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Induction of chitinase and β-1,3-glucanase in tobacco plants infected with Pseudomonas tabaci and Phytophthora parasitica var. Nicotianae
Plant Science, 61 (1989} 1 5 5 - 1 6 1
155
Elsevier Scientific Publishers Ireland Ltd.
I N D U C T I O N OF C H I T I N A S E A N D f3-1,3-GLUCANASE IN TOBACCO P L A N T S INFECTED WITH PSEUDOMONAS TABACI AND PHYTOPHTHORA PARASITICA var. N I C O T I A N A E
FREDERICK MEINS, JR. ~'* and PATRICIA AHL b
°Friedrich Miescher Institu~ P.O. Box 2543, CH-4002, Basel and bCiba-Geigy, A G, CH-4002, Basel (Switzerland) (Received October 25th, 1988) (Revision received December 8th, 1988) (Accepted December 8th, 1988)
We have measured by rocket immunoassay the glucan endo-l,3-/~-glucosidase (/~-l,3-glucanase, EC 3.2.1.39) and endochitinase (chitinase, EC 3.2.1.14) contents of Nicotiana tabacum cv. "Havana 425" plants following infection with the bacterial pathogen Pseudomonas tabaci and the fungal pathogen Phytophthora parasitica var. nicotianae. Both enzymes were induced, relative to non-infected controls, in leaves infected with P. tabaci£nd in stems and leaves infected with P. parasitica, Increases in enzyme content were confined to infected parts of the plant indicating t h a t this response is local r a t h e r than systemic. The ~-l,3-glucanase- and chitinase-mRNA content of tissues was measured by RNA blot hybridization using the inserts of cDNA clones for the tobacco mRNAs as probes. P. tabaci and P. parasitica infection increased the mRNA content relative to controls indicating t h a t both pathogens act at least in part at the level of mRNA accumulation. As judged by immunological tests and RNA hybridization, the/3-1,3-glucanase and chitinase induced by P. tabaci~andP:parasitica are the same basic isoforms known to be induced by tobacco mosaic virus. Therefore the induction appears to be a non-specific response of the host to pathogens.
Key words: auxin; cytnkinin; mRNA (chitinase,/~-l,3-glucanase); pathogen-related proteins; Phytophthora parasitica var. nicotianae; Pseudomonas tabaci
Introduction
The glucanohydrolases glucan endo-l,3-f3glucosidase (f3-1,3-glucanase, EC 3.2.1.39) and endochitinase (chitinase, EC 3.2.1.14} are induced in parts of plants infected with viral, bacterial, and fungal pathogens [1]. These enzymes can hydrolyze the cell walls of fungi [2,3] and, depending on the fungus, chitinase or combinations of chitinase and f3-1,3-glucanase exhibit pronounced fungicidal activity in vitro [3,4]. It has been proposed that induction of ~1,3-glucanase and chitinase is part of the biochemical defense of plants against pathogenic fungi [5,6]. Acidic and basic isoforms of/3-1,3-glucanase *To whom all correspondence should be addressed. Abbreviations: chitinase, endochitinase; fi-l,3-glucanase, glucan endo-l,3-f~-glucosidase; IgG, immunoglobulin G; kb, kilobase(s); TMV, tobacco mosaic virus.
and chitinase are induced in tobacco leaves infected with tobacco mosaic virus (TMV) [7--9]. The basic isoforms also exhibit complex regulation in the uninfected tobacco plant. They are major components (approx. 5O/o of the soluble protein) of the lower leaves and roots, but are either not detectable or present at very low concentrations in leaves near the top of the plant [10,11]. The induction of f3-1,3-glucanase in cultured cells requires ethylene [12]; and/3-1,3glucanase and chitinase are coordinately downregulated in cultured cells by combinations of auxin and cytokinin added to the medium
[11,13]. TMV appears to act at the mRNA level to induce the basic isoforms of fi-l,3-glucanase and the basic and acidic isoforms of chitinase [9,14]. It is not known if bacterial and fungal pathogens act in a similar way. The present report deals with the induction of glucanohydrolases
0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
156
in tobacco infected with a bacterial leaf pathogen, Pseudomonas tabaci~ which causes wildfire disease, and a fungal wilt pathogen, Phytophthora parasitica var. nicotianae, which causes backshank disease [15]. We show that basic isoforms of/3-1,3-glucanase and chitinase encoded by host genes are locally induced in response to both pathogens and that this induction is, at least in part, at the level of mRNA. Materials and methods
Culture of host and pathogens Nicotiana tabacum cv. "Havana 425" plants were raised from seed in a greenhouse and used when 8 weeks old and approx. 15 ,cm tall. The virulent strains of Pseudomonas tabaci and Phytophthora parasitica var. nicotianae were from the Ciba-Geigy, AG collection (Basel, Switzerland). P. tabaci was maintained on Nutrient Agar (Difco Laboratories, Detroit, MI, U.S.A.). P. parasitica was maintained on 10% (v/v) V-8 vegetable juice (Campbell's Soups, Felegara, Italy) sterilized by autoclaving. To induce sporangia formation, 10-day-old cultures were incubated in water for 3 days. The mycelium was macerated, incubated for 30 min at 6 °C and then 60 min at room temperature and zoospores collected by filtering through a 500 ~m-pore size copper sieve.
Inoculation of plants and sampling Suspensions of P. tabaci (5 × 105 colonyforming units/ml) in sterile water were injected into the proximal halves of the two first fully developed leaves. Mock inoculated plants were injected in the same way with sterile water. The plants were incubated at 22 °C in a growth chamber (14 h light) for 2 days at 100% relative humidity and thereafter at 20°C at 3 0 - 4 0 % relative humidity. Tissues from the inoculated halves of the leaves, the uninoculated halves of the same leaves and the next upper leaf were sampled at the times indicated, frozen in liquid N 2 and stored at - 2 0 ° C (protein) and - 8 0 ° C (RNA). Suspensions of P. parasitica zoospores (7.0 × 104 zoospores in 3.5 ml of sterile water) were pipetted around the base of the
stem of the tobacco plants and washed into the soil by flooding with 50 ml of water. Uninoculated plants were used as controls. Plants were incubated in a growth chamber (14 h light) at 2 4 - 2 6 °C and 100% relative humidity. Segments of the lower stem, 2--3 cm from the base of the plants, and upper leaves were sampled at the times indicated, frozen in liquid Ne, and stored as indicated above. Three replicate plants were used for each treatment. Experiments were repeated at least twice.
Analysis of proteins Extracts of frozen plants tissues were prepared as described by Mohnen et al. [13]. Extracts were prepared from P. tabaci and P. parasitica by grinding the material at 0 °C in a mortar and pestle with powdered glass and 2 vols. of the homogenization medium used for plant tissues [13]. Chitinase and/3-1,3-glucanase in the extracts were measured by a rocket immunoelectrophoresis method using authentic samples of the tobacco enzymes as standards [11,16]. Protein was measured by the method of Bradford [17]. Ouchterlony double immunodiffusion was performed with 5 gl of sample in each well. The wells contained extracts of tobacco tissue, 100 ~g • m1-1 purified tobacco ~-l,3-glucanase, 100 ~g • m1-1 purified tobacco chitinase, 10 mg • m1-1 rabbit anti-tobacco/3-1,3-glucanase IgG, and 10 mg • m1-1 rabbit anti-tobacco chitinase IgG [11,16].
Analysis of mRNA Total RNA was isolated from frozen tissues by the method of Short and Torrey [18] as modified by VSgeli-Lange et al. [9]. The method for RNA blot analysis was that of Thomas [19]. The amount of RNA applied to agarose gels and the integrity of the ribosomal RNA fraction were confirmed by electrophoresing RNA samples, staining the gels with ethidium bromide, and comparing the position of bands and their intensity of fluorescence with RNA standards [20]. The size of the RNA species was estimated from DNA molecular weight standards (New England Biolabs, Beverly, MA, U.S.A.) applied to the same gel. The probes used for hybridiza-
157
tion were the Pst I inserts of cDNA clones of tobacco ~-l,3-glucanase (pGL43) [13] and chitinase (pCHN50) [11] mRNA. The probes were labeled using a random primer labeling kit (Boehringer, Mannheim, F.R.G.). Slot blots with 8, 4, 1, and 0.5 ~g of each RNA sample were hybridized as described above. Intensity of hybridization was measured by scanning the autoradiograms with a densitometer. The amount of mRNA was estimated from a standard curve of total RNA prepared from cultured Havana 425 tissue induced to produce ]%l,3-glucanase and chitinase [13]. The data are expressed in relative units defined as the slope of the least-squares fit of the mRNA content estimated from standards vs. ~g of RNA applied to the filter. Results
Induction of fl-l,3-glucanase and chitinase by infection Plants inoculated with P. parasitica started to wilt after 4--5 days. By day 7 plants showed visible necrosis inside the stem, and by day 11 necrosis was also visible on the outside of the stem. Figure 1 shows the time course of glucanohydrolase induction in upper-leaf and stem tissues from infected and control plants. The data are expressed on a fresh weight (fresh wt.) basis. Similar pattern of induction were observed when the data were expressed on a soluble protein basis. The fl-l,3-glucanase and chitinase content of stem tissue from uninfected control plants remained at a low level, approx. 6 ~g. g-1 fresh wt., which is at the limit of detection (Fig. 1A). In contrast, there was a marked, parallel induction of both enzymes in the infected plants. This was already evident 4 days after inoculation when the plants began to wilt and reached a peak 7 days after inoculation to give a maximum induction of 95 ~g- g-' for/% 1,3-glucanase and 56 ~g • g-1 for chitinase. A parallel increase in both enzymes was also observed in the upper leaves (Fig. 1B). Because infectious material could be recovered from these leaves, it is likely that the enzyme induc-
60i®
I
u~ 20
~ ~ .... =-4
I
o
.....
t
u
®
o u
2 o
60
:1
4O 2O 2
4
6 Days
8
10
12
Fig. 1. Glucanohydrolase content of P. parasiticainfected stems (A) and upper leaves (B) of tobacco. Chitinase (O);/3-1,3-glucanase (El); infected plants, open symbols; control plants, filled symbols. E r r o r bars, _ S.E.M. for three replicate plants.
tion in leaves, like that of the stem, is a local response to pathogen infection. Infection with P. parasitica causes wilting. To find out if enzyme induction is an indirect effect of water stress, uninfected tobacco plants were grown in the greenhouse without watering and the glucanohydrolase content of upper leaves, lower leaves and the lower stem was assayed after 0 and 4 days, when there was extensive wilting of the leaves. Although the fl1,3-glucanase and chitinase content of the lower-leaf tissue increased somewhat by day 4, water stress did not induce the accumulation of the enzymes in the upper-leaf and stem tissues that exhibited a dramatic response to the pathogen (Table I). Plants inoculated with P. tabaci showed the first symptoms of infection, slight yellowing of the inoculated regions of the leaf, 2 - 3 days after inoculation. With increasing time, the yellowing increased and necrotic zones formed. The infection was highly localized. No symptoms were evident in the uninfected halves of the lower leaves or upper leaves nor could P.
158
60[-¢
I. Effect of water stress on the ~-l,3-glucanase and chitinase content of tobacco leaf and stem tissues.
Table
Tissue
Days"
~g Enzyme/g. fresh wt. ~-l,3-Glucanase
0 ~"
Chitinase LL
~
,
~I
i------,
~
i
,
L
,
ov®
0~
Lower leaf
0 4
25.5 ± 2.10~ 45.2 ± 14.3
23.7 ± 5.50 44.6 ± 12.5
0 4
<6.0 ° <6.0
6.75 ± 0.75 <6.0
0 4
< 6.0 < 6.0
2o.=~.~/-"--• . 0 ]
Upperleaf
.C
co o
o
Stem
< 6.0 < 6.0
Days after watering of plants discontinued. b Mean ± S.E.M. for two replicate plants. c Belowthe limit of detection.
. . .
~
~
.....
T
O
180
,
~
,
[
®
160 ~ao
~ 120
/
100 go 60
tabaci be re-isolated f r o m t h e a s y m p t o m a t i c regions. Chitinase and f~-l,3-glucanase w e r e m e a s u r e d in t h e infected and u n i n f e c t e d halves of a l o w e r leaf and in t h e n e x t u p p e r leaf of t h e s a m e plant. T h e r e s u l t s e x p r e s s e d on a f r e s h wt. basis a r e s h o w n in Fig. 2. As r e p o r t e d earlier for u n t r e a t e d p l a n t s [10,11], leaf t i s s u e s f r o m mock-inoculated p l a n t s contained high c o n c e n t r a t i o n s of chitinase and ~-l,3-glucanase r e l a t i v e to s t e m tissues. T h e s e c o n c e n t r a t i o n s i n c r e a s e d slightly o v e r t h e 12-day period of t h e e x p e r i m e n t . Both e n z y m e s w e r e induced in t h e t). tabaci infected l e a v e s b y d a y 2 w h e n t h e first s y m p t o m s w e r e e v i d e n t . Chitinase concentrat i o n s r e a c h e d a m a x i m u m 8 d a y s a f t e r inoculation; w h e r e a s , f3-1,3-glucanase r e a c h e d a m a x i m u m earlier, 4 d a y s a f t e r inoculation. T h e m a g n i t u d e of t h e induction, 105 Mg" g-1 for p-l,3g l u c a n a s e and 150 Mg • g-1 for chitinase, was c o m p a r a b l e to t h a t o b s e r v e d in s t e m s e g m e n t s infected w i t h P. parasitica, T h e induction of chitinase and f3-1,3-glucanase w a s localized in t h e infected regions of t h e leaf s h o w i n g s y m p toms. Although the glucanohydrolase content of l e a v e s at d a y 0 v a r i e d c o n s i d e r a b l y f r o m exp e r i m e n t to e x p e r i m e n t , e.g. c o m p a r e Figs. 1B and 2C, no i n c r e a s e in t h e e n z y m e s r e l a t i v e to mock-infected controls w a s found in t h e uninfected half of infected l e a v e s or in t h e n e x t
4o 2o 2
4
6 Doys
8
0
12
Fig. 2. Glucanohydrolase content of tobacco leaves infected with P. tabacL (A) the infected half of lower leaves; (B) the uninfected half of infected lower leaves; (C) untreated upper leaves. Chitinase (O); fl-l,3-glucanase (E]); infected plants, open symbols; mock-infected control plants, filled symbols. Error bars, ± S.E.M. for three replicate plants. u p p e r leaves. P. tabaci infection did not induce a s y s t e m i c i n c r e a s e in t h e e n z y m e s . Control e x p e r i m e n t s w e r e p e r f o r m e d to v e r ify t h a t the f3-1,3-glucanase and chitinase induced b y infection with t h e p a t h o g e n s a r e t h e s a m e immunological f o r m as t h e e n z y m e s purified f r o m c u l t u r e d t o b a c c o tissues. No/3-1,3-gluc a n a s e or chitinase a n t i g e n could be d e t e c t e d in p r o t e i n e x t r a c t s p r e p a r e d f r o m c u l t u r e s of P. parasitica and P. tabacL O u c h t e r l o n y double immunodiffusion t e s t s w e r e m a d e with plant e x t r a c t s containing high c o n c e n t r a t i o n s of f3-1,3glucanase and chitinase as j u d g e d b y r o c k e t i m m u n o a s s a y . T h e induced e n z y m e s a p p e a r to be immunologically identical to t h e basic isof o r m s of/3-1,3-glucanase and chitinase p r e p a r e d f r o m cultured tissues.
159
(~)13-1,3-Glucanase (~Chitinase 1
2
3
1
2
3
Fig. 3. RNA blot analysis of total RNA prepared from P. tabaci-infected leaf tissue and P. parasitica-infected stem tissue of tobacco plants. (1) RNA from P. tabaci-infected leaf tissue 4 days post-inoculation; (2) RNA from P. parasitica-infected stem tissue 4 days post-inoculation; (3) RNA standard from cultured tobacco tissues induced to produce chitinase and/3-1,3-glucanase. Hybridization probes: (A) tobacco/~-l,3-glucanase cDNA; (B) tobacco chitinase cDNA. Numbers on the left, approx, size of the RNA hybridizing with the probe in kb.
R N A blot analysis An RNA blot analysis was performed with total RNA isolated from tissues assayed for glucanohydrolases. The Pst I inserts of cDNA clones for tobacco/3-1,3-glucanase and chitinase mRNA were used as hybridization probes. When mRNAs were detectable, they gave a Table II. Days
[11,131.
The/1-1,3-glucanase- and chitinase-mRNA content of tobacco stem tissue infected with P. parasitica. m R N A c o n t e n ta ~-l,3-Glucanase
0 2 4 7
single hybridization band with each probe (Fig. 3). These bands correspond in size to the approx. 1.7-kb mRNA for /3-1,3-glucanase and the approx. 1.3-kb mRNA for chitinase found in cultured tissues and in tissues from the lower leaves and roots of untreated tobacco plants
Chitinase
Control
Infected
Control
Infected
0b 42_+28 ~ 0 0
0 225_+ 29 278 _ 37 1230 ___ 123
6.0 _ 1.0 8.0_+2.0 0 2.0 _+ 0.0
6.0 57.0 337.0 1390.0
" Relative to an RNA standard prepared from cultured cells induced to produce/3-1,3-glucanase and chitinase ( × 103). b Not detected. c Slope of the regression line of plots of absorbance vs. amount of RNA applied _+ S.E.M.
+ 1.0 _+ 7.0 _+ 28.0 _+ 63.0
160 Table III.
Days
The/~-l,3-glucanase- and chitinase-mRNA content of tobacco leaf tissue infected with P. tabaci. mRNA content s fl-l,3-Glucanase
0 3 6 9
Chitinase
Control
Infected
Control
Infected
66_+ 22b 142_+ 15 0¢ 30_+ 0.0
50_+ 10 150_+ 49 93_ 21 748_+ 270
4.0_+ 0.4 6.7_+ 0.2 3.0_+ 2.0 4.0 +_ 0.0
9.0 + 3.0 58.0 _+ 1.0 9.0 _+ 1.0 25.0 _+ 4.0
Relative to an RNA standard prepared from cultured cells induced to produce ~-l,3-glucanase and chitinase ( × 103). b Slope of the regression line of plots of absorbance vs. amount of RNA applied _+ S.E.M. Not detected. T h e m R N A c o n t e n t of t i s s u e s w a s m e a s u r e d b y slot-blot h y b r i d i z a t i o n and e x p r e s s e d relat i v e to a s t a n d a r d R N A p r e p a r e d f r o m c u l t u r e d t i s s u e s p r o d u c i n g fl-l,3-glucanase and chitinase. T h e r e w a s a m a r k e d induction of fl-l,3-glucanase and chitinase m R N A in t h e l o w e r s t e m of plants infected w i t h P. parasitica {Table II). T h e m R N A for t h e t w o e n z y m e s i n c r e a s e d in parallel with t i m e a f t e r infection to m a x i m u m values of a p p r o x . 1200 and a p p r o x . 1400 relative units on d a y 7 for fl-l,3-glucanase and chitinase r e s p e c t i v e l y . In c o n t r a s t t h e m R N A c o n t e n t of t h e l o w e r s t e m of control plants r e m a i n e d at a v e r y low level or w a s u n d e t e c t a ble. T h e t i m e course for m R N A a c c u m u l a t i o n in P. tabaci infected and m o c k infected l e a v e s varied f r o m e x p e r i m e n t to e x p e r i m e n t ; h o w e v e r , t h e r e w a s a c o n s i s t e n t induction of m R N A for b o t h e n z y m e s following infection. T h e r e s u l t s of one e x p e r i m e n t a r e s h o w n in T a b l e I I I . Relat i v e to controls, t h e m a x i m u m induction of m R N A w a s 25- and 9-fold for fl-l,3-glucanase and chitinase r e s p e c t i v e l y . As e x p e c t e d f r o m t h e enzyme measurements, mRNA for b o t h e n z y m e s w a s also found in t h e mock-infected controls. Discussion
Our r e s u l t s p r o v i d e s t r o n g e v i d e n c e t h a t fl-l,3-glucanase and chitinase encoded b y host g e n e s a r e induced in p a r t s of tobacco plants infected w i t h P. tabaci and P. parasitic~ First, e x t r a c t s of t h e t w o p a t h o g e n s did not contain a n t i g e n s which r e a c t with t h e a n t i b o d i e s u s e d
in our a s s a y for t h e tobacco e n z y m e s . Second, no immunological differences w e r e d e t e c t e d in e n z y m e s induced by t h e p a t h o g e n s and t h e e n z y m e s purified to h o m o g e n e i t y f r o m uninfected tobacco tissues. Finally, high s t r i n g e n c y R N A hybridization s h o w e d t h a t R N A f r o m p a t h o g e n - i n f e c t e d t i s s u e s contains one m R N A encoding each e n z y m e with t h e s a m e size as the m R N A for t h e e n z y m e s p r e p a r e d f r o m h e a l t h y tissues (Fig. 3). The r o c k e t i m m u n o e l e c t r o p h o r e s i s m e t h o d we used does not d e t e c t acidic isoforms of fll-3glucanase and chitinase. Thus, t h e p r e s e n t r e s u l t s and earlier r e s u l t s o b t a i n e d with T M V infection [9] indicate t h a t viral, b a c t e r i a l and fungal p a t h o g e n s induce t h e s a m e basic isof o r m s of the tobacco e n z y m e s . This p r o v i d e s f u r t h e r evidence for t h e v i e w t h a t fl-l,3-glucanase and chitinase induction is p a r t of a s t e r e o t y p i c r e s p o n s e of the p l a n t to infection r a t h e r t h a n a r e s p o n s e tailored to a specific pathogen. In principle, p a t h o g e n s could i n c r e a s e t h e c o n c e n t r a t i o n of e n z y m e s b y i n c r e a s i n g e n z y m e production, d e c r e a s i n g e n z y m e d e g r a d a t i o n , or b y a combination of t h e s e m e c h a n i s m s . E f f e c t s on e n z y m e p r o d u c t i o n could be at the level of t r a n s l a t i o n or at t h e level of m R N A t r a n s c r i p tion, processing, t r a n s p o r t , or d e g r a d a t i o n . A l t h o u g h multiple effects of infection cannot at p r e s e n t be ruled out, our r e s u l t s f a v o r the h y p o t h e s i s t h a t P. tabaci and P. parasitica act, at least in p a r t , at t h e level of m R N A accumulation. F o r u n k n o w n r e a s o n s , r e s u l t s of R N A blot e x p e r i m e n t s with P. tabaci infected l e a v e s w e r e highly variable. Infection did, h o w e v e r ,
161
consistently increase the relative concentrations of m R N A for both enzymes (Tables II and III). The results were more convincing in the case of P. parasitica infected stems where the magnitude of mRNA induction was far greater than in P. tabaci infected leaves and the background level of enzymes in control plants was very low. Accumulation of the two mRNAs was coordinated and closely paralleled the increases in enzyme concentration. Tobacco chitinases and /3-1,3-glucanases are encoded by small gene families, each with approx, four members [21,22]. The possibility that these genes are coordinately induced in response to pathogens and the mechanism of induction at the m R N A level are interesting areas for further study. Acknowledgements We thank Alfred Milani and Monique Seldrun for technical help and Thomas Boller and Witek Filipowicz for their critical comments.
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20
21
nases. Proc. Natl. Acad. Sci. U.S.A., 84 (1987} 6 7 5 0 6754. S. Kauffmann, M. Legrand, P. Geoffroy and B. Fritig, Biological function of 'pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-~-glucanase activity. EMBO J., 6 (1987} 3 2 0 9 - 3212. R. V6geli-Lange, A. Hansen-Gehri, T. Boller and F. Meins, Jr., Induction of the defense-related glucanohydrolases, /~-l,3-glucanase and chitinase, by tobacco mosaic virus infection of tobacco leaves. Plant Sci., 54 (1988) 171 -- 176. G. Felix and F. Meins, Jr., Developmental and hormonal regulation of//-1,3-giucanase in tobacco. Planta, 167 (1986)206-- 211. H. Shinshi, D. Mohnen and F. Meins, Jr., Regulation of a plant pathogenesis-relatcd enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8 9 - 9 3 . G. Felix and F. Meins, Jr., Ethylene regulation of ]~-l,3glucanase in tobacco. Planta, 172 (1987) 386--392. D. Mohnen, H. Shinshi, G. Felix and F. Meins, Jr., Hormonal regulation of /3-1,3-glucanase messenger RNA levels in cultured tobacco tissues. EMBO J., 4 (1985) 1631 -- 1635. R.A.M. Hooft van Huijsduijnen, S. Kauffmann, F.T. Brederode, B.J.C. Cornelissen, M. Legrand, B. Fritig and J.F. Bol, Homology between chitinases t h a t are induced by TMV infection of tobacco. Plant Mol. Biol. 9 (1987) 411 -- 420. G.B. Lucas, Diseases of tobacco, 3rd. edn., Biological Consulting Associates, Raleigh, NC, U.S.A., 1975. G. Felix and F. Meins, Jr., Purification, immunoassay and characterization of an abundant, cytokinin-regulated polypeptide in cultured tobacco tissues. Evidence the protein is p-l,3-glucanase. Planta, 164 (1985) 423 -- 428. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Bioehem., 72 (1976) 248-- 254. K.C. Short and J.G. Torrey, Cytokinins in seedling roots of pea. Plant Physiol., 49 (1972) 155-- 160. P.S. Thomas, Hybridization of denatured RNA transferred or dotted to nitrocellulose paper. Methods Enzymol., 100 (1983) 255-- 266. T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, New York. H. Shinshi, H. Wenzler, J.-M. Neuhaus, G. Felix, J. Hofsteenge and F. Meins, Jr., Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-{3-1,3glucanase. Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 5541 5545. J.-M. Neuhaus, Characterization of tobacco chitinase genes. Abstract, 2nd International Congress of Plant Molecular Biology, Jerusalem, 1988, in press. -
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Elsevier Scientific Publishers Ireland Ltd.
I N D U C T I O N OF C H I T I N A S E A N D f3-1,3-GLUCANASE IN TOBACCO P L A N T S INFECTED WITH PSEUDOMONAS TABACI AND PHYTOPHTHORA PARASITICA var. N I C O T I A N A E
FREDERICK MEINS, JR. ~'* and PATRICIA AHL b
°Friedrich Miescher Institu~ P.O. Box 2543, CH-4002, Basel and bCiba-Geigy, A G, CH-4002, Basel (Switzerland) (Received October 25th, 1988) (Revision received December 8th, 1988) (Accepted December 8th, 1988)
We have measured by rocket immunoassay the glucan endo-l,3-/~-glucosidase (/~-l,3-glucanase, EC 3.2.1.39) and endochitinase (chitinase, EC 3.2.1.14) contents of Nicotiana tabacum cv. "Havana 425" plants following infection with the bacterial pathogen Pseudomonas tabaci and the fungal pathogen Phytophthora parasitica var. nicotianae. Both enzymes were induced, relative to non-infected controls, in leaves infected with P. tabaci£nd in stems and leaves infected with P. parasitica, Increases in enzyme content were confined to infected parts of the plant indicating t h a t this response is local r a t h e r than systemic. The ~-l,3-glucanase- and chitinase-mRNA content of tissues was measured by RNA blot hybridization using the inserts of cDNA clones for the tobacco mRNAs as probes. P. tabaci and P. parasitica infection increased the mRNA content relative to controls indicating t h a t both pathogens act at least in part at the level of mRNA accumulation. As judged by immunological tests and RNA hybridization, the/3-1,3-glucanase and chitinase induced by P. tabaci~andP:parasitica are the same basic isoforms known to be induced by tobacco mosaic virus. Therefore the induction appears to be a non-specific response of the host to pathogens.
Key words: auxin; cytnkinin; mRNA (chitinase,/~-l,3-glucanase); pathogen-related proteins; Phytophthora parasitica var. nicotianae; Pseudomonas tabaci
Introduction
The glucanohydrolases glucan endo-l,3-f3glucosidase (f3-1,3-glucanase, EC 3.2.1.39) and endochitinase (chitinase, EC 3.2.1.14} are induced in parts of plants infected with viral, bacterial, and fungal pathogens [1]. These enzymes can hydrolyze the cell walls of fungi [2,3] and, depending on the fungus, chitinase or combinations of chitinase and f3-1,3-glucanase exhibit pronounced fungicidal activity in vitro [3,4]. It has been proposed that induction of ~1,3-glucanase and chitinase is part of the biochemical defense of plants against pathogenic fungi [5,6]. Acidic and basic isoforms of/3-1,3-glucanase *To whom all correspondence should be addressed. Abbreviations: chitinase, endochitinase; fi-l,3-glucanase, glucan endo-l,3-f~-glucosidase; IgG, immunoglobulin G; kb, kilobase(s); TMV, tobacco mosaic virus.
and chitinase are induced in tobacco leaves infected with tobacco mosaic virus (TMV) [7--9]. The basic isoforms also exhibit complex regulation in the uninfected tobacco plant. They are major components (approx. 5O/o of the soluble protein) of the lower leaves and roots, but are either not detectable or present at very low concentrations in leaves near the top of the plant [10,11]. The induction of f3-1,3-glucanase in cultured cells requires ethylene [12]; and/3-1,3glucanase and chitinase are coordinately downregulated in cultured cells by combinations of auxin and cytokinin added to the medium
[11,13]. TMV appears to act at the mRNA level to induce the basic isoforms of fi-l,3-glucanase and the basic and acidic isoforms of chitinase [9,14]. It is not known if bacterial and fungal pathogens act in a similar way. The present report deals with the induction of glucanohydrolases
0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
156
in tobacco infected with a bacterial leaf pathogen, Pseudomonas tabaci~ which causes wildfire disease, and a fungal wilt pathogen, Phytophthora parasitica var. nicotianae, which causes backshank disease [15]. We show that basic isoforms of/3-1,3-glucanase and chitinase encoded by host genes are locally induced in response to both pathogens and that this induction is, at least in part, at the level of mRNA. Materials and methods
Culture of host and pathogens Nicotiana tabacum cv. "Havana 425" plants were raised from seed in a greenhouse and used when 8 weeks old and approx. 15 ,cm tall. The virulent strains of Pseudomonas tabaci and Phytophthora parasitica var. nicotianae were from the Ciba-Geigy, AG collection (Basel, Switzerland). P. tabaci was maintained on Nutrient Agar (Difco Laboratories, Detroit, MI, U.S.A.). P. parasitica was maintained on 10% (v/v) V-8 vegetable juice (Campbell's Soups, Felegara, Italy) sterilized by autoclaving. To induce sporangia formation, 10-day-old cultures were incubated in water for 3 days. The mycelium was macerated, incubated for 30 min at 6 °C and then 60 min at room temperature and zoospores collected by filtering through a 500 ~m-pore size copper sieve.
Inoculation of plants and sampling Suspensions of P. tabaci (5 × 105 colonyforming units/ml) in sterile water were injected into the proximal halves of the two first fully developed leaves. Mock inoculated plants were injected in the same way with sterile water. The plants were incubated at 22 °C in a growth chamber (14 h light) for 2 days at 100% relative humidity and thereafter at 20°C at 3 0 - 4 0 % relative humidity. Tissues from the inoculated halves of the leaves, the uninoculated halves of the same leaves and the next upper leaf were sampled at the times indicated, frozen in liquid N 2 and stored at - 2 0 ° C (protein) and - 8 0 ° C (RNA). Suspensions of P. parasitica zoospores (7.0 × 104 zoospores in 3.5 ml of sterile water) were pipetted around the base of the
stem of the tobacco plants and washed into the soil by flooding with 50 ml of water. Uninoculated plants were used as controls. Plants were incubated in a growth chamber (14 h light) at 2 4 - 2 6 °C and 100% relative humidity. Segments of the lower stem, 2--3 cm from the base of the plants, and upper leaves were sampled at the times indicated, frozen in liquid Ne, and stored as indicated above. Three replicate plants were used for each treatment. Experiments were repeated at least twice.
Analysis of proteins Extracts of frozen plants tissues were prepared as described by Mohnen et al. [13]. Extracts were prepared from P. tabaci and P. parasitica by grinding the material at 0 °C in a mortar and pestle with powdered glass and 2 vols. of the homogenization medium used for plant tissues [13]. Chitinase and/3-1,3-glucanase in the extracts were measured by a rocket immunoelectrophoresis method using authentic samples of the tobacco enzymes as standards [11,16]. Protein was measured by the method of Bradford [17]. Ouchterlony double immunodiffusion was performed with 5 gl of sample in each well. The wells contained extracts of tobacco tissue, 100 ~g • m1-1 purified tobacco ~-l,3-glucanase, 100 ~g • m1-1 purified tobacco chitinase, 10 mg • m1-1 rabbit anti-tobacco/3-1,3-glucanase IgG, and 10 mg • m1-1 rabbit anti-tobacco chitinase IgG [11,16].
Analysis of mRNA Total RNA was isolated from frozen tissues by the method of Short and Torrey [18] as modified by VSgeli-Lange et al. [9]. The method for RNA blot analysis was that of Thomas [19]. The amount of RNA applied to agarose gels and the integrity of the ribosomal RNA fraction were confirmed by electrophoresing RNA samples, staining the gels with ethidium bromide, and comparing the position of bands and their intensity of fluorescence with RNA standards [20]. The size of the RNA species was estimated from DNA molecular weight standards (New England Biolabs, Beverly, MA, U.S.A.) applied to the same gel. The probes used for hybridiza-
157
tion were the Pst I inserts of cDNA clones of tobacco ~-l,3-glucanase (pGL43) [13] and chitinase (pCHN50) [11] mRNA. The probes were labeled using a random primer labeling kit (Boehringer, Mannheim, F.R.G.). Slot blots with 8, 4, 1, and 0.5 ~g of each RNA sample were hybridized as described above. Intensity of hybridization was measured by scanning the autoradiograms with a densitometer. The amount of mRNA was estimated from a standard curve of total RNA prepared from cultured Havana 425 tissue induced to produce ]%l,3-glucanase and chitinase [13]. The data are expressed in relative units defined as the slope of the least-squares fit of the mRNA content estimated from standards vs. ~g of RNA applied to the filter. Results
Induction of fl-l,3-glucanase and chitinase by infection Plants inoculated with P. parasitica started to wilt after 4--5 days. By day 7 plants showed visible necrosis inside the stem, and by day 11 necrosis was also visible on the outside of the stem. Figure 1 shows the time course of glucanohydrolase induction in upper-leaf and stem tissues from infected and control plants. The data are expressed on a fresh weight (fresh wt.) basis. Similar pattern of induction were observed when the data were expressed on a soluble protein basis. The fl-l,3-glucanase and chitinase content of stem tissue from uninfected control plants remained at a low level, approx. 6 ~g. g-1 fresh wt., which is at the limit of detection (Fig. 1A). In contrast, there was a marked, parallel induction of both enzymes in the infected plants. This was already evident 4 days after inoculation when the plants began to wilt and reached a peak 7 days after inoculation to give a maximum induction of 95 ~g- g-' for/% 1,3-glucanase and 56 ~g • g-1 for chitinase. A parallel increase in both enzymes was also observed in the upper leaves (Fig. 1B). Because infectious material could be recovered from these leaves, it is likely that the enzyme induc-
60i®
I
u~ 20
~ ~ .... =-4
I
o
.....
t
u
®
o u
2 o
60
:1
4O 2O 2
4
6 Days
8
10
12
Fig. 1. Glucanohydrolase content of P. parasiticainfected stems (A) and upper leaves (B) of tobacco. Chitinase (O);/3-1,3-glucanase (El); infected plants, open symbols; control plants, filled symbols. E r r o r bars, _ S.E.M. for three replicate plants.
tion in leaves, like that of the stem, is a local response to pathogen infection. Infection with P. parasitica causes wilting. To find out if enzyme induction is an indirect effect of water stress, uninfected tobacco plants were grown in the greenhouse without watering and the glucanohydrolase content of upper leaves, lower leaves and the lower stem was assayed after 0 and 4 days, when there was extensive wilting of the leaves. Although the fl1,3-glucanase and chitinase content of the lower-leaf tissue increased somewhat by day 4, water stress did not induce the accumulation of the enzymes in the upper-leaf and stem tissues that exhibited a dramatic response to the pathogen (Table I). Plants inoculated with P. tabaci showed the first symptoms of infection, slight yellowing of the inoculated regions of the leaf, 2 - 3 days after inoculation. With increasing time, the yellowing increased and necrotic zones formed. The infection was highly localized. No symptoms were evident in the uninfected halves of the lower leaves or upper leaves nor could P.
158
60[-¢
I. Effect of water stress on the ~-l,3-glucanase and chitinase content of tobacco leaf and stem tissues.
Table
Tissue
Days"
~g Enzyme/g. fresh wt. ~-l,3-Glucanase
0 ~"
Chitinase LL
~
,
~I
i------,
~
i
,
L
,
ov®
0~
Lower leaf
0 4
25.5 ± 2.10~ 45.2 ± 14.3
23.7 ± 5.50 44.6 ± 12.5
0 4
<6.0 ° <6.0
6.75 ± 0.75 <6.0
0 4
< 6.0 < 6.0
2o.=~.~/-"--• . 0 ]
Upperleaf
.C
co o
o
Stem
< 6.0 < 6.0
Days after watering of plants discontinued. b Mean ± S.E.M. for two replicate plants. c Belowthe limit of detection.
. . .
~
~
.....
T
O
180
,
~
,
[
®
160 ~ao
~ 120
/
100 go 60
tabaci be re-isolated f r o m t h e a s y m p t o m a t i c regions. Chitinase and f~-l,3-glucanase w e r e m e a s u r e d in t h e infected and u n i n f e c t e d halves of a l o w e r leaf and in t h e n e x t u p p e r leaf of t h e s a m e plant. T h e r e s u l t s e x p r e s s e d on a f r e s h wt. basis a r e s h o w n in Fig. 2. As r e p o r t e d earlier for u n t r e a t e d p l a n t s [10,11], leaf t i s s u e s f r o m mock-inoculated p l a n t s contained high c o n c e n t r a t i o n s of chitinase and ~-l,3-glucanase r e l a t i v e to s t e m tissues. T h e s e c o n c e n t r a t i o n s i n c r e a s e d slightly o v e r t h e 12-day period of t h e e x p e r i m e n t . Both e n z y m e s w e r e induced in t h e t). tabaci infected l e a v e s b y d a y 2 w h e n t h e first s y m p t o m s w e r e e v i d e n t . Chitinase concentrat i o n s r e a c h e d a m a x i m u m 8 d a y s a f t e r inoculation; w h e r e a s , f3-1,3-glucanase r e a c h e d a m a x i m u m earlier, 4 d a y s a f t e r inoculation. T h e m a g n i t u d e of t h e induction, 105 Mg" g-1 for p-l,3g l u c a n a s e and 150 Mg • g-1 for chitinase, was c o m p a r a b l e to t h a t o b s e r v e d in s t e m s e g m e n t s infected w i t h P. parasitica, T h e induction of chitinase and f3-1,3-glucanase w a s localized in t h e infected regions of t h e leaf s h o w i n g s y m p toms. Although the glucanohydrolase content of l e a v e s at d a y 0 v a r i e d c o n s i d e r a b l y f r o m exp e r i m e n t to e x p e r i m e n t , e.g. c o m p a r e Figs. 1B and 2C, no i n c r e a s e in t h e e n z y m e s r e l a t i v e to mock-infected controls w a s found in t h e uninfected half of infected l e a v e s or in t h e n e x t
4o 2o 2
4
6 Doys
8
0
12
Fig. 2. Glucanohydrolase content of tobacco leaves infected with P. tabacL (A) the infected half of lower leaves; (B) the uninfected half of infected lower leaves; (C) untreated upper leaves. Chitinase (O); fl-l,3-glucanase (E]); infected plants, open symbols; mock-infected control plants, filled symbols. Error bars, ± S.E.M. for three replicate plants. u p p e r leaves. P. tabaci infection did not induce a s y s t e m i c i n c r e a s e in t h e e n z y m e s . Control e x p e r i m e n t s w e r e p e r f o r m e d to v e r ify t h a t the f3-1,3-glucanase and chitinase induced b y infection with t h e p a t h o g e n s a r e t h e s a m e immunological f o r m as t h e e n z y m e s purified f r o m c u l t u r e d t o b a c c o tissues. No/3-1,3-gluc a n a s e or chitinase a n t i g e n could be d e t e c t e d in p r o t e i n e x t r a c t s p r e p a r e d f r o m c u l t u r e s of P. parasitica and P. tabacL O u c h t e r l o n y double immunodiffusion t e s t s w e r e m a d e with plant e x t r a c t s containing high c o n c e n t r a t i o n s of f3-1,3glucanase and chitinase as j u d g e d b y r o c k e t i m m u n o a s s a y . T h e induced e n z y m e s a p p e a r to be immunologically identical to t h e basic isof o r m s of/3-1,3-glucanase and chitinase p r e p a r e d f r o m cultured tissues.
159
(~)13-1,3-Glucanase (~Chitinase 1
2
3
1
2
3
Fig. 3. RNA blot analysis of total RNA prepared from P. tabaci-infected leaf tissue and P. parasitica-infected stem tissue of tobacco plants. (1) RNA from P. tabaci-infected leaf tissue 4 days post-inoculation; (2) RNA from P. parasitica-infected stem tissue 4 days post-inoculation; (3) RNA standard from cultured tobacco tissues induced to produce chitinase and/3-1,3-glucanase. Hybridization probes: (A) tobacco/~-l,3-glucanase cDNA; (B) tobacco chitinase cDNA. Numbers on the left, approx, size of the RNA hybridizing with the probe in kb.
R N A blot analysis An RNA blot analysis was performed with total RNA isolated from tissues assayed for glucanohydrolases. The Pst I inserts of cDNA clones for tobacco/3-1,3-glucanase and chitinase mRNA were used as hybridization probes. When mRNAs were detectable, they gave a Table II. Days
[11,131.
The/1-1,3-glucanase- and chitinase-mRNA content of tobacco stem tissue infected with P. parasitica. m R N A c o n t e n ta ~-l,3-Glucanase
0 2 4 7
single hybridization band with each probe (Fig. 3). These bands correspond in size to the approx. 1.7-kb mRNA for /3-1,3-glucanase and the approx. 1.3-kb mRNA for chitinase found in cultured tissues and in tissues from the lower leaves and roots of untreated tobacco plants
Chitinase
Control
Infected
Control
Infected
0b 42_+28 ~ 0 0
0 225_+ 29 278 _ 37 1230 ___ 123
6.0 _ 1.0 8.0_+2.0 0 2.0 _+ 0.0
6.0 57.0 337.0 1390.0
" Relative to an RNA standard prepared from cultured cells induced to produce/3-1,3-glucanase and chitinase ( × 103). b Not detected. c Slope of the regression line of plots of absorbance vs. amount of RNA applied _+ S.E.M.
+ 1.0 _+ 7.0 _+ 28.0 _+ 63.0
160 Table III.
Days
The/~-l,3-glucanase- and chitinase-mRNA content of tobacco leaf tissue infected with P. tabaci. mRNA content s fl-l,3-Glucanase
0 3 6 9
Chitinase
Control
Infected
Control
Infected
66_+ 22b 142_+ 15 0¢ 30_+ 0.0
50_+ 10 150_+ 49 93_ 21 748_+ 270
4.0_+ 0.4 6.7_+ 0.2 3.0_+ 2.0 4.0 +_ 0.0
9.0 + 3.0 58.0 _+ 1.0 9.0 _+ 1.0 25.0 _+ 4.0
Relative to an RNA standard prepared from cultured cells induced to produce ~-l,3-glucanase and chitinase ( × 103). b Slope of the regression line of plots of absorbance vs. amount of RNA applied _+ S.E.M. Not detected. T h e m R N A c o n t e n t of t i s s u e s w a s m e a s u r e d b y slot-blot h y b r i d i z a t i o n and e x p r e s s e d relat i v e to a s t a n d a r d R N A p r e p a r e d f r o m c u l t u r e d t i s s u e s p r o d u c i n g fl-l,3-glucanase and chitinase. T h e r e w a s a m a r k e d induction of fl-l,3-glucanase and chitinase m R N A in t h e l o w e r s t e m of plants infected w i t h P. parasitica {Table II). T h e m R N A for t h e t w o e n z y m e s i n c r e a s e d in parallel with t i m e a f t e r infection to m a x i m u m values of a p p r o x . 1200 and a p p r o x . 1400 relative units on d a y 7 for fl-l,3-glucanase and chitinase r e s p e c t i v e l y . In c o n t r a s t t h e m R N A c o n t e n t of t h e l o w e r s t e m of control plants r e m a i n e d at a v e r y low level or w a s u n d e t e c t a ble. T h e t i m e course for m R N A a c c u m u l a t i o n in P. tabaci infected and m o c k infected l e a v e s varied f r o m e x p e r i m e n t to e x p e r i m e n t ; h o w e v e r , t h e r e w a s a c o n s i s t e n t induction of m R N A for b o t h e n z y m e s following infection. T h e r e s u l t s of one e x p e r i m e n t a r e s h o w n in T a b l e I I I . Relat i v e to controls, t h e m a x i m u m induction of m R N A w a s 25- and 9-fold for fl-l,3-glucanase and chitinase r e s p e c t i v e l y . As e x p e c t e d f r o m t h e enzyme measurements, mRNA for b o t h e n z y m e s w a s also found in t h e mock-infected controls. Discussion
Our r e s u l t s p r o v i d e s t r o n g e v i d e n c e t h a t fl-l,3-glucanase and chitinase encoded b y host g e n e s a r e induced in p a r t s of tobacco plants infected w i t h P. tabaci and P. parasitic~ First, e x t r a c t s of t h e t w o p a t h o g e n s did not contain a n t i g e n s which r e a c t with t h e a n t i b o d i e s u s e d
in our a s s a y for t h e tobacco e n z y m e s . Second, no immunological differences w e r e d e t e c t e d in e n z y m e s induced by t h e p a t h o g e n s and t h e e n z y m e s purified to h o m o g e n e i t y f r o m uninfected tobacco tissues. Finally, high s t r i n g e n c y R N A hybridization s h o w e d t h a t R N A f r o m p a t h o g e n - i n f e c t e d t i s s u e s contains one m R N A encoding each e n z y m e with t h e s a m e size as the m R N A for t h e e n z y m e s p r e p a r e d f r o m h e a l t h y tissues (Fig. 3). The r o c k e t i m m u n o e l e c t r o p h o r e s i s m e t h o d we used does not d e t e c t acidic isoforms of fll-3glucanase and chitinase. Thus, t h e p r e s e n t r e s u l t s and earlier r e s u l t s o b t a i n e d with T M V infection [9] indicate t h a t viral, b a c t e r i a l and fungal p a t h o g e n s induce t h e s a m e basic isof o r m s of the tobacco e n z y m e s . This p r o v i d e s f u r t h e r evidence for t h e v i e w t h a t fl-l,3-glucanase and chitinase induction is p a r t of a s t e r e o t y p i c r e s p o n s e of the p l a n t to infection r a t h e r t h a n a r e s p o n s e tailored to a specific pathogen. In principle, p a t h o g e n s could i n c r e a s e t h e c o n c e n t r a t i o n of e n z y m e s b y i n c r e a s i n g e n z y m e production, d e c r e a s i n g e n z y m e d e g r a d a t i o n , or b y a combination of t h e s e m e c h a n i s m s . E f f e c t s on e n z y m e p r o d u c t i o n could be at the level of t r a n s l a t i o n or at t h e level of m R N A t r a n s c r i p tion, processing, t r a n s p o r t , or d e g r a d a t i o n . A l t h o u g h multiple effects of infection cannot at p r e s e n t be ruled out, our r e s u l t s f a v o r the h y p o t h e s i s t h a t P. tabaci and P. parasitica act, at least in p a r t , at t h e level of m R N A accumulation. F o r u n k n o w n r e a s o n s , r e s u l t s of R N A blot e x p e r i m e n t s with P. tabaci infected l e a v e s w e r e highly variable. Infection did, h o w e v e r ,
161
consistently increase the relative concentrations of m R N A for both enzymes (Tables II and III). The results were more convincing in the case of P. parasitica infected stems where the magnitude of mRNA induction was far greater than in P. tabaci infected leaves and the background level of enzymes in control plants was very low. Accumulation of the two mRNAs was coordinated and closely paralleled the increases in enzyme concentration. Tobacco chitinases and /3-1,3-glucanases are encoded by small gene families, each with approx, four members [21,22]. The possibility that these genes are coordinately induced in response to pathogens and the mechanism of induction at the m R N A level are interesting areas for further study. Acknowledgements We thank Alfred Milani and Monique Seldrun for technical help and Thomas Boller and Witek Filipowicz for their critical comments.
8
9
10
11
12 13
14
References 15 1
2
3
4
5
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