Expression of a Human Lactoferrin Cdna in Tobacco Cells Produces Antibacterial Protein(S)'

Plant Physiol. (1994) 106: 977-981

Expression of a Human Lactoferrin cDNA in Tobacco Cells Produces Antibacterial Protein(s)'

Amitava Mitra* and Zhanyuan Zhang Department of Plant Pathology and the Center for Biotechnology (A.M.) and Department of Horticulture (Z.Z.), University of Nebraska, Lincoln, Nebraska 68583-0722

crop plants. Lactofenin has been known as an antibacterial A suspension tobacco (Nicotiana tabacum 1.) cell line was trans- agent for a long time. Since the initial inoculum required for formed to express human ladoferrin, an iron-binding glycoprotein. development of plant bacterial disease is usually small, we The transgenic calli produced a protein that was significantly thought that high-level, constitutive expression of an anti- smaller than the full-length lactoferrin protein. Total protein ex- bacterial agent such as lactofemn might be able to reduce or tracts made from transgenic tobacco callus exhibited much higher control plant bacterial diseases. As a first step in achieving antibacterial activity than,commercially available purified lacto- this objective, an attempt was made to express a human ferrin as determined by the decrease of colony-forming units when tested with four phytopathogenic species of bacteria. Introduction lactofemn cDNA in tobacco (Nicotiana tabacum L.) suspen- of the ladoferrin gene in crop plants may provide resistance against sion cells. In this study we demonstrate the expression of the phytopathogenic bacteria. lactoferrin protein in plant cells. Furthermore, it appears that the gene product undergoes posttranslational processing, because only a truncated protein is detected in plant cells. The protein extract from transgenic calli exhibited a strong bac- Lactoferrin, originally found in milk, is a member of a tericidal activity. family of iron-binding glycoproteins that includes transferrin and melanotransfemn found in polymorphonuclear leuko- MATERIALS AND METHODS cytes and in mucosal and epithelial secretions. It is the major iron-binding protein of human mammary secretions. Lacto- Construction of Plasmids femn genes from various sources show significant homology The original plasmid pLACMODC/l8.1 containing a mod- and code for a single polypeptide chain of about 700 amino ified human lactofemn cDNA was kindly provided by Dr. acids (Mr approximately 80,000), with an iron-binding site in M. Powell (Delta Biotechnology Limited, Nottingham, UK) each of the similar amino- and carboxy-terminal regions. (Powell and Ogden, 1990). The plasmid contained the 2082- High levels of lactofemn expression have been shown in bp mature lactofemn protein sequence preceded by a syn- neutrophils (Masson et al., 1969), in lactating mammary thetic ATG but without the sequence encoding the signal glands (Green and Pastewka, 1978), and in the uterus under peptide. The lactoferrin cDNA was subsequently subcloned different types of regulation, whereas a low level of consti- into the binary plant expression vector pAM1400 using the tutive expression is found in many biological fluids (Liu and BamHI or Hind111 sites downstream from the cauliflower Teng, 1991). Lactoferrin has been associated with the pro- mosaic virus 35s promoter to achieve a transcriptional fusion motion of cell growth, regulation of hematopoiesis, and pro- between the cauliflower mosaic virus 35s promoter and the tection against microbial infection and immune modulating lactoferrin cDNA. The resultant binary plasmid vector was properties in vitro. Little, however, is known about the func- named pAM1401 (Fig. 1, top). The plant expression vector tion of lactofemn in vivo. Extracellular lactofenin has been pAM1400 was constructed by replacing the neomycin phos- reported to have both antibacterial and antiviral activities photransferase-selectablemarker in the plasmid pGA643 (An (Arnold et al., 1977; Lu et al., 1987). et al., 1988) with the bar-selectable marker (Fromm et al., Diseases caused by phytopathogenic bacteria are of major 1990). This allowed selection of transgenic calli using the economic importance and result in losses of more than $100 herbicide bialaphos and its derivatives. The binary plasmid million annually (Kennedy and Alcom, 1980; Perombelon also contained a transcriptional termination signal from the and Kelman, 1980). Advancement in plant genetic engineer- T-DNA gene nopaline synthase immediately following the ing methodologies has allowed introduction of foreign genes lactofemn stop codon. in plants, creating the potential for development of disease- resistant plants. Expression of an antibacterial gene in a plant, Transformation of Tobacco Cells in theory, could produce resistance or tolerance in transgenic A suspension tobacco cell line NT-1 (Nicotiana tabacum L. Supported by the Center for Biotechnology, University of cv Bright Yellow) (Nagata et al., 1981) was transformed by Nebraska. This manuscript has been assigned Joumal Series No. the Agrobacterium co-cultivation method (Horsch et al., 1985; 10759, Agricultural Research Division, University of Nebraska. Mitra and An, 1989). Transformed tobacco cells were selected * Corresponding author; fax 1-402-472-2853. on Murashige and Skoog (1962) plates containing 40 mg/L 977 978 Mitr Zhand aan g Plant Physiol. Vol. 106, 1994

Ignite. Ignite, a commercial formulation of glufosinate-am- To determine expression levels of the lactoferrin cDNA in monium, was a gift from the Hoechst-Roussel Agri-Vet Co the transformed NT-1 calli, total RNA was prepared from (Loland, MS). Individual transformants were pooled to form 0 group1 e f transformanto th seac f o h s describea s y b d 10 groups, each group consisting of 10 independently trans- Logeman . (1987) al extractio t A ne .RN dons buffea nwa n ei r formed calli. containing 8 M guanidinium chloride, 20 HIM Mes, 20 mM EDTA, and 50 mM /3-mercaptoethanol, pH 7.0. RNA samples Verificatio f Inserno t were separate formaldehydn do e gel, transferre Zetae th -o dt probe membranes d probean , s aboven internaa da s A . l Integratio e plasmi th ints A f o nwa DN d callu A DN s control, a labeled /3-tubulin fragment was used to probe total determine Southery db n blot analysis (Southern, 1975). Five RNA from transgeni controd can l tobacco calli. gram fresf so h callus tissue fro group0 1 m e eacth s f werho e use describes a preparo dt A Guillemauy DN eb d - Ma d an t Extraction of Total Protein from the NT-1 Calli rechal-Drouard (1992). Briefly, callus tissu s grounwa e n di liquid nitrogen with extraction buffer (100 HIM sodium ace- A total cellular protein extract was prepared by homoge- tate, pH 4.8, 50 mM EDTA, pH 8.0, 500 HIM NaCI, 2% PVP- nizatio transformee th f no d callu protein si n extraction buffer 40, mixture adjuste s 5.5 H 1.4%o p Cy t ,o d thedt S an , nSD (0.SueM 5 , 0.1% ascorbic acid, 0.1% Cys-HCl, 0.0 Tris1M - HIM)0 1 centrifugedd o ,t an incubate n mi 0 t 65°2 a d. r Cfo HCI 7.5H p , ) (Mitr t al.ae ,presenc e 1989th Mf 5 n gi o )1 f eo One-half potassiuvolumM 3 f eo m acetat 4.8H s (p e )wa PMSF in liquid nitrogen. The homogenate was centrifuged added to the supernatant and incubated at 0°C for 1 h. The supernatane th t 4°C a d an tan , 14,000mi 0 t3 containinr gfo g DN s recovereAwa ethanoy db l precipitation, dissolven i d soluble th e protein recovereds swa . Protein extracts contain- Tris-EDTA (10 mM Tris-Cl, 1 HIM EDTA, pH 8.0), and centri- ing 100 ng of total protein were separated on a 12.5% (w/v) fuged overnigh a cesiu n i t m s digestegradientwa A d DN . acrylamide gel (Laemmli, 1970) along with 200 ng of com- with Hindlll, (w/vseparate% 1 a ) n agaroso d e geld ,an mercially available lactoferrin (Sigma a standard s a ) e Th . transferre Zeta-proba o dt e membrane (Bio-Rad) accordino gt resolved proteins were transferred to a nitrocellulose mem- manufacturer's recommendations. The membrane was hy- brane using a Bio-Rad rransblot apparatus at 250 mA for 1 bridized with a randomly labeled DNA probe using 2082 bp membrane Th . h immerses ewa blockina n di g solutio% n(5 of human lactoferrin cDNtemplatee th As a . nonfat dry milk in Tris-buffered saline) and incubated at

g\—— ^\ •••-• '" '...... -____....,-:,:,„.....,.. 7 * ;1 K ->-l RB Pnos bar Tnos W.F ^ 1 Tnos LB 1 i PROBE

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2 3 5 6

4.0 hLf

3.0

2.0

1.6 tub

0.5

Figure 1. Transformation of tobacco suspension culture cells with the gene encoding human lactoferrin. Top, Schematic diagram of the plant expression cassette from the plasmid pAM1401. RB and LB, T-DNA right and left border sequences, respectively; bar, herbicide resistance gene; P35S, cauliflower mosaic virus 35S promoter; Tnos, T-DNA nopaline synthase gene terminator. Bottom left, Southern blot of transgenic calli. Total genomic DNA was digested with H/ndlll, transferred to a membrane, and hybridized with 32P-labeled randomly primed fragments generated from 2082 bp of human lactoferrin cDNA (hLf) probe as marked in the diagram. Numbers at right are size markers in kb. Bottom right, Northern blot analysis of the transgenic calli. Total cellular RNA was isolated and electrophoresed on a 1%agarose-formaldehydegel, transferred a membrane o t d hybridizean , d wite samth h e n i bottoprob s a em left panele bottoTh . m panel shows j3-tubulin (tufa) transcripts as an internal control. Lane 1, Control calli; lanes 2 to 6, transgenic calli. Expressio Antibacterian a f no l Gen Tobaccn ei o 979

28°C for 1 h. The membrane was washed and incubated immunoreactive lactoferrin levels did not differ significantly with 1:1000 dilution of a commercially available (Sigma) among all the transgenic calli tested. The plant-produced polyclonal antibody prepared against human lactoferrin an- lactoferrin was compared to the full-length human lactoferrin tigen. Following incubation in the lactoferrin antibody, the (Fig lan, versu2B e.2 lactoferrie s lanTh . e3) n cDNA thas wa t membrane was placed in the biotinylated goat anti-rabbit expresse tobacce th dn i o2082-ba calld iha p coding sequence, antibod yt 28° a solutio h C 1 witr nfo h gentle agitatione Th . indicating thamature th t e protein shoul approximatele db y membran washes ewa incubated an dstreptavidine th n i d - 80 kD. The full-length protein was never detected in trans- biotinylated alkaline phosphatase comple , followeh 1 r xfo d genic tobacco callus, although a total of 50 independently by immersion in the color development solution until the transformed calli were screened. Furthermore calle th if o l al , purple bands became visible, usually withi min0 n1 . produced a truncated lactoferrin protein of the same size. This implie a posttranslarionas l processin e proteinth f o g , Assay of Antibacterial Activity since the northern blot analysis indicated generation of a single 2.3-kb t knotranscriptno present wo a d e W .t whether For determinations of antibacterial activity, total protein truncatio aminoe th t r a carboxy-termina o -s ni e th f o d en l extract was made from the transgenic callus in protein ex- protein immunobloe Th . t showe majoda r protein ban8 4 f do traction buffer as stated above except that no PMSF was used kD in extracts from transgenic calli. A faint but slightly larger and the protein extracts were concentrated using a concen- trator. Four different phytopathogenic bacterial strains, Xan- thomonas campestris v phaseoli,p Pseudomonas syringaev p phaseolicola, Pseudomonas syringae pv syringae, and Clavibac- M CL T1 T2 T3 TC T4 T5 CL M

ter flaccumfaciens pv flaccumfaciens, were grown to an A6oo of 0.4, and aliquots of 2 mL were transferred to culture tubes. Various amounts of callus extract, ranging from 0 to 500 /ig of total protein, were added to each culture tube and incubate t 27°da C e controunti th e A 60f th lt>o l cell suspension -107 reached to 1.00. The final volume of suspensions in each - 76 sample of all treatments was adjusted to 2.5 mL by adding - 52 appropriate amount culturf so e medium. Commercially avail- 9 able lactoferrin solutio extracd nan t from tobacco calli trans- - 37 formed with the plasmid pAM1400 were used as positive - 27 and negative controls, respectively. After the incubation, the l total numbe f colony-formino r g units determinewa s y b d - 19 serial dilutio nutrienn no t agar plate countind san nume gth - coloniesf o r be .

RESULT D DISCUSSIOAN S N Tobacco Cell Transformation with Lactoferrin cDNA Transformed tobacco calli were selecte 40-mg/n do L Ignite and subculrured three times on herbicide-supplemented Mu- rashige-Skoog plates transformee Th . d transgenica calld ha i normal appearance and had similar growth characteristics as the control calli. Southern blot analysis of Hmdlll-digested -PL genomi froA mDN c several independently transformed transgenic calli showe e expecteth d d 2.4-kb bands (Fig, 1 . bottom left) herbicide-resistanl Al . t calli teste thid dsha band. Northern blot analysis of the same calli indicated the presence of 2.3-kb transcripts (Fig. 1, bottom right).

Expressio f Lactoferrino n Protei Transgenin i c B Tobacco Calli Figur . eImmunodetectio2 f lactoferrio n n protei transgenin i c calli. The transgenic tobacco calli were tested for the expression Total protein extract fro transgenie mth c fractionatecalls wa i y db e lactoferrioth f n protei y westerb n n blot analysise Th . SDS-PACE and stained with Coomassie blue (A) or blotted onto a amount of total protein in the extract was quantified by the nitrocellulose membran probed ean d wit commerciallha y available Bradford (1976) method. A known amount of protein from anti-lactoferrin antibody reagent (B). Numbers at the right are protein size markers in kD. Lane M, Marker; lane CL, commercially transformed calli was loaded on a gel and transferred to a available lactoferrin protei, ;T5 laneo t n1 extractsT s from transgenic nitrocellulose membrane for immunoblotting. All transgenic tobacco calli; , lanproteiTC e n extract from control tobacco calli expressed lactoferrin protein, but the control calli extract calli; PL, immunoreactive lactoferrin protein bands from transgenic t reacno td wit di anti-lactoferrie hth n antibodye (FigTh . 2) . tobacco calli. 980 Mitra and Zhang Plant Physiol. Vol , 106, 1994 protein band was consistently detected in all transgenic calli and is mediated by iron chelation. Commercially available tested. Since a human lactofemn gene was successfully ex- lactoferrin was only marginally antibacterial. A fairly high pressed in Aspergillus nidulans with the production of large concentration (500 pg) of commercially available lactofemn amounts of full-length lactofemn (Ward et al., 1992), it is not was required to achieve only 10% of the antibacterial activity known why full-length lactoferrin is not produced in tobacco conferred by the transgenic tobacco cell extract. The antibac- cells. It is possible that the plant-produced lactofemn protein terial activity of purified lactofemn was totally abolished does not undergo proper folding and the unfolded part is when the culture medium was supplemented with 0.1 rn~ degraded. It is also not known whether the protein is glyco- FeS04. However, FeS04 had no effect on the plant cell sylated in tobacco. extract-induced antibacterial activity. This indicates that there must be some other mechanism for the bactericidal activity Antibacterial Properties of the Transgenic Calli Extract of the transgenic tobacco extract. Because transformed calli were grown in the presence of 40 mg/L herbicide, the bac- To test the antibacterial activities of the transformed to- terial species were also tested in medium Supplemented with bacco calli, total protein extracts from these calli were made the herbicide, but no effect on the growth of bacteria was in a protein extraction buffer as mentioned earlier. The pro- observed (data not shown). tein extracts were further concentrated using a filtration Tomita et al. (1991) reported that the low mol wt peptides apparatus. Antibacterial activity was determined by adding a generated by enzymatic hydrolyzation of bovine lactofemn known amount of protein to a 2-mL culture of bacteria grown showed strong broad-spectrum antibacterial activity against to an A600of 0.4. Control extracts were prepared from tobacco a number of Gram-negative and Gram-positive bacterial calli and used at the same concentration as the protein species. The antibacterial activities of the enzymitic hydrol- extracts from the transgenic calli. Protein extracts from the ysates were at least 8-fold greater than that of undigested transformed tobacco calli clearly had significant antibacterial lactoferrin. They hypothesized that the smaller peptides had activity (Table I). Furthermore, the protein extracts were better access to the target sites on the bacterial membrane bactericidal for all of the bacterial species tested. Extracts and, hence, were more effective. In transgenic tobacco cells from control calli had no antibacterial activity. Since lactofer- we detected only a single peptide of 48 kD, although gener- rin produced in the tobacco cells was much more effective ation of smaller antibacterial peptides that either ran off the than the full-length lactofemn, it is possible that the trunca- gel or remained undetected by immunoblot canriot be ruled tion of the lactofemn in plant cells made it more toxic. A out. Yamauchi et al. (1993) showed that a pepsin-derived similar observation was made with the Bacillus thuringiensis bovine lactofemn peptide fragment had a strong bactericidal crystal protein toxin, in which the strongest insecticidal activ- effect, whereas bovine lactoferrin was at most bacteriostatic. ity was found in the truncated protein. Actual cytosolic The protein extracts from the transformed tobacco calli were concentration of lactofemn in individual transformed calli effective against both Gram-negative and Gram-positive varied from 0.6 to 2.5% of the total cellular protein as phytopathogenic bacteria. Among the species tested, the determined by ELISA (data not shown). Extract prepared extract was almost equally effective against P. syringae pv from pooled calli had an average of 1.8% lactofenin protein. phaseolicola, X. campestris, and C. flaccumfaciens pv flaccum- !$ince protein extracts were concentrated for the antibacterial faciens, whereas P. syringae pv syringae was slightly less assay, the effectiveness of cytosolic lactofemn levels is not sensitive. Icnown. Amold et al. (1982) reported that the antibacterial In conclusion, it is evident from our data that thc expression activity of lactoferrin was dependent on its iron-free state of a human lactofemn gene produces a potent antibacterial

Table 1. Antibacterial activity of total protein extract from transgenic tobacco calli expressing a human lactoferrin cDNA - Data are from seven replications. Different letters within a column for a bacterial species indicate sienificant differences (P 5 0.05) as determined bv Duncan’s multide range test.

~~~ Protein Control Callus Lactoferrin Transgenic Bacterial Species Concentration Extract (commercial) Callus Extract fig“ log,,, colony-forming unitslml X. campestris pv phaseoli O 7.43” 7.44” 7.34“ 150 7.48” 6.94ab 6.43b 500 7.50a 6.70b 2.67‘ P. syringae pv phaseolicola O 6.80” 6.81a 6.72” 150 6.75” 6.30b 5.04b 500 6.64” 6.1Ob 2.52‘ P. syringae pv syringae O 7.21” 7.18” 7.32” 1 O0 7.32” 6.96” 5.18b 500 7.31“ 6.71” 3.78‘ C. f/accumfaciens pv O 7.30” 7.34” 7.05” flaccum faciens 150 7.2 1 a 6.84b 5.06b 500 7.28a 6.72b 2.73‘ Expression of an Antibacterial Gene in Tobacco 98 1 protein(s) in transgenic tobacco calli. Expression of the lac- RT (1985) A simple and general method for transferring genes tofemn gene in transgenic crop plants may introduce a broad- into plants. Science 227: 1229-1231 Kennedy BW, Alcorn SM (1980) Estimates of U.S. crop losses to spectrum resistance against phytopathogenic bacteria. prokaryote plant pathogens. Plant Dis 64 674-676 Laemmli UK (1970) Cleavage of structural proteins during the ACKNOWLEDGMENTS assembly of the head of bacteriophage T4. Nature 227: 680-685 Liu Y, Teng CT (1991) Characterization of estrogen-responsive We would like to thank Dr. Mike Powell for making the lactoferrin mouse lactofenin promoter. J Biol Chem 266 21880-21885 cDNA available to us, Dr. Anne K. Vidaver and Pat Lambrecht for Logemann J, Schell J, Willmitzer L (1987) Improved method for providing the bacterial strains and their generous help with the the isolation of RNA from plant tissues. Anal Biochem 163 16-20 antibacterial activity assay, and Hoechst Roussell Agri-Vet for the Lu L, Hangoc G, Oliff A, Chen LT, Shen R-N, Broxmeyer HE gift of Ignite. We are grateful to Drs. M.B. Dickman and A.E. Zipf (1987) Antiviral activity of cloned lactoferrin protein. Cancer Res 47: 4184-4188 for comments concerning the manuscript. The technical assistance of Masson PL, Heremans JF, Schonne E (1969) Lactoferin, an iron- Dan Higgins and the assistance of S. Mitra with plant tissue culture binding protein in neutrophilic leukocytes. J Exp Med 130 are sincerely appreciated. 643-658 Mitra A, An G (1989) Three distinct regulatory elements comprise Received May 4, 1994; accepted July 11, 1994. the upstream promoter region of the nopaline synthase gene. Mol Gen Genet 294-299 Copyright Clearance Center: 0032-0889/94/106/0977/05. 215: Mitra A, Choi HK, An G (1989) Structural and functional analyses of Arabidopsis thaliana chlorophyll a/b-binding protein (cab) pro- LITERATURE CITED moters. Plant Mol Biol 12: 169-179 Murashige T, Skoog F (1962) A revised medium for rapid growth An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In SB Gelvin, and bio-assays with tobacco tissue cultures. Physiol Plant 15 RA Schilperoort, eds, Plant Molecular Biology Manual. Martinus 473-497 Nijhoff, Dordrecht, The Netherlands, pp A3:1-A3:19 Nagata T, Okada K, Takebe I, Matsui C (1981) Delivery of TMV- Arnold RR,.Cole MF, McGhee JR (1977) A bactericidal effect for RNA into plant protoplasts by reverse phase evaporation vesicles. human lactofemn. Science 197: 263-265 Mol Gen Genet 184 161-165 Arnold RR, Russell JE, Champion WJ, Brewer M, Gauthier JJ Perombelon MCM, Kelman A (1980) Ecology of soft rot Erwinias. (1982) Bacteriocidal activity of human lactofemn. Infect Immun Ann Rev Phytopatholl8 361-387 35 792-796 Powell MJ, Ogden JE (1990) Nucleotide sequence of a human Bradford M (1976) A rapid and sensitive method for the quantitation lactoferrin cDNA. Nucleic Acids Res 18: 4013-4018 of microgram quantities of protein utilizing the principle of protein- Southern EM (1975) Detection of specific sequences among DNA dye binding. Anal Biochem 72: 248-254 fragments separated by gel electrophoresis.J Mol Biol98: 503-517 Fromm M, Morrish F, Armstrong C, Williams R, Thomas J, Klein Tomita M, Bellamy W, Takase M, Yamauchi K, Wakabayshi H, TM (1990) Inheritance and expression of chimeric genes in the Kawase K (1991) Potent antibacterial peptides generated by pepsin progeny of transgenic maize plants. Biotechnology 8: 833-839 digestion of bovine lactofemn. J Dairy Sci 74 4137-4142 Green MR, Pastewka JV (1978) High levels of lactofemn expression Ward PP, May GS, Headon DS, Conneely OM (1992) An inducible in lactating mammary glands. Endocrinology 103 1510-1513 expression system for the production of human lactoferrin in Guillemaut P, Maréchal-Drouard L (1992) Isolation of plant DNA: Aspergillus nidulans. Gene 122: 219-223 a fast, inexpensive and reliable method. Plant Mol Biol Rep 10 Yamauchi K, Tomita M, Giehl TJ, Ellison RT (1993) Antibacterial 60-65 activity of lactofemn and a pepsin-derived lactofemn peptide Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley fragment. Infect Immun 61: 719-728