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The pathogenesis-related proteins of tobacco: their labelling from [14C] amino acids in leaves reacting hypersensitively to infection by tobacco mosaic virus
Physiological
Plant Pathologll
(1985)
27,29941
The pathogenesis-related proteins of tobacco: their labelling from [‘“Cl amino acids in leaves reacting hypersensitively to infection by tobacco mosaic virus E. JAMET, Laboratoire France
M. KOPP and B. FRITrGt de Virologie,
!..lcceptedfbrpublication
Institut
February
de Biologic
Molkulaire
et Cellulaire
du C.N.R.S.,
15 rue Descartes,
67000 Strasbourg,
1985)
Extracts from leaves of Ncotiana tabacum cv. Samsun NN which have developed a hypersensitive response to infection by tobacco mosaic virus (TMV), contain at least 10 pathogenesis-related (PR) proteins which are absent from or present in very small amounts in uninfected leaves. When [‘“Cl amino acids were injected into leaves which were still attached to the plants and which had been inoculated with TMV 3 days earlier, a significant radioactivity became associated with all PR-proteins that were resolvable from other host proteins on non-denaturing gels. The incorporation of labelled amino acids into the individual polypeptides was investigated by a procedure involving two successive electrophoretic migrations, first under non-denaturing and then under denaturing conditions. This procedure, when applied to those PR-proteins whose composition is known, PR-la, PR-lb, PR-lc, PR-2 and PR-N showed that they all accumulated significant radiolabel within 3 h of feeding the leaves with the [‘*Cl amino acids. Significa,nt radioactivity was also associated with PR-proteins in inoculated leaves within a few hours of feeding the [‘%I amino acids to detached leaves through the petiole, but this method was much less efficient than the injection procedure. Specific radioactivities of the PR-proteins were compared with those of other host proteins and changes were followed during further incubation with unlabelled amino acids in order to investigate the possibility that the PR-proteins are stable end-products from proteolytic cleavage of constitutive proteins. The results indicate that de ~DZJDsynthesis rather than proteolytic cleavage is responsible for the production and accumulation of PR-proteins in hypersensitively reacting leaves of tobacco.
INTRODUCTION The induction and accumulation of pathogenesis-related proteins (PR-proteins) in tobacco mosaic virus (TMV) infected tobacco was originally demonstrated by Van Loon & Van Kammen [30], and subsequently confirmed by Gianinazzi et al. [13]. Such proteins have since been shown to accumulate in other plant species infected by viruses, viroids, bacteria and fungi (for reviews see [12,27-291). These proteins are characteristic of the host and are of host origin. They are commonly produced in association with hypersensitive necrosis and in the case of viruses are found in both inoculated and neighbouring uninoculated, virus-free leaves [1.5,22,24,25,30]. A role for these proteins tTo whom all correspondence Abbreviations used in text: sodium dodecyl sulphate; TMV, 0048&4059/85/010029+
should be addressed. PAGE, polyacrylamide tobacco mosaic virus.
13 $03.00/O
gel electrophoresis; 0 1985 Academic
PR, pathogenesis-related; Press Inc. (London)
SDS, Limited
E. Jamet, M. Kopp and 6. Fritig
30
in the limitation of virus multiplication or spread and in acquired systemic resistance has been suggested but not proven [I, 2, 10, 11, 1.5, 20, 26, 271. However, since the accumulation of PR-proteins represents a major activity of plant tissues responding hypersensitively to virus infection, their production should provide useful biochemical markers for studies on the regulation of gene expression in plants. So far most studies have involved the four proteins produced during the hypersensitive reactions of the two tobacco cultivars Samsun NN and Xanthi-nc. Three of these proteins, termed PR-la, PR-I b and PR-Ic, appear to be charged isomers with molecular weights near 15 000 [3]. The fourth, termed PR-2, is about twice as big. All four proteins exhibit rather high electrophoretic mobilities under non-denaturing conditions but are clearly resolved by electrophoresis of a total soluble protein fraction obtained at neutral pH from hypersensitively reacting tobacco leaves. However, the PR-proteins are more or less selectively extracted at low pH since they remain soluble at low pH whereas the majority of the leaf proteins do not. From extracts obtained at low pH, at least six additional PR-proteins, provisionally designated N, 0, P, Q R and S (in order of decreasing electrophoretic mobility) can be resolved by electrophoresis under non-denaturing conditions [271. Very little is known about the origin of PR-proteins. All ten from tobacco are resistant to trypsin and chymotrypsin as well as to endogenous tobacco proteases. Since protease activity strongly increases in hypersensitively reacting tobacco, it has been suggested that the PR-proteins could simply be the stable end-products of proteolysis. This hypothesis is supported by the fact that PR-proteins do not appear to incorporate label when radioactive amino acids are fed to the leaves of hypersensitively reacting plants [21] although they accumulate in these leaves to up to 5-10% of the total soluble protein fraction. We earlier reported on the regulatory mechanisms controlling the increases in the activities of several enzymes which occur during the hypersensitive response [S, 7, 91. These investigations involved the application of density labelling rather than radiolabelling since feeding detached leaves with [3H] leucine through the petioles led to an abnormal distribution of radioactivity between veins and mesophyll in leaves carrying local lesions [9]. The present paper describes experiments involving feeding radioactive amino acids to leaves which were still attached to the plant by injecting them into the interveinal areas. This method gave significant radiolabelling of all PR-proteins in inoculated Samsun NN leaves within a few hours of incubation.
MATERIALS
AND METHODS
Plant material and virus inoculation Three-month-old tobacco plants (Nicotiana tabacum cv Samsun NN) with six or seven leaves, grown in a greenhouse, were used in all experiments. The temperature was maintained below 20 “C at night and, depending on the season, between 20 and 25 “C during the day. The two first fully expanded leaves at the top of each plant were inoculated by rubbing them with an aqueous suspension of highly purified TMV (Ul strain) in the presence of Celite. The inoculated plants were incubated in a growth chamber at 22 + 1 “C (16 h photoperiod), and under these conditions, local lesions appeared about 33 + 2 h after inoculation.
Radiolabelling
of tobacco
PR-proteins
31
Radioactive labelling The following radioactive amino acids were used: L-[(U)-~~C] leucine, 335 Ci mole-’ (New England Nuclear); L-[(U)-14C] phenylalanine, 504 Ci mole-‘; and ~-[(U)-r~c] aspartic acid, 224 Ci mole- ’ (Amersham International plc). They were supplied to the leaves in phosphate buffer (pH 7.0) without dilution of specific radioactivity. Two methods of feeding the 14C-amino acids were used. In the first, the leaves were detached from the plant at different times after inoculation and the cut ends of the petioles were placed in vials containing the radioactive solution. The radioactive solution (50 pCi in 400 ~1 per leaf) was absorbed within 2 h and after absorption the petioles were placed in buffer until the end of the incubation period. In the second method leaves were injected while still attached to the plant. A syringe fitted with a very fine needle was used to inject radioactive solution (50-100 l.tCi in 3 ml per leaf) into the interveinal areas of the leaf. Injection was continued slowly until the leaf tissues between the secondary veins became uniformly dark green. About 20-30 min was usually necessary to achieve compIete injection of a leaf. After the required treatment, the leaves were frozen in liquid nitrogen and stored at -20 “C. Extraction of leaf proteins Leaves were ground in a mortar with a pestle at 4 “C, in two volumes of buffer (pH2.8) containing 84 mM citric acid, 32 mM Na,HPO,, 14 mM 2-mercaptoethanol and 6 mM ascorbic acid. Occasionally a sample of the frozen leaves was extracted in the presence of buffer (pH 8.0) containing 100 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 14 mM sodium diethyl dithiocarbamate and 14 mM 2-mercaptoethanol. The extracts were centrifuged at 15 OOOg for 15 min, and the supernatants chromatographed on a column (22.0 x 2.5 cm) of Sephadex G-25 equilibrated with 50 mM Tris, pH 8.0, containing 1 mM EDTA and 3 mM 2-mercaptoethanol. Fractions of the eluate which contained proteins, as judged by optical density at 280 nm, were pooled, freeze-dried and then dissolved in water. Their protein content was estimated by the method described by Bradford [4] and radioactivity was measured in 20 ~1 samples in a scintillation counter after the addition of 10 ml Beckman Ready-Solv MP scintillation cocktail. The ratio of radioactivity to protein content was calculated to obtain the mean specific radioactivity of soluble proteins in the leaf extract. Polyacrylamide gel electrophoresis and staining Electrophoresis under denaturing conditions on slab gels was performed using the discontinous system and buffers described by Laemmli [17]. Stacking and resolving gels contained 5 and 14Ti0 (w/v) acrylamide, respectively. The same method was used for non-denaturing conditions, except that sodium dodecyl sulphate (SDS) was omitted and an ll:‘,, (w/v) acrylamide gel was used as the resolving gel. Gels were usually loaded with 10 pl of extract containing 30 pg of protein. blue Serva Gels were stained for 20 min with 0.15% (w/v) C oomassie brilliant G250 in a mixture of methanol, acetic acid and water (45 : 10 : 45) and then destained overnight in a mixture of methanol, acetic acid and water (5.0 : 7.5 : 87.5). Some gels, after staining with Coomassie blue were then stained with silver according to the method of Morrissey [19] except that the fixation stage in glutaraldehyde was omitted.
32
E. Jamet, M. Kopp and B. Fritig
Detection and measurement of radioactivity after slab gel electrophoresis The slab gels were dried under vacuum on Whatman 3 mm chromatography paper. Autoradiographs were then produced by exposing the dried gels to Fuji X-ray film for 3 weeks or each lane was analysed for radioactivity with a linear Berthold thin layer chromatogram radioactivity analyser equipped with a gas flow detector possessing 4 channels per millimetre. The apparent specific radioactivity of the proteins was calculated from measurements obtained with a Shimadzu CS-930 densitometer. The stained and dried gels were scanned first using the reflection mode at 600 nm and then the corresponding autoradiograms were scanned using the transmission mode at 600 nm. The ratio between the areas of the corresponding peaks of the autoradiographic and staining densitometric scans was taken as measurement of the apparent specific radioactivity. Using the linear Berthold thin layer chromatogram radioactivity analyser we found a linear relationship between the radioactivity of the protein bands on the gels and the areas of their peaks obtained from the autoradiographic densitometer scans, provided the optical density was below 2.0 units. The same condition was required to obtain a linear relationship between the amount of a given protein loaded on the slab gel and the area of the peak of densitometer scan after staining with Coomassie blue. For comparisons between a given protein present in different lanes, the standard deviation of the apparent specific radioactivity measurements was estimated to be 20%. RESULTS Radiolabelling of the leaf proteins extractable at low pH with [14C] amino acids Detached or attached leaves of Samsun NN plants were supplied with a mixture of [‘“Cl leucine and [‘“Cl phenylalanine 3 days after inoculation with TMV or mockinoculation with water. The feeding was performed either by absorption through the petioles or by the injection procedure. After 5 h of incubation with the radiochemicals the leaves were extracted under acidic conditions; occasionally a sample, after freezing, was also extracted at pH 8.0. The overall specific radioactivity of the proteins extracted by both procedures (Table 1) was lower in infected leaf extracts than in healthy leaf extracts when the label was supplied through the cut petiole (probably as a consequence of the disturbance in the distribution of the label between the veins and the mesophyll [9]). In contrast, the overall specific radioactivity of the proteins was higher in infected leaf extracts, especially in the pH 2.8 extracts, when the label was supplied by injecting attached leaves. Furthermore, the mean specific radioactivity of soluble proteins in the acidic extract from injected attached leaves was about twice that of the proteins in the corresponding pH 8.0 extract, although the amount of protein extractable at pH 8.0 was 10 times greater than that extractable at pH 2.8. The proteins extracted at low pH were separated by electrophoresis under nondenaturing conditions to determine if the PR-proteins were labelled. The results are illustrated in Fig. 1. Extracts from healthy leaves contained a slowly migrating protein which appeared to be the major one from staining reactions and autoradiography. This protein, as yet unidentified, was also present in similar amounts in extracts from hypersensitively reacting leaves but appeared to contain less label than that in the healthy amino acids were fed to detached leaves through material, especially when the [‘“Cl
Radiolabelling
of tobacco
PR-proteins
33 1
TABLE
.Mean specific radioactivity
ofproteins
(cl min- ’ ng-
‘) afterlabel&g
Experiment A Mode of labelling Absorption through petioles Injection into attached leaves
Plant material Healthy Infected Healthy Infected
pH 2.8 2.74 1.11 1.41 2.25
and conditions
mith i “C]
ofextraction
B pH 2.8
0.54 0.82
amino acids
C pH 8.0
pFi 2.8
pH 8.0
0.24 0.38
4;47 2.15 2.05 2.32
2.05 1.14 1.01 1.16
Leaves of tobacco, Samson NN, were inoculated with tobacco mosaic virus or mock-inoculated with water (healthy) and supplied 3 days later with 4 : 1 (experiments A and CI or 1 : 9 (experiments B) mixtures of [‘%I leucine : [‘%I phenylalanine. The amounts of label supplied were 30 pCi g-’ fresh weight in all experiments. After 5 h incubation the proteins were extracted at the pH indicated, chromatographed on Sephadex G-25 and their mean specific radioactivity measured as indicated in the Materials and Methods Section.
the petiole [lane I,, (A) in Fig. 11. The major differences in staining reactions between the gel separations of healthy and infected leaf proteins was due to the PR-proteins of the infected extracts. All 10 PR-proteins became significantly labelled when the radioactive precursors were supplied by injection into attached leaves [lane Ii (A)] but became slightly labelled only when the precursors were fed to detached leaves through the petioles [lane I,, (A)]. Thus, all further radiolabelling experiments were carried out by injecting the labelled amino acids into attached leaves. A more detailed examination of the electrophoretic patterns obtained from extracts of healthy and infected leaves using this procedure (the four lanes left most in Fig. 1) indicated that most of the PR-proteins (PR-la, PR-1 b, PR-lc, PR-2, PR-P, PR-R and PR-S) were labelled. The results were less clear for PR-N, PR-0 and PR-Qwhich migrated very close to labelled proteins which were also present in healthy leaves. The same extracts were electrophoresed under denaturing conditions. Again, all additional bands in extracts from infected leaves that were revealed by staining carried a significant [14C] label revealed by autoradiography [see lanes I(S) and I(A) of Fig. 21. Only two of these protein bands found under denaturing conditions were clearly identified. One band of mol. wt 16 000 corresponded to the subunits of PR-la, PR-1 b and PR-lc which are charge isomers [3]. It has been suggested [29] that PR-2 and PR-N are also charge isomers, and we have confirmed this by high performance liquid chromatography followed by SDS-PAGE W amet et al., unpublished results]. The positions of the subunits of PR-2 and PR-N were heavily labelled [lane I(A) of Fig. 21 and preliminary results obtained using high pressure liquid chromatography suggest that the two labelled bands of mol. wt about 25 000 could be PR-poteins P and Q The labelling at the positions of the PR-protein bands might be due to contaminating proteins of very high specific radioactivity migrating at the same positions and
E. Jamet, M. Kopp and B. Fritig
. lb
4
“‘
-A s
A
s
A
S
A
S
A
u--u
FIG. 1, Incorporation of lab&d amino acids into pathogenesis-related proteins of Tobacco lSamsun NN) leaves. Three days after inoculation with tobacco mosaic virus or mock-inoculation with water (healthy! each leaf was supplied with a mixture of40 pCi [“%I leucine and of 10 pCi [“Cl phenylalanine either by injection while still attached to the plant or through the cut end of the petiole after detachment. The leaves were then incubated for 5 h with the [‘%I amino acids before their proteins were extracted at low pH. The extracted proteins were fractionated by electrophoresis under non-denaturing conditions on 1 l’& acrylamide slab gels and the radioactive proteins detected by autoradiography using the methods described in the Materials and Methods Section. H = healthy, I = infected, i = label supplied by injection of attached leaves, ap = label supplied by absorption through the cut petioles, S =electrophoretogram stained with Coomassie blue, A = autoradiograph.
so the following experiment was carried out to examine this possibility. The proteins in acidic extracts from infected leaves which had been fed a mixture of [14C] leucine and [ 14C] aspartic acid were separated in acrylamide gels under non-denaturing conditions. The gels were then sliced and the proteins extracted with 5 mM Tris-HCl buffer at pH 6.8. The fractions were concentrated and divided into two parts, one part being electrophoresed under non-denaturing conditions in order to select those fractions containing single PR-proteins, and the other part of these selected fractions was treated with SDS and subjected to SDS-PAGE. These two successive electrophoretic separations were applied to the PR-proteins PR-la, PR-1 b, PR-lc, PR-2 and PR-N whose compositions were known. The results of silver staining and of the analysis of radioactivity in the SDS slab gels are given in Fig. 3. The gels were first stained with Coomassie brilliant blue which gave very faint spots in the positions of the different
Radiolabelling
of tobacco
PR-proteins
35
kD
94 67 43
2N 30
I a,b,c
S
A
i
A
S
i
FIG. 2. Electrophoretograms in 14% acrylamide slab gels under denaturing conditions of acidic extracts of uninfected and tobacco mosaic virus infected leaves supplied with [‘%I amino acids. The same procedures were followed as for Fig. 1 except that the [‘“Cl amino acids were supplied only by injection into attached leaves and that proteins were electrophoresed on 14”,, acrylamide slab gels under denaturating conditions as described in the Materials and Methods Section. H = Healthy, I = infected, S = electrophoretogram stained with Coomassie blue, h = autoradiograph. The positions and molecular weights (kD) of marker proteins are indicated in the left-hand side. The marker proteins were: phosphorylase b (94 000 mol. wt), bovine serum albumin (67 000 mol. wt), ovalbumin (43 000 mol. wt), carbonic anhydrase (30 000 mol. wt), soybean trypsin inhibitor (20 000 mol. wtj and a-lactalbumin (14 000 mol. wt).
PR-protein bands while the second staining procedure with silver nitrate enhanced the intensity of these bands. The intensity of the silver nitrate staining reaction with proteins is not directly proportional to protein concentration and so the method cannot be used to quantify protein levels. Further bands which were probably minor contaminants of the first electrophoretic migration, except in the case of PR-la, were revealed. The important result of this experiment was that radioactivity was associated with each of the PR-proteins examined, thus showing that within 3 h of feeding [14C] leucine and [‘“Cl aspartic acid significant radioactivity had been incorporated into the PR-proteins. Rate q/ radiolabelling as evidencefor de novo synthesis of PR-proteins Infected and uninfected leaves were injected with [‘“Cl amino acids and the apparent specific. radioactivity of the proteins after 5 h incubation was estimated. In Experiment
2 IC
lb
la
FIG. 3. Incorporation of radioactivity into the subunits of different pathogenesis-related (PR) proteins in hypersensitively reacting Tobacco (Samsun NN) leaves. Three days after inoculation with tobacco mosaic virus each leaf was injected with a mixture containing 30 pCi [‘%I leucine and 20 pCi [“‘Cl aspartic acid. After 3 h incubation, the leaves were harvested and frozen in liquid nitrogen. The proteins were then extracted at pH 2.8 and chromatographed on Sephadex G-25, as described in the Materials and Methods Section. Two millilitres of the protein containing fractions obtained, 750 pg protein, were loaded onto a preparative 12% acrylamide slab gel, 4 mm thick, and electrophoresed under non-denaturing conditions. The gel was sliced into bands 2 mm wide and the proteins extracted from each band in Tris buffer. The extracts were concentrated under vacuum and a sample of each fraction was electrophoresed again under non-denaturing conditions in order to select the fractions containing PR-la, PR-1 b, PR-lc, PR-2 and PR-N. A sample of each selected fraction was then treat&l with SDS and electrophoresed on 14”/, acrylamide slab gels containing SDS. The electrophoretograms were stained successively with Coomassie blge and silver to locate the PR-proteins. Scanning to locate the peaks ofactivity was carried out using a Berthold radioactivity analyser. ‘l‘he pbsitions and molecular weights ikD) ofmolecular weight marker proteins (see legend to Fig. 2 for details) are indicated.
N
kD
- 14
- 20
- 30
- 43
- 67
- 94
Radiolabelling
of tobacco
37
PR-proteins TABLE
2
Apparent speciJic radioactiui~ ofpathogenesis-related (PR) proteins labelled by injecting mixtures amino acids into infected leaves while still attached to theplane
of 1“%,y
Experiment
Experimental conditions Period with label (h) Incubation with non-labelled amino acids Results PR-proteins la lb 1C
2
A
B
C
D
5.0
3.0
3.5
3.5
0.0
0.0
16.0
40.0
3.3b 1.6 2.0 4.2
1.50 0.67 1.00 3.10
1.10 0.62 0.55 2.10
0.84 0.54 0.45 1.30
“Three days after inoculation with tobacco mosaic virus, leaves carrying local lesions were each injected with a solution containing 4OpCi [“Cl leucine and 1OpCi [“‘Cl phenylalanine (Experiment A) or with a solution containing 30 pCi [“Cl leucine and 20 pCi I’%] aspartic acid (Experiments B, C and D). Plants from the same batch were used in Experiments B, C and D and from a separate batch for Experiment A. After 3 h of incubation the leaves were harvested and immediately frozen in liquid nitrogen (Experiment B) or re-injected with a solution containing unlabelled leucine and aspartic acid at concentrations 50-fold higher than those supplied in the label (Experiments C and D). After a further 16 h (Experiment C) or 40 h (Experiment D) the leaves were harvested and frozen in liquid nitrogen. In all four experiments, the proteins were extracted and separated on gels as described in the Materials and Methods Section. The non-denaturing 11 Sb acrylamide slab gels were stained with Coomassie blue G250, dried and autoradiographed. The dried gels and the autoradiographic films were scanned with a Shimadzu densitometer. %pecific radioactivities are expressed in relative units calculated as indicated in Materials and Methods.
11 (Table 2) acidic extracts from healthy leaves were subjected to electrophoresis under the same conditions as extracts from hypersensitively reacting leaves and found to contain several labelled proteins which did not correspond to any of the PR-proteins. The specific radioactivities of these constitutive proteins were estimated and found to be in the range 0.7-3.8. The specific activities of the PR-proteins PR-la, PR-lb, PR-Ic and PR-2, which were widely separated, were in the range 1.64.2. Furthermore, the mean specific radioactivities of proteins extracted at pH 8.0 from these healthy and infected leaves, were 0.90 and 1.5, respectively. These results demonstrate that the PR-proteins are as efficiently and rapidly labelled as any of the proteins in the uninfected and infected plants. The possibility that PR-proteins are produced by proteolytic cleavage of constitutive proteins which are normally present in healthy leaves was investigated further with the experiments illustrated by experiments B, C and D in Table 2. The rationale of Experiment B was as follows. If large pools of constitutive proteins are precursors of the PR-proteins, then these pools should incorporate the radioactive amino acids very efficiently and very rapidly and exhibit a much higher specific radioactivity than the PR-proteins themselves; this is because the low amounts of radioactive PR-proteins
38
E. Jamet, M. Kopp and B. Fritig
produced during a 3 h incubation would be necessarily diluted by the unlabelled PRproteins which were present before injection of radioactive amino acids into the leaf. Tn Experiments C and D, there was an additional 0.5 h period of incubation in the presence of the radioactive amino acids (which corresponded to the time required to inject the unlabelled amino acids) followed by a 16 h (C) or a 40 h (D) incubation after injection with unlabelled amino acids. The PR-proteins produced in C during the further 16.5 h, and in D with the longer incubation period, if they were derived by proteolytic cleavage would be of nearly the same high specific radioactivity as the pool of precursor protein and thus of a significantly higher specific radioactivity than in B. The data of Table 2 indicate that this was not the case and that the specific radioactivities were even lower in C than in B and also lower in D than in C. This progressive decrease in specific radioactivity of the PR-proteins is exactly what would be expected if they were produced by de nouo synthesis from a pool of radioactive amino acids which is later progressively diluted by unlabeled amino acids.
DISCUSSION This is the first report of rapid and efficient incorporation of labelled amino acids into PR-proteins in tobacco leaves during the hypersensitive response to TMV infection. A recent publication [21] describes the results of attempts to label the PR-proteins of TMV-infected Xanthi-nc tobacco leaves by petiolar uptake of [3H] leucine or by exposure of attached leaves to [‘“Cl CO,. Th e experiments with [‘“Cl CO, indicated that radioactivity was probably incorporated into PR-la, PR-1 b, PR-lc and PR-2. Furthermore, detached leaves, both infected and uninfected, incorporated [3H] leucine into proteins. Surprisingly the radioactivity was not as clearly associated with specific protein peaks in acrylamide gels as that incorporated from [‘“Cl CO,. Some radioactivity was associated with PR-la, and probably with PR-R as well, but the incubation periods with the radiochemicals were rather long; 3 days after exposure to [‘“Cl CO, and 4-6 days after petiolar uptake of [3H] leucine. It is surprising that exposure to [‘“Cl CO, labelled the PR-proteins more efficiently than leucine. The poor labelling achieved with [3H] leucine contrasts with our results obtained by feeding [14C] amino acids in the same way. We found radioactivity associated with all the PR-proteins which could be clearly resolved from other host proteins. Although the radioactivity associated with the PR-proteins was always lower from petiolar uptake compared to that incorporated after injection into attached leaves, it was still significant. Differences in labelling efficiency obtained by different workers using the same procedure are probably due to differences in the specific radioactivities of the precursors used and/or the duration of the incubation period. Concerning the latter factor, the data of Table 2 (experiments B, C and D) show that a progressive dilution of label occurs with increasing length of the incubation period and a similar dilution probably occurred during the 446 days incubation without continuous feeding used by Pierpoint in his experiments [21]. However, the main reason for the poor incorporation of [3H] leucine was probably due to its very high specific radioactivity and to abnormal patterns of incorporation which are known to result from petiolar uptake of labelled precursors by detached leaves. It has already been mentioned that tissues near the cut end of the petiole may
Radiolabelling
of tobacco
PR-proteins
39
act as a sink for labelled metabolites [.?3], so that only a small proportion of that which is fed actually reaches the mesophyll tissues. This distortion of distribution appears to be enhanced by the presence of enlarging local lesions on the leaves [S, 9,14,18]. Even though petiolar uptake, in our conditions, led to significant incorporation of radioactivity into PR-proteins, injection into leaves was the preferred method in our experiments. An obvious advantage of this procedure is that it can be applied to leaves still attached to the plant. But the major advantage is that it gave high efficiency of incorporation due to the more uniform distribution of labelled amino acids throughout the mesophyll tissue. This injection procedure had been shown to be very efficient in previous studies and enabled us to detect very low levels of virus replication around individual infection sites [16]. In the present work, radioactivity was clearly associated with PR-proteins which were separated on either denaturing or non-denaturing gels after a 3 h incubation with labelled amino acids. But an unambiguous demonstration of the incorporation of radioactive amino acids into the PR-proteins was obtained from successive electrophoretic migrations under non-denaturing and denaturing conditions /Fig. 3). Figures 1 and 2 illustrate the electrophoretic migrations of total acidic leaf protein extracts. One cannot exclude the possibility that minor contaminating proteins or polypeptides of very high specific radioactivity were present in these crude extracts which co-migrated with the PR-proteins. Such contamination might not be detectable by staining the gels but could account for most of the label shown by autoradiography. Any radioactive contaminants of each PR-protein should not be the same under both non-denaturing and denaturing conditions of electrophoretic separation. The results of the successive electrophoretic migrations (Fig. 3) demonstrated clearly the incorporation of amino acids into PR-proteins whose composition is known and similar experiments will be performed with PR-0, PR-P, PR-Q PR-R and PR-S once their composition is known. The use of short incubation periods, which are possible with the injection procedure, enabled estimates to be made of incorporation rates of radiolabel into the PRproteins so that comparisons could be made with incorporation rates into other host proteins. The aim of these experiments was to investigate the possibility that PR-proteins are produced solely by proteolytic cleavage of constitutive proteins. Production by proteolytic cleavage cannot be distinguished from de novo synthesis by the use of long incubation periods following exposure to [14C] CO,. In contrast, the use of short incubation periods should enable isotopic dilution effects to be detectable, should they occur. The data presented here exclude the possibility of large pools of precursor proteins being degraded by proteolysis to the PR-proteins as stable end-products. Whilst proteolysis cannot be totally excluded, at the most it can only involve shortlived precursor proteins that were being actively synthesized de novo in hypersensitively reacting leaves. In such a case, proteolytic cleavage would not involve the removal of a large fragment, at least in the case of PR- 1 a, since this protein was found to be labelled during in vitro translation of messenger RNA extracted from both healthy and TMVinfected Xanthi-nc leaves [g. We are indebted to Mrs Pierrette Geoffroy for skilful technical assistance. We also thank Professor L. Hirth, Head of the Institute, for providing facilities. This work was de 1’Industrie et de la Recherche” supported by grant 82Lllll from the “Ministere
40
E. Jamet, M. Kopp and B. Fritig
and by grant
ATP 5482 (Biologie Molkculaire Vkgktale) from the “Centre National de la Recherche Scientifique”.
REFERENCES P. & GIANINAZZI, S. (1982). B-protein as constitutive component in highly (TMV) resistant interspecific hybrids ofkotiana glutinosa x Nicotiana debneyi. Plant Science Letters 26, 173-181. 2. ANTONIW, J. F. & WHITE. R. F. (1980). The effect of aspirin and polyacrylic acid on soluble leaf proteins and resistance to virus infection in five cultivars of tobacco. Phytopathologirche xeitschrift 98,33 1-341. 3. ANTOHIW, J. F., RITTER, C. E., PIERPOINT, W. S. & VAN LOON, L. C. (1980). Comparison of three pathogenesis-related proteins from plants of two cultivars of tobacco infected with TMV. Journal of 1. AHL,
General
Virology 47, 79-87.
4. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry 72,248-254. 5. CARR, J. P., ANTONIW, J. F., WHITE, R. F. & WILSON, T. M. A. (1982). Latent messenger RNA in tobacco (Nicotiana tabacum~~. Biochemical Socieg Transactions 10,353S354. 6. COLLENDAVELLOO, J,, LEGRAND, M. & FRITIG, B. (1982). Plant disease and regulation of enzymes involved in lignification. De nouo synthesis controls o-methyltransferase activity in hypersensitive tobacco leaves infected by tobacco mosaic virus. Physiological Plant Pathology 21,27 l-28 1. 7. COLLENDAVELLOO, J., LEGRAND, M. & FRITIG, B. (1983). Plant disease and the regulation of enzymes involved in lignification. Increased rate of de nova synthesis of the three tobacco o-methyltransferases during the hypersensitive response to infection by tobacco mosaic virus. Plant Physiology 73,550-554. 8. DE LAAT, A. M. M. & VAN LOON, L. C. (1981). Complications in interpreting precursor/product relationships by labeling experiments. Methionine as the precursor of ethylene in tobacco leaves.
Radiolabelling 23. 24.
25.
26. 27.
28. 29. 30.
PRUT,
of tobacco
M. J. & MATTHEWS,
41
PR-proteins R. E. F. (1971).
Non-uniformities
in the metabolism
of excised
leaves
and
lrafdiscs. Plan~a 99,21-36. ROHLOFF, H. & LERCH, B. (1977). Soluble leafproteins in virus infected plants and acquired resistance 1. Investigations on Ncotiana tabacum CVS. “Xanthi-nc’ and ‘Samsun’. Phytopathologische
Plant Pathologll
(1985)
27,29941
The pathogenesis-related proteins of tobacco: their labelling from [‘“Cl amino acids in leaves reacting hypersensitively to infection by tobacco mosaic virus E. JAMET, Laboratoire France
M. KOPP and B. FRITrGt de Virologie,
!..lcceptedfbrpublication
Institut
February
de Biologic
Molkulaire
et Cellulaire
du C.N.R.S.,
15 rue Descartes,
67000 Strasbourg,
1985)
Extracts from leaves of Ncotiana tabacum cv. Samsun NN which have developed a hypersensitive response to infection by tobacco mosaic virus (TMV), contain at least 10 pathogenesis-related (PR) proteins which are absent from or present in very small amounts in uninfected leaves. When [‘“Cl amino acids were injected into leaves which were still attached to the plants and which had been inoculated with TMV 3 days earlier, a significant radioactivity became associated with all PR-proteins that were resolvable from other host proteins on non-denaturing gels. The incorporation of labelled amino acids into the individual polypeptides was investigated by a procedure involving two successive electrophoretic migrations, first under non-denaturing and then under denaturing conditions. This procedure, when applied to those PR-proteins whose composition is known, PR-la, PR-lb, PR-lc, PR-2 and PR-N showed that they all accumulated significant radiolabel within 3 h of feeding the leaves with the [‘*Cl amino acids. Significa,nt radioactivity was also associated with PR-proteins in inoculated leaves within a few hours of feeding the [‘%I amino acids to detached leaves through the petiole, but this method was much less efficient than the injection procedure. Specific radioactivities of the PR-proteins were compared with those of other host proteins and changes were followed during further incubation with unlabelled amino acids in order to investigate the possibility that the PR-proteins are stable end-products from proteolytic cleavage of constitutive proteins. The results indicate that de ~DZJDsynthesis rather than proteolytic cleavage is responsible for the production and accumulation of PR-proteins in hypersensitively reacting leaves of tobacco.
INTRODUCTION The induction and accumulation of pathogenesis-related proteins (PR-proteins) in tobacco mosaic virus (TMV) infected tobacco was originally demonstrated by Van Loon & Van Kammen [30], and subsequently confirmed by Gianinazzi et al. [13]. Such proteins have since been shown to accumulate in other plant species infected by viruses, viroids, bacteria and fungi (for reviews see [12,27-291). These proteins are characteristic of the host and are of host origin. They are commonly produced in association with hypersensitive necrosis and in the case of viruses are found in both inoculated and neighbouring uninoculated, virus-free leaves [1.5,22,24,25,30]. A role for these proteins tTo whom all correspondence Abbreviations used in text: sodium dodecyl sulphate; TMV, 0048&4059/85/010029+
should be addressed. PAGE, polyacrylamide tobacco mosaic virus.
13 $03.00/O
gel electrophoresis; 0 1985 Academic
PR, pathogenesis-related; Press Inc. (London)
SDS, Limited
E. Jamet, M. Kopp and 6. Fritig
30
in the limitation of virus multiplication or spread and in acquired systemic resistance has been suggested but not proven [I, 2, 10, 11, 1.5, 20, 26, 271. However, since the accumulation of PR-proteins represents a major activity of plant tissues responding hypersensitively to virus infection, their production should provide useful biochemical markers for studies on the regulation of gene expression in plants. So far most studies have involved the four proteins produced during the hypersensitive reactions of the two tobacco cultivars Samsun NN and Xanthi-nc. Three of these proteins, termed PR-la, PR-I b and PR-Ic, appear to be charged isomers with molecular weights near 15 000 [3]. The fourth, termed PR-2, is about twice as big. All four proteins exhibit rather high electrophoretic mobilities under non-denaturing conditions but are clearly resolved by electrophoresis of a total soluble protein fraction obtained at neutral pH from hypersensitively reacting tobacco leaves. However, the PR-proteins are more or less selectively extracted at low pH since they remain soluble at low pH whereas the majority of the leaf proteins do not. From extracts obtained at low pH, at least six additional PR-proteins, provisionally designated N, 0, P, Q R and S (in order of decreasing electrophoretic mobility) can be resolved by electrophoresis under non-denaturing conditions [271. Very little is known about the origin of PR-proteins. All ten from tobacco are resistant to trypsin and chymotrypsin as well as to endogenous tobacco proteases. Since protease activity strongly increases in hypersensitively reacting tobacco, it has been suggested that the PR-proteins could simply be the stable end-products of proteolysis. This hypothesis is supported by the fact that PR-proteins do not appear to incorporate label when radioactive amino acids are fed to the leaves of hypersensitively reacting plants [21] although they accumulate in these leaves to up to 5-10% of the total soluble protein fraction. We earlier reported on the regulatory mechanisms controlling the increases in the activities of several enzymes which occur during the hypersensitive response [S, 7, 91. These investigations involved the application of density labelling rather than radiolabelling since feeding detached leaves with [3H] leucine through the petioles led to an abnormal distribution of radioactivity between veins and mesophyll in leaves carrying local lesions [9]. The present paper describes experiments involving feeding radioactive amino acids to leaves which were still attached to the plant by injecting them into the interveinal areas. This method gave significant radiolabelling of all PR-proteins in inoculated Samsun NN leaves within a few hours of incubation.
MATERIALS
AND METHODS
Plant material and virus inoculation Three-month-old tobacco plants (Nicotiana tabacum cv Samsun NN) with six or seven leaves, grown in a greenhouse, were used in all experiments. The temperature was maintained below 20 “C at night and, depending on the season, between 20 and 25 “C during the day. The two first fully expanded leaves at the top of each plant were inoculated by rubbing them with an aqueous suspension of highly purified TMV (Ul strain) in the presence of Celite. The inoculated plants were incubated in a growth chamber at 22 + 1 “C (16 h photoperiod), and under these conditions, local lesions appeared about 33 + 2 h after inoculation.
Radiolabelling
of tobacco
PR-proteins
31
Radioactive labelling The following radioactive amino acids were used: L-[(U)-~~C] leucine, 335 Ci mole-’ (New England Nuclear); L-[(U)-14C] phenylalanine, 504 Ci mole-‘; and ~-[(U)-r~c] aspartic acid, 224 Ci mole- ’ (Amersham International plc). They were supplied to the leaves in phosphate buffer (pH 7.0) without dilution of specific radioactivity. Two methods of feeding the 14C-amino acids were used. In the first, the leaves were detached from the plant at different times after inoculation and the cut ends of the petioles were placed in vials containing the radioactive solution. The radioactive solution (50 pCi in 400 ~1 per leaf) was absorbed within 2 h and after absorption the petioles were placed in buffer until the end of the incubation period. In the second method leaves were injected while still attached to the plant. A syringe fitted with a very fine needle was used to inject radioactive solution (50-100 l.tCi in 3 ml per leaf) into the interveinal areas of the leaf. Injection was continued slowly until the leaf tissues between the secondary veins became uniformly dark green. About 20-30 min was usually necessary to achieve compIete injection of a leaf. After the required treatment, the leaves were frozen in liquid nitrogen and stored at -20 “C. Extraction of leaf proteins Leaves were ground in a mortar with a pestle at 4 “C, in two volumes of buffer (pH2.8) containing 84 mM citric acid, 32 mM Na,HPO,, 14 mM 2-mercaptoethanol and 6 mM ascorbic acid. Occasionally a sample of the frozen leaves was extracted in the presence of buffer (pH 8.0) containing 100 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 14 mM sodium diethyl dithiocarbamate and 14 mM 2-mercaptoethanol. The extracts were centrifuged at 15 OOOg for 15 min, and the supernatants chromatographed on a column (22.0 x 2.5 cm) of Sephadex G-25 equilibrated with 50 mM Tris, pH 8.0, containing 1 mM EDTA and 3 mM 2-mercaptoethanol. Fractions of the eluate which contained proteins, as judged by optical density at 280 nm, were pooled, freeze-dried and then dissolved in water. Their protein content was estimated by the method described by Bradford [4] and radioactivity was measured in 20 ~1 samples in a scintillation counter after the addition of 10 ml Beckman Ready-Solv MP scintillation cocktail. The ratio of radioactivity to protein content was calculated to obtain the mean specific radioactivity of soluble proteins in the leaf extract. Polyacrylamide gel electrophoresis and staining Electrophoresis under denaturing conditions on slab gels was performed using the discontinous system and buffers described by Laemmli [17]. Stacking and resolving gels contained 5 and 14Ti0 (w/v) acrylamide, respectively. The same method was used for non-denaturing conditions, except that sodium dodecyl sulphate (SDS) was omitted and an ll:‘,, (w/v) acrylamide gel was used as the resolving gel. Gels were usually loaded with 10 pl of extract containing 30 pg of protein. blue Serva Gels were stained for 20 min with 0.15% (w/v) C oomassie brilliant G250 in a mixture of methanol, acetic acid and water (45 : 10 : 45) and then destained overnight in a mixture of methanol, acetic acid and water (5.0 : 7.5 : 87.5). Some gels, after staining with Coomassie blue were then stained with silver according to the method of Morrissey [19] except that the fixation stage in glutaraldehyde was omitted.
32
E. Jamet, M. Kopp and B. Fritig
Detection and measurement of radioactivity after slab gel electrophoresis The slab gels were dried under vacuum on Whatman 3 mm chromatography paper. Autoradiographs were then produced by exposing the dried gels to Fuji X-ray film for 3 weeks or each lane was analysed for radioactivity with a linear Berthold thin layer chromatogram radioactivity analyser equipped with a gas flow detector possessing 4 channels per millimetre. The apparent specific radioactivity of the proteins was calculated from measurements obtained with a Shimadzu CS-930 densitometer. The stained and dried gels were scanned first using the reflection mode at 600 nm and then the corresponding autoradiograms were scanned using the transmission mode at 600 nm. The ratio between the areas of the corresponding peaks of the autoradiographic and staining densitometric scans was taken as measurement of the apparent specific radioactivity. Using the linear Berthold thin layer chromatogram radioactivity analyser we found a linear relationship between the radioactivity of the protein bands on the gels and the areas of their peaks obtained from the autoradiographic densitometer scans, provided the optical density was below 2.0 units. The same condition was required to obtain a linear relationship between the amount of a given protein loaded on the slab gel and the area of the peak of densitometer scan after staining with Coomassie blue. For comparisons between a given protein present in different lanes, the standard deviation of the apparent specific radioactivity measurements was estimated to be 20%. RESULTS Radiolabelling of the leaf proteins extractable at low pH with [14C] amino acids Detached or attached leaves of Samsun NN plants were supplied with a mixture of [‘“Cl leucine and [‘“Cl phenylalanine 3 days after inoculation with TMV or mockinoculation with water. The feeding was performed either by absorption through the petioles or by the injection procedure. After 5 h of incubation with the radiochemicals the leaves were extracted under acidic conditions; occasionally a sample, after freezing, was also extracted at pH 8.0. The overall specific radioactivity of the proteins extracted by both procedures (Table 1) was lower in infected leaf extracts than in healthy leaf extracts when the label was supplied through the cut petiole (probably as a consequence of the disturbance in the distribution of the label between the veins and the mesophyll [9]). In contrast, the overall specific radioactivity of the proteins was higher in infected leaf extracts, especially in the pH 2.8 extracts, when the label was supplied by injecting attached leaves. Furthermore, the mean specific radioactivity of soluble proteins in the acidic extract from injected attached leaves was about twice that of the proteins in the corresponding pH 8.0 extract, although the amount of protein extractable at pH 8.0 was 10 times greater than that extractable at pH 2.8. The proteins extracted at low pH were separated by electrophoresis under nondenaturing conditions to determine if the PR-proteins were labelled. The results are illustrated in Fig. 1. Extracts from healthy leaves contained a slowly migrating protein which appeared to be the major one from staining reactions and autoradiography. This protein, as yet unidentified, was also present in similar amounts in extracts from hypersensitively reacting leaves but appeared to contain less label than that in the healthy amino acids were fed to detached leaves through material, especially when the [‘“Cl
Radiolabelling
of tobacco
PR-proteins
33 1
TABLE
.Mean specific radioactivity
ofproteins
(cl min- ’ ng-
‘) afterlabel&g
Experiment A Mode of labelling Absorption through petioles Injection into attached leaves
Plant material Healthy Infected Healthy Infected
pH 2.8 2.74 1.11 1.41 2.25
and conditions
mith i “C]
ofextraction
B pH 2.8
0.54 0.82
amino acids
C pH 8.0
pFi 2.8
pH 8.0
0.24 0.38
4;47 2.15 2.05 2.32
2.05 1.14 1.01 1.16
Leaves of tobacco, Samson NN, were inoculated with tobacco mosaic virus or mock-inoculated with water (healthy) and supplied 3 days later with 4 : 1 (experiments A and CI or 1 : 9 (experiments B) mixtures of [‘%I leucine : [‘%I phenylalanine. The amounts of label supplied were 30 pCi g-’ fresh weight in all experiments. After 5 h incubation the proteins were extracted at the pH indicated, chromatographed on Sephadex G-25 and their mean specific radioactivity measured as indicated in the Materials and Methods Section.
the petiole [lane I,, (A) in Fig. 11. The major differences in staining reactions between the gel separations of healthy and infected leaf proteins was due to the PR-proteins of the infected extracts. All 10 PR-proteins became significantly labelled when the radioactive precursors were supplied by injection into attached leaves [lane Ii (A)] but became slightly labelled only when the precursors were fed to detached leaves through the petioles [lane I,, (A)]. Thus, all further radiolabelling experiments were carried out by injecting the labelled amino acids into attached leaves. A more detailed examination of the electrophoretic patterns obtained from extracts of healthy and infected leaves using this procedure (the four lanes left most in Fig. 1) indicated that most of the PR-proteins (PR-la, PR-1 b, PR-lc, PR-2, PR-P, PR-R and PR-S) were labelled. The results were less clear for PR-N, PR-0 and PR-Qwhich migrated very close to labelled proteins which were also present in healthy leaves. The same extracts were electrophoresed under denaturing conditions. Again, all additional bands in extracts from infected leaves that were revealed by staining carried a significant [14C] label revealed by autoradiography [see lanes I(S) and I(A) of Fig. 21. Only two of these protein bands found under denaturing conditions were clearly identified. One band of mol. wt 16 000 corresponded to the subunits of PR-la, PR-1 b and PR-lc which are charge isomers [3]. It has been suggested [29] that PR-2 and PR-N are also charge isomers, and we have confirmed this by high performance liquid chromatography followed by SDS-PAGE W amet et al., unpublished results]. The positions of the subunits of PR-2 and PR-N were heavily labelled [lane I(A) of Fig. 21 and preliminary results obtained using high pressure liquid chromatography suggest that the two labelled bands of mol. wt about 25 000 could be PR-poteins P and Q The labelling at the positions of the PR-protein bands might be due to contaminating proteins of very high specific radioactivity migrating at the same positions and
E. Jamet, M. Kopp and B. Fritig
. lb
4
“‘
-A s
A
s
A
S
A
S
A
u--u
FIG. 1, Incorporation of lab&d amino acids into pathogenesis-related proteins of Tobacco lSamsun NN) leaves. Three days after inoculation with tobacco mosaic virus or mock-inoculation with water (healthy! each leaf was supplied with a mixture of40 pCi [“%I leucine and of 10 pCi [“Cl phenylalanine either by injection while still attached to the plant or through the cut end of the petiole after detachment. The leaves were then incubated for 5 h with the [‘%I amino acids before their proteins were extracted at low pH. The extracted proteins were fractionated by electrophoresis under non-denaturing conditions on 1 l’& acrylamide slab gels and the radioactive proteins detected by autoradiography using the methods described in the Materials and Methods Section. H = healthy, I = infected, i = label supplied by injection of attached leaves, ap = label supplied by absorption through the cut petioles, S =electrophoretogram stained with Coomassie blue, A = autoradiograph.
so the following experiment was carried out to examine this possibility. The proteins in acidic extracts from infected leaves which had been fed a mixture of [14C] leucine and [ 14C] aspartic acid were separated in acrylamide gels under non-denaturing conditions. The gels were then sliced and the proteins extracted with 5 mM Tris-HCl buffer at pH 6.8. The fractions were concentrated and divided into two parts, one part being electrophoresed under non-denaturing conditions in order to select those fractions containing single PR-proteins, and the other part of these selected fractions was treated with SDS and subjected to SDS-PAGE. These two successive electrophoretic separations were applied to the PR-proteins PR-la, PR-1 b, PR-lc, PR-2 and PR-N whose compositions were known. The results of silver staining and of the analysis of radioactivity in the SDS slab gels are given in Fig. 3. The gels were first stained with Coomassie brilliant blue which gave very faint spots in the positions of the different
Radiolabelling
of tobacco
PR-proteins
35
kD
94 67 43
2N 30
I a,b,c
S
A
i
A
S
i
FIG. 2. Electrophoretograms in 14% acrylamide slab gels under denaturing conditions of acidic extracts of uninfected and tobacco mosaic virus infected leaves supplied with [‘%I amino acids. The same procedures were followed as for Fig. 1 except that the [‘“Cl amino acids were supplied only by injection into attached leaves and that proteins were electrophoresed on 14”,, acrylamide slab gels under denaturating conditions as described in the Materials and Methods Section. H = Healthy, I = infected, S = electrophoretogram stained with Coomassie blue, h = autoradiograph. The positions and molecular weights (kD) of marker proteins are indicated in the left-hand side. The marker proteins were: phosphorylase b (94 000 mol. wt), bovine serum albumin (67 000 mol. wt), ovalbumin (43 000 mol. wt), carbonic anhydrase (30 000 mol. wt), soybean trypsin inhibitor (20 000 mol. wtj and a-lactalbumin (14 000 mol. wt).
PR-protein bands while the second staining procedure with silver nitrate enhanced the intensity of these bands. The intensity of the silver nitrate staining reaction with proteins is not directly proportional to protein concentration and so the method cannot be used to quantify protein levels. Further bands which were probably minor contaminants of the first electrophoretic migration, except in the case of PR-la, were revealed. The important result of this experiment was that radioactivity was associated with each of the PR-proteins examined, thus showing that within 3 h of feeding [14C] leucine and [‘“Cl aspartic acid significant radioactivity had been incorporated into the PR-proteins. Rate q/ radiolabelling as evidencefor de novo synthesis of PR-proteins Infected and uninfected leaves were injected with [‘“Cl amino acids and the apparent specific. radioactivity of the proteins after 5 h incubation was estimated. In Experiment
2 IC
lb
la
FIG. 3. Incorporation of radioactivity into the subunits of different pathogenesis-related (PR) proteins in hypersensitively reacting Tobacco (Samsun NN) leaves. Three days after inoculation with tobacco mosaic virus each leaf was injected with a mixture containing 30 pCi [‘%I leucine and 20 pCi [“‘Cl aspartic acid. After 3 h incubation, the leaves were harvested and frozen in liquid nitrogen. The proteins were then extracted at pH 2.8 and chromatographed on Sephadex G-25, as described in the Materials and Methods Section. Two millilitres of the protein containing fractions obtained, 750 pg protein, were loaded onto a preparative 12% acrylamide slab gel, 4 mm thick, and electrophoresed under non-denaturing conditions. The gel was sliced into bands 2 mm wide and the proteins extracted from each band in Tris buffer. The extracts were concentrated under vacuum and a sample of each fraction was electrophoresed again under non-denaturing conditions in order to select the fractions containing PR-la, PR-1 b, PR-lc, PR-2 and PR-N. A sample of each selected fraction was then treat&l with SDS and electrophoresed on 14”/, acrylamide slab gels containing SDS. The electrophoretograms were stained successively with Coomassie blge and silver to locate the PR-proteins. Scanning to locate the peaks ofactivity was carried out using a Berthold radioactivity analyser. ‘l‘he pbsitions and molecular weights ikD) ofmolecular weight marker proteins (see legend to Fig. 2 for details) are indicated.
N
kD
- 14
- 20
- 30
- 43
- 67
- 94
Radiolabelling
of tobacco
37
PR-proteins TABLE
2
Apparent speciJic radioactiui~ ofpathogenesis-related (PR) proteins labelled by injecting mixtures amino acids into infected leaves while still attached to theplane
of 1“%,y
Experiment
Experimental conditions Period with label (h) Incubation with non-labelled amino acids Results PR-proteins la lb 1C
2
A
B
C
D
5.0
3.0
3.5
3.5
0.0
0.0
16.0
40.0
3.3b 1.6 2.0 4.2
1.50 0.67 1.00 3.10
1.10 0.62 0.55 2.10
0.84 0.54 0.45 1.30
“Three days after inoculation with tobacco mosaic virus, leaves carrying local lesions were each injected with a solution containing 4OpCi [“Cl leucine and 1OpCi [“‘Cl phenylalanine (Experiment A) or with a solution containing 30 pCi [“Cl leucine and 20 pCi I’%] aspartic acid (Experiments B, C and D). Plants from the same batch were used in Experiments B, C and D and from a separate batch for Experiment A. After 3 h of incubation the leaves were harvested and immediately frozen in liquid nitrogen (Experiment B) or re-injected with a solution containing unlabelled leucine and aspartic acid at concentrations 50-fold higher than those supplied in the label (Experiments C and D). After a further 16 h (Experiment C) or 40 h (Experiment D) the leaves were harvested and frozen in liquid nitrogen. In all four experiments, the proteins were extracted and separated on gels as described in the Materials and Methods Section. The non-denaturing 11 Sb acrylamide slab gels were stained with Coomassie blue G250, dried and autoradiographed. The dried gels and the autoradiographic films were scanned with a Shimadzu densitometer. %pecific radioactivities are expressed in relative units calculated as indicated in Materials and Methods.
11 (Table 2) acidic extracts from healthy leaves were subjected to electrophoresis under the same conditions as extracts from hypersensitively reacting leaves and found to contain several labelled proteins which did not correspond to any of the PR-proteins. The specific radioactivities of these constitutive proteins were estimated and found to be in the range 0.7-3.8. The specific activities of the PR-proteins PR-la, PR-lb, PR-Ic and PR-2, which were widely separated, were in the range 1.64.2. Furthermore, the mean specific radioactivities of proteins extracted at pH 8.0 from these healthy and infected leaves, were 0.90 and 1.5, respectively. These results demonstrate that the PR-proteins are as efficiently and rapidly labelled as any of the proteins in the uninfected and infected plants. The possibility that PR-proteins are produced by proteolytic cleavage of constitutive proteins which are normally present in healthy leaves was investigated further with the experiments illustrated by experiments B, C and D in Table 2. The rationale of Experiment B was as follows. If large pools of constitutive proteins are precursors of the PR-proteins, then these pools should incorporate the radioactive amino acids very efficiently and very rapidly and exhibit a much higher specific radioactivity than the PR-proteins themselves; this is because the low amounts of radioactive PR-proteins
38
E. Jamet, M. Kopp and B. Fritig
produced during a 3 h incubation would be necessarily diluted by the unlabelled PRproteins which were present before injection of radioactive amino acids into the leaf. Tn Experiments C and D, there was an additional 0.5 h period of incubation in the presence of the radioactive amino acids (which corresponded to the time required to inject the unlabelled amino acids) followed by a 16 h (C) or a 40 h (D) incubation after injection with unlabelled amino acids. The PR-proteins produced in C during the further 16.5 h, and in D with the longer incubation period, if they were derived by proteolytic cleavage would be of nearly the same high specific radioactivity as the pool of precursor protein and thus of a significantly higher specific radioactivity than in B. The data of Table 2 indicate that this was not the case and that the specific radioactivities were even lower in C than in B and also lower in D than in C. This progressive decrease in specific radioactivity of the PR-proteins is exactly what would be expected if they were produced by de nouo synthesis from a pool of radioactive amino acids which is later progressively diluted by unlabeled amino acids.
DISCUSSION This is the first report of rapid and efficient incorporation of labelled amino acids into PR-proteins in tobacco leaves during the hypersensitive response to TMV infection. A recent publication [21] describes the results of attempts to label the PR-proteins of TMV-infected Xanthi-nc tobacco leaves by petiolar uptake of [3H] leucine or by exposure of attached leaves to [‘“Cl CO,. Th e experiments with [‘“Cl CO, indicated that radioactivity was probably incorporated into PR-la, PR-1 b, PR-lc and PR-2. Furthermore, detached leaves, both infected and uninfected, incorporated [3H] leucine into proteins. Surprisingly the radioactivity was not as clearly associated with specific protein peaks in acrylamide gels as that incorporated from [‘“Cl CO,. Some radioactivity was associated with PR-la, and probably with PR-R as well, but the incubation periods with the radiochemicals were rather long; 3 days after exposure to [‘“Cl CO, and 4-6 days after petiolar uptake of [3H] leucine. It is surprising that exposure to [‘“Cl CO, labelled the PR-proteins more efficiently than leucine. The poor labelling achieved with [3H] leucine contrasts with our results obtained by feeding [14C] amino acids in the same way. We found radioactivity associated with all the PR-proteins which could be clearly resolved from other host proteins. Although the radioactivity associated with the PR-proteins was always lower from petiolar uptake compared to that incorporated after injection into attached leaves, it was still significant. Differences in labelling efficiency obtained by different workers using the same procedure are probably due to differences in the specific radioactivities of the precursors used and/or the duration of the incubation period. Concerning the latter factor, the data of Table 2 (experiments B, C and D) show that a progressive dilution of label occurs with increasing length of the incubation period and a similar dilution probably occurred during the 446 days incubation without continuous feeding used by Pierpoint in his experiments [21]. However, the main reason for the poor incorporation of [3H] leucine was probably due to its very high specific radioactivity and to abnormal patterns of incorporation which are known to result from petiolar uptake of labelled precursors by detached leaves. It has already been mentioned that tissues near the cut end of the petiole may
Radiolabelling
of tobacco
PR-proteins
39
act as a sink for labelled metabolites [.?3], so that only a small proportion of that which is fed actually reaches the mesophyll tissues. This distortion of distribution appears to be enhanced by the presence of enlarging local lesions on the leaves [S, 9,14,18]. Even though petiolar uptake, in our conditions, led to significant incorporation of radioactivity into PR-proteins, injection into leaves was the preferred method in our experiments. An obvious advantage of this procedure is that it can be applied to leaves still attached to the plant. But the major advantage is that it gave high efficiency of incorporation due to the more uniform distribution of labelled amino acids throughout the mesophyll tissue. This injection procedure had been shown to be very efficient in previous studies and enabled us to detect very low levels of virus replication around individual infection sites [16]. In the present work, radioactivity was clearly associated with PR-proteins which were separated on either denaturing or non-denaturing gels after a 3 h incubation with labelled amino acids. But an unambiguous demonstration of the incorporation of radioactive amino acids into the PR-proteins was obtained from successive electrophoretic migrations under non-denaturing and denaturing conditions /Fig. 3). Figures 1 and 2 illustrate the electrophoretic migrations of total acidic leaf protein extracts. One cannot exclude the possibility that minor contaminating proteins or polypeptides of very high specific radioactivity were present in these crude extracts which co-migrated with the PR-proteins. Such contamination might not be detectable by staining the gels but could account for most of the label shown by autoradiography. Any radioactive contaminants of each PR-protein should not be the same under both non-denaturing and denaturing conditions of electrophoretic separation. The results of the successive electrophoretic migrations (Fig. 3) demonstrated clearly the incorporation of amino acids into PR-proteins whose composition is known and similar experiments will be performed with PR-0, PR-P, PR-Q PR-R and PR-S once their composition is known. The use of short incubation periods, which are possible with the injection procedure, enabled estimates to be made of incorporation rates of radiolabel into the PRproteins so that comparisons could be made with incorporation rates into other host proteins. The aim of these experiments was to investigate the possibility that PR-proteins are produced solely by proteolytic cleavage of constitutive proteins. Production by proteolytic cleavage cannot be distinguished from de novo synthesis by the use of long incubation periods following exposure to [14C] CO,. In contrast, the use of short incubation periods should enable isotopic dilution effects to be detectable, should they occur. The data presented here exclude the possibility of large pools of precursor proteins being degraded by proteolysis to the PR-proteins as stable end-products. Whilst proteolysis cannot be totally excluded, at the most it can only involve shortlived precursor proteins that were being actively synthesized de novo in hypersensitively reacting leaves. In such a case, proteolytic cleavage would not involve the removal of a large fragment, at least in the case of PR- 1 a, since this protein was found to be labelled during in vitro translation of messenger RNA extracted from both healthy and TMVinfected Xanthi-nc leaves [g. We are indebted to Mrs Pierrette Geoffroy for skilful technical assistance. We also thank Professor L. Hirth, Head of the Institute, for providing facilities. This work was de 1’Industrie et de la Recherche” supported by grant 82Lllll from the “Ministere
40
E. Jamet, M. Kopp and B. Fritig
and by grant
ATP 5482 (Biologie Molkculaire Vkgktale) from the “Centre National de la Recherche Scientifique”.
REFERENCES P. & GIANINAZZI, S. (1982). B-protein as constitutive component in highly (TMV) resistant interspecific hybrids ofkotiana glutinosa x Nicotiana debneyi. Plant Science Letters 26, 173-181. 2. ANTONIW, J. F. & WHITE. R. F. (1980). The effect of aspirin and polyacrylic acid on soluble leaf proteins and resistance to virus infection in five cultivars of tobacco. Phytopathologirche xeitschrift 98,33 1-341. 3. ANTOHIW, J. F., RITTER, C. E., PIERPOINT, W. S. & VAN LOON, L. C. (1980). Comparison of three pathogenesis-related proteins from plants of two cultivars of tobacco infected with TMV. Journal of 1. AHL,
General
Virology 47, 79-87.
4. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry 72,248-254. 5. CARR, J. P., ANTONIW, J. F., WHITE, R. F. & WILSON, T. M. A. (1982). Latent messenger RNA in tobacco (Nicotiana tabacum~~. Biochemical Socieg Transactions 10,353S354. 6. COLLENDAVELLOO, J,, LEGRAND, M. & FRITIG, B. (1982). Plant disease and regulation of enzymes involved in lignification. De nouo synthesis controls o-methyltransferase activity in hypersensitive tobacco leaves infected by tobacco mosaic virus. Physiological Plant Pathology 21,27 l-28 1. 7. COLLENDAVELLOO, J., LEGRAND, M. & FRITIG, B. (1983). Plant disease and the regulation of enzymes involved in lignification. Increased rate of de nova synthesis of the three tobacco o-methyltransferases during the hypersensitive response to infection by tobacco mosaic virus. Plant Physiology 73,550-554. 8. DE LAAT, A. M. M. & VAN LOON, L. C. (1981). Complications in interpreting precursor/product relationships by labeling experiments. Methionine as the precursor of ethylene in tobacco leaves.
Radiolabelling 23. 24.
25.
26. 27.
28. 29. 30.
PRUT,
of tobacco
M. J. & MATTHEWS,
41
PR-proteins R. E. F. (1971).
Non-uniformities
in the metabolism
of excised
leaves
and
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