Agrobacterium tumefaciens Ribonucleic Acid Induction - Journal of ...

2 downloads 0 Views 620KB Size Report
han, Anker, and Sheikh, in press). ' Present address: Faculty of Medicine, .... of bacterialDNA (8), plant DNA (8), and plant RNA (2), thein vitro RNA-DNA hybridi-.
Vol. 106, No. 2

JOURNAL OF BACTERIOLOGY, May 1971, p. 634-639 Copyright © 1971 American Society for Microbiology

Printed in U.S.A.

Agrobacterium tumefaciens Ribonucleic Acid Synthesis in Tomato Cells and Crown Gall Induction M. STROUN, P. ANKER, P. GAHAN,' A. ROSSIER, AND H. GREPPIN Department of Plant Physiology, University of Geneva, Geneva, Switzerland

Received for publication I February 1971

Purified Agrobacterium tumefaciens deoxyribonucleic acid (DNA) does not produce crown gall tumors in growing plants, conditioned by wounding, as the living bacteria do. Purified bacterial DNA migrates in the plant and replicates, but it is not transcribed in our experimental conditions. On the contrary, when DNA is released naturally from bacteria into plant cells, a bacterial ribonucleic acid (RNA) can be found in these cells. There seems to be a direct relation between the appearance of A. tumefaciens RNA in the plant cells and the induction of the tumor.

Crown gall (cancerous plant tumor) arises when a virulent strain of Agrobacterium tumefaciens is introduced into growing plants conditioned by wounding. When purified A. tumefaciens deoxyribonucleic acid (DNA), instead of bacteria, is introduced, no tumor develops. However, DNA isolated from a temperate phage (PS 8) of A. tumefaciens can induce a tumor, whereas the phage itself cannot (6). After the introduction of purified DNA of bacterial origin into tomato plant cells, it is observed that (i) the bacterial DNA retains its primary and secondary structure while in the host cells (11); (ii) after more than 2 days in the host, the bacterial DNA combines with that of the host cells, and the molecules thus formed replicate (12); and (iii) only plant ribonucleic acid (RNA) is synthesized in the tomato cells (15). However, after plants have been in contact with a suspension of bacteria, one finds the following in plant cells: (i) bacterial DNA having retained its primary and secondary structure (Stroun, Gahan, Anker, and Sheikh, Symposium on Uptake of Informative Molecules by Living Cells, 1970, North-Holland Publishing Co., in press); (ii) after less than I day in the host, self-replicating bacterial DNA and replicating molecules formed of bacterial DNA combined with plant DNA (Stroun, Gahan, Anker, and Sheikh, in press); (iii) the synthesis of large amounts of bacterial RNA (13-15); and (iv) the partial or total shut-off (reversible phenomenon) of the transcription of the host cell DNA (14; Stroun, Gahan, Anker, and Sheikh, in press). ' Present address: Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada.

634

The present communication concerns the possible involvement in tumor induction of the different factors present after the release of nucleic acids from A. tumefaciens into plant cells and absent after the uptake by plants of purified DNA of the same bacteria. MATERIALS AND METHODS A. tumefaciens strain B. (kindly supplied by Dr. Manigault of the Institut Pasteur, Paris) was grown for 24 hr in nutrient broth and collected by centrifugation. The bacteria were used either for the feeding of the plants or for the preparation of DNA. The terminal portions of the shoots of Lycopersicum esculentum var. "Poire" were removed; the shoots, after their cut ends were thoroughly washed, were placed in a suspension of A. tumefaciens (109 bacteria/ml of 0.1 SSC: 0.015 M sodium chloride, 0.0015 M sodium citrate) for 21 hr. After feeding under these conditions, bacteria are found only in the xylem and on the surface of the epidermis (15). The surface of the epidermis was washed free of bacteria in successive baths: 5% hypochlorite, 70% ethanol, sterile water, and 100 gg of chloramphenicol per ml. (Bacterial nutrient medium put in contact with plant epidermis treated in such a way remained sterile.) The stem was then wounded with a sterile needle to initiate the formation of the tumor. The transcription of bacterial DNA in plant cells was blocked by rifamycin SV (Lepetit, Milan), an inhibitor of DNA-dependent RNA polymerase. Ten micrograms of rifamycin per milliliter, which is harmless to plants, was given for 15 hr to the cut portion of the shoot at various times after wounding the plants fed with bacteria. This not only' blocks RNA synthesis in the plant cells but also kills A. tumefaciens (14). A culture of rifamycin (10 ug/ml)-treated A. tumefaciens does not grow. Electron microscope observations show

A. TUMEFACIENS RNA SYNTHESIS

VOL. 106, 1971

that these bacteria are completely disrupted. As a control, similar experiments were performed in which the rifamycin was replaced by sterile water. The state of tumor growth was observed after a period of 6 weeks. Other assays were carried out with A. tumefaciens strain B6 DNA. The shoots were placed with their cut ends in a solution of DNA (400 ug/ml) for I day (sufficient time for the penetration of the DNA in the cells; II) or for more than 2 days (sufficient time to allow the bacterial DNA to combine with that of the host cells and the molecules so formed to replicate; 12). Biochemical experiments were carried out on the shoots dipped in A. tumefaciens suspension or in A. tumefaciens DNA solution, after the wounding or just after the rifamycin treatments. Cut shoots were washed under sterile conditions and 0.2 mCi of 3H-thymidine was applied for 7 hr or 2 mCi of 3H-uridine was applied for 3 hr. (During these periods, not only the messenger RNA but also the ribosomal and the transfer RNA are labeled.) Control plants were dipped in 0.1 SSC instead of the bacterial suspension or the DNA solution. The labeled nucleic acids extracted from tomato cells were differentiated from nucleic acids of the bacteria in the xylem vessels by autoradiographic and electron microscope techniques. They showed that, after sterile washing of the plants which are dipped in a bacterial suspension, bacteria are present only in the xylem. When these plants are subsequently transferred to a solution of 3H-uridine or of 3H-thymidine, .the bacteria in the xylem are not, in most cases, labeled whereas most plant cells from all tissues are (15). To eliminate further the possibility that some biochemical results could be due to the contamination of the labeled nucleic acids in plant cells by some isolated labeled bacteria present in the xylem, identical biochemical experiments were performed with eggplants (Solanum melongena) (Stroun, Gahan, Anker, and Sheikh, in press). Of course eggplants are not very suitable for the study of crown gall tumors. But it is a perfect material to check the site of the synthesis of bacterial nucleic acids in plant cells. Indeed, the cortex of this plant can easily be separated from the central cylinder which contains the xylem. Therefore, by eliminating the bacteria with the xylem just before labeling, it is possible not only to study which RNA and DNA species are present but also to determine whether they are synthesized in the plant cells. The extraction of bacterial DNA (8), plant DNA (8), and plant RNA (2), the in vitro RNA-DNA hybridization (3), and DNA ultracentrifugation in CsCl gradient (12) were performed by methods already described. All radioactivity measurements were carried out in toluene-based scintillation solution in a Beckmann

Tri-Carb counter.

RESULTS

Tumor emergence. If rifamycin is applied immediately after the bacterial treatment and the wounding process, no tumor develops (Fig. I B). When rifamycin is replaced by water, the tumor develops normally (Fig. IA). Although they are smaller, tumors also develop if there is a delay of 10 hr between the wounding and the application

635

of rifamycin (Fig. IC). If the delay is 24 hr, the tumors are not in the least inhibited (Fig. ID). If the bacterial treatment is replaced by A. tumefaciens DNA, no tumor develops (Fig. IF, G). Biochemical data. We shall present the data obtained with the tomato plants only, since th-e

results with eggplants are similar. After CsCl ultracentrifugation of DNA from plants dipped in A. tumefaciens for 21 hr, wounded, and labeled for 7 hr in 3H-thymidine, three peaks of radioactive molecules are found (Fig. 2A): the first peak had the density of the tomato DNA (6 = 1.692 g/ml), a second peak had the density of A. tumefaciens DNA (6 = 1.718 g/ml), and a third peak had an intermediate density (6 = 1.702 g/ml). The intermediate density may range up to a density of 1.712 g/ml. After heat denaturation, the fractions corresponding to the intermediate peak have a buoyant density of 1.716 g/ml, thus showing an increase of density of 14 mg/ml (Fig. 2B). If the fractions corresponding to the intermediate density are ultrasonically treated and then ultracentrifuged in the presence of CsCI, two radioactive peaks appear (Fig. 2C): one in the region of plant DNA (1.694 g/ml), the other in the region of A. tumefaciens DNA (1.716 g/ml), thus showing, as already discussed (7, 12), that the newly formed molecules contain double-stranded fragments of both bacterial and plant DNA. On the other hand, the fractions corresponding to the density of A. tumefaciens DNA, ultrasonically treated once and again ultracentrifuged in CsCI, sediment at the same place as before (1.718 g/ml), thus showing that these molecules were formed of self-replicating bacterial DNA. With plants dipped in A. tumefaciens DNA, one observes either only one intermediate peak or two peaks of radioactive molecules (Fig. 2D): one in the position of tomato DNA (6 = 1.692 g/ml) and one in an intermediate position (6 = 1.705 g/ml). The intermediate density may range up to a density of 1.709 g/ml. After the rifamycin treatments (Fig. I B-D), the DNA extracted from plants dipped in A. tumefaciens sediment was in the same three positions as before the rifamycin treatment: one in the position of tomato DNA, one in an intermediate position, and one in the position of A. tumefaciens DNA. It should be stressed that with control plants, dipped only in 0.1 SSC, radioactive molecules sediment at the level of the density of tomato DNA. Typical data on RNA-DNA hybridization show that when plants are wounded after feeding with bacteria for 21 hr (Fig. IA-D), newly synthesized bacterial RNA appears, whereas host cell DNA transcription is partially shut off (Fig.

636

STROUN ET AL.

FIG. 1. Effects of rifamycin treatments on the formation of crown gall tumor.

J. BACTERIOL.

637

A. TUMEFACIENS RNA SYNTHESIS

VOL. 106, 1971

I1X2

a Ia

1716

A

1702 1692

Sol0

a

1.73

0

1.63

300.

MO° 3

op

0

-,

.-

VW I so

so

1.742 1.7

13

1.694 1.63

3".

3 4d

..o.o.w

so

FIG. 2. CsCl ultracentrifugation diagram of the DNA extracted from tomato plants conditioned by wounding. (A) Incubation for 21 hr in the presence of A. tumefaciens followed by 7 hr in a solution of 3H-thymidine. Symbols: 0, ultraviolet absorption (tomato DNA: 6 1.692 g/ml; reference, denatured M. lysodeikticus DNA: 6 1.742 glml); 0, radioactivity. (B) Denaturation at 100 C and cooling (on dry ice) of the radioactive molecules of intermediate density in part A. Symbols: 0, ultraviolet absorption (reference, Clostridium perfringens DNA: 6 = 1.691 g/ml; reference, denatured M. lysodeikticus DNA: 6 = 1.742 g/ml); *, radioactivity. (C) Ultrasonic oscillation of the radioactive molecules of intermediate density in part A. Symbols: 0, ultraviolet absorption (reference, C. perfringens DNA: 6 1.691 glml; reference, denatured M. lysodeikticus DNA: 6 = 1.742 g/ml); 0, radioactivity. (D) Incubation for 65 hr in the presence of A. tumefaciens DNA followed by 7 hr in a solution of 3H-thymidine. Symbols: 0, ultraviolet absorption (tomato DNA: 6 = 1.692 g/ml; reference, denatured M. lysodeikticus DNA: 6 1.742 g/ml); 0, radioactivity. =

=

=

=

638

STROUN ET AL.

'I

B

2 a--

./^,. ..." '..

A"X, ,^

-,AL,, ^ 100

so

jig

N3 RNA

50

ipt

FIG. 3. Saturation curves with 3H-RNA extracted from plants dipped for 21 hr in 0.1 SSC (0), for 21 hr in 0.1 SSC and then for 15 hr in rifamycin (@),for 21 hr in A. tumefaciens (A), or for 21 hr in A. tumefaciens and then for 15 hr in rifamycin (A) and then labeled with 3H-uridine. (A) A. tumefaciens DNA (50 ,ug) was trapped on the filters; (B) tomato DNA (20 /ig) was trapped on the filters. The per cent expressed is the relation between the micrograms of DNA trapped on the filters and the micrograms of 3H-RNA hybri-

dized.

3). It should be stressed that, when plants

are

in

contact with bacteria for a longer time (Fig. 1 E), the tomato DNA transcription is completely shut

off. After the administration of rifamycin (treatment of Fig. 1B-D), there is always (14) disappearance of the bacterial RNA and a restoration of the transcription of the host DNA (Fig. 3). If rifamycin is replaced by water (Fig. IA), there is the same amount of bacterial RNA as just after the wounding. DISCUSSION Two factors seem necessary for the induction of crown gall tumor in plants: (i ) wounding of the plant, (ii) the presence of bacterial information in the plant. Wounding of the plant alone does not produce a tumor. A. tumefaciens information present in the plant is not sufficient in itself for the emergence of crown gall (13). We shall discuss in plants conditioned by wounding the role of A. tumefaciens information on crown gall formation. The presence of A. tumefaciens DNA alone is not sufficient to stimulate tumor formation. Indeed one finds, after rifamycin treatment which prevents tumor formation, that the bacterial DNA is in the same state as before the treatment. Moreover, after the uptake by plants of purified A. tumefaciens DNA, no transcription is observed and no crown gall is formed. It seems that in a plant conditioned by wounding the crown gall induction is dependent on the synthesis of bacterial RNA in plant cells. The rifamycin treatment, administered immediately after the wounding, stops the formation of

J. BACTERIOL.

bacterial RNA in plant cells and prevents the formation of crown gall. Since after a delay of 10 hr after wounding, the rifamycin treatment does not prevent the crown gall induction, it is clear that the time for the necessary interactions between the factors induced by the wound and those induced by bacterial RNA is short. It is difficult to evaluate the exact influence, if any, of the plant DNA transcription shut off on the crown gall induction. The suppression of the synthesis of plant RNA is not in itself a cause of crown gall since other bacteria produce the same shut off (14) without provoking the appearance of crown gall. We wonder whether, on the basis of these results, the apparent contradictions which seem to exist between the possibility to induce crown gall by A. tumefaciens, and not by A. tumefaciens DNA, and by A. tumefaciens temperate phage (PS 8) DNA, and not by whole phage (6), could be explained on the basis of one of the following hypothesis. (i) The tumor-inducing principle is determined by bacterial genes. Living bacteria release their DNA which reaches the cells in a state more biologically active than purified DNA (1). The self-replicating bacterial DNA, which is absent in plants treated with purified DNA, might act as a kind of episome. Moreover, the spontaneously released bacterial DNA might be accompanied by its own polymerase, thus allowing transcription. The phage PS 8 DNA could also induce tumor formation after having integrated the necessary fragment of the bacterial chromosome. It is well known that free viral nucleic acids can be active in hosts highly resistant to the intact virus (4, 5, 9). (ii) The tumor-inducing principle is determined by phage PS 8 genes. The possibility of phage DNA to be transcribed in plant cells was demonstrated when complete phage were recovered from leaf extracts of tobacco plants inoculated with Escherichia coli phage DNA (9, 10). If the tumor-inducing principle is determined by phage PS 8 genes, it is not surprising that tumor formation was obtained with purified PS 8 DNA. However, in normal conditions, PS 8 DNA is integrated into A. tumefaciens DNA, and its expression is probably dependent on the bacterial DNA transcription. Purified bacterial DNA is not transcribed in plant cells. However, when bacterial DNA is spontaneously released into plant cells, PS 8 DNA is expressed with the rest of the bacterial genes and thus induces crown gall. Further work is being carried on to determine the exact part of the bacteria and the phage in the tumor-inducing process.

VOL. 106, 1971

A. TUMEFA CIENS RNA SYNTHESIS

ACKNOWLEDGMENTS This investigation was supported by the Fonds National Suisse de la Recherche Scientifique. We are indebted to A. Cattaneo for excellent technical assistance in these investigations. LITERATURE CITED 1. Borenstein, S., and E. Ephrati-Elizur. 1969. Spontaneous release of DNA in sequential genetic order by Bacillus subtilis. J. Mol. Biol. 45:137-152. 2. Gigot, C., G. Philipps, and L. Hirth. 1968. Quelques proprietes des RNA a marquage rapide extraits de cellules foliaires. J. Mol. Biol. 35:311-331. 3. Gillespie, D., and S. Spiegelman. 1965. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12:829-846. 4. Gordon, M. P., and C. Smith. 1961. The infection of Rhoeo discolor by tobacco mosaic virus ribonucleic acids. J. Biol. Chem. 236:2762-2763. 5. Holland, J. J., L. C. Maclaren, and L. T. Syverton. 1959. Mammalian cell-virus relationship. IV. Infection of naturally insusceptible cells with entero virus RNA. J. Exp. Med. 110:65-80. 6. Leff, J., and R. E. Beardsley. 1970. Action tumorigene de l'acide nucleique d'un bacteriophage present dans les cultures de tissu tumoral de toumesol (Helianthus annus).

639

C. R. Acad. Sci. Paris 270:2505-2507. 7. Ledoux, L., and R. Huart. 1969. Fate of exogenous bacterial deoxyribonucleic acids in barley seedlings. J. Mol. Biol. 43:243-262. 8. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid for micro-organisms. J. Mol. Biol. 3:208218. 9. Sander, E. 1964. Evidence of the synthesis of a DNA phage in leaves of tobacco plants. Virology 24:545-551. 10. Sander, E. 1967. Alteration of Fd phage in tobacco leaves, Virology 33:121-130. 11. Stroun, M., P. Anker, P. Charles, and L. Ledoux. 1967. Translocation of DNA of bacterial origin in Lycopersicum esculentum by ultracentrifugation in caesium chloride gradient. Nature (London) 215:975-976. 12. Stroun M., P. Anker, and L. Ledoux. 1967. DNA replication in Solanum lycopersicum esc. after absorption of bacterial DNA. Curr. Mod. Biol. 1:231-234. 13. Stroun, M., P. Gahan, and S. Sarid. 1969. Agrobacterium tumefaciens RNA in non-tumorous tomato cells. Biochem. Biophys. Res. Commun. 37:652-657. 14. Stroun, M. 1970. The natural release of nucleic acids from bacteria into plant cells and the transcription of host cell DNA. FEBS Lett. 8:349-352. 15. Stroun, M., P. Anker, and G. Auderset. 1970. Natural release of nucleic acids from bacteria into plant cells. Nature (London) 227:607-608.