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.     

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.     

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.     

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.     

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.     

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.     

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.     

The Influence of Plant-Growth Factors on the Initiation and Growth of Crown-Gall Tumours on Primary Pinto Bean Leaves

The number of tumours developing on primary pinto bean leaves(Phaseolus vulgaris L. variety Pinto) inoculated with Agrobacteriumtumefaciens (Smith and Town.) Conn, strain B6, was increasedby as much as 100 per cent through the addition of several plant-growthfactors and antagonists of some of these factors to the freshlyinoculated leaves. Naphthylacetic acid, gibberellic acid, (2-chloroethyl)trimethylammonium chloride, and adenine gave a biphasic responsewith optimal promotions of tumour initiation at 10^-5 to 10^-4mg/ml. Tri-iodobenzoic acid and 4-chlorophenoxyisobutyric acidwere most active at 10^-1 mg/ml. Mean tumour diameter showeda direct correlation with tumour number in these experiments.The results show tumour initiation to be sensitive to growthfactor changes, possibly through a heightened traumatic responseof the wounded leaves. The growth of the tumours was unaffectedby these additions except as they altered tumour number andthis secondarily affected tumour growth.     

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.     

Polymyxin resistance in Agrobacterium tumefaciens and its effect on crown gall tumor induction.

Polymyxin-resistant (PBLr) mutants of Agrobacterium tumefaciens A6, B6, and B6M were isolated from polymyxin-sensitive (PBLs) parent strains in a defined medium containing 600 microgram of polymyxin B sulfate per millilitre. The weight and number of tumors induced by PBLr mutants on a variety of host plants such as carrot, potato, and pinto bean were 45--75% less than those induced by PBLs wild types. The crude cell envelopes (CCE) prepared from both PBLs and PBLr bacteria were inhibitory for tumor initiation when they were applied before or during the inoculation of viable tumorigenic bacteria, but not when they were applied 30 min after the inoculation of infectious bacteria. The potency to inhibit the tumor initiation by the CCE prepared from PBLs cells was approximately 50% higher than that by the equal amount of the CCE prepared from PBLr cells. The concentration of CCE preparations required to reduce tumor induction 50% in carrot and pinto bean was determined to be 2.6 mg/mL and 4.0--6.2 mg/mL for the CCE derived from PBLs and PBLr cells, respectively. These data suggest that the envelope structure or composition of PBLs and PBLr cells is distinct, and that the acquisition of resistance to polymyxin by agrobacteria modifies envelope structure or components which are essential for tumor initiation.     

Fixation of carbon dioxide in the dark by the malic enzyme of bean and oat stem rust uredospores

Malic enzyme was found in both bean rust and cat stem rust uredospores. In bean rust uredospores it was shown to catalyze the formation of pyruvic acid from l-malic acid and to synthesize malic acid from pyruvic acid and CO₂. The malic enzyme from bean rust uredospores was specific for NADP and dependent on manganous ions for activity. The specific activity of the bean rust malic enzyme in crude extracts of ungerminated uredospores was approximately 6 times greater than that found in crude extracts obtained from germinated uredospores. The malic enzyme was also found in extracts obtained from healthy and rust-infected bean leaves. The specific activity of the enzyme was approximately 2 to 5 times greater in partially purified extracts obtained from the infected bean tissue at 6 days after inoculation. The specific activity of the malic enzyme in crude extracts obtained from oat stem rust uredospores was 2 times greater than the specific activity of this enzyme in crude extracts obtained from bean rust uredospores. Phosphoenolpyruvate carboxylase activity could not be demonstrated in crude extracts obtained from the ungerminated uredospores of the bean rust fungus.     

Regulatory aspects of chloroplast growth in leaves ofXanthium pensylvanicum and etiolated red kidney bean seedling leaves

Summary The average chloroplast size was studied as a function of leaf growth in leaves of cocklebur (Xanthium, pensylvanicum) and the primary leaves of 9-day old seedlings of red kidney bean (Phaseolus vulgaris).Diameters of chloroplasts were measured in crude tissue homogenates with the aid of a fluorescence microscope. Chlorophyll content of the leaves was determined spectrophotometrically in acetone extracts.For cocklebur, data are presented to show the relationship of average chloroplast diameter to morphological age of leaves (Leaf Plastochron Index) and are discussed in relation to the available leaf growth analyses. In bean, the increase in chloroplast diameter in response to illumination of etiolated leaves of various size was studied as a function of the duration of continuous illumination. The size of the etiolated bean leaves was varied experimentally by exposing the seedlings in darkness to low energy red light. Average diameter of the chloroplasts was found to be related to the size of leaf lamina.In both cocklebur and bean, a definite relationship of chloroplast size to leaf area and morphological age was established. The observed patterns of chloroplast size increase are interpreted to be a reflection of the integration of growth at three levels of organization: the leaf, its cells and the chloroplasts.This study was performed during the tenure of a U. S. Public Health Service postdoctoral fellowship by the senior author and was supported in part by research grant number GM-08145, from the National Institutes of Health, Public Health Service.Predoctoral fellow of the National Science Foundation.     

Bacterial Attachment to a Specific Wound Site as an Essential Stage in Tumor Initiation by Agrobacterium tumefaciens

The number of tumors initiated by Agrobacterium tumefaciens strain B6 on primary pinto bean leaves was decreased when cells of an avirulent strain (IIBNV6) were included in the inoculum. With sufficient B6 cells to initiate ca. 50% of the maximal number of tumors per leaf, inhibition was detected at a 1:1 ratio of B6 to IIBNV6 cells and increased linearly with the logarithm of the number of IIBNV6. Varying the number of B6 in the presence of a constant number of IIBNV6 or varying the number of both, while maintaining a constant ratio of B6 to IIBNV6, showed that the inhibition was a function of the absolute concentration of each cell type. The data fit a one-particle dose response curve, which indicates that a single IIBNV6 cell can prevent tumor initiation by a single B6 cell. Inhibition was obtained with mixed inocula and when the addition of IIBNV6 preceded B6, but not when B6 preceded IIBNV6. Heat-inactivated IIBNV6 inhibited, as did ultraviolet or heat-inactivated B6. Several unrelated bacteria and certain strains of Agrobacterium failed to inhibit, whereas other related strains gave inhibition. Attachment of IIBNV6 to a specific would site, thus excluding B6 from the site, is proposed to account for these data. A specific complementary binding of a virulent bacterium to a host wound site exposed by the inoculation procedure is suggested as an essential early event in the crown-gall tumor initiation process.     

Promotion of crown-gall tumor growth by lysopine, octopine, nopaline, and carnosine

The growth of crown-gall tumors on primary bean leaves (Phaseolus vulgaris L. cv. “Pinto”) was promoted by the addition of d-lysopine, d-octopine, l-carnosine, or nopaline. Assayed on tumors induced by Agrobacterium tumefaciens strain B6, the relative activity was octopine = carnosine > lysopine nopaline; assayed on tumors induced by A. tumefaciens strain T-37, which induces tumors which form nopaline, the relative activity was nopaline = octopine = carnosine > lysopine. From one to three applications of carnosine or octopine gave equal additive increments in tumor growth, showing that a continual supply of these substances is required to maintain an increased rate of growth. At concentrations above 0.1 mm, pairs of these growth-promoting substances were less active than when applied singly. Inhibition of octopine-induced growth was obtained by applying 0.01 mm carnosine with 1 mm octopine and partial inhibition was obtained when carnosine was added 10 hr after octopine. Equimolar mixtures of lysopine, octopine, and carnosine, however, were at least as active in promoting tumor growth as any of the compounds added singly at equivalent concentrations. The activity of 0.1 to 0.5 mm lysopine, octopine, and carnosine was inhibited, respectively, by 1 mml-lysine, l-arginine, and l-histidine and this inhibition was limited in each case to the basic amino acid corresponding to that of the growth factor. Arginine fully inhibited octopine-induced tumor growth when applied as much as 6 hr after octopine, indicating that this inhibition was not due to prevention of octopine uptake. Although four separate substances were found which promoted tumor growth, the molecular specificity required for activity of each compound was high. Evidence is presented which suggests that a tumor growth-promoting substance extracted from tumorous leaves is a carnosine-like derivative of l-histidine.     

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.     

Effect of seed and root exudates on the growth ofXanthomonas phaseoli var.fuscans

In a medium containing bean, barley and wheat seed exudates,Xanthomonas phaseoli var.fuscans (Burk.) Starr et Burk. grew substantially better than in that containing root exudates of these plants. When the bacteria were cultivated in a medium containing root exudates of bean plants deprived of cotyledons after eleven days of growth, growth was slower than in the presence of root exudates of control plants. On the other hand, the growth was stimulated in a medium containing root exudates of bean plants deprived of leaves. It was found that seed exudates of these plants contained biologically active peptides stimulating the growth of the microorganism. These peptides were not found in root exudates. These findings suggest a relationship between the survival ofXanthomonas phaseoli var.fuscans in the rhizosphere of bean and the exudation of biologically active peptides originating from the stock substances of seeds and cotyledons.     

Partial purification of a legume nodulation factor present in coconut water

The nodulation of adventitious roots growing from segments of bean hypocotyl tissue was used as a bioassay for the material present in coconut water which stimulated nodulation. The active material in coconut water is acidic, but it was not possible to extract it from an acid solution with organic solvents. A purification of approximately 70-fold (on a dry wt basis) was obtained using activated charcoal, but at least 10 different compounds were present in the active fractions. A purified fraction of coconut water, which is stimulatory to the growth of carrot root explants, was active in the nodulation assay at a concentration of 2 μg/ml. This represents a 4000-fold purification of the diffusible fraction of coconut water. The charcoal fractionation procedure can be applied to the active material present in extracts of bean leaves.     

The Inhibition of Rust Fungus Growth in Allopurinol-Treated Bean Plants: Indirect Evidence for the Involvement of Xanthine Oxidoreductase in the Biotrophism of Uromyces phaseoli

Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine], a specific, potent inhibitor of xanthine oxidoreductase, effective in vitro and in vivo, was applied to bean plants as soil drench at a 400 μM concentration 8–10 days before inoculation and strongly reduced the development of Uromyces phaseoli in bean leaves. Allopurinol was ineffective on uredospore germination, presumably due to the absence of any xanthine oxidoreductase activity in the extract of germinated uredospores. The concentration of allopurinol used for the treatment did not significantly influence the level of ureides in leaves mainly because low concentration of these compounds were found in leaves and also probably because allopurinol-insensitive biosynthetic route/s of these compounds are active in bean plants. This paper examines the possibility that host xanthine oxidase is in some way involved in the biotrophic nutritional process leading to the growth of bean rust fungus.     

OZONE-INDUCED MEMBRANE PERMEABILITY CHANGES

The effect of 0.5 ppm ozone for 0.5-1 hr on plant cell membrane permeability was ascertained. Permeabilities to both water and solutes were estimated by measuring leaf disc weight changes and following tritiated water and ⁸⁶Rb fluxes. Measurements were made immediately after ozone exposure and 24 hr after exposure. The reflection coefficient, σ, an index of solute permeability, decreased in ozone-treated primary leaves of pinto bean (Phaseolus vulgaris). The latter indicates an increase in membrane solute permeability or internal solute leakage. Water and THO flux estimates both indicated a decrease in membrane permeability to water; both the hydraulic conductivity (L_p) and the water diffusional coefficient (L_D) apparently decreased, an anomaly which is discussed. These data indicate that ozone has a direct effect on membrane function by altering permeability characteristics. We assume from these data that cell membranes are primary target sites for ozone injury.