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.     

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.     

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.     

Agrobacterium tumefaciens Site Attachment as a Necessary Prerequisite for Crown Gall Tumor Formation on Potato Discs

The infectivity of Agrobacterium tumefaciens strain B6 was inhibited about 50% when these bacteria were inoculated on potato discs with equal viable cell counts of a weakly virulent strain of A. tumefaciens (B-48) or autoclaved strains of B6 or B-48. Inhibition by B-48 or autoclaved B6 could still be obtained when these cells were added up to a maximum of 10 minutes after the addition of viable B6. Maximum inhibition occurred when these cells were added 10 minutes prior to the addition of B6. There was no inhibition observed when equal cell counts of B6 were added along with a Gram-positive bacterium or yeast cell, while inhibition was observed when these B6 cells were added simultaneously with other Gram-negative cells. These results suggest that a physical, specific bacterial attachment that occurs within 10 minutes is necessary for tumor formation on potato discs.     

Agrobacterium adherence involves the pectic portion of the host cell wall and is sensitive to the degree of pectin methylation

Cell walls isolated from dicotyledon tissues compete with natural plant host sites for Agrobacterium tumefaciens (strain B6) when co-inoculated with infectious bacteria, thereby reducing tumor initiation. Removal of the pectic fraction from the cell walls results in loss of inhibition and the soluble pectic fraction is inhibitory. On treatment with pectin methyl transferase plus S-adenosyl-L-methionine these cell walls become less inhibitory and this change is reversible by pectinesterase. Cell walls isolated from monocotyledons, crown gall tumors or embryonic dicotyledons do not compete for Agrobacterium in the infection assay. These cell walls become inhibitory on treatment with pectinesterase and this is partially reversed by pectin methyl transferase. These data indicate that the pectic portion of the host cell wall is involved in the Agrobacterium -host adherence which is essential for tumor initiation and that the degree of methylation of polygalacturonic acid is critical to this adherence.     

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.     

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.     

Susceptibility of Agrobacterium tumefaciens Strains to Two Agrocin-Producing Agrobacterium Strains

Sixty-five strains and isolates of Agrobacterium tumefaciens representing each of the known biotypes, were tested for in vitro and in vivo susceptibility to the agrocin-producing strains Agrobacterium radiobacter 84 and A. tumefaciens D286. No biotype 3 strain was susceptible to the effects of either of the agrocinogenic strains in vitro. On datura and tobacco, the best inhibition of tumor formation was obtained when the agrocinogenic strains were applied to wounds 24 h before the pathogens and by the concomitant application of agrocin producer and pathogen at cell ratios of 10:1 or 3:1; inhibition of infection tended to decrease progressively as the cell ratio decreased from 10:1 to 3:1 to 1:1. Generally, strain 84 was superior to D286 in inhibiting tumor formation. A combined cell suspension of 84 and D286 was as effective as 84 alone. The overall pattern of inhibition of tumor formation by biotype 1 and 2 pathogens resistant to the agrocinogenic strains in vitro was similar to that obtained with strains that were susceptible in vitro.     

Agrobacterium tumehciens After in vivo Inoculation of Potato (Sohnum tuberosum L.) (Scanning Electron Microscopy)

Five weeks after the in vivo inoculation of potatoes ( Solanum tuberosum L.) with Agrobacterium. tumefaciens strain B6S3, bacteria were found in the non-differentiated cells of tumors (formed from xylem parenchyma or other living cells), in xylem cells at the site of inoculation, as well as in xylem cells of the adjacent stem.
Bacteria were attached by fibrillar aggregates to the tumor cell walls. They were also attached to a fibrillar mass which arose from agrobacteria connected to this mass in the tumor. Agrobacteria, singly or in pairs, were attached to an electron dense formation (possibly bacterial extracellular polysaccharides) found both inside the xylem cells of the stem adjacent to the tumor and at the site of inoculation. Some A. tumefaciens cells were attached by means of a pedestal-like structure at the inoculation site.
A possible function of the different means of attachment of A. tumefaciens in both nontransformed plant cells and tumors is discussed.     

In Vitro Binding of Agrobacterium tumefaciens to Plant Cells from Suspension Culture

In vitro binding experiments were carried out using (32)P-labeled cells of the virulent Agrobacterium tumefaciens strain B6 and Datura innoxia cells from suspension culture. Binding kinetics showed that adherence of bacteria to Datura cells increased gradually during the first 60 minutes and attained a maximum level within 120 minutes of incubation. Maximum binding occurred at pH 6.0. The presence of Ca(2+) and Mg(2+) reduced binding slightly and EDTA had little effect at concentrations of 0.1 to 10 millimolar. The binding of bacteria to Datura cells was temperature-dependent. Escherichia coli, Salmonella typhimurium, Rhizobium japonicum, and Micrococcus lysodeikticus did not compete with virulent A. tumefaciens strain B6 for binding to Datura cells. The admixture of avirulent A. tumefaciens strain IIBNV6 enhanced adherence of virulent A. tumefaciens strain B6 to Datura cells. Octopine had no effect on the binding of virulent A. tumefaciens strain B6 to Datura cells, but 10 millimolar canavanine was inhibitory. Arginine enhanced the adherence of the bacteria at concentrations higher than 0.1 millimolar. Incubation with DNase, RNase, and lipase did not affect the binding, but protease stimulated the adherence of bacteria to Datura cells. Concanavaline A and soybean lectin had little effect whereas lecithin and lysolecithin enhanced binding slightly. Poly-l-lysine markedly stimulated the bacteria-plant cell adherence. Cells from suspension cultures of pea, vetch, and soybean had a 2- to 3-fold higher binding capacity than Datura cells, whereas cells from wheat, corn, rice, and sorghum had a considerably lower affinity for binding with virulent A. tumefaciens strain B6. Bacterial adherence to plant cells was confirmed by autoradiography and electron microscopy. Autoradiographic analysis showed that bacteria were associated with the cell wall, and that often binding of bacteria was localized. Electron micrographs clearly illustrated a tight association of virulent A. tumefaciens strain B6 cells to the Datura cell wall.     

Observations on the effect of radiotherapy,followed by injection of pig lymph node cells,in reducing the number of pulmonary tumors induced by intravenous injection of tumor cells into isogenic mice

Summary Pulmonary tumors were produced in A. strain mice by intravenous injection of A. strain mammary carcinoma cells. The mesenteric lymph nodes of pigs were immunized by implantation of fragments from the same tumors into the pig mesentery.Tumor-immune pig lymph node cells when injected IV 7 days after tumor cells did not reduce the number of tumors, counted on day 14. However, when preceded by 200 rad thoracic irradiation on day 3 (which increased the number of pig cells settling in the lungs) tumor-immune cells given IV reduced the number of tumors compared with the effect of irradiation alone, or in combination with nonimmune pig cells.When tumor-immune pig cells were injected IP on day 7 (following thoracic irradiation on day 3), no antitumor effect was observed. Thus immediate pig cell/tumor cell contact is important in order to obtain an antitumor effect.Pig cells immunized against a human bladder carcinoma did not reduce pulmonary tumor formation by one of the mouse tumors.     

Changes in cyclic-AMP phosphodiesterase activity during the formation of crown-gall tumors on potato discs

Cyclic-AMP phosphodiesterase activity was found in crown-galltumor tissue obtained from the inoculation of potato discs withAgrobacterium tumefacicns strain B6. Consistently more enzymeactivity was found, in this tumor tissue than in the correspondingnormal tissue. This difference in enzyme activity was observedwhen strains other than B6 were used to induce tumors, or whennormal and tumor potato tissue grown in culture were examinedfor phosphodiesterase activity. (Received January 27, 1978; )     

Correlation between binding of Agrobacterium tumefaciens by root cap cells and susceptibility of plants to crown gall

We compared the binding of Agrobacterium tumefaciens by freshly isolated root cap cells with susceptibility of plants to crown gall tumorigenesis. A high binding reaction was strongly correlated with susceptibility to tumorigenesis in a survey of the binding of strain B6 to cells from 48 species in 17 families. In reciprocal experiments with nine virulent A. tumefaciens strains, tumors developed in plant-bacteria combinations that gave a high binding response in the root cap cell assay. Binding was quantified by direct measurement of the number of bacteria bound to the periphery of individual cells. Root cap cells from six susceptible species bound significantly more bacteria than did cells from five resistant species.     

A new site-specific endonuclease showing phenotypical crypticity in a tumorigenic strain of Agrobacterium tumefaciens.

AtuBVI, an endonuclease showing new site-specificity, has been isolated from the tumorigenic strain IIBV7 of Agrobacterium tumefaciens, and is undetectable in the non-tumorigenic sister strain IIBNV6. AtuBVI degrades IIBV7 DNA in vitro and should, therefore, be regarded as being phenotypically cryptic in the bacterial cell; it also shows anomalous behavior under cerain incubation conditions. These properties point to a possible role for this enzyme in the insertion of exogenous Ti-plasmid DNA into plant tissues during tumorigenesis.     

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.     

Temperature-sensitive loss of tumor characteristics in a tobacco crown gall strain

Summary Three independently isolated tobacco crown gall strains incited byAgrobacterium tumefaciens C58 required phytohormone (auxin and cytokinin) supplements in the basal medium to grow, at 37°C. Six other tobacco crown gall strains incited, respectively, byA. tumefaciens IIBV7, B6, CGIC, A6NC, 27 and AT4 expressed, at 37°C, the tumor characteristic of ability to grow in vitro on medium lacking phytohormones. Nopaline was not detectable in C58 tumors cultured at 37°C, but octopine was produced by B6 tumor tissues incibated at the elevated temperature. C58 tumor strains kept at 37°C for 1 week or more lost the ability to express tumor characteristics at 27°C such as tissue morphology, growth on basal medium lacking phytohormones and nopaline production. Heat-treated C58 tissues also differed from the original tumor strain in regeneration ability and phytohormone requirements of explants; i.e. explants from regenerated, heart-treated C58 tumors required both auxin and cytokinin for growth in vitro.     

The use of immunologically competent cells from pig mesenteric lymph nodes to treat pulmonary tumors in mice

Summary Pulmonary tumors were produced in A strain mice by intravenous injection of 1×10⁶ A strain mammary carcinoma cells. The mice were killed on day 14, their lungs fixed in Bouin's fluid, and the number of tumors counted.The mesenteric lymph node chains of pigs were immunized by implantation of tissue into the mesentery. In all animals the middle segment of node chain was excised. The remaining segments of node proximal and distal to the resected segment were, in separate pigs, nonimmunized or immunized against mouse tumor, immunized against mouse tumor or mouse skin, or immunized against human tumor or mouse tumor. All segments of node chain were removed 7 days after immunization for preparation of cell suspensions.When tumor cells were combined in a ratio of 1 : 10 or 1 : 60 with mouse tumor-immune pig cells, there was a significant reduction in tumor formation compared to that in mice receiving tumor cells alone.Injection of mouse tumor-immune pig cells on day 7, to treat tumors inoculated on day 0, was ineffective. However, when the mice received, in addition, 200 rad thoracic irradiation on day 3, immune pig cells reduced the number of tumors compared to that in animals receiving irradiation alone, or irradiation and nonimmune pig cells.In further experiments, in order to increase the number of pig cells reaching the lungs, a splenectomy was performed on day 6, prior to intravenous injection of immune cells on day 7. A comparison was made of the antitumor effect of pig cells immunized against mouse tumor, mouse skin, or human tumor. Cells immunized against either mouse tissue were equally effective in reducing the number of tumors compared to the number in animals receiving tumor cells alone. However, cells immune to human tumor were ineffective.     

Studies of allogeneic tumor transplants: induced rejection of advanced tumors by immune alteration of recipients

In the present experiments, a methylcholanthrene-induced sarcoma (S-702) of B10.D2 origin was found to grow rapidly in B6AF1 mice leading to the death of all recipients in 5 to 9 wk. Nevertheless, immunity to MHC antigens presented by the tumor was readily demonstrable in tumor-bearing mice by their responses to donor strain skin grafts until late in the course of tumor growth, when a nonspecific form of immune suppression developed. In addition, B6AF1 mice preimmunized by exposure to B10.D2 donor strain antigens did not permit tumor growth. Treatment of tumor-bearing B6AF1 mice with CY at 18 days, when the tumors measured over 12-mm in diameter, followed by the i.p. injection of B10.D2 lymphoid cells (at a dosage of from 1.2 to 2.5 X 10(8) cells) resulted in the complete regression of 100% of these large tumors. CY treatment combined with localized immune stimuli in the form of donor strain skin grafts or secondary tumor implants was incapable of producing a sufficiently heightened immune response to cause tumor rejection. A dose of CY temporarily retarded tumor growth in most mice, and in a minority of animals so treated (less than 25%) tumors regressed completely. In syngeneic (B10.D2) animals, CY also temporarily slowed tumor growth, but total regression was never observed. An effective B10.D2 cell inoculum could consist not only of living lymphoid cells but of irradiated (1000 rad) cells as well. Tumor cell suspensions (after irradiation, 10,000 rad) were also effective. These observations suggest local immune factors at the host-tumor interface may have been of importance in the survival of these allogeneic tumor transplants and that CY influenced this state, perhaps through an influence on suppressor cells, allowing subsequent administration of donor strain cellular antigens to induce an effective tumor rejection response.     

TGFβ secreted by B16 melanoma antagonizes cancer gene immunotherapy bystander effect

Tumor-targeted delivery of immune stimulatory genes, such as pro-inflammatory cytokines and suicide genes, has shown to cure mouse models of cancer. Total tumor eradication was also found to occur despite subtotal tumor engineering; a phenomenon coined the "bystander effect". The bystander effect in immune competent animals arises mostly from recruitment of a cancer lytic cell-mediated immune response to local and distant tumor cells which escaped gene modification. We have previously described a Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin 2 (IL2) fusokine (aka GIFT2) which serves as a potent anticancer cytokine and it here served as a means to understand the mechanistic underpinnings to the immune bystander effect in an immune competent model of B16 melanoma. As expected, we observed that GIFT2 secreted by genetically engineered B16 tumor cells induces a bystander effect on non modified B16 cells, when admixed in a 1:1 ratio. However, despite keeping the 1:1 ratio constant, the immune bystander effect was completely lost as the total B16 cell number was increased from 10(4) to 10(6) which correlated with a sharp reduction in the number of tumor-infiltrating NK cells. We found that B16 secrete biologically active TGFbeta which in turn inhibited GIFT2 dependent immune cell proliferation in vitro and downregulated IL-2R beta expression and IFN gamma secretion by NK cells. In vivo blockade of B16 originating TGFbeta significantly improved the immune bystander effect arising from GIFT2. We propose that cancer gene immunotherapy of pre-established tumors will be enhanced by blockade of tumor-derived TGFbeta.     

我国葡萄根癌农杆菌Ti质粒的特征

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