays, most determinations showing standard errors of 20% of the mean tumor per leaf value. The assay has the advantages of speed and practicality. The efficiency of the system is low, typically requiring between 10⁵ and 10⁶ bacteria for each tumor initiated. Infectivity titers of 10³-10⁴, however, are readily obtained from stationary phase cultures. Statistical analysis of the infectivity titration curve indicates that a single bacterium is the usual infectious unit. The assay is specific within the family Rhizobiaceae to the species Agrobacterium tumefaciens and Agrobacterium rubi. A. tumefaciens strains IIBNV6 and ATCC # 11095 were non-infectious, while strain B6 was the most infectious of the strains tested. The infectivity of the latter strain is shown to decrease about 4-fold between early log and stationary phases of growth. Changes in the growth medium or in the dilution-inoculation medium failed to alter the infectivity of the bacterium.     

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved.     

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.     

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.     

Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.     

Tumor induction by agrobacterium involves attachment of the bacterium to a site on the host plant cell wall

Cell wall preparations from primary bean leaves were found to inhibit tumor initiation by Agrobacterium tumefaciens strain B6 when inoculated with the bacteria on bean leaves. Membrane fractions from these same leaves were noninhibitory. The cell walls were effective when applied prior to or with bacteria, but application of cell walls about 15 minutes after bacteria did not affect the number of tumors initiated. Much of the inhibitory activity of the plant cell walls was eliminated by pretreatment with dead site-attaching bacteria or with lipopolysaccharide from these bacteria. Cells and lipopolysaccharide from non-site-attaching agrobacteria had no effect on the activity of the plant cell walls. About 30% inhibition of tumor initiation was obtained with plant cell walls at 50 μg/ml dry weight, and at 10 mg/ml dry weight about 70% inhibition was typical. Both early and late appearing tumors were affected by the cell walls, indicating that they do not exclusively affect tumors arising from either small or large wounds. These data show that plant cell walls but not membranes contain surfaces to which A. tumefaciens adheres and these exhibit the specificity typical of the host site to which virulent agrobacteria must attach to induce tumors. It is concluded that some portion of wound-exposed plant cell wall constitutes the host adherence site in Agrobacterium infections.     

Physiological Comparisons of Pith Callus With Crown-Gall and Genetic Tumors of Nicotiana glauca, N. langsdorffii, and N. glauca-langsdorffii Grown in Vitro. I. Tumor Induction and Proliferation

Agrobacterium tumefaciens B-6 and T-37 strains, inoculated into Nicotiana glauca, N. langsdorffii, and their interspecific hybrid, which forms genetic (spontaneous) tumors as well, initiate amorphous tumors from the B-6 strain and organoid tumors (aberrant roots, stems, and buds) from the T-37 strain. In the hybrid, the critical point was to induce crown gall tumors at the site of wounding and not spontaneous genetic tumors. To succeed, this inoculation had to be made at a very early (5-6 leaf stage of development). It is observed that genetic organoid tumors readily formed at the nodes following flowering or leaf abscission. Furthermore, it was noted that genetic tumor derivatives are obtainable from hybrid pith callus or hybrid seedlings cultured in vitro.     

Evidence for a Tumor-associated Factor Active in the Promotion of Crown-gall Tumor Growth on Primary Pinto Bean Leaves

The growth of crown-gall tumors on primary pinto bean leaves (Phaseolus vulgaris L. cv. “Pinto”) between day 3 and day 6 after inoculation was found to be proportional to the number of tumors on the leaves. Similar differences observed in the growth of tumors induced by adenine, methionine and asparagine requiring mutants of Agrobacterium tumefaciens (Smith and Town.) Conn appear to be due to the same phenomenon. Tumors induced by these auxotrophs thus show no obvious growth differences from those induced by the prototrophic strain despite the lower specific infectivity and the existence of a mutational lesion in these bacteria. A diffusible growth factor(s) produced by the tumor tissue is proposed to account for the relation between tumor number and early tumor growth.     

Rapid and Efficient Identification of Agrobacterium Species by recA Allele Analysis

The analysis of housekeeping recA gene sequences from 138 strains from 13 species or genomic species of Agrobacterium, nine being biovar 1 genomospecies, and the others Agrobacterium larrymoorei, Agrobacterium rubi, Agrobacterium sp. NCPPB 1650, and Agrobacterium vitis and one “former” Agrobacterium species, Rhizobium rhizogenes, led to the identification of 50 different recA alleles and to a clear delineation of the 14 species or genomospecies entirely consistent with that obtained by amplified fragment length polymorphism (AFLP) analysis. The relevance of a recA sequencing approach for epidemiological analyses was next assessed on agrobacterial Tunisian isolates. All Tunisian isolates were found to belong to the Agrobacterium tumefaciens/biovar 1 species complex by both biochemical tests and rrs sequencing. recA sequence analysis further permitted their unambiguous assignment to A. tumefaciens genomospecies G4, G6, G7, and G8 in total agreement with the results of an AFLP-based analysis. At subspecific level, several Tunisian recA alleles were novel, indicating the power and accuracy of recA-based typing for studies of Agrobacterium spp.     

THE INDUCTION OF LEAF TUMORS BY AGROBACTERIUM TUMEFACIENS

Using carborundum as an abrasive and light rubbing with a culture of Agrobacterium tumefaciens, leaves of various species of bean and tobacco develop tumors on the leaf lamina. The induction of these tumors requires wounding, the presence of a virulent strain of the bacterium and is due to the bacterium, not substances released into the bacterial culture medium during growth. Observations of the histology and cytology of these tumors on the primary leaves of pinto bean show no significant differences from the more commonly studied stem tumors. The tumors on pinto beans first appear as chlorotic nests of dividing cells which gradually accumulate chlorophyll, eventually becoming dark green in color as opposed to the surrounding leaf tissue which is completely chlorotic at this stage. Tumor development is enhanced by a dark period following inoculation while growth of the leaf is essentially stopped. The tumors thus exhibit a pattern of growth and development independent of that of the normal leaf. The number of tumors obtained on pinto bean leaves was found to depend on the concentration of bacteria in the inoculum and on the age of the plants. A sharp peak in response was observed at about 7 days from planting. Best results were obtained by adding the bacterium at the time of wounding. The tumors were shown to differ from IAA-induced leaf proliferations with respect to their point of origin on the leaf, morphology, physiology and development.     

Strain and cultivar specificity in the Agrobacterium-soybean interaction

The response of Glycine max., G. soja and G. canescens genotypes to inoculation with different Agrobacterium strains was assessed. Percent visual tumor formation and tumor size varied widely among species and genotypes. Susceptible genotypes displayed a heightened response to nopaline strains of A. tumefaciens, relative to octopine, agropine, and A. rhizogenes strains. A nopaline strain engineered to contain a chimeric neomycin phosphotransferase II gene conferred kanamycin resistance to soybean tissue at kanamycine levels as high as 300 g/ml.     

Crown Gall on Castor Bean Leaves: II. FORMATION OF SECONDARY TUMOURS

Secondary tumours were formed on the cotyledonary leaf petiole,the hypocotyl, and first true leaf of castor bean seedlingsafter inoculating the blades of the cotyledonary leaves withAgrobacterium tumefaciens. Depending on the strain of bacteriaemployed, 0 to 80 per cent of the plants developed secondarytumours. The ability of different strains to initiate secondarytumours was not obviously correlated with their relative effectivenessin initiating primary tumours. Though all produced primary tumours,five out of ten auxotrophic strains failed to yield secondarytumours, whereas only one out of 14 prototrophic strains failedto do so. Both the number of plants developing secondary tumoursand the frequency with which these tumours occurred on differentparts of the plant were positively correlated with the concentrationof the primary inoculum. Tumours also developed on the cotyledonaryleaf petiole and on the hypocotyl after vacuum infiltrationof A. tumefaciens into the blade of cotyledonary leaves. Inmost instances (9 out of 11 plants) no tumours were formed onthe blade of the infiltrated leaf. Thus, tumour formation equivalentto secondary tumours can occur in the absence of a primary tumouror an overt primary wound. Excision of inoculated leaves showedthat the stimulus for secondary tumour formation moves fromthe blade to the hypocotyl within 24 h. Attempts to demonstratethe presence of a sub-cellular tumour-initiating agent in homogenatesof inoculated leaves were unsuccessful. A. tumefaciens, however,was found in the petiole of the cotyledonary leaf and in thehypocotyl within 24 h of inoculation. The migrating agent responsiblefor secondary tumour formation in castor beans is concludedto be intact bacteria.