Octopine Synthase Mrna Isolated from Sunflower Crown Gall Callus Is
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Proc. Nati Acad. Sci. USA Vol. 79, pp. 86-90, January 1982 Biochemistry Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens (recombinant plasmid/hybridization/mRNA size/in vitro translation/immunoprecipitation) NORIMOTO MURAI AND JOHN D. KEMP Department of Plant Pathology, University ofWisconsin, and Plant Disease Research Unit, ARS, USDA, Madison, Wisconsin 53706 Communicated by Folke Skoog, September 14, 1981 ABSTRACT We have shown that the structural gene for oc- families have been identified. The enzymes responsible for the topine synthase (a crown gall-specific enzyme) is located in a cen- synthesis of the first two have been purified and characterized tral portion ofthe T-DNA that came from the Ti plasmid ofAgro- (17, 18). bacterium tumefaciens and is expressed after it has been trans- The particular opine family present in a crown gall cell cor- ferred to the plant cells. Polyadenylylated RNA was prepared relates with a particular Ti plasmid rather than with the host from polysomes isolated from an octopine-producing crown gall plant (19, 20). Analysis ofdeletion mutants of an octopine-type callus and purified by selective hybridization to one offive recom- Ti plasmid has shown that a gene controlling octopine produc- binant plasmids. Each such plasmid contained a different frag- tion is located on a 14.6-kbp fragment ofthe T-DNA generated ment ofT-DNA ofpTi-15955 (octopine-type Ti plasmid). Purified by Sin 1 (21) (Fig. 1). Detailed examination ofthe physical or- mRNA was translated in vitro in rabbit reticulocyte lysates, and ganization of T-DNA in octopine-type tumor lines has shown the translation products were immunoprecipitated with antibody that octopine production is correlated with the presence ofthe against octopine synthase. Total and immunoprecipitated prod- left portion ofa 2.8-kbp fragment generated by EcoRI (5). These ucts were characterized by their molecularweights. A polypeptide production of Mr 40,000 (the same as authentic octopine synthase) was syn- results indicate that the gene controlling octopine thesized in vitro by crown gall mRNA selectively hybridized to is located on the T-DNA contained in the left half of the 14.6- three of the five fragments of T-DNA and precipitated with an- kbp Sin I fragment. However, the question remains as to tibody against octopine synthase. This polypeptide was not im- whether or not the controlling gene is the structural gene for munoprecipitated with normal rabbit antibody nor was it synthe- octopine synthase. sized when mRNA from the habituated callus was substituted. A The demonstration that the controlling gene on the T-DNA mRNA 1500 bases long was detected when total mRNA was frac- is the structural gene for octopine synthase is of importance if tionated on an agarose gel, transferred to nitrocellulose, and used the Ti plasmid is to be developed as a vector for genetic mod- for hybridization to three of the five 32P-labeled T-DNA frag- ification of crop plants. In this report, we show that the struc- ments. This apparent mRNA for octopine synthase hybridized to tural gene for octopine synthase is located in a central portion the same three fragments of T-DNA as the mRNA for the Mr of the T-DNA ofA. tumefaciens Ti plasmid and expressed as a 40,000 polypeptide and was not detected in the habituated callus. 1500-base mRNA and a polypeptide of Mr 40,000 in sunflower crown gall callus. Virulence of Agrobacterium tumefaciens (the causal organism ofcrown gall tumors) (1) is correlatedwith the presence ofa large MATERIALS AND METHODS tumor-inducing (Ti) plasmid (2). During tumorigenesis, a 12- to 23-kilobase pair (kbp) portion (T-DNA) of the Ti plasmid is Isolation of Sunflower Polyadenylylated RNA. The estab- transferred to plant cells (3-5) and covalently linked to their lishment and maintenance ofthe primary sunflower (Helianthus nuclear DNA (6, 7). Specific sections ofthe integrated T-DNA annuus cv. Mammoth Russian) crown gall tissue PSCG-15955 are transcribed to RNA by tobacco (8) and sunflower (9) tumor (22) and ofhabituated sunflower stem section culture HSSS (12) cell cultures. Furthermore, T-DNA transcripts are translated have been described. PSCG-15955 was isolated from a primary in vitro into polypeptides (10), but the polypeptides have not crown gall tumor incited by A. tumefaciens (E. F. Sm. and been identified as crown gall-specific proteins. Therefore, there Town.) Conn (1) strain 15955. PSCG-15955 synthesizes octo- is still a question as to whether expression ofthe T-DNA genes pine synthase (17), accumulates octopine (12), and contains the to functional proteins in the plant cell is a requirement for the T-DNA (unpublished data). induction and maintenance of crown gall tumors. Polysomes were prepared from 3-week-old tissue cultures by In addition to the presence ofT-DNA, crown gall tissues are a modification ofthe method ofSun et aL (23). Harvested tissues characterized by their ability to grow in culture in the absence were frozen instantly with liquid N2 and ground to afine powder of auxin and cytokinin (11) and by their ability to accumulate in a Waring blendor. Ground tissues were homogenized in 2 crown gall-specific opines (12). These opines are in turn used vol of 150 mM Tris acetate, pH 8.6/200 mM sucrose/20 mM for nutrition by A. tumefaciens (13). The hormone autonomy KC1/5 mM Mg (OAc)2/5 mM 2-mercaptoethanol/0.4% Noni- of crown gall tissue presumably results from the activation of det P40 (British Drug House, Poole, England). The homoge- auxin and cytokinin biosynthesis. However, little is known nates were centrifuged at 30,000 X g for 20 min (4°C). The su- about individual enzymes involved in the biosynthesis ofthese pernatant was layered onto 1.5 M sucrose-containing 50 mM compounds. On the other hand, opine biosynthesis is well char- Tris acetate, pH 8.5/20 mM KC1/5 mM Mg (OAc)2 (5 ml per acterized for crown gall tissues. Three families of opines, the tube) and centrifuged at 218,000 x g for 2 hr (4°C). The sedi- octopine (12, 14), the nopaline (12, 15), and the agropine (16) mented polysomes were rinsed three times with sterile H20 and suspended in 100 mM Tris-HC1, pH 9.0/0.5% NaDodSO4 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: T-DNA, portion ofTi plasmid that is transferred to plant ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. cells; kbp, kilobase pairs. 86 Downloaded by guest on September 27, 2021 Biochemistry: Murai and Kemp Proc. Natl. Acad. Sci. USA 79 (1982) 87 0.23/0335 with ethanol and dissolved in sterile water for in vitro Eco RI IS 113IK69/o- 2.8$/'55 w.0 translation. Banm HI , /, 4 8.X/ 6.8 In Vitro Translation and Immunoprecipitation. Rabbit re- Hind II 132 !261201049 12.3 0.69-1 94 ticulocyte lysate (Green Hectares, Madison, WI) was treated qmSin 2.12.5 62 L 14.6 with micrococcal nuclease (Worthington) for preparation ofthe _ ~~~~~~~~~_ mRNA-dependent in vitro translation system (30). Poly(A)+RNA CONSERVED REGON Os was translated in vitro at 30'C for 90 min in 50 1.l ofa mixture (31) containing 25 pl ofrabbit reticulocyte lysate and 30-70 Ci C c,o 2022 ofL-[3S]methionine (1000 Ci/mmol; 1 Ci = 3.7 X 1010 becque- o 203_ rels; Amersham). Polysomes that formed were removed from 3303 the translation mixture by centrifuging at 106,000 X g for 2 hr o 403 (40C). The supernatant fraction was then assayed for octopine synthase by immunoprecipitation. FIG. 1. Restriction endonuclease map of the T-DNA region of pTi- Antibody against octopine synthase (anti-octopine synthase 15955 and location of cloned fragments. Each fragment is designated IgG) was prepared as described (32) and purified by chroma- by its size in kilobase pairs. Solid bars, T-DNA fragments contained in pBR322 recombinants; shaded areas, region of T-DNA common to tography on a protein A-Sepharose 4B column. Antibody from most Ti plasmids (4) and location of the octopine synthase (OS) gene normal rabbit serum was purified as above. as deduced by this study. The order of the BamHI 1.1- and 0.49-kbp Total products from in vitro translation were immunoprecip- fragments has not been determined. itated with anti-octopine synthase IgG and Staphylococcus au- reus strain-Cowan I (33). Forty micrograms each lot of anti-oc- (1 ml per tube). The suspensions were deproteinized twice by topine synthase IgG was added to the supernatant oftranslation mixing with buffer-saturated phenol followed by centrifugation mixtures and the mixture was incubated at 370C for 1 hr and for 5 min at 12,000 X g. The recovered aqueous phases were then at 40C overnight. A 20-,ul aliquot of10% (vol/vol) S. aureus twice mixed with chloroform/isoamyl alcohol (24:1) followed by cell suspension was then incubated with the above mixture at centrifugation as above. Polysomal RNA was precipitated from 25°C for 30 min. Immunocomplex bound to S. aureus cells was the aqueous phase by the addition of ethanol, dissolved in 0.4 purified by centrifuging the cells through 15% (wt/vol) sucrose M NaOAc, pH 6.0, reprecipitated, and finally dissolved in 10 at 5000 x g for 5 min (4°C) followed by washing as described mM Tris HCl, pH 7.5/1 mM EDTA. by Kessler (33). Finally, the immunocomplexes were eluted Poly(A)+RNA was purified from polysomal RNA by two pas- from the cells by incubating at 100°C for 2 min in 50,ul ofLaem- sages through an oligo(dT)-cellulose column (Collaborative Re- mli's application buffer (34).