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Identification of a basic pathogenesis-related, thaumatin-like protein of virus-infected tobacco as osmotin
Physiological and Molecular Plant Pathology (1991) 38, 137-146
1 37
Identification of a basic pathogenesis-related, thaumatin-like protein of virus-infected tobacco osmotin A.
STINTZI,T.
HEITZ, S.
KAUFFMANN,hi.
as
LEGRAND~ a n d B. FRITIG
Institut de Biologic ,ttoldculaire des Plantes du CaVRS, Unirersitd Louis Pasteur, 12 rue du GgnEralZimmer, 67084 Strasbourg Cidex, France (Acceptedfor publication aVanuao' 1991)
A basic PR protein was isolated and characterized from Samsun NN tobacco leaves infected with tobacco mosaic virus. The protein is serologically-related to the pathogenesis-related (PR) proteins R and S, the thaumatin-like (TL) proteins so-called because of their high level of homology with thaumatin, a sweet-tasting protein from the West African shrub Thaumatococcus daniellii Benth. Its amino acid composition and its NH2-terminal sequence indicate that this PR protein is in fact osmotin, a protein known to accumulate in tobacco cells in response to osmotic stress. A specific serum was obtained and used in immunoblotting experiments to study the serological relationships of TL-proteins of tobacco and to compare the induction of osmotin and acidic PR proteins R and S during the hypersensitive reaction of tobacco to tobacco mosaic virus.
INTRODUCTION After infection b y fungi, bacteria, viruses or viroids, plants a c c u m u l a t e soluble proteins called P R (pathogenesis-related) proteins. P R proteins were first detected in tobacco cultivars reacting hypersensitively to tobacco mosaic virus ( T M V ) [8, 25, 27, 41, 42] b u t m a n y P R proteins have now been found in a large v a r i e t y o f plants [2-4, 1 6 , 2 3 , 2 9 , 3 5 , 3 9 , 4 4 ] . I n tobacco ten m a j o r acidic P R proteins have been identified a n d designated P R p r o t e i n la, l b , le, 2, N, O, P, Q , R a n d S [27, 42]. R e c e n t l y five o f them have been shown to be hydrolytic e n z y m e s : P R proteins P a n d Q are chitinases a n d proteins 2, N a n d O a r e 1,3-/J'-glucanases [12, 18]. T h e biological function o f the r e m a i n i n g five acidic P R proteins is still unknown. Proteins R and S have been d e m o n s t r a t e d to share sequence h o m o l o g y with t h a u m a t i n [5, 11, 26, 28, 38], with a m a i z e trypsin/0c-amylase bifunctional inhibitor [30], with osmotin, a protein i n d u c e d in tobacco d u r i n g salt a d a p t a t i o n [33, 34], and to a lesser extent with trypsin a n d a g r o u p of p i c o r n a v i r a l proteases [1, 3 / ] but no biological activity was found in purified p r e p a r a t i o n s by ourselves [13] or by others [28]. Moreover, the constitutive expression o f P R - S in tobacco has been shown to have no effect on virus infection [19]. P R proteins R a n d S belong to the s a m e serological g r o u p [13] a n d correspond to ]-To whom correspondence should be addressed. 'Abbreviations used in text: PR-protein, pathogenesis-related protein; TL protein, thaumatin-like protein; TMV, tobacco mosaic virus; ABA, absclssic acid; FPLC, fast protein liquid chromatography. 0885-5765/91]020137+ 10 S03.00/0
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proteins 5a and 5b in the nomenclature of Van Loon et al. [42]. PR proteins with a higher isoelectric point are also produced in TMV-infeeted tobacco [9, 10, 12, 18, 40]. In particular, two basic chitinases and one basic 1,3-fl-glucanase have been isolated and shown to be serologically related to their acidic counterparts [12, 18]. The acidic isoforms accumulate predominantly in extracellular spaces [7, 10, 25] whereas the basic ones have been found inside ihe cell [9, 37, 40]. Concerning PR proteins R and S no basic counterpart has been shown to accumulate in tobacco leaves upon infectioni Here we report the isolation and characterization of a new PR protein of TMV-infected tobacco which is serologically related to proteins -R and -S but has a higher isoelectric point. Its amino acid composition and its N H 2 terminus have been shown to be sinfilar to those of osmotin. Specific antibodies were used to compare the kinetics of accumulation of the three thaumatin-Iike PR proteins of tobacco during the hypersensitive reaction to T M V . MATERIALS AND METHODS
Plant material Tobacco plants (Wicotiana tabacum cv. Samsun NN) were grown in a greenhouse under controlled conditions. T h e first three fully expanded leaves of 3-month-old plants were inoculated with a suspension of purified T M V . Plants were then incubated in a growth chamber at 2 2 + 1 °C with a 16 h photoperiod. T h e leaves bearing -,- 200 lesions were harvested 7 days after inoculation~ frozen in liquid nitrogen and stored at --80 °C.
Extraction of proteins 100 g of infected leaves were ground in a Waring Blendor at 4 °C in 150 ml of 0"5 M sodium acetate buffer, p H 5-2, containing 15 ms~ 2-mercaptoethanol and l g of charcoal. After filtration through a double layer of cheesecloth, the extract was centrifuged at 15000g for 30 min. In the case of salt-adapted tobacco cells 130 mg of material were ground in liquid nitrogen and then extracted with 1"6 ml of sodium acetate buffer. The protein fraction issued from a Sephadex G-25 column was analysed without further purification.
Purification procedure The 15000g supernatant was desalted on a Sephadex G-25 column ( 4 x 7 0 cm Pharmacia) equilibrated with 20mM sodium acetate buffer, pH 5"2, containing 5 mM 2-mercaptoethanol. Tile protein fraction was allowed to stand at 4 °C for a few hours and then centrifuged at 2 0 0 0 0 g for 30 min. The clear supernatant was applied to a S-Sepharose column (2"2 x 9 cm F a s t Flow Pharmacia) equilibrated with 20 mM sodium acetate buffer, p H 5"2. The nonadsorbed protein fraction was used to isolate PR-proteins R and S as previously described [13]. The adsorbed protein fraction'.,was eluted with 500 ml of a linear gradient from 0-0"25 ~f NaCI in the same buffer; The pooled fractions were concentrated to 200 lal on Centricon 10 concentrators (Am!con), filtered through 0-22 lam filters and injected onto a Superose 12 column (Pharmacia). Elution was carried out with 0"1 ~f sodium acetate buffer, pH 5"2, containing 0"2 xf NaC1 at a flow rate of 0"I ml min -l (FPLC system, Pharmacia). Pooled fractions were dialysed against 20 mM sodium acetate
Identification of protein of virus-infected tobacco
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buffer pH 5"2, filtered and loaded onto a T S K - C M SW column (0"75 x 7"5 cm, Altex) equilibrated in the same buffer.: Elution was performed by a linear gradient from 60-180 m~t NaC1 in the sodium acetate buffer.
Pol)'acrylamide gel electrophoresis and protein staining Electrophoresis of denatured proteins was carried out on slab gels by the method of Laemmli [17] with a 5 % stacking gel and a 12"5% resolving gel. Gels were fixed for 1 h in a 12% trichloroacetic acid solution and then stained according to the method of Neuhoff [24]. In some cases the gels were subsequently stained with silver [22].
Amino acid analysis and aVH2-terminus sequence determination Amino acid analysis and sequence determinations were carried out in the laboratory of Y. Boulanger and J. Reinbolt (IBMC, Strasbourg). An H P L C - W I S P 712 system (Waters Associates, Milforn, U.S.A.) was used for amino acid analysis. NHz-terminal sequence was determined by Edman degradation using an Applied Biosystems (Foster City, USA) 470A sequencer and its on-line phenylthiohydantoin amino acid analyser.
Production ofsera The purified protein preparation was emulsified with either Freund's complete adjuvant for the first injection or incomplete adjuvant for all the following boost injections. Fifty lag of antigen was used in the first injection and 20 lag in the others. Ten days after boost immunization serum was collected. After clot removal, the serum was clarified by centrifugation and stored in small batches at --20 °C. The production of the sera raised against proteins R and S was described previously [13].
hnmunoblotting The basic procedure of Towbin et al. [36"] was used; with modifications [18]. In addition, 3 % gelatin was used for the saturation of the membrane and 1% defatted milk was added to diluted serum solutions to decrease the background [21]. The band intensities on the blots were estimated by densitometry using a CS930 Shimadzu densitometer. The relative band intensities were taken as a measure of the relative amounts of immunologically reactive proteins. For protein staining amidoblack was used as described previously [13]. RESULTS
Occurrence of a basic counterpart lo PR proteins R and S Tile purification to homogeneity of PR proteins R and S h a s been achieved recently and specific antibodies have been obtained [13]. Ifi a search for a putative basic counterpart to PR proteins R and S, proteins from TMV-infected tobacco leaves were fractionated by ion exchange and probed independently:with the anti-S and anti-R sera. As expected, both sera recognized proteins R and S pi'esent in acidic fraction (Fig. 1, lanes 2A and 3A). Two proteins of the basic fractionwere also recognized by the anti-R serum (Fig. 1, lane 2B) and to a lesser extent by tile anti-S serum (Fig. 1, 3B). One of these has a molecular mass of 22 kD and was: isolated and characterized further.
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Fxo. 1. Immunoblot of acidic and basic protein fractions of tobacco. Protein extract from tobacco was fractionated in basic (B) and acidic (A) proteins by ion exchange chromatography. After electophoresis under denaturing conditions proteins were transferred onto nitrocellulose sheets and either stained with amidoblack (1) or immuno-rcvealed with the anti-R serum (2) or the anti-S serum (3). The position of marker proteins is indicated on the left.
Isolation of the 22 kD-basic protein serologically related to R and S Protein extract was loaded onto a S-Sepharose column and was eluted with a linear gradient of salt:'concentration (see Materials and Methods). The fractions were analysed by electrophoresis followed by immunoblotting with the anti-R serum. Fractions containing the protein serologically related to R were pooled, concentrated and injected onto a Supcrose 12 column. Eluted fractions containing the protein of interest were pooled and submitted to a final cationic exchange chromatography under high performance conditions (see Materials and Methods). At each step of purification the fractions were analysed by electrophoresis under denaturing conditions (Fig. 2). At
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Fro. 2. Electrophoretic analysis of the fractions containing the 22 kD protein. Fractions at different stages of purification were analysed by electrophoresis under denaturing conditions: (1) Sephadex G-25 fraction, (2) CM-Sepharose fraction, (3) Superose 12 fraction and (4) TSK-CM fraction. Proteins were stained with Coomassie Blue G250 (1,2,3) or with silver (4). The molecular mass (kD) of marker proteins (M) is indicated on the left.
Identification of protein of virus-infected tobacco
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the final stage o f the purification, the p r e p a r a t i o n a p p e a r e d homogeneous, even after silver staining (Fig. 2, lane 4).
Amino acid composition and aV-terminus sequence determination A m i n o acid analysis was performed as described in M a t e r i a l s a n d "Methods. T h e results a r e presented in T a b l e 1 a n d they a r e indistinguishable from those o b t a i n e d with osmotin [33]. T h e purified P R protein was sequenced from the N - t e r m i n u s through 20 TABLE 1
Comparisonof amino acidcompositionsof protein 22 kD and osmotin Residue Asx Thr Ser Glx Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp
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11"4 9"0 4"2 6"6 8"9 12'8 6"9 8"3 3"6 1"0 3"9 3"6 2"8 5"7 0-8 2"4 5"4 3-2
The values for osmotin were taken from [33]. cycles. No p h e n y h h i o h y d a n t o i n derivative was identified in the 9th cycle of E d m a n d e g r a d a t i o n suggesting that the residue (X) at position 9 is a cysteine. Tile other 19 a m i n o acid residues of the N - t e r m i n u s were identified as those of osmotin (Fig. 3). These d a t a , together with a m i n o acid composition, strongly suggest that the basic P R p r o t e i n serologically related to R a n d S is, in fact, osmotin. 22kD: Ala Thr Ile Glu Val Arg Asn Asn X Pro Tyr Thr Val Trp Ala Ala Ser Thr Pro lie Osm : Ala Thr Ile Glu Val Arg Asn Asn Cys Pro Tyr Thr Val Trp Ala Ala Ser Thr Pro Ile FIG. 3. Comparison ofNH z terminal sequences of protein 22 kD and osmotin. Osmotin sequence is from ref. [M].
Serological relationships between the thaumatin-like proteins of tobacco Purified p r e p a r a t i o n s o f this virus-induced osmotin were injected into rabbits a n d polyclonal antibodies were o b t a i n e d . T h e specificity o f the serum was studied in i m m u n o b l o t t i n g experiments. T o t a l protein fractions from infected tobacco leaves a n d from s a l t - a d a p t e d tobacco cells were electrophoresed ufider d e n a t u r i n g conditions.
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Fxo. 4. Immunodetection ofosmotin in tobacco extracts. After electrophorcsis under denaturing conditions proteins were transferred onto nitrocellulose sheets and stained with amidoblack (1) or immunodetected with the anti-osmotin serum (2,3,4). Sephadex G-25 fractions from T M V infected tobacco leaves (1, 2) or from salt-adapted tobacco cells (3) and purified osmotin (4) were tested.
After transfer onto nitrocellulose, proteins were either stained with a protein stain or immunorevealed with the antibodies (Fig. 4). Among all the proteins extracted from infected leaves and detected on the nitrocellulose sheet (Fig. 4, lane 1), only one band was immunodetected with the antibodies (Fig. 4, lane 2). This illustrates the specificity of the serum. On the other hand, a band with the same mobility was also revealed in the crude extract ofsalt-adapted cells (Fig. 4, lane 3) and in a purified preparation of osmotin (Fig. 4, lane 4). These data confirm that the same protein, osmotin, is induced in tobacco upon hypersensitive reaction and during salt adaptation. The three sera raised against the three different thaumatin-like proteins of tobacco were used in immunoblotting experiments to study their serological relationships. Purified p~'eparations of each PR protein were electrophoresed, transferred onto nitrocellulose and immunodetected with each serum (Fig. 5). As expected, anti-R and anti-S sera strongly recognized proteins R and S which showed their characteristic broad pattern (Fig. 5, lanes I and 2). Anti-R serum cross-reacted with osmotin more readily thal~ anti-S serum whereas anti-osmotin antibodies strongly cross-reacted with both prote!ns R and S (Fig. 5, C). Thus, although the two acidic forms (R and S) appear to be more closely related to each other than to the basic form (osmotin), the three thau.rnatin-like proteins which were known to have sequence homologies [5, 26, 28, if,4, 38], share also common epitopes. It is worth noting that an immunological cross-reactiveness of a TL-protein of tobacco with thaumatin itself has also been demonstrated [7].
I£inetics of induction of the thaumatin-like PR-proteins in TMV-infected tobacco leaves Since the sera raised against each protein displayed various levels of cross-reactivity with tile otfier T L proteins which have similar molecular masses (Fig. 5), these proteins cannot be distinguished by immunoblotting after one-dimensional electrophoresis of crude extracts. Therefore, the basic and the acidic proteins were separated in a first
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Fxo. 5. Immunoblots of the 3 TL-proteins of tobacco. 0-I pg of a purified preparation of PRR (1), PR-S (2) or osmotin (3) was electrophoresed under denaturing conditions. After transfer onto nitrocellulose the proteins were incubated with a 2000-fold diluted solution o f a polyclonal serum raised against P R - R (A), or against PR-S (B) or against osmotin (C).
dimension by ion exchange chromatography, electrophoresed under denaturing conditions and immunorevealed after transfer onto nitrocellulose. The relative amounts of osmotin and acidic counterparts were estimated by measuring the intensity of the bands on immunoblots. The data are presented in Fig. 6. Acidic thaumatin-like PR-
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proteins were undetectable in healthy leaves but were strongly induced in infected leaves with kinetics resembling those described for other tobacco PR-proteins [12, 18]. In contrast, osmotin was already present in detectable amounts in health), tissues (zero time of Fig. 6) but its quantity was dramatically increased by infection and reached a
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maximum by day 6 of infection. This is 4 days before the maximum level of the acidic counterparts R and S was reached.
DISCUSSION
The results presented here demonstrate that osmotin which has been shown at first to be induced in tobacco during the adaptation to water stress, is also strongly induced during the hypersensitive response to TMV. Osmotin can be considered, therefore, as a basic PR protein of tobacco. It appears that in tobacco T M V infection triggers the synthesis of a set of TL-proteins which includes acidic (proteins R and S) and basic (osmotin) forms. This situation has been found to occur for several groups of PR proteins of tobacco: basic and acidic isoforms offl-l,3 glucanase and chitinase have been described [12, 18] and cDNAs and genes encoding a basic counterpart to acidic PR proteins of group I have been characterized [6]. Acidic forms are exported outside the cell and accumulate in intercellular spaces [10, 25, I. Dore, unpubished results] whereas their basic counterparts have an intracellular localization [9, 37, 40]. Osmotin has been found, indeed, in the vacuole of tobacco cells [33] and this contrasts with the extracellular localization of PR proteins R and S [25, 40, I. Dore, unpublished results]. In the case ofglucanases and chitinases it has been proposed that the dual localization of these enzymes which have a lyric action on fungi and bacteria is important in plant defense [20]. For TL-proteins the discovery of their function(s) is a prerequisite to the understanding of the physiological significance oftheir differential cellular localization. Proteins with a high degree of homology with thaumatin have been characterized in various plant species: PR-R, -S and osmotin in tobacco [5, 14, 26, 28, 32-34, 38], NP24 in tomato [15], protein C in potato [29], HVI1 protein in barley [3], a bifunctional trypsin/~z-amylase inhibitor in maize [30]. Related proteins have also been detected in petunia, datura, alfalfa and bean [14] but have not been fully characterized. Some of these proteins (the maize inhibitor and thaumatin) are constitutively expressed while others like HV-1 and PR-R and -S are highly inducible by pathogen infection. Osmotin and NP24 were detected in stems, roots and leaves of greenhouse grown plants [14] but are also highly inducible by osmotic stress, ABA treatment or infection. Some studies investigated whether the same stress proteins are produced upon different forms of stress. For instance, proteins associated with adaptation to salt or water stress (like osmotin) have not been found after heat shock or anoxia [32]. In cowpea and cucumber, however, similarities in protein patterns have been noticed after plasmolysis or virus infection [43]. In tobacco, polypeptides which accumulate in cultured mesophyll protoplasts have been identified as the basic fl-l,3 glucanase and chitinases which are also induced by T M V infection [9]. Here, we show that osmotin is induced by pathogenic and osmotic stress. This may suggest that, at least in some cases, the same transduction signals are involved in plant response to various forms of stress. We thank Dr Y. Meyer (Universit6 de Perpignan, France) for the gift ofsah-adapted tobacco cells. We are indebted to Dr M. H. Metz and Mrs M. Le Ret (IBMC, Strasbourg) for amino acid analysis and sequence determinations.
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24. NEultovv, V., AROLD, N., TAUBE, D. & EttRIIARDT, ~,V. (1988). Improved staining of proteins in polyacrylamide gels including isoclectric focusing gels with clear background at nanogram sensitivity using Coomassie brilliant blue G-250 and R-250. Electrophoresis9, 255-262. 25. P,XRrNT,J. G. & ASSELtN,A. (1984). Detection of pathogenesis-related proteins (PR or b) and of other proteins in the intercellular fluid &hypersensitive plants infected with tobacco mosaic virus. Canadian Journal of Botany 62, 564-569. 26. I'A'e.~E, G., .MIDDLEST~ADa',W., WIH-m.~s, S., DESAI, N., PARKS, T. D., DINCHE~, S., CARNES, M. & Rvat.s, J. (1988). Isolation and nucleotide sequence of a novel eDNA encoding the major form of pathogenesis-related protein R. Plant Molecular Biology 11,223-224. 27. PIE~POIN'r, W.S. (1986). The pathogenesis-related proteins of tobacco leaves. Ph.)'toehemistry 25, 1595-1601. 28. PIERPOINT,~V. S., TATHAM, A. S. & PAPPIN, D.J.C. (1987). Identification of the virus-induced protein of tobacco leas'es that resembles the sweet-protein thaumatiu. Physiologicaland Molecular Plant Pathology 31, 291-298. 29. PIrRPOINa', W. S.,JAcKsoN, M.J. & Evans, R. M. (1990). The presence ofa thaumatin-like protein, a chitinase and a glucanase among the pathogenesis-related proteins of potato (Solanum tuberosum). Physiological and Molecular Plant Pathology36, 325-338. 30. Rm~IARDSON, M., VALDEs-RonRmUEZ, S. & BLANco-LAnRA, A. (1987). A possible function for thaumatin and a TMV-induced protein suggested by homology to a maize inhibitor. Nature 327, 432--434. 31. SKERN, T., ZORN, M., BLAre, D., KUECnLER, E. & SO.~L~XERCaUBER,W. (1990). Protease or protease inhibitor? Nature 344, 26. 32. SINGH,N. K., HANDA,A. K., HASEGAWA,P.M. & BRESSAN,R.A. (1985). Proteins associated with adaptation ofcuhured tobacco cells to NaCI. Plant Physiology79, 126-137. 33. SINGH, N. K., BRACKER,C.A., HASEGAWA,P. M., HANDA,A. K., BUCKEL,S., HERMODSON,,XI.A., PFANKOCH, E., REGNIER, F. E. & BRESSAN,R. A. (1987). Characterization ofosmotin. A thaumatinlike protein associated with osmotic adaptation in plant ceils. Plant Physiology85, 529-536. 34. SINOH, N. K., NELSON, D. E., KvttN, D., HASEGAWA,P..~1. & BRESSAN,R. A. (1989). ,Xlolecular cloning of osmotln and regfilation of its expression by ABA and adaptation to low water potential. Plant Physiology 90, 1096--1101. 35. SoMssIctt, I. E.~"SCttMELZER, E., BOLLMAN,J. & HAHLBROCK, K. (1986). Rapid activation by fungal elicitor of genes encoding "pathogenesis-related" proteins in cultured parsley ceils. Proceedingsof the National Academy of Sciences of the U.S.A. 83, 2427-2430. 36. Towm.~, H., S'rAEHELIN, T. & Goano.% J. (1979). Electrophoretlc transfer of proteins from polyaerylamlde gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences of the U.S.A. 76, 4350~-354. 37. VA.'~DrN BULCKE, ~I., BAUW, G., CASTRESANA, C., XrAN .~IONTAGU, ,'~I.& XfANDEKERCKHOVE, j. (1989). Characterization of vacuolar and extracellular]~(l,3)-glucanases of tobacco: Evidence for a strictly compartmentalized plant defense system. Proceedingsoft& National Academy of Sciences of the U.S.A. 86, 2673-2677. 38. 'VAN KAY, J. A. L., VAN DE RHEE, *I. D., ZUIDEMA,D., CORNELISSEN, B.J.C. & BOL,j . F. (1989). Structure of tobacco genes encoding thaumatin-like proteins. Plant Molecular Biology 12, 153-155. 39. VA.~ LOON, L. C. (1985). Pathogenesis-related proteins. Plant ,ilolecular Biology 4, 111-116. 40. VAN LOON, L.C. & GERRXTSE.N, Y. A..Xl. (1989). Localization of pathogenesis-related proteins in infected and non-infected leaves ofSamsun NN tobacco during the hypersensitive reaction to tobacco mosaic virus. Plant Science 63, 131-140. 41. \rAN LOON, L. C. & VAN KAMMEN, A. (1970). Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum vat. "Samsun" and "Samsun NN ". Changes in protein constitution after infection with T , I V . Virology40, 199-201. 42. \rAN LOON, L.C., GERRrrSEN, Y. A. *l. & RXTTER, C.E. (1987). Identification, purification and characterization ofpathogenesis-related proteins from virus-infected Samsun NN tobacco leaves. Plant Molecular Biology 9, 593-609. 43. XX~AGIH,E. E. & COUTTS,R. H. A. (1982). Comparison ofvirus elicited and other stresses on the soluble protein fraction of cucumber cotyledons. Ph.ytopathologischeZeitschrift 104, 364-374. 44. WroTE, R. F., RYBmKL E. P., VON ~VEGII.MAR,.Xl. B., DEKKER,J. L. & ANTONIW,J. F. (1987). Detection of PR 1-type proteins in Amaranthaceae, Chenopodiaceae, Gramineae and Solanaceae by immunoelectroblotting. Journal of General Virology68, 2043-2048.
1 37
Identification of a basic pathogenesis-related, thaumatin-like protein of virus-infected tobacco osmotin A.
STINTZI,T.
HEITZ, S.
KAUFFMANN,hi.
as
LEGRAND~ a n d B. FRITIG
Institut de Biologic ,ttoldculaire des Plantes du CaVRS, Unirersitd Louis Pasteur, 12 rue du GgnEralZimmer, 67084 Strasbourg Cidex, France (Acceptedfor publication aVanuao' 1991)
A basic PR protein was isolated and characterized from Samsun NN tobacco leaves infected with tobacco mosaic virus. The protein is serologically-related to the pathogenesis-related (PR) proteins R and S, the thaumatin-like (TL) proteins so-called because of their high level of homology with thaumatin, a sweet-tasting protein from the West African shrub Thaumatococcus daniellii Benth. Its amino acid composition and its NH2-terminal sequence indicate that this PR protein is in fact osmotin, a protein known to accumulate in tobacco cells in response to osmotic stress. A specific serum was obtained and used in immunoblotting experiments to study the serological relationships of TL-proteins of tobacco and to compare the induction of osmotin and acidic PR proteins R and S during the hypersensitive reaction of tobacco to tobacco mosaic virus.
INTRODUCTION After infection b y fungi, bacteria, viruses or viroids, plants a c c u m u l a t e soluble proteins called P R (pathogenesis-related) proteins. P R proteins were first detected in tobacco cultivars reacting hypersensitively to tobacco mosaic virus ( T M V ) [8, 25, 27, 41, 42] b u t m a n y P R proteins have now been found in a large v a r i e t y o f plants [2-4, 1 6 , 2 3 , 2 9 , 3 5 , 3 9 , 4 4 ] . I n tobacco ten m a j o r acidic P R proteins have been identified a n d designated P R p r o t e i n la, l b , le, 2, N, O, P, Q , R a n d S [27, 42]. R e c e n t l y five o f them have been shown to be hydrolytic e n z y m e s : P R proteins P a n d Q are chitinases a n d proteins 2, N a n d O a r e 1,3-/J'-glucanases [12, 18]. T h e biological function o f the r e m a i n i n g five acidic P R proteins is still unknown. Proteins R and S have been d e m o n s t r a t e d to share sequence h o m o l o g y with t h a u m a t i n [5, 11, 26, 28, 38], with a m a i z e trypsin/0c-amylase bifunctional inhibitor [30], with osmotin, a protein i n d u c e d in tobacco d u r i n g salt a d a p t a t i o n [33, 34], and to a lesser extent with trypsin a n d a g r o u p of p i c o r n a v i r a l proteases [1, 3 / ] but no biological activity was found in purified p r e p a r a t i o n s by ourselves [13] or by others [28]. Moreover, the constitutive expression o f P R - S in tobacco has been shown to have no effect on virus infection [19]. P R proteins R a n d S belong to the s a m e serological g r o u p [13] a n d correspond to ]-To whom correspondence should be addressed. 'Abbreviations used in text: PR-protein, pathogenesis-related protein; TL protein, thaumatin-like protein; TMV, tobacco mosaic virus; ABA, absclssic acid; FPLC, fast protein liquid chromatography. 0885-5765/91]020137+ 10 S03.00/0
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proteins 5a and 5b in the nomenclature of Van Loon et al. [42]. PR proteins with a higher isoelectric point are also produced in TMV-infeeted tobacco [9, 10, 12, 18, 40]. In particular, two basic chitinases and one basic 1,3-fl-glucanase have been isolated and shown to be serologically related to their acidic counterparts [12, 18]. The acidic isoforms accumulate predominantly in extracellular spaces [7, 10, 25] whereas the basic ones have been found inside ihe cell [9, 37, 40]. Concerning PR proteins R and S no basic counterpart has been shown to accumulate in tobacco leaves upon infectioni Here we report the isolation and characterization of a new PR protein of TMV-infected tobacco which is serologically related to proteins -R and -S but has a higher isoelectric point. Its amino acid composition and its N H 2 terminus have been shown to be sinfilar to those of osmotin. Specific antibodies were used to compare the kinetics of accumulation of the three thaumatin-Iike PR proteins of tobacco during the hypersensitive reaction to T M V . MATERIALS AND METHODS
Plant material Tobacco plants (Wicotiana tabacum cv. Samsun NN) were grown in a greenhouse under controlled conditions. T h e first three fully expanded leaves of 3-month-old plants were inoculated with a suspension of purified T M V . Plants were then incubated in a growth chamber at 2 2 + 1 °C with a 16 h photoperiod. T h e leaves bearing -,- 200 lesions were harvested 7 days after inoculation~ frozen in liquid nitrogen and stored at --80 °C.
Extraction of proteins 100 g of infected leaves were ground in a Waring Blendor at 4 °C in 150 ml of 0"5 M sodium acetate buffer, p H 5-2, containing 15 ms~ 2-mercaptoethanol and l g of charcoal. After filtration through a double layer of cheesecloth, the extract was centrifuged at 15000g for 30 min. In the case of salt-adapted tobacco cells 130 mg of material were ground in liquid nitrogen and then extracted with 1"6 ml of sodium acetate buffer. The protein fraction issued from a Sephadex G-25 column was analysed without further purification.
Purification procedure The 15000g supernatant was desalted on a Sephadex G-25 column ( 4 x 7 0 cm Pharmacia) equilibrated with 20mM sodium acetate buffer, pH 5"2, containing 5 mM 2-mercaptoethanol. Tile protein fraction was allowed to stand at 4 °C for a few hours and then centrifuged at 2 0 0 0 0 g for 30 min. The clear supernatant was applied to a S-Sepharose column (2"2 x 9 cm F a s t Flow Pharmacia) equilibrated with 20 mM sodium acetate buffer, p H 5"2. The nonadsorbed protein fraction was used to isolate PR-proteins R and S as previously described [13]. The adsorbed protein fraction'.,was eluted with 500 ml of a linear gradient from 0-0"25 ~f NaCI in the same buffer; The pooled fractions were concentrated to 200 lal on Centricon 10 concentrators (Am!con), filtered through 0-22 lam filters and injected onto a Superose 12 column (Pharmacia). Elution was carried out with 0"1 ~f sodium acetate buffer, pH 5"2, containing 0"2 xf NaC1 at a flow rate of 0"I ml min -l (FPLC system, Pharmacia). Pooled fractions were dialysed against 20 mM sodium acetate
Identification of protein of virus-infected tobacco
139
buffer pH 5"2, filtered and loaded onto a T S K - C M SW column (0"75 x 7"5 cm, Altex) equilibrated in the same buffer.: Elution was performed by a linear gradient from 60-180 m~t NaC1 in the sodium acetate buffer.
Pol)'acrylamide gel electrophoresis and protein staining Electrophoresis of denatured proteins was carried out on slab gels by the method of Laemmli [17] with a 5 % stacking gel and a 12"5% resolving gel. Gels were fixed for 1 h in a 12% trichloroacetic acid solution and then stained according to the method of Neuhoff [24]. In some cases the gels were subsequently stained with silver [22].
Amino acid analysis and aVH2-terminus sequence determination Amino acid analysis and sequence determinations were carried out in the laboratory of Y. Boulanger and J. Reinbolt (IBMC, Strasbourg). An H P L C - W I S P 712 system (Waters Associates, Milforn, U.S.A.) was used for amino acid analysis. NHz-terminal sequence was determined by Edman degradation using an Applied Biosystems (Foster City, USA) 470A sequencer and its on-line phenylthiohydantoin amino acid analyser.
Production ofsera The purified protein preparation was emulsified with either Freund's complete adjuvant for the first injection or incomplete adjuvant for all the following boost injections. Fifty lag of antigen was used in the first injection and 20 lag in the others. Ten days after boost immunization serum was collected. After clot removal, the serum was clarified by centrifugation and stored in small batches at --20 °C. The production of the sera raised against proteins R and S was described previously [13].
hnmunoblotting The basic procedure of Towbin et al. [36"] was used; with modifications [18]. In addition, 3 % gelatin was used for the saturation of the membrane and 1% defatted milk was added to diluted serum solutions to decrease the background [21]. The band intensities on the blots were estimated by densitometry using a CS930 Shimadzu densitometer. The relative band intensities were taken as a measure of the relative amounts of immunologically reactive proteins. For protein staining amidoblack was used as described previously [13]. RESULTS
Occurrence of a basic counterpart lo PR proteins R and S Tile purification to homogeneity of PR proteins R and S h a s been achieved recently and specific antibodies have been obtained [13]. Ifi a search for a putative basic counterpart to PR proteins R and S, proteins from TMV-infected tobacco leaves were fractionated by ion exchange and probed independently:with the anti-S and anti-R sera. As expected, both sera recognized proteins R and S pi'esent in acidic fraction (Fig. 1, lanes 2A and 3A). Two proteins of the basic fractionwere also recognized by the anti-R serum (Fig. 1, lane 2B) and to a lesser extent by tile anti-S serum (Fig. 1, 3B). One of these has a molecular mass of 22 kD and was: isolated and characterized further.
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Isolation of the 22 kD-basic protein serologically related to R and S Protein extract was loaded onto a S-Sepharose column and was eluted with a linear gradient of salt:'concentration (see Materials and Methods). The fractions were analysed by electrophoresis followed by immunoblotting with the anti-R serum. Fractions containing the protein serologically related to R were pooled, concentrated and injected onto a Supcrose 12 column. Eluted fractions containing the protein of interest were pooled and submitted to a final cationic exchange chromatography under high performance conditions (see Materials and Methods). At each step of purification the fractions were analysed by electrophoresis under denaturing conditions (Fig. 2). At
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Fro. 2. Electrophoretic analysis of the fractions containing the 22 kD protein. Fractions at different stages of purification were analysed by electrophoresis under denaturing conditions: (1) Sephadex G-25 fraction, (2) CM-Sepharose fraction, (3) Superose 12 fraction and (4) TSK-CM fraction. Proteins were stained with Coomassie Blue G250 (1,2,3) or with silver (4). The molecular mass (kD) of marker proteins (M) is indicated on the left.
Identification of protein of virus-infected tobacco
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the final stage o f the purification, the p r e p a r a t i o n a p p e a r e d homogeneous, even after silver staining (Fig. 2, lane 4).
Amino acid composition and aV-terminus sequence determination A m i n o acid analysis was performed as described in M a t e r i a l s a n d "Methods. T h e results a r e presented in T a b l e 1 a n d they a r e indistinguishable from those o b t a i n e d with osmotin [33]. T h e purified P R protein was sequenced from the N - t e r m i n u s through 20 TABLE 1
Comparisonof amino acidcompositionsof protein 22 kD and osmotin Residue Asx Thr Ser Glx Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp
22 kD protein
Osmotin
11"2 10"9 3"9 6"2 8"5 12"5 6"5 8"3 3-2 1"2 3"6 3"5 2'8 5"6 0"6 2-6 5"7 3"2
11"4 9"0 4"2 6"6 8"9 12'8 6"9 8"3 3"6 1"0 3"9 3"6 2"8 5"7 0-8 2"4 5"4 3-2
The values for osmotin were taken from [33]. cycles. No p h e n y h h i o h y d a n t o i n derivative was identified in the 9th cycle of E d m a n d e g r a d a t i o n suggesting that the residue (X) at position 9 is a cysteine. Tile other 19 a m i n o acid residues of the N - t e r m i n u s were identified as those of osmotin (Fig. 3). These d a t a , together with a m i n o acid composition, strongly suggest that the basic P R p r o t e i n serologically related to R a n d S is, in fact, osmotin. 22kD: Ala Thr Ile Glu Val Arg Asn Asn X Pro Tyr Thr Val Trp Ala Ala Ser Thr Pro lie Osm : Ala Thr Ile Glu Val Arg Asn Asn Cys Pro Tyr Thr Val Trp Ala Ala Ser Thr Pro Ile FIG. 3. Comparison ofNH z terminal sequences of protein 22 kD and osmotin. Osmotin sequence is from ref. [M].
Serological relationships between the thaumatin-like proteins of tobacco Purified p r e p a r a t i o n s o f this virus-induced osmotin were injected into rabbits a n d polyclonal antibodies were o b t a i n e d . T h e specificity o f the serum was studied in i m m u n o b l o t t i n g experiments. T o t a l protein fractions from infected tobacco leaves a n d from s a l t - a d a p t e d tobacco cells were electrophoresed ufider d e n a t u r i n g conditions.
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Fxo. 4. Immunodetection ofosmotin in tobacco extracts. After electrophorcsis under denaturing conditions proteins were transferred onto nitrocellulose sheets and stained with amidoblack (1) or immunodetected with the anti-osmotin serum (2,3,4). Sephadex G-25 fractions from T M V infected tobacco leaves (1, 2) or from salt-adapted tobacco cells (3) and purified osmotin (4) were tested.
After transfer onto nitrocellulose, proteins were either stained with a protein stain or immunorevealed with the antibodies (Fig. 4). Among all the proteins extracted from infected leaves and detected on the nitrocellulose sheet (Fig. 4, lane 1), only one band was immunodetected with the antibodies (Fig. 4, lane 2). This illustrates the specificity of the serum. On the other hand, a band with the same mobility was also revealed in the crude extract ofsalt-adapted cells (Fig. 4, lane 3) and in a purified preparation of osmotin (Fig. 4, lane 4). These data confirm that the same protein, osmotin, is induced in tobacco upon hypersensitive reaction and during salt adaptation. The three sera raised against the three different thaumatin-like proteins of tobacco were used in immunoblotting experiments to study their serological relationships. Purified p~'eparations of each PR protein were electrophoresed, transferred onto nitrocellulose and immunodetected with each serum (Fig. 5). As expected, anti-R and anti-S sera strongly recognized proteins R and S which showed their characteristic broad pattern (Fig. 5, lanes I and 2). Anti-R serum cross-reacted with osmotin more readily thal~ anti-S serum whereas anti-osmotin antibodies strongly cross-reacted with both prote!ns R and S (Fig. 5, C). Thus, although the two acidic forms (R and S) appear to be more closely related to each other than to the basic form (osmotin), the three thau.rnatin-like proteins which were known to have sequence homologies [5, 26, 28, if,4, 38], share also common epitopes. It is worth noting that an immunological cross-reactiveness of a TL-protein of tobacco with thaumatin itself has also been demonstrated [7].
I£inetics of induction of the thaumatin-like PR-proteins in TMV-infected tobacco leaves Since the sera raised against each protein displayed various levels of cross-reactivity with tile otfier T L proteins which have similar molecular masses (Fig. 5), these proteins cannot be distinguished by immunoblotting after one-dimensional electrophoresis of crude extracts. Therefore, the basic and the acidic proteins were separated in a first
Identification of protein of virus-infected tobacco A
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dimension by ion exchange chromatography, electrophoresed under denaturing conditions and immunorevealed after transfer onto nitrocellulose. The relative amounts of osmotin and acidic counterparts were estimated by measuring the intensity of the bands on immunoblots. The data are presented in Fig. 6. Acidic thaumatin-like PR-
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proteins were undetectable in healthy leaves but were strongly induced in infected leaves with kinetics resembling those described for other tobacco PR-proteins [12, 18]. In contrast, osmotin was already present in detectable amounts in health), tissues (zero time of Fig. 6) but its quantity was dramatically increased by infection and reached a
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maximum by day 6 of infection. This is 4 days before the maximum level of the acidic counterparts R and S was reached.
DISCUSSION
The results presented here demonstrate that osmotin which has been shown at first to be induced in tobacco during the adaptation to water stress, is also strongly induced during the hypersensitive response to TMV. Osmotin can be considered, therefore, as a basic PR protein of tobacco. It appears that in tobacco T M V infection triggers the synthesis of a set of TL-proteins which includes acidic (proteins R and S) and basic (osmotin) forms. This situation has been found to occur for several groups of PR proteins of tobacco: basic and acidic isoforms offl-l,3 glucanase and chitinase have been described [12, 18] and cDNAs and genes encoding a basic counterpart to acidic PR proteins of group I have been characterized [6]. Acidic forms are exported outside the cell and accumulate in intercellular spaces [10, 25, I. Dore, unpubished results] whereas their basic counterparts have an intracellular localization [9, 37, 40]. Osmotin has been found, indeed, in the vacuole of tobacco cells [33] and this contrasts with the extracellular localization of PR proteins R and S [25, 40, I. Dore, unpublished results]. In the case ofglucanases and chitinases it has been proposed that the dual localization of these enzymes which have a lyric action on fungi and bacteria is important in plant defense [20]. For TL-proteins the discovery of their function(s) is a prerequisite to the understanding of the physiological significance oftheir differential cellular localization. Proteins with a high degree of homology with thaumatin have been characterized in various plant species: PR-R, -S and osmotin in tobacco [5, 14, 26, 28, 32-34, 38], NP24 in tomato [15], protein C in potato [29], HVI1 protein in barley [3], a bifunctional trypsin/~z-amylase inhibitor in maize [30]. Related proteins have also been detected in petunia, datura, alfalfa and bean [14] but have not been fully characterized. Some of these proteins (the maize inhibitor and thaumatin) are constitutively expressed while others like HV-1 and PR-R and -S are highly inducible by pathogen infection. Osmotin and NP24 were detected in stems, roots and leaves of greenhouse grown plants [14] but are also highly inducible by osmotic stress, ABA treatment or infection. Some studies investigated whether the same stress proteins are produced upon different forms of stress. For instance, proteins associated with adaptation to salt or water stress (like osmotin) have not been found after heat shock or anoxia [32]. In cowpea and cucumber, however, similarities in protein patterns have been noticed after plasmolysis or virus infection [43]. In tobacco, polypeptides which accumulate in cultured mesophyll protoplasts have been identified as the basic fl-l,3 glucanase and chitinases which are also induced by T M V infection [9]. Here, we show that osmotin is induced by pathogenic and osmotic stress. This may suggest that, at least in some cases, the same transduction signals are involved in plant response to various forms of stress. We thank Dr Y. Meyer (Universit6 de Perpignan, France) for the gift ofsah-adapted tobacco cells. We are indebted to Dr M. H. Metz and Mrs M. Le Ret (IBMC, Strasbourg) for amino acid analysis and sequence determinations.
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