ant to proteolytic enzymes (Pierpoint et al., 1981; Van Loon,. 1982), and resolved by electrophoresis in native polyacrylamide gels. They have been detected ...
The EMBO Journal vol.6 no.11 pp.3209-3212, 1987
Biological function of 'pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-f-glucanase activity
Serge Kauffmann, Michel Legrand, Pierrette Geoffroy and Bernard Fritig Institut de Biologie Moleculaires des Plantes, 12, rue du Gendral Zimmer, 67000 Strasbourg, France Communicated by L.Hirth
Three of the ten acidic 'pathogenesis-related' (PR) proteins known to accumulate in Nicofiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely °0, -N and -2, have been shown to have 1,3-4-glucanase (EC 3.2.1.39) activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross-reacted with a basic protein which is also a 1,3-f-glucanase induced by virus infection and which can be considered as a new basic pathogenesis-related protein of tobacco. Protein PR-O and the basic 1,3-3-glucanase display about the same specific enzymatic activity, i.e. 50-fold and 250-fold higher than specific activities of proteins PR-N and -2 respectively. Key words: 1,3-3-glucanases/purification/specific activities/antibodies/serological relationships
Introduction Soluble proteins, referred to as 'pathogenesis-related' (PR) proteins accumulate in plants infected by viruses, viroids, fungi or bacteria (Gianinazzi et al., 1970, 1980; Van Loon and Van Kammen, 1970; Kassanis et al., 1974;. De Wit and Bakker, 1980; Ahl et al., 1981; Camacho Henriquez and Singer, 1982). Such proteins were first detected in tobacco leaves reacting hypersensitively to tobacco mosaic virus (TMV) (Van Loon and Van Kammen, 1970; Gianinazzi et al., 1970) and have now been found in numerous plant species under various circumstances (Van Loon, 1985). These PR proteins share common distinguishing properties. For example, they are selectively extractable at low pH (Van Loon, 1976; Gianinazzi et al., 1977), highly resistant to proteolytic enzymes (Pierpoint et al., 1981; Van Loon, 1982), and resolved by electrophoresis in native polyacrylamide gels. They have been detected predominantly in the intercellular spaces (Parent and Asselin, 1984; Carr et al., 1987). In Nicotiana tabacum cv Samsun NN 10 major acidic PR proteins have been purified and characterized (Van Loon, 1982; Jamet and Fritig, 1986; Pierpoint, 1986; S.Kauffmann et al., unpublished data) and are referred to as proteins PR-la, -lb, -Ic, -2, -N, -0, -P, -Q, -R and -S. Synthesis of proteins PR-la, -lb and -Ic has been shown to be regulated at the mRNA level (Hooft van Huijsduijnen et al., 1985, 1986). Recently proteins PR-P and -Q have been identified as acidic endochitinases serologically related to two basic chitinases which are basic PR proteins of tobacco (Legrand et al., 1987). cDNA clones coresponding to the chitinases have been identified (Hooft van Huijsduijnen et al., 1987). Preliminary work indicated that other PR proteins of tobacco have 1,3-3-glucanase activity and (D IRL Press Limited, Oxford, England
the present report deals with the identification of PR-O, -N and -2 as 1,3-,B-glucanases. In addition, we have purified and characterized from TMV-infected tobacco leaves another 1,3-3-glucanase which is a basic protein and can be considered as a new basic PR protein. The serological relationships between the four enzymes have been investigated.
Results
Induction of 1,3-,B-glucanase activity during the hypersensitive reaction of Samsun NN tobacco plants Figure 1 shows the time course of 1,3-,B-glucanase induction in Samsun NN tobacco leaves in response to TMV infection. About 36 h after inoculation, lesions became visible and 1,3-,3-glucanase activity increased. Enzyme activity increased by 30-fold by the seventh day of infection. The kinetics of induction are very similar to that described for 1,3-3-glucanase activity in N. glutinosa infected with TMV (Moore and Stone, 1972a). 1,3-f-glucanases of tobacco In a search for proteins responsible for 1,3-f-glucanase activity in TMV-infected tobacco leaves we analysed protein extracts by
x
C.) -i
Ci) z 0
IdL
C.)
r---0
0
I
-o-
-
-_
-
-
5 4 3 DAYS AFTER INOCULATION
course curve of TMV-induced 1,3-,-glucanase activity. Extracts from tobacco leaves inoculated with water (0--O) or with purified TMV (0-0) were assayed for 1,3-(3-glucanase activity as described in Materials and methods. Enzyme activity was measured on void volume fractions of the Sephadex G-25 column and is expressed on a fresh weight basis.
Fig. 1. Time
3209
S.Kauffmann et al.
0.1 E 0 Go
.= 1.1
C.)
w
z
(0co cn
CO)
co
I
0.1
-0 N -2
-0 -N -2
E
c
0
-o
F
-dm
w
-~,
N
z w w U]~
1 5>
20
60
100
FRACTION NUMBER
NON
Fig. 2. Elution profiles after successive cation and anion exchange chromatographies. CM-cellulose and Q-Sepharose columns were equilibrated, loaded and eluted as described in Materials and methods. 1,3-,3-glucanase activity (0 ) was assayed by incubating 10 Al of each 6.5-ml fraction for 10 min at 37°C. The absorbance (-) was monitored at 280 nm. -0
A f.
B r-
Kdd 94
-
67
-
43-
-00. R -00-
S
Q p 0
30-
N 2
1c 20-
1b
1
4
-*
6.40
-10.
4. .0
-00.
.
Table I. Amounts and specific activities of the four 1,3-,B-glucanases purified from TMV-infected tobacco leaves
PR-O PR-N PR-2 Basic
Amounts
(/g/g)
Sp. act. (nKat/mg)
1.0 1.5 2.3 2.0
1300 23 5 1100
Amounts are expressed on a fresh weight basis. Protein in the purified preparation was estimated using Coomassie Blue (Bradford, 1976). Proteins PR-O, -N, -2 were extracted at pH 2.8; the basic isoform was purified from a pH 5.2 extract. Enzyme activity was assayed as described in Materials and methods.
-10-00-00-
-.01
Fig. 4. Electrophoretic analysis of purified PR proteins under non-denaturing and denaturing conditions. Proteins were stained with Coomassie Blue.
Isoform
,
I."C'
DNTRN
DENATURING
.10
1a -_Fig. 3. Electrophoretic analysis of active fractions obtained after cation and anion-exchange chromatographies. (A) Fractions from Sephadex G-25 (C), CM-cellulose (1) and PBE118 (2) were electrophoresed on SDS-polyacrylamide slab gels; proteins were stained with silver. (B) Fractions from Sephadex G-25 (C) and Q-Sepharose (3) were electrophoresed on polyacrylamide slab gels under native conditions; proteins were stained with Coomassie Blue; the position of acidic PR proteins in (C) is indicated.
successive cationic and anionic exchange chromatographies. The enzymatic extract was first loaded onto a CM-cellulose column. The unbound protein fraction was dialysed against 20 mM Tris HCl buffer, pH 7.8, and then applied to a Q-Sepharose column. 3210 -
Figure 2 presents the elution profiles obtained -from the CMcellulose and Q-Sepharose columns. All the fractions issuing from both columns were assayed for 1,3-,B-glucanase activity and one major peak of activity was detected in each case. Active fractions were pooled, concentrated and analysed by electrophoresis on polyacrylamide gels (Figure 3). The basic 1,3-13-glucanase fraction which bound to CM-cellulose was electrophoresed in the presence of SDS. A major protein band was revealed with very few contaminants (Figure 3A, lane 1). Further purification was easily achieved by an additional chromatofocusing step as demonstrated by electrophoretic analysis (Figure 3A, lane 2). The relative mobility of this protein corresponds to a mol. wt of 33 000 4 1000. The acidic proteins eluted from the Q-Sepharose column were analysed by electrophoresis on basic native gels (Figure 3B). The major protein band had a mobility similar to that of protein PR0. This prompted us to purify protein PR-O to homogeneity in order to determine unequivocally its catalytic activity. In addition, the purification of proteins PR-N and -2 which have been proposed to belong to the same group of PR proteins as PR-O (Jamet and Fritig, 1986) was also carried out.
Four tobacco PR proteins are
tobacco tissues cultured on auxin-containing medium (Felix and Meins, 1985). In this material it has been shown that glucanase is regulated by auxin and cytokinin at the mRNA level
A
1,3-3-
(Shinshi et al., 1987). Thus at least four isoforms of 1,3-13-glucanase account for the high level of enzymatic activity in tobacco Samsun NN leaves bearing necrotic lesions. Moore and Stone (1972a) have shown a drastic increase in 1 ,3-,3-glucanase activity of N. glutinosa upon
S -*
R -_ P
-_
N -_
2 1c 1b =-_ 1
a
--*
AB
S2 SN So
AB
S
Fig. 5. Serological relationships between the four 1,3-0-glucanases of tobacco. (A) Aliquots of Sephadex G-25 fraction containing the two acidic PR proteins have been electrophoresed under non-denaturing conditions. (B) Aliquots of purified basic 1,3-,3-glucanase have been electrophoresed on SDS-polyacrylamide slab gels. At the end of electrophoresis proteins were transferred onto nitrocellulose sheets and were either revealed by staining with amidoblack (AB) or immunodetected with the sera raised against protein PR-O (lanes SO) or protein PR-N (lanes SN) or protein PR-2 (lanes
S2).
Enzymatic activity of PR proteins Proteins PR-O, -N and -2 were purified to homogeneity by procedures described in Materials and methods. At the final stage of purification each protein appeared homogeneous under native and denaturing conditions (Figure 4). These preparations were assayed for 1,3-3-glucanase activity. Protein was estimated by the method of Bradford (1976) and the values of specific enzyme activity of the proteins PR-O, -N and -2, together with that of the basic 1,3-,B-glucanase, are given in Table I. Protein PR-O and the basic 1,3-f-glucanase displayed about the same specific activity. Values measured for proteins PR-N and -2 were 50-fold and 250-fold lower, respectively. No exoglucanase activity could be detected in the purified enzyme preparations, indicating that the four 1,3-0-glucanases are endoenzymes. Serological relationships between tobacco 1,3-fl-glucanases Antisera against each of the three proteins PR-O, -N and -2 were raised in rabbits and used in immunoblotting experiments presented in Figure 5. Each serum cross-reacted with the three proteins -0, -N and -2 but no significant reactivity with any of the other acidic PR proteins of tobacco could be detected. Furthermore, the basic 1,3-f-glucanase was recognized by the three sera (Figure SB). Thus the acidic proteins PR-O, -N, -2 and the basic 1,3-,B-glucanase share common antigenic sites although they display marked differences in their physico-chemical properties. Discussion We have shown that proteins PR-0, -N and -2 purified to homogeneity have 1,3-0-glucanase activity. Protein PR-O displays the highest specific enzyme activity and accounts for the major
of the acidic 1,3-f-glucanase activity of tobacco. In addition, have detected a basic 1,3-3-glucanase with specific activity as high as that of protein PR-0 and with a mol. wt of 33 000. This basic 1,3-,B-glucanase is localized in intercellular spaces of TMV-infected tobacco leaves (M.Legrand, unpublished results) as are the acidic PR proteins (Parent and Asselin, 1984) and can be considered as a new basic PR protein. Many of its features resemble those described for the 1,3-,B-glucanase produced in
part we
1,3-0-oglucanases
TMV infection. The same authors (Moore and Stone, 1972b,c) purified and characterized a 1,3-(3-glucanase which was present in healthy and infected material and had some features similar to those of protein PR-O, one of the four isoforms we have characterized. However, this point remains to be clarified since the PR proteins of group I have been shown to vary in different Nicotiana species (Ahl et al., 1985). activity seems to be ubiquitous in higher plants (Clarke and Stone, 1962) and multiple forms of the enzyme have been reported in several cases (Wong and Maclachlan, 1979a,b; Young and Pegg, 1981; Cline and Albersheim, 198 la; Keen and Yoshikawa, 1983). We
1,3-3-glucanase
have raised antisera against each of the acidic isoforms of tobacco (i.e. proteins PR-O, -N and -2) and have shown that each serum cross-reacted with the four 1,3-,B-glucanases but not with any of the other PR proteins of tobacco. These data confirm the serological relationships between proteins PR-O, -N and -2 etwhich have been detected by using one anti-PR-2 serum (Fortin al.,
1985).
,B-1,3 glucan and chitin, a polymer of N-acetylglucosamine, major cell wall components of many fungi (Bartnicki-Garcia, 1968). Since 1,3-f-glucanases and chitinases have been shown to be capable of attacking the cell wall of pathogens in vitro, these enzymes have been proposed as direct defence enzymes of plants (Abeles et al., 1970; Wargo, 1975; Netzer et al., 1979; Young and Pegg, 1981, 1982; Boller et al., 1983). Another potential role of 1,3-0-glucanase is related to the biological activity of cell wall constituents which were isolated from various of the plants fungi and can act as elicitors of defence reactionsfrom and soybean (Darvill Albersheim, 1984). 1,3-3-glucanases have been shown to release active elicitors from Phytophthora cell walls (Keen and Yoshikawa, 1983). It has also been proposed that 1,3-f-glucanase may be involved in the processing of high mol. wt fl-glucans into more active forms or in the turnover of elicitor molecules (Cline and Albersheim, 1981a,b; Darvill and Albersheim, 1984). reported in Deposition of callose, a f-1,3 glucan,tohaslocalbeen lesion in relation combinations -virus host developmany ment and virus localization (Shimomura and Dijkstra, 1975). However, the functional significance of high levels of 1,3-,Bremains unclear. glucanase activity in virus-infected tissues Nevertheless the identification of eight PR proteins of tobacco as chitinases and 1,3-fl-glucanases (Legrand et al., 1987; and this paper) indicates that induction of glucanhydrolase activities is a general plant response to stress and disease. are
Materials and methods Plant material Three-month-old tobacco plants (N. tabacum cv Samsun NN), grown in a glassat the house, were used in all experiments. The three first fully expanded leaves inocuThe TMV. of a with inoculated were purified each of suspension plant top lated plants were then incubated in a growth chamber at 22 1° C with a 16 h lesions were harvested 6 days after photoperiod. The leaves bearing - 200-300 inoculation, frozen in liquid nitrogen and stored at -80'C. Extraction and purification of (3-glucanases
of leaves was ground in 150 ml of 0.5 M sodium acetate, pH 5.2, containing 15 mM 2-mercaptoethanol. After centrifugation at 14 000 g, the super-
About 100
g
3211
S.Kauffmann et al. natant was desalted on a Sephadex G-25 column (4.5 x 60 cm) equilibrated with 20 mM sodium acetate, pH 5.2. The protein fraction was kept at 4°C overnight, centrifuged at 20 000 g and the precipitate was discarded. The supematant was loaded on a CM-cellulose column (2.2 x 16 cm) equilibrated with 20 mM sodium acetate, pH 5.2. Elution was performed with 1I1 of a linear gradient from 0 to 0.5 M NaCI. Active fractions were pooled, concentrated and adsorbed on a column (1 x 40 cm) of PBE1 18 (Pharmacia) equilibrated with 25 mM triethylamine-HCI buffer, pH 11.4. Elution was carried out with a pH gradient generated with a Pharmalyte 8-10.5 solution (Pharmacia) adjusted to pH 8.0 with HCl. The protein fraction which passed through the CM-cellulose column was dialysed successively against water and 20 mM Tris-HCI, pH 7.8 (2 x 5 1 of each) and loaded on a Q-Sepharose (Pharmacia, Sweden) column (3.2 x 10 cm). After washing with 20 mM Tris-HC1 buffer, pH 7.8, elution was carried out with 1 1 of a linear gradient from 0 to 400 mM NaCl in the same buffer. Most active fractions were concentrated on Centricon 10 concentrators (Amicon, MA) and analysed by electrophoresis. Extraction and purification of PR proteins Proteins PR-O, -N and -2 were extracted and purified from 400 g of TMV-infected tobacco leaves by using a procedure previously described (Jamet and Fritig, 1986) with the following modifications. The extract was desalted on a Sephadex G-25 column (10 x 49 cm) equilibrated with 20 mM Tris-HC1 buffer, pH 8.0. Then the DEAE-cellulose chromatography was carried out on a 4.8 x 16 cm column. Elution with a 2 1 linear gradient from 0 to 0.25 M NaCI yielded proteins PR-O, -N and -2 separated from each other. PR-O was further purified by chromatofocusing on a 1.6 x 40 cm column of PBE94 (Pharmacia) equilibrated with Bis Tris-HCI buffer, pH 7.4. Elution was performed by a pH gradient generated with 25 mM Polybuffer 74 (Pharmacia) adjusted to pH 4.0 with HCI. Fractions containing protein PR-O were pooled, adjusted to 1.2 M (NH4)2SO4 with solid salt and the solution was filtered through 0.22-1zm filters. After injection onto a TSK-Phenyl-5 PW column (LKB, Sweden), elution was carried out by a decreasing gradient of salt concentration obtained by diluting a 1.2 M (NH4)2SO4 solution with water under the control of the programmer of a f.p.l.c. system (Pharmacia, Sweden). Fractions containing protein PR-O were pooled, concentrated to 200 p1 on Centricon 10 concentrators (Amicon, USA), filtered and injected onto a TSK G-2000 SW column (0.75 x 60 cm, Beckman, USA). Elution with 0.1 M sodium phosphate buffer, pH 6.9, containing 0.2 M NaCl yielded purified protein PR-O. The purification procedure used for protein PR-N was the same as that used for protein PR-O except that the final exclusion chromatography was omitted. Protein PR-2 was further purified after the DEAE-cellulose chromatography by successive hydrophobic and exclusion chromatographies under the highperformance conditions described above for protein PR-O. Polyacrylamide gel electrophoresis and staining Electrophoresis was performed on polyacrylamide slab gels according to the method of Laemmli (1970). The same system was used for native gels, except that SDS was omitted. Gel fixation and staining have been described previously (Legrand et al., 1987). Immunoblotting The basic procedure of Towbin et al. (1979) was used with modifications previously described (Legrand et al., 1987). 1,3-(3-glucanase assay 1,3-fl-glucanase activity was assayed by measuring the rate of reducing sugar production with laminarin (Sigma, USA) as the substrate. The assay mixture consisted of 0.5 ml of 0.1 M acetate buffer, pH 5.2, containing 1 mg/mi laminarin and of various volumes of enzymatic solution. After a 10-30-min incubation at 37°C, 0.5 ml of the alkaine copper reagent was added (Ashwell, 1957) and the mixture was heated at 100°C for 15 min. After cooling, 0.5 ml of Nelson's chromogenic reagent was added and the absorbance measured at 660 nm (Ashwell, 1957). Standards of glucose and enzyme or substrate blanks were included. A Katal (Kat) was defined as the enzyme activity catalysing the formation of 1 mol glucose equivalents/s. Exo-glucanase activity was measured by the glucose oxidase method (Li6nart et al., 1986).
Acknowledgements We thank Dr K.Richards for reading the manuscript. This work was supported by a grant from the Ministere de la Recherche et de l'Enseignement Supenor
(No. 86.C.0952).
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