Gene. 87 (1990) 139-143 Elsevier GENE 03431

Expression of Agrobacteriumrhizogenes roIB gene fusions in Escherichia coli: production of antibodies against the RolB protein (Recombinant DNA; E. coil host; plant genetics; expression vector; rabbit reticulocyte system for protein synthesis; Pt. and ptac promoters)

M. Trovato', M. Cianfriglia b, P. Filetiei ~, M.L. Mauro" and P. Costantino" ° Dipartimento di Genetica e Biologia Molecolare, Universitd di Roma "La Sapienza: Rome (Italy); b Laboratorio di Immunolo~a, Istituto Superiore di Sanitd, Rome (Italy) Tel. (6)4990891, and c Centro per io Studio degli Acidi Nucleici, CNR, Rome (Italy) Tel. (6)491183 Received by J.L. Slightom: 25 April 1989 Revised: 15 September 1989 Accepted: 2 October 1989

SUMMARY

Expression of the rolB gene ofAgrobacterium rhizogenes TL-DNA is sufficient to trigger root differentiation in transformed plant cells. To investigate the role of RolB in differentiation, a large portion of rolB, comprising about 90% of its C~terminal coding sequence, was cloned into vectors pEX34 and pEA305 in frame with the truncated N termini of the pL--MS2 phage DNA polymerase and, respectively, the ptac-clts phage ~.represser gene. Hybrid proteins were expressed from both fusions and the one from pMTBEX1 was utilized to raise antibodies. These antibodies specifically recognize the RolB moiety in both pL-MS2-roiB and ptac-cl-roIB fusions. Unfused, complete RolB protein was obtained by in vitro translation in a rabbit reticulocyte system of a transcript obtained by in vitro transcription of rolB. RolB protein is specifically immunoprecipitated by the antibodies raised against the hybrid protein MS2-RolB.

INTRODUCTION

Agrobacterium rhizogenes is responsible for the hairy root syndrome of dicotyledonous plants (Elliot, 1951). The Correspondenceto: Dr. P. Costantino, Dip. Genetica e Biologia Molecolare, Universit~ 'La Sapienza', P. le A.Moro 5, 00185 Rome (Italy) Tel. (6)4455344; Fax (6)4040812. Abbreviations: A., Agrobacterium; aa, amino acid(s); Ap, ampicillin; bp, base pair(s); BSA, bovine serum albumin; IPTG, isopropyl-/~-D.thiogalactopyranoside; kb, kilobase(s) or 1000bp; LB, Luria-Bertani (broth); nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PL, major leftward promoter of phage ,I; Pollk, Klenow (large) fragment ofE. coliDNA polymerase I; ptac, lac/trp hybrid promoter; Ri, root-inducing plasmid; RolB, protein encoded by A. rhizogenes,capable of inducing root differentiation in plant cells; rolB, gene encoding RolB; SDS, sodium dodecyl sulfate; Tc, tetracycline; TL-DNA, left-transferred DNA of the Ri plasmid ofA. rhizogenes; [ ], denotes plasmid-carrier state. 0378-1 ! 19/90/503.50 © 1990 Elsevier Science Publishers B.V.(Biomedical Division)

growth of neoplastic roots is due to expression in the plant cells of a portion of bacterial DNA (T-DNA) transferred from the large A. rhizogenes Pd plasmid into the host-plant cell genome (Chilton etal., 1982; White etal., 1982; Willmitzer et al., 1982; Span6 et al., 1982). The transferred DNA of the Ri plasmid pRi1855 consists of two separate segments, designated TL- and TR-DNA. Sequencing ofthe whole TL-DNA revealed the presence of 18 ORFs (Slightom et al., 1986), most of which have been cloned in plant vectors and transferred to plant cells. Of all the TL-DNA genes only ORF 11, the genetic locus roIB (White et al., 1985), is capable alone of eliciting strong hairy-root response on all plant tissues tested. The morphogenetic action of rolB is manifested only if a long noncoding segment at its 5' end is included in the construction and provided an adequate supply of auxin is available (Capone et al., 1989a,b): this indicates the presence of an upstream activating sequence and suggests a role for auxin in the

140 regulation of ro/B (Capone et al., 198%). Thus rolB seems to be the key gene in the differentiation mechanism triggered by Ri plasmid TL-DNA. So far, however, nothing is known about the gene product of to/B: its role in the modifcafion of the auxin reception-transduction system (Cardarelli etal., 1987; Shen et ai., 1988), as well as its involvement in the biosynthesis o f ' a substance with auxin-like activity but which is not transported in plant tissues' (Schmulling et al., 1988) are compatible with the presently available data. Antibodies specific against the roIB gene product will be a major tool in the clarification of its function. We describe here the cloning and expression in Escher~chia coli of different gene fusions which yielded hybrid RolB proteins, allowing to raise antibodies that specifically recognize the rolB gene product.

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EXPERIMENTALAND DISCUSSION (a) Gene fusions and expression in Escherichia coli The restriction map of the TL region of the Ri plasmid, pRi1855, encompassing rol genes A, B and C is shown in Fig. 1. The entire coding sequence of rolB and some 300 nt at its 5' end are contained in the HindIII fragment 37b (Slightom et al., 1986). The TL-DNA segment shown below the map, between the left HindIIl site to the BarnHI site internal to HindIII 37b, was cloned in vectors pEx34 (Strebel et al., 1986) and pEA305 (Amann et al., 1983) as described t,t the legend to Fig. 1; plasmids pMTBEkl and pMTBEAI, respectively, were thus obtained (Fig. 1). In constructing pMTBExl, hosted by E. coli K12AH1Atrp, the C terminus of rolB (89~o of its coding sequence) is fused in-frame with the first 297 nt of the gene encoding the DNA polymerase of phage MS2 (Fiers et al., 1976). This fusion is placed under the control of the PL promoter of phage ~. Liquid cultures of K-12AHIAtrp[pMTBExl] were thus overproducing the MS2-RolB hybrid protein when induced by a 2.5-h temperature shift from 28 o to 42°C. Total proteins from induced and uninduced cultures were analyzed by SDS-PAGE. As shown in Fig. 2A, following induction (lane 2) a substantial increase of a protein component around 38 kDa was observed, in good agreement with the 37.5 kDa expected for the MS2-RolB protein (11 kDa contributed by the truncated MS2 N-terminal and 26.5 kDa by the RolB moiety). In Fig. 2A, lane 3, total proteins from uninduced K12AH IAtrp[pMTBExl ] cells are represented. Since fusion proteins overexpressed in E. coli are often insoluble, induced cells were sonicaily disrupted and insoluble proteins were analyzed by SDS-PAGE. As shown in Fig. 2A, lane 1, the major component in the insoluble fraction of induced cells containing plasmid pMTBEx ! is represented by the hybrid MS2-RoIB protein.

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Fig. 1. Constructionof plasmids for expression in E. coli and in vitro. (Top)Restriction map of the roM,B,C region of pRi1855 TL-DNA. Cloningsteps:(1) The Hindlll-Dral fragmentcontainingroiBwas iigated between the Hindlll and Smal sites of pSP65 to yield pMTBSP2. (2) Hindlll + BamHl digest of pMTBSP2 yielded a 689-bp fragment (89% of roiB coding sequence) which was cloned between the HindlIl and BamHl sites of pEg34 to yield pMTBExl. (3)The same 689-bp Hindlll-BamHl fragment was blunted with mung-bean nuclease and ligated between the Pollk-filled Hindlll sites of pEA30$ to yield pMTBEAI. (4)Hindill digestion. (S)Pollk fill-in. (6)T4 ligation. (7)T4

blunt-endiigation.Standardcloningtechniqueswereutilizedin all steps. H, Hindlll; B, BamHl; D, DraL Open box with arrow, rolB-coding region; open box, truncated toM-codingregion; hatched box, intergenic regionof TL-DNA;stripedbox, truncated MS2 DNA polymerasegene; box withplus symbols,~.repressergene(cI). Promoters are represented as pointedboxesindicatingdirectionof transcription: stippledbox, 2.PL promoter; blackenedbox, SP6 promoter; crosshatched box, ptac promoter. In the other construction, pMTBEAI, the same C-terminal portion of RolB is fused in-frame with the first 480 nt of the gene encoding the ~ represser (Sauer, 1978); the fused gene is under the control ofptac (De Boer et al., 1983; Amann et al., 1983). E. coli W31 lOlacl Q L8 (a strain which overproduces the lac represser; Brent and Ptashne, 1981) cells harboring plasmid pMTBEA1 were thus induced for 4 h with IPTG and total and insoluble proteins were analyzed by SDS-PAGE. The 43-kDa CI-RolB hybrid protein corresponds fairly well to what is expected from the size of the truncated CI (17.5 kDa) and RolB (26.5 kDa) moieties; it can be detected in the total protein pattern of

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Fig. 2. Expression ofrolB 8ene fusions. 0.1% SDS-12~ PAGE (Laemmli, 1970) of E. coil protein extracts. (Panel A) PL-MS2-ro/B 8ene fusion. Lanes: 1, insoluble protein fraction from temperature-induced K-12AHIAtrp[pMTBExl] cells; 2, total protein from temperature-induced cells; 3, total protein from uninduced cells. K-12~lHl~trp cells (Bernard et al., 1979) harbouring pMTBExl, 8town overnight in LB with 30 At8 Ap/mi at 28"C, were diluted 1:40, incubated for 2 h at 28"C and shifted to 42°C for 2.5 h (for induction ofthe PL promoter), centrifuged, resuspended in Laemmli (1970) buffer and analyzed by SDS-PAGE (total proteins). Insoluble protein fraction was obtained from induced cultures by centrifusation, resuspension in 20% sucrose/50 mM Tris pH 8, treatment with 100/As/mlof iysozyme and sonication. Disrupted cells were centrifuged 30 rain at 7500 x g, resuspended m 20~ sucrose, 3 mM EDTA pH 7.6, recentrifuged, resuspended in Laemmli (1970)buffer and analyzed by SDS-PAGE. (Panel B) ptac-cl-re/B 8ene fusion. Lanes: 1, insoluble protein fraction from IPTG-induced W31101acl°L8 pMTBEAI cells; 2, total protein from I PTG-induced cells; 3, total protein from uninduced cells; 4, total protein from lPTG-treated plasmidless W31101aclOL8cells. Cells 8rown in LB/Ap to a density ofA6oo = 0.6 were supplemented for 4 h with I mM IPTG for induction of the ptae promoter. Total and insoluble proteins were obtained as above. Lanes M in both panels: markers (in kDa).

induced cells (Fig. 2B, lane 2) and represents virtually the sole component of the insoluble fraction (Fig. 2B, lane 1). Lanes 3 and 4 represent electrophoretograms ofuninduced W31 IOlaclQLS[pMTBEAI] and IPTG-induced plasmidless W31 lOlaclQL8 host cells, respectively. Antibodies and Western analysis in Escherichia coil To raise antibodies against RolB, the MS2-RolB fusion protein from the insoluble fraction of temperature-induced K-12AHIAtrp[pMTBExl] cells was excised from a polyacrylamide gel and utilized for rabbit immunization. The antiserum thus obtained was immunoadsorbed by incubation for 30 min at 37°C with a total protein extract from temperature-induced K-12AHIAtrp[pEx34] cells, producing the unfused 1l-kDa MS2 fragment (Fig. 3A, lane 3). The immunoadsorption was carried out to remove most of (b)

the unspecific antibodies present in the preimmune serum which might recognize E. coli proteins and to eliminate the newly synthesized antibodies specific against the MS2 moiety of the fusion protein. Western blots (Towbin et el., 1979) of total and insoluble proteins from temperature-induced K-12AHIAtrp[pMTBExl] (Fig. 3A, lanes 4 and 1, respectively) and of total proteins from temperature-induced K-12AHIAtrp[pEx34] cells (Fig. 3A, lane 3) were incubated with a 1 : 1000 dilution of immunoadsorbed antiserum and subsequently immunostained (Fig. 3, legend). The antiserum reacts with the MS2-RolB fusion protein both in the total and insoluble fractions from cells containing pMTBExI (Fig. 3B, lanes 4 and 1, respectively) but not with the unfused MS2 fragrnent from pEx34-containing cells (Fig. 3B, lane 3). No immunostaining can be detected by

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Fig. 3. lmmunodetection of RolB hybrid proteins. (Panel A) 0.1% SDS-12% PAGE of: (I)insoluble protein fraction from temperatureinduced K-12AHIAtrp[pMTBExl] cells; (2)total protein from K12AHIAtrp cells; (3)total protein from temperature-induced K12dHIdtrp[pEx34] cells; (4)total protein from temperature-induced K-12AHIAtrp[pMTBExl] cells. (Panel B)Western blot of the gel in panel A, immunostalnedwith antiserum anti MS2-RolB after immunoadsorption of the serum as described in sectionb. Bacterial proteins, fractionated by SDS-PAGE were electroblotted onto nitrocellulose membranes (Towbin et al., 1979). Membranes were preincubated 4 h with 3% BSA then incubated overnight with 1:1000 dilutions of the antiserumantiMS2-RolBand subsequentlyfor 3 h with a 1 : 1000dilution of commercial antibodiesagainstrabbit Ig conjugatedwith horseradish peroxidase. Membranes were then transferred to a solution 0.6 mg 4-¢hloronaphthol per ml/0.01% H202/50mM Tris pH 6.8 for immunostaining. (PanelC) 0.1% SDS-1270 PAGE of: (1) insoluble protein fraction from IPTG-induced W31101acQLgpMTBEAI cells; (2) total protein from samecells;(3) and (4) Westernblots of(I) and (2), respectively, imm,mostained with antiserum anti-MS2-RolB as in panel B. (Panel D) Immunoprecipitationofthe productofroIB in vitrotranslation. 0.1% SDS-12% PAGEof [3SS]methionine-labeledproteins.Lanes: i, 2, 3, proteins translated by rabbit retieulocytes(NEN) from, respectively, yeast polyadenylated RNA, no added RNA and from rolB RNA transcribedin vitro frompMTBSP2(see Fig. 1); 4, S, proteins immunoprecipitated from reticulocyte-translatedrolB transcript(see lane 3) by antiserum anti-MS2-RolB and preimmune serum. The in vitro translation mix was incubatedfor I h with protein A-Sepharose(Pharmacia) and the decanted solutionimmunoprecipitatedwith an antiserumantiMS2-RolB as describedby Kessler(1975). The immunoprecipitatewas analyzedby SDS-PAGE and gelswerefluorographedwith the Amplify reagent (Amersham).

reaction of the antiserum with total protein extract from plasmid-less K-12AHIAtrp host cells (Fig. 3B, lane 2). Thus, the recognition of the MS2-RolB hybrid protein by the antiserum rests on the specificity of the latter for the RolB moiety. To confum this, the serum was incubated with Western blots of total (Fig. 3C, lane 2) and insoluble (Fig. 3C, lane 1) proteins from IPTG-induced E. coli W31 lOlacI QLg[pMTBEA1]. The ~mmunostained blots reported in Fig. 3C clearly show that the antiserum raised against the MS2-Rolb fusion protein specifically recognizes the CIRolB protein in both cases (Fig. 3C, lanes 4 and 3), strongly pointing to the presence of antibodies against RolB. The possibility that the recognition of the CI-RolB hybrid is due to cross-reaction of the CI moiety with unspecific antibodies present in the serum is in fact ruled out by the lack of reaction of this latter with the protein extract from IPTGinduced cells containing plasmid pEA305, which produce the unfused 17.5-kDa CI N-terminal fragment (not shown). Altogether, these data demonstrate that the anti-MS2-RolB serum contains antibodies specific against the RolB moiety which specifically recognize the same protein portion also in the CI-RolB fusion. (c) In vitro transcription and translation of rolB

To test the specificity of the antiserum for RoiB in unfused form, the above described construction pMTB SP2, where the whole of the rolB-coding sequence is under the control of the SP6 phage promoter (Fig. 1), was utilized for in vitro transcription directed by phage-specific SP6 RNA polymerase (Melton et al., 1984). The in vitro transcript, checked for size by agarose-gel electrophoresis (not shown), was translated in vitro in a commercial rabbit reticulocyte lysate. As shown in Fig. 3D, lane 3, the major product of this in vitro translation is a 3SS-labeled protein around 30 kDa, matching the size (29.5 kDa) calculated for full-length RolB. The antiserum against the MS2-RolB fusion protein was utilized to immunoprecipitate the in vitro translation product as described in the legend to Fig. 3: as shown in Fig. 3D, lane 4, the antiserum specifically precipitates the 30 kDa RolB and a smaller translation product, presumably from a prematurely terminated roIB transcript. No labeled protein was precipitated by preimmune serum (Fig. 3D, lane 5). Thus, antibodies raised against a rolB gene fusion expressed in E. coli are not only capable of recognizing the same RolB moiety in a different E. coli fusion but also specifically react against the unfused, complete RolB protein synthesized in vitro.

ACKNOWLEDGEMENTS

Thanks are due to W. Rohde and R. Rappuoli for providing E. coZ~ strains and expression vectors and to

143 C. Pisano for helpful discussions. This work was partially supported by Fondazione 'Istituto Pasteur-Fondazione Cenci-Bolognetti' and by EEC contract BAP-0018-I(S).

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