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A reliable strategy for the study of disease and hypersensitive reactions induced by Erwinia amylovora
Plant Science, 85 (1992) 171-177 Elsevier ScientificPublishers Ireland Ltd.
171
A reliable strategy for the study of disease and hypersensitive reactions induced by Erwinia amylovora Marie-Nofille Brisset a and Jean-Pierre Paulin b aSchool of Biological Sciences, University of Bath, Bath BA2 7.4 Y ( UK) and blNRA, Station de Pathologie V~g~tale, 42, Rue Georges Morel, B.P. 57, 49071 Beaucouze (France)
(Received March 23rd, 1992; revision receivedMay 25th, 1992; accepted May 26th, 1992) Virulent strains of Erwinia amylovora (CFBP 1430), an apple pathogen and Pseudomonas syringae pv. tabaci (CFBP 2106), a tobacco pathogen, induced differentialreactions when co-cultivatedwith apple and tobacco cell suspensions, dependenton the compatibility of the interaction. Typical patterns of disease or hypersensitivereactions (slow and progressiveor fast and intense respectively) were found, specially with E. amylovora. Mortality and electrolyte loss of plant cells were correlated. The use of three transposon mutants (hrp-, dsp- and EPS- mutants) of CFBP 1430confirmed the specificityof the reaction and provides further evidence on the role of pathogenicitygenes of E. amylovora. This straightforwardexperimentalmethod describedin this paper can be proposed for further analysis of the first events associated with disease and hypersensitivereactions induced by E. amylovora. Key words: Erwinia amylovora; tobacco/appleplant cell suspension; electrolyteleakage; compatible/incompatiblesituations
Introduction Erwinia amylovora (Burrill) Winslow et al. is the causal agent of fire blight, a disease of Pomoideae, characterized by a progressive necrosis of aerial parts o f host plants and ooze production [1]. Two pathogenicity determinants are presently recognized in all virulent strains: the bacterial exopolysacchafide (EPS) [2] and another yet uncharacterized factor which could be an electrolyte leakage factor [3,4], also called 'induced toxin' [5]. Genetic studies have led to the identification of dusters of genes involved in the pathogenicity o f E. amylovora: hrp genes [6,7], dsp genes [6] and rcsA Correspondence to: Jean-Pierre Paulin, INRA, Station de Pathologie Vtgttale, 42, rue Georges Morel, B.P. 57, 49 071 Beaucouze, France Abbreviations: CFBP, Collection Fran¢aise de Baettries Phytopatho~nes; ev., cultivar; dsp, disease specific; EPS, exopolysacclmride; hrp, hypersensitive reaction pathogenicity; HR, hypersensitivereaction; MES, morpholinoethane sulfonic acid; PCV, packed cell volume; rcsA, regulation capsule synthesis A; PMV, Pathologie Moldeulalre et Vtgttale; pv., pathovar; YPA, yeast peptone agar.
genes [8,9]. Expression of the hrp cluster has been shown to be involved in electrolyte leakage induction from both host and non-host plant cells [10], while rcsA genes are involved in the regulation of EPS production by the bacteria [11]. A simple, reliable bioassay, which involves both the plant and the pathogen, is strongly needed for a better understanding of the biological mechanisms affecting the plant-pathogen interaction and also to establish a link between genetical and physiological studies and to characterize the pathogenecity determinants. Bioassays have already been described for studies on E. amylovora, using leaf discs [10,12,13], fruit discs [4], protoplasts [14] or cell suspensions [5,15,16]. Among these, the best candidate for biochemical mechanism studies could be the one using cell suspensions because it provides populations of plant cells (small aggregates or single cells) that are easily manipulated and propagated. Numerous studies were reported, mainly on the expression of the hypersensitive reaction (HR) of cell suspensions co-cultivated with several plant pathogens [17-22]. Conversely, for E.
0168-9452/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
172
amylovora, cell suspensions were used in compatible situations, for the analysis of toxic ability of the bacteria or of bacterial products [5,15,16,23,24]. However none of the described methods has allowed a reproducible production of the electrolyte leakage agent of E. amylovora or its isolation as yet. Because both disease and hypersensitive reactions are controlled by the hrp cluster of E. amylovora, as for many other phytopathogenic bacteria [25], it is of particular interest to conduct comparative experiments between compatible and incompatible situations. In this paper, we describe a straightforward and fast method of co-culture of E. amylovora with host (apple) and non-host (tobacco) cell suspensions, associated with electrolyte leakage measurements. We established that the reactions of plant cells to two bacteria, E. amylovora and Pseudomonas syringae pv. tabaci, were specific, i.e. disease-like or hypersensitivelike reactions and expressed within 20 h. The use of well-characterized transposon mutants of E. amylovora in this bioassay induced different reactions according to their insertion, or their phenotype. Material and Methods
Plant cells Cells of Nicotiana tabacum cv. Xanthi were obtained from M. Spencer and G. Warren (University of Sheffield, UK) and were grown in liquid B5 basal medium [26] with 2-4-dichlorophenoxyacetic acid (1 mg 1-1), kinetin (0.1 mg l-l), sucrose (3%), at 25°C, in 250 Erlenmeyer flasks, on a rotary shaker (150 rev./min) under fluorescent lighting (16 h photoperiod). Cells were subeultured every 7 days, by adding 20 ml of cell suspensions to 80 ml of fresh medium. Cells of Malus X domestica Borkh. cv. M26 were obtained from R. Cooper and C. Brown (University of Bath, UK) and were grown in liquid Murashige and Skoog basal medium [27] with 2-4-dichlorophenoxyacetic acid (0.5 mg l-l), kinetin (1 mg 1-1), sucrose (2%) in the same conditions as tobacco cells. Apple cells were subcultured every 7 days by adding 30 rnl of cell suspensions to 70 ml of fresh medium.
Bacterial strains Strains of E. amylovora and P. syringae pv. tabaci are listed on Table I. Bacteria were cultured on YPA medium (yeast, 3 g 1-1, bacteriological peptone, 5 g l -l, agar, 14 g 1-1), which was supplemented with chloramphenicol (20 ppm) for the transposon mutants. Cells were harvested from plates after growing for 24 h at 25°C. They were washed in the following assay medium: MES 5 mM + mannitol 3%, adjusted to pH 6 with NaOH, by centrifugation at 2000 × g for 10 min and resuspended in the assay medium to an inoculum density of - 10 l° bacteriaJml. Co-culture assays Cell suspensions were used in their exponential growth phase (4-5 days old). They were first adjusted to a concentration of 10% PCV, calculated after centrifugation of 10 ml of cell suspensions at 600 × g for 10 min in graduated tubes), with two different assay media according to the plant species. For tobacco cells, MES 5 mM + CaCl 2 0.5 mM + mannitol 30, adjusted to pH 6 with NaOH, but for apple cells, MES 5 mM + mannitol 3%, adjusted to pH 6 with NaOH were used. Cells were washed by centrifugation, at 150 × g for 3 min and resuspended in their respective assay medium. Cells suspensions were incubated for 1 h in test tubes (4.5 ml/tube) in the rotary shaker used for their growth (tubes at an angle to keep cells in suspension). They were then inoculated with 0.5 ml bacterial suspension (or 0.5 ml of assay medium for controls), giving a final concentration of approximately 109 bacteria/ml and incubated, in the same conditions as above, for 2 h. Co-cultures were then washed by centrifugation at 150 × g for 3 min, resuspended in their respective assay medium, dispensed into Eppendorf tubes (1 ml per Eppendorf, 3 replicates per co-culture) and incubated at 25°C in a shaking water bath (60 strokes/min) under constant fluorescent lighting. Cell death and electrolyte loss Viability of plant cells was determined by staining with fluorescein diacetate [28]. Results are expressed in percentage of net mortality, i.e. difference between percentage'of mortality at 20 h and percentage of mortality at time 0 (beginning of
173
TaWieI. E. amyiovoraand P. syringaepv. tabaci strains used in this study Source
Characteristics
Type
Reaction on
E. amylovora
Host
Non-host
CFBP 1430 INRA-Angers, France
Wild-type
Disease reaction
HR
+
PMV 6046a INA-PG, Paris, France
Mu transposon mutant, hrp insertion Mu transposon mutant, dsp insertion Mu transposon mutant, unknown insertionb
_c
_
+
-
HR
+
-
HR
-
Disease reaction
HR
NTd
PMV 6070a INA-PG, Paris, France PMV 6089a INA-PG, Paris, France P
EPS production
syringae, pv. CFBP 2106 INRA-Angers, France tabaci
Wild-type
* R e f . 6.
bMenggad, pers. commun. =No reaction, or no production (EPS). dNot tested.
incubation in the shaking water bath). Electrolyte loss was assessed by measuring directly in the Eppendorfs, the conductivity of the plant cell suspensions, with a conductivity cell (Tacussel XC 170), suitable for conductivity measurements in small volumes (1 ml). Before each reading, each Eppendorf was shaken to allow the resuspension of the plant cells. For experiments comparing cell death and electrolyte loss, the latter is expressed as (C20 - C0)/(CT - Co) where C2o is the conductivity at 20 h, Co the conductivity at time 0 and CT the total conductivity, obtained by freezing-unfreezing each sample. Other data are presented as the numerical differences of conductivity between bacterial treatments and sterile controls. All experiments were performed at least twice and results were statistically analysed using Seheffe's test [29], with a level of significance of 5%.
Bacterial populations in plant cell culture Aliquots (5 ~ ) were removed from the Eppendoffs containing the inoculated plant cell suspensions and dilution-plated on YPA medium to determine concentrations of viable bacteria. The
experiment was repeated once. Results were statistically analyzed as described above. Results
Mortality and electrolyte loss of tobacco and apple cells co-cultured with E. amylovora and P. syringae pv. tabaci Viability of plant cells were 82.0 ~- 4.9 and 81.0 4- 5.0 for tobacco and apple cells, respectively, at the beginning of the experiment. Controls showed a limited mortality within 20 h (Table II). A significant mortality was induced in incompatible interactions, i.e.E, amylovora/tobacco and syringae pv. tabaci/apple while this was less pronounced for compatible interactions, specially in the E. amylovora/apple interaction. Electrolyte loss appeared to be highly correlated with cell death, as shown on Table II. Total conductivities were - 5 8 0 ItS and 330 ItS for apple and tobacco cells, respectively, while they were 210 its and 190 itS, respectively at time 0. After 20 h, mortality and electrolyte loss of control cells increased significantly, particularly for tobacco cells (results
174 Table H. Mortality and electrolyte loss of apple and tobacco cell suspensions co-cultured with CFBP 1430 E. amylovora and CFBP 2106 P. syringae pv. tabaci for 20 li
20o
apple
~ a
"" 150 % Net mortality a
% Net conductivity b
Control CFBP 1430 CFBP 2106
14.8±8.2a 28.4±4.2b 66.0±8.3e
19.4±0.2a 28.44-1.3b 66.9 ± 7.0c
Tobacco Control CFBP 1430 CFBP 2106
23.9 ± 3.3a 73.8 a- 2. lc 56.9 a- 3.8b
18.4 ±2.6a 74.4 ± 8.9c 54.1 ±4.2b
Apple
aPercentage of net mortality determined as described in Material and Methods. Means and standard deviations of four replicates (total of at least 250 cells per treatment). Means with the same letter are not significantly different (P = 0.05). bpercentage of net conductivity determined as described in Material and Methods. Means and standard deviations of three replicates. Means with the same letter are not significanily different (P = 0.05).
not shown). Bacterial concentrations at time 0 were, for both strains, of around l0 s bacteria/ ml for tobacco cell co-cultures and of 8" l0 s bacteria/ml for apple cell co-cultures.
Electrolyte loss induced by mutants of E. amylovora, as compared with the wild-type and P. syringae pv~ tabaci No significant electrolyte losses were induced by the hrp- mutant, PMV 6046 (Fig. 1). PMV 6070, the dsp- mutant, induced electrolyte loss only with tobacco cells. PMV 6089, the EPS- mutant, behaved like CFBP 1430 in both interactions. On apple cells, these increases of conductivity were much slower and more limited than the increase of conductivity induced by CFBP 2106 P. syringae pv. tabaci (within 20 h, the latter induced a net loss of 70% of total electrolytes, when the formers induced a net loss of 25%). On tobacco cells, no significant differences were obtained between leakage inducing ability of CFBP 1430, PMV 6089; PMV 6070 and CFBP 2106. Data showed that electrolyte losses were induced within 5 h from time 0. Bacterial concentration at time 0 were comprised between 4" l0 s and 8" l0 s bacteria/ml of cell suspensions.
~'~"~ 100£ ' ">
50
e~ o
v
t.)
C C
I
I
I
tobacco
125
!
~t a
100 r~
"~
75
•-...
50
= 0
25 iv
!
!
i
!
O
5
l0
15
20
Time(hrs) 1 ~ . 1. Electrolyte loss induced by CFPB 1430 E. amylovora (ID, PMV 6046 (O), PMV 6070 (O), PMV 6089 03), its transposon mutants and CFPB 2106 P. syringae pv. tabaci (lX), on apple and tobacco cell suspensions. Datum points are the means of three replicates; vertical bars represent standard errors of the means. Points at 20 h followed by the same letters represent conductivities that axe not significantly different
(e = 0.05).
Bacterial populations in plant cell culture The tested bacteria, CFBP 1430 E. amylovora, its hrp- mutant PMV 6046 and CFBP 2106 P. syringae pv. tabaci, did not differ significantly in their ability to multiply in plant cell cultures (Table III). Bacterial populations remained stable within 20 h. Discussion
We have demonstrated that plant cells suspensions (tobacco and apple) express fast but differential reaction to E. amylovora. Results described here are in complete agreement with our previous data [10], describing electrolyte leakage from leaf tissue of host and non host plants of this
175 Table m .
Bacterial
populations a of CFBP
1430 E.
amylovora, PMV 6046 hrp transposon mutant and CFBP 2106 P. syringae pv. tabaci co-cultured with apple and tobacco cells Time (h) of incubation b
Apple
CFBP 1430 PMV 6046 CFBP 2106
Tobacco CFBP 1 4 3 0 PMV 6046 CFBP 2106
0
20
7.79 ±0.20d 7.99±0.22bcd 8.35 ±0.22ab
8.08-4-0.19bed 8.02-4-0.18bed 8.33 ±0.26ab
7.904-0.12cd 8.05 .,'-0.45bcd 8.25.4-0.20abc
8.24-t-0.15abc 8.14.4-0.15abc 8.45-*-0.14a
aData expressed in logl0 of total bacterial population. Means and standard deviations of three replicates. Means with the same letter are not significantly different (P = 0.05). bTiming began with incubation in the shaking water bath (see Material and Methods).
bacterium. This applies to both swiftness of the reaction and specific patterns for hypersensitive and disease reactions: faster and more severe effiux (and cell death) in incompatible than in compatible situations. On apple cells, E. amylovora induced a weak but significant leakage. This is consistent with the slow progression of the disease in planta, contrasting with the fast and intense reaction obtained either with tobacco cells or tobacco leaf infiltration, which characterize the hypersensitive reaction induced by this bacterium. In our experiments, P. syringae pv. tabaci was used as a control of incompatibility with apple cells and of compatibility with tobacco cells. It induced a typical HR on apple cells; on tobacco cells, it also often induced a reaction similar to HR. This is consistent with the type of symptoms produced by this bacterium on its host plant: leaf spots similar to local HR, experimentally induced by infiltration of a dense bacterial suspension in tobacco leaves [30] which suggested that HR and disease expression could result from the same mechanism. Our results actually show that P. syringae pv tabaci is probably not the best candidate to be used as a control for disease reaction with tobacco cells.
The use in our experiments of transposon mutants, each lacking a known (EPS) or alleged (leakage factors) pathogenicity determinant of E. amylovoraconfirmed the specificity of the reaction described above and provided further evidence on the role of these determinants. Results obtained with the hrp- and dsp- mutants of E. amylovora (no leakage with the hrp- mutant in both compatible and incompatible situations and a reaction only in compatible situation with the ~ p - mutant) are in agreement with previous work showing that the hrp cluster is involved in electrolyte leakage induction in HR, as well as in disease reaction, whereas the dsp region is involved in electrolyte leakage only in the compatible situation [10]. It has been hypothesized that the dsp region could be under the control of the hrp region, suggesting an indirect role of the hrp region in the electrolyte leakage process in compatible situations. The EPS- mutant behaved like the wild-type in its ability to induce electrolyte leakage. With apple cells, the reaction was clearly not an hypersensitive-like reaction, when compared with the electrolyte loss induced by the incompatible pathogen P. syringae pv. tabaci. This is in contradiction with the hypothesis suggesting a role of EPS of virulent E. amylovora in avoiding the hypersensitive response in host tissues [4,31]. Our results showed that, in our experimental conditions, disease reaction in the compatible situation E. amylovora/apple cells is not an HR attenuated by the presence of EPS and rather suggest a passive role of EPS in disease reaction, or at least in the electrolyte leakage process. Typical patterns of bacterial growth in compatible or incompatible situation in leaf tissues are not recovered with this bioassay. This result could be expected because of the artificial conditions (liquid medium, poor oxygenation, dilution...) and has already been described with cell suspensions [18]. This could alter the validity of this bioassay. However, the use of suitable controls (well defined mutants of pathogenicity of E. amylovora and P. syringae pv. tabacOdemonstrated the specificity of the reaction induced by E. amylovora on cell suspensions. We can therefore postulate that our experimental conditions allow the initiation of the
176
first events of a compatible or an incompatible situation, which was our aim. Results described here were obtained within 20 h of incubation. During this period of time, controis (sterile cell suspensions) showed light electrolyte loss or cell death, which became substantial after 20 h. This can be explained by the very low rate of shaking, not allowing the cells to remain in suspension. Higher rates of shaking (rotary shaker, 150 rev./min, in flasks) gave very stable controls after 48 h, even in the assay media, but did not result in any reaction of the plant cells when co-cultured with the bacteria (results not shown), thereby contrasting with previous reports
[5,18]. The correlation between cell death and electrolyte loss has been already described on tobacco cell suspensions during a hypersensitive reaction [18,32]. However, cell death assessment is the method generally used to monitor the reaction of plant cells to phytopathogenic bacteria. Our results confirmed that this method, which is time consuming and often subjective, can be successfully replaced by an objective, quick and non destructive method of measurement of extracellular conductivity, for incompatible situations as well as for compatible situations. Furthermore, the method described here showed that this conductivity can be easily determined from very small samples of cell cultures (0.5-1 ml). Co-cultures of cell suspensions with E. amylovora, associated with electrolyte leakage measurements can be considered as a fast, reliable and straightforward technique for the study of the first events associated with plant-bacteria interaction. In addition, they should provide a useful tool for the study of physiological, biochemical and molecular mechanisms of hypersensitive and disease reactions induced by this bacterium. Acknowledgements Most of this work took place at the University of Bath, UK The authors are grateful to Dr. R.M. Cooper for access to the laboratory facilities at the School of Biological Sciences (University of Bath). The authors are particularly grateful to Dr. S. Ochatt for very fruitful discussions at the origin
and during the course of this work and for reviewing the manuscript. M.N. Brisset held a fellowship BRIDGE from ECC (DG XII). References 1 T. van der Zwet and H.A. Keil, Fire bright, a bacterial disease of Rosaceous plants. USDA Agricultural Handbook, 510 (1979) 1-200. 2 R.A. Bennett and E. Billing, Capsulation and virulence in Erwinia amylovora. Ann. Appl. Biol., 89 (1978) 41-45. 3 R.A. Bennett, Evidence for two virulence determinants in the fire blight pathogen Erwinia amylovora. L C-en. Microbiol., 116 (1980) 351-356. 4 R.C. Higuett and A.L. Roberts, A possible regulatory function for bacterial outer surface components in fire blight disease. Physiol. Plant Pathol., 27 (1985) 235-243. 5 D. Youle and R.M. Cooper, Possible determinants of pathogenicity of Erwinia amylovora; evidence for an induced toxin. Acta Holt., 217 (1987) 161-166. 6 M.A. Barny, M.H. Guinebretiere, B. Marcais, E. Coissac, J.P. Paulin and J. Laurent, Cloning of a large gene cluster involved in Erwiniaamylovora CFBP 1430 virulence. Mol. Microbiol., 4 (1990) 777-786. 7 D.W. Bauer and S.V. Beer, Further characterization of an hrp gene cluster of Erwinia amyiovora. Mol. PlantMicrobe Interact., 4 (1991) 493-499. 8 F. Bernhard, K. Poetter, K. G-eider and D.L. Coplin, The rcsA gene from Erwinia amylovora: identification, nucleotide sequence and regulation of exopolysaccharide biosynthesis. Mol. Plant-Microbe Interact., 3 (1990) 429-437. 9 A. Chatterjee, W. Chun and A.K. Chatterje¢, Isolation and characterization of an rcsA-like gene of Erwinia amylovora that activates extracellular polysaccharide production in Erw/n/a species Escherichiacoli and Salmonella typhimurium. Mol. Plant-Microbe Interact., 3 0990) 144-148. 10 M.N. Brisset and J.P. Paulin, Relationships between electrolyte leakage from Pyrus communis and virulence of Erwinia amylovora. Physiol. Mol. Plant Pathol., 39 (1991) 443-453. 11 A. Torres-Cabassa and S. Gottesman, Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. J. Bacteriol., 169, (1987) 981-989. 12 A. Burkowicz and R.N. Goodman, Permeability alterations induced in apple leaves by virulent and avirulent strains of Erwinia amylovora. Phytopathology, 59 (1969) 314-318. 13 S.K. Addy, Leakage of electrolytes and phenols from apple leaves caused by virulent and avirulent strains of Erwinia amylovora. Phytopathology, 66 (1976) 1403-1405. 14 M.N. Brisset, S.J. Ochatt and J.P. Paulin, Evidence for quantitative responses during co-culture of Pyrus communis protoplasts and Erwiniaamylovora. Plant Cell Pep., 9 (1990) 272-275.
177 15 G. Feistner and C.M. Staub, 6-Thioguanine from Erwinia amylovora. Curr. Microbiol., 13 (1986) 95-101. 16 T. Schwartz, F. Bernhard, R. Theiler and K. G-eider, Diversity of the fire blight pathogen in production of dihydrophenylalanine, a virulence factor of some Erwinia amylovora strains. Phytopathology, 81 (1991) 873-878. 17 J.P. Duvick and L. Sequeira, Interaction of Pseudomonas solanacearum with suspension cultured tobacco ceils and tobacco leaf cell walls in vitro. Appl. Environ. Microbiol., 48 (1984) 199-205. 18 M.M. Atkinson, J. Huang and J.A. Knopp, Hypersensitivity of suspension-cultured tobacco cells to pathogenic bacteria. Phytopathology, 75 (1985) 1270-1274. 19 L.D. Keppler, M.M. Atkinson and C.J. Baker, Plasma membrane alteration during bacteria-induced hypersensitive reaction in tobacco suspension cells as monitored by intracellular accumulation of fluorescein. Physiol. Mol. Plant Pathol., 32 (1987) 209-219. 20 A.G. Matthysse, Interactions of P. syringae pv. phaseolicola with non host suspension culture cells, in: Proc. 3rd Int. Work. Gr. on Pseudomonas syringae pvs. Lisbon, 1-4 Sept. 1987. 21 Y. Huang, J.P. Helgeson and L. Sequeira, Isolation and purification of a factor from Pseudomonas solanacearum that induces a hypersensitive-like response in potato cells. Mol. Plant-Microbe Interact., 2 (1989) 132-138. 22 M. Rickauer, J. Fournier, M.L. Pouenat, E. BerthaIon, A. Bottin and M.T. Esquerre-Tugaye, Early changes in ethylene synthesis and lipoxygenase activity during defense induction in tobacco cells. Plant Physiol. Biochem., 28 (1990) 647-653. 23 G. Feistner, (L)-2, 5-dihydrophenylalanine from the fire
24
25
26
27
28
29 30
31
32
blight pathogen Erwinia amylovora. Phytocheraistry, 27 (1988) 3417-3422. R.M. Cooper, D. Youle, A. Katerinas and C. Fox, Pathogenicity factors in Erwinia amylovora. Acta Hortic., 273 (1990) 267-271. D.K. Willis, J.J. Rich and E.M. Hrabak, hrp genes of phytopathogenic bacteria. Mol. Plant-Microbe interact., 4 (1991) 132-138. O.L. Gamborg, R.A. Miller and K. Ojima, Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res., 50 (1968) 151-158. T. Murashige and F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15 (1962) 473-497. J.M. Widholm, The use of fluorescein diacctate and phenosafranine for determining viability of cultured plant cells. Stain Technol., 47 (1972) 189-194. J Scheffe, The Analysis of Variance, J. Wiley and Sons, London, 1959. Z. Klement, M. Hevesi and M. Sasser, Mechanism of the development of wildfire disease in susceptible tobacco plant, in: Proc. 4th Int. Conf. Plant Pathogenic Bacteria, Angers, France, 1978, 679-685. R.C. Hignett, Effects of growth conditions on the surface structures and extraceUular products of virulent and avirulent forms of Erwinia amylovora. Physiol. Mol. Plant Pathol., 32 (1988) 387-394. L.D. Keppler and C.J. Baker, O2=-initiated lipid peroxidation in a bacteria-induced hypersensitive reaction in tobacco cell suspensions, Phytopathoiogy, 79 (1989) 555-562.
171
A reliable strategy for the study of disease and hypersensitive reactions induced by Erwinia amylovora Marie-Nofille Brisset a and Jean-Pierre Paulin b aSchool of Biological Sciences, University of Bath, Bath BA2 7.4 Y ( UK) and blNRA, Station de Pathologie V~g~tale, 42, Rue Georges Morel, B.P. 57, 49071 Beaucouze (France)
(Received March 23rd, 1992; revision receivedMay 25th, 1992; accepted May 26th, 1992) Virulent strains of Erwinia amylovora (CFBP 1430), an apple pathogen and Pseudomonas syringae pv. tabaci (CFBP 2106), a tobacco pathogen, induced differentialreactions when co-cultivatedwith apple and tobacco cell suspensions, dependenton the compatibility of the interaction. Typical patterns of disease or hypersensitivereactions (slow and progressiveor fast and intense respectively) were found, specially with E. amylovora. Mortality and electrolyte loss of plant cells were correlated. The use of three transposon mutants (hrp-, dsp- and EPS- mutants) of CFBP 1430confirmed the specificityof the reaction and provides further evidence on the role of pathogenicitygenes of E. amylovora. This straightforwardexperimentalmethod describedin this paper can be proposed for further analysis of the first events associated with disease and hypersensitivereactions induced by E. amylovora. Key words: Erwinia amylovora; tobacco/appleplant cell suspension; electrolyteleakage; compatible/incompatiblesituations
Introduction Erwinia amylovora (Burrill) Winslow et al. is the causal agent of fire blight, a disease of Pomoideae, characterized by a progressive necrosis of aerial parts o f host plants and ooze production [1]. Two pathogenicity determinants are presently recognized in all virulent strains: the bacterial exopolysacchafide (EPS) [2] and another yet uncharacterized factor which could be an electrolyte leakage factor [3,4], also called 'induced toxin' [5]. Genetic studies have led to the identification of dusters of genes involved in the pathogenicity o f E. amylovora: hrp genes [6,7], dsp genes [6] and rcsA Correspondence to: Jean-Pierre Paulin, INRA, Station de Pathologie Vtgttale, 42, rue Georges Morel, B.P. 57, 49 071 Beaucouze, France Abbreviations: CFBP, Collection Fran¢aise de Baettries Phytopatho~nes; ev., cultivar; dsp, disease specific; EPS, exopolysacclmride; hrp, hypersensitive reaction pathogenicity; HR, hypersensitivereaction; MES, morpholinoethane sulfonic acid; PCV, packed cell volume; rcsA, regulation capsule synthesis A; PMV, Pathologie Moldeulalre et Vtgttale; pv., pathovar; YPA, yeast peptone agar.
genes [8,9]. Expression of the hrp cluster has been shown to be involved in electrolyte leakage induction from both host and non-host plant cells [10], while rcsA genes are involved in the regulation of EPS production by the bacteria [11]. A simple, reliable bioassay, which involves both the plant and the pathogen, is strongly needed for a better understanding of the biological mechanisms affecting the plant-pathogen interaction and also to establish a link between genetical and physiological studies and to characterize the pathogenecity determinants. Bioassays have already been described for studies on E. amylovora, using leaf discs [10,12,13], fruit discs [4], protoplasts [14] or cell suspensions [5,15,16]. Among these, the best candidate for biochemical mechanism studies could be the one using cell suspensions because it provides populations of plant cells (small aggregates or single cells) that are easily manipulated and propagated. Numerous studies were reported, mainly on the expression of the hypersensitive reaction (HR) of cell suspensions co-cultivated with several plant pathogens [17-22]. Conversely, for E.
0168-9452/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
172
amylovora, cell suspensions were used in compatible situations, for the analysis of toxic ability of the bacteria or of bacterial products [5,15,16,23,24]. However none of the described methods has allowed a reproducible production of the electrolyte leakage agent of E. amylovora or its isolation as yet. Because both disease and hypersensitive reactions are controlled by the hrp cluster of E. amylovora, as for many other phytopathogenic bacteria [25], it is of particular interest to conduct comparative experiments between compatible and incompatible situations. In this paper, we describe a straightforward and fast method of co-culture of E. amylovora with host (apple) and non-host (tobacco) cell suspensions, associated with electrolyte leakage measurements. We established that the reactions of plant cells to two bacteria, E. amylovora and Pseudomonas syringae pv. tabaci, were specific, i.e. disease-like or hypersensitivelike reactions and expressed within 20 h. The use of well-characterized transposon mutants of E. amylovora in this bioassay induced different reactions according to their insertion, or their phenotype. Material and Methods
Plant cells Cells of Nicotiana tabacum cv. Xanthi were obtained from M. Spencer and G. Warren (University of Sheffield, UK) and were grown in liquid B5 basal medium [26] with 2-4-dichlorophenoxyacetic acid (1 mg 1-1), kinetin (0.1 mg l-l), sucrose (3%), at 25°C, in 250 Erlenmeyer flasks, on a rotary shaker (150 rev./min) under fluorescent lighting (16 h photoperiod). Cells were subeultured every 7 days, by adding 20 ml of cell suspensions to 80 ml of fresh medium. Cells of Malus X domestica Borkh. cv. M26 were obtained from R. Cooper and C. Brown (University of Bath, UK) and were grown in liquid Murashige and Skoog basal medium [27] with 2-4-dichlorophenoxyacetic acid (0.5 mg l-l), kinetin (1 mg 1-1), sucrose (2%) in the same conditions as tobacco cells. Apple cells were subcultured every 7 days by adding 30 rnl of cell suspensions to 70 ml of fresh medium.
Bacterial strains Strains of E. amylovora and P. syringae pv. tabaci are listed on Table I. Bacteria were cultured on YPA medium (yeast, 3 g 1-1, bacteriological peptone, 5 g l -l, agar, 14 g 1-1), which was supplemented with chloramphenicol (20 ppm) for the transposon mutants. Cells were harvested from plates after growing for 24 h at 25°C. They were washed in the following assay medium: MES 5 mM + mannitol 3%, adjusted to pH 6 with NaOH, by centrifugation at 2000 × g for 10 min and resuspended in the assay medium to an inoculum density of - 10 l° bacteriaJml. Co-culture assays Cell suspensions were used in their exponential growth phase (4-5 days old). They were first adjusted to a concentration of 10% PCV, calculated after centrifugation of 10 ml of cell suspensions at 600 × g for 10 min in graduated tubes), with two different assay media according to the plant species. For tobacco cells, MES 5 mM + CaCl 2 0.5 mM + mannitol 30, adjusted to pH 6 with NaOH, but for apple cells, MES 5 mM + mannitol 3%, adjusted to pH 6 with NaOH were used. Cells were washed by centrifugation, at 150 × g for 3 min and resuspended in their respective assay medium. Cells suspensions were incubated for 1 h in test tubes (4.5 ml/tube) in the rotary shaker used for their growth (tubes at an angle to keep cells in suspension). They were then inoculated with 0.5 ml bacterial suspension (or 0.5 ml of assay medium for controls), giving a final concentration of approximately 109 bacteria/ml and incubated, in the same conditions as above, for 2 h. Co-cultures were then washed by centrifugation at 150 × g for 3 min, resuspended in their respective assay medium, dispensed into Eppendorf tubes (1 ml per Eppendorf, 3 replicates per co-culture) and incubated at 25°C in a shaking water bath (60 strokes/min) under constant fluorescent lighting. Cell death and electrolyte loss Viability of plant cells was determined by staining with fluorescein diacetate [28]. Results are expressed in percentage of net mortality, i.e. difference between percentage'of mortality at 20 h and percentage of mortality at time 0 (beginning of
173
TaWieI. E. amyiovoraand P. syringaepv. tabaci strains used in this study Source
Characteristics
Type
Reaction on
E. amylovora
Host
Non-host
CFBP 1430 INRA-Angers, France
Wild-type
Disease reaction
HR
+
PMV 6046a INA-PG, Paris, France
Mu transposon mutant, hrp insertion Mu transposon mutant, dsp insertion Mu transposon mutant, unknown insertionb
_c
_
+
-
HR
+
-
HR
-
Disease reaction
HR
NTd
PMV 6070a INA-PG, Paris, France PMV 6089a INA-PG, Paris, France P
EPS production
syringae, pv. CFBP 2106 INRA-Angers, France tabaci
Wild-type
* R e f . 6.
bMenggad, pers. commun. =No reaction, or no production (EPS). dNot tested.
incubation in the shaking water bath). Electrolyte loss was assessed by measuring directly in the Eppendorfs, the conductivity of the plant cell suspensions, with a conductivity cell (Tacussel XC 170), suitable for conductivity measurements in small volumes (1 ml). Before each reading, each Eppendorf was shaken to allow the resuspension of the plant cells. For experiments comparing cell death and electrolyte loss, the latter is expressed as (C20 - C0)/(CT - Co) where C2o is the conductivity at 20 h, Co the conductivity at time 0 and CT the total conductivity, obtained by freezing-unfreezing each sample. Other data are presented as the numerical differences of conductivity between bacterial treatments and sterile controls. All experiments were performed at least twice and results were statistically analysed using Seheffe's test [29], with a level of significance of 5%.
Bacterial populations in plant cell culture Aliquots (5 ~ ) were removed from the Eppendoffs containing the inoculated plant cell suspensions and dilution-plated on YPA medium to determine concentrations of viable bacteria. The
experiment was repeated once. Results were statistically analyzed as described above. Results
Mortality and electrolyte loss of tobacco and apple cells co-cultured with E. amylovora and P. syringae pv. tabaci Viability of plant cells were 82.0 ~- 4.9 and 81.0 4- 5.0 for tobacco and apple cells, respectively, at the beginning of the experiment. Controls showed a limited mortality within 20 h (Table II). A significant mortality was induced in incompatible interactions, i.e.E, amylovora/tobacco and syringae pv. tabaci/apple while this was less pronounced for compatible interactions, specially in the E. amylovora/apple interaction. Electrolyte loss appeared to be highly correlated with cell death, as shown on Table II. Total conductivities were - 5 8 0 ItS and 330 ItS for apple and tobacco cells, respectively, while they were 210 its and 190 itS, respectively at time 0. After 20 h, mortality and electrolyte loss of control cells increased significantly, particularly for tobacco cells (results
174 Table H. Mortality and electrolyte loss of apple and tobacco cell suspensions co-cultured with CFBP 1430 E. amylovora and CFBP 2106 P. syringae pv. tabaci for 20 li
20o
apple
~ a
"" 150 % Net mortality a
% Net conductivity b
Control CFBP 1430 CFBP 2106
14.8±8.2a 28.4±4.2b 66.0±8.3e
19.4±0.2a 28.44-1.3b 66.9 ± 7.0c
Tobacco Control CFBP 1430 CFBP 2106
23.9 ± 3.3a 73.8 a- 2. lc 56.9 a- 3.8b
18.4 ±2.6a 74.4 ± 8.9c 54.1 ±4.2b
Apple
aPercentage of net mortality determined as described in Material and Methods. Means and standard deviations of four replicates (total of at least 250 cells per treatment). Means with the same letter are not significantly different (P = 0.05). bpercentage of net conductivity determined as described in Material and Methods. Means and standard deviations of three replicates. Means with the same letter are not significanily different (P = 0.05).
not shown). Bacterial concentrations at time 0 were, for both strains, of around l0 s bacteria/ ml for tobacco cell co-cultures and of 8" l0 s bacteria/ml for apple cell co-cultures.
Electrolyte loss induced by mutants of E. amylovora, as compared with the wild-type and P. syringae pv~ tabaci No significant electrolyte losses were induced by the hrp- mutant, PMV 6046 (Fig. 1). PMV 6070, the dsp- mutant, induced electrolyte loss only with tobacco cells. PMV 6089, the EPS- mutant, behaved like CFBP 1430 in both interactions. On apple cells, these increases of conductivity were much slower and more limited than the increase of conductivity induced by CFBP 2106 P. syringae pv. tabaci (within 20 h, the latter induced a net loss of 70% of total electrolytes, when the formers induced a net loss of 25%). On tobacco cells, no significant differences were obtained between leakage inducing ability of CFBP 1430, PMV 6089; PMV 6070 and CFBP 2106. Data showed that electrolyte losses were induced within 5 h from time 0. Bacterial concentration at time 0 were comprised between 4" l0 s and 8" l0 s bacteria/ml of cell suspensions.
~'~"~ 100£ ' ">
50
e~ o
v
t.)
C C
I
I
I
tobacco
125
!
~t a
100 r~
"~
75
•-...
50
= 0
25 iv
!
!
i
!
O
5
l0
15
20
Time(hrs) 1 ~ . 1. Electrolyte loss induced by CFPB 1430 E. amylovora (ID, PMV 6046 (O), PMV 6070 (O), PMV 6089 03), its transposon mutants and CFPB 2106 P. syringae pv. tabaci (lX), on apple and tobacco cell suspensions. Datum points are the means of three replicates; vertical bars represent standard errors of the means. Points at 20 h followed by the same letters represent conductivities that axe not significantly different
(e = 0.05).
Bacterial populations in plant cell culture The tested bacteria, CFBP 1430 E. amylovora, its hrp- mutant PMV 6046 and CFBP 2106 P. syringae pv. tabaci, did not differ significantly in their ability to multiply in plant cell cultures (Table III). Bacterial populations remained stable within 20 h. Discussion
We have demonstrated that plant cells suspensions (tobacco and apple) express fast but differential reaction to E. amylovora. Results described here are in complete agreement with our previous data [10], describing electrolyte leakage from leaf tissue of host and non host plants of this
175 Table m .
Bacterial
populations a of CFBP
1430 E.
amylovora, PMV 6046 hrp transposon mutant and CFBP 2106 P. syringae pv. tabaci co-cultured with apple and tobacco cells Time (h) of incubation b
Apple
CFBP 1430 PMV 6046 CFBP 2106
Tobacco CFBP 1 4 3 0 PMV 6046 CFBP 2106
0
20
7.79 ±0.20d 7.99±0.22bcd 8.35 ±0.22ab
8.08-4-0.19bed 8.02-4-0.18bed 8.33 ±0.26ab
7.904-0.12cd 8.05 .,'-0.45bcd 8.25.4-0.20abc
8.24-t-0.15abc 8.14.4-0.15abc 8.45-*-0.14a
aData expressed in logl0 of total bacterial population. Means and standard deviations of three replicates. Means with the same letter are not significantly different (P = 0.05). bTiming began with incubation in the shaking water bath (see Material and Methods).
bacterium. This applies to both swiftness of the reaction and specific patterns for hypersensitive and disease reactions: faster and more severe effiux (and cell death) in incompatible than in compatible situations. On apple cells, E. amylovora induced a weak but significant leakage. This is consistent with the slow progression of the disease in planta, contrasting with the fast and intense reaction obtained either with tobacco cells or tobacco leaf infiltration, which characterize the hypersensitive reaction induced by this bacterium. In our experiments, P. syringae pv. tabaci was used as a control of incompatibility with apple cells and of compatibility with tobacco cells. It induced a typical HR on apple cells; on tobacco cells, it also often induced a reaction similar to HR. This is consistent with the type of symptoms produced by this bacterium on its host plant: leaf spots similar to local HR, experimentally induced by infiltration of a dense bacterial suspension in tobacco leaves [30] which suggested that HR and disease expression could result from the same mechanism. Our results actually show that P. syringae pv tabaci is probably not the best candidate to be used as a control for disease reaction with tobacco cells.
The use in our experiments of transposon mutants, each lacking a known (EPS) or alleged (leakage factors) pathogenicity determinant of E. amylovoraconfirmed the specificity of the reaction described above and provided further evidence on the role of these determinants. Results obtained with the hrp- and dsp- mutants of E. amylovora (no leakage with the hrp- mutant in both compatible and incompatible situations and a reaction only in compatible situation with the ~ p - mutant) are in agreement with previous work showing that the hrp cluster is involved in electrolyte leakage induction in HR, as well as in disease reaction, whereas the dsp region is involved in electrolyte leakage only in the compatible situation [10]. It has been hypothesized that the dsp region could be under the control of the hrp region, suggesting an indirect role of the hrp region in the electrolyte leakage process in compatible situations. The EPS- mutant behaved like the wild-type in its ability to induce electrolyte leakage. With apple cells, the reaction was clearly not an hypersensitive-like reaction, when compared with the electrolyte loss induced by the incompatible pathogen P. syringae pv. tabaci. This is in contradiction with the hypothesis suggesting a role of EPS of virulent E. amylovora in avoiding the hypersensitive response in host tissues [4,31]. Our results showed that, in our experimental conditions, disease reaction in the compatible situation E. amylovora/apple cells is not an HR attenuated by the presence of EPS and rather suggest a passive role of EPS in disease reaction, or at least in the electrolyte leakage process. Typical patterns of bacterial growth in compatible or incompatible situation in leaf tissues are not recovered with this bioassay. This result could be expected because of the artificial conditions (liquid medium, poor oxygenation, dilution...) and has already been described with cell suspensions [18]. This could alter the validity of this bioassay. However, the use of suitable controls (well defined mutants of pathogenicity of E. amylovora and P. syringae pv. tabacOdemonstrated the specificity of the reaction induced by E. amylovora on cell suspensions. We can therefore postulate that our experimental conditions allow the initiation of the
176
first events of a compatible or an incompatible situation, which was our aim. Results described here were obtained within 20 h of incubation. During this period of time, controis (sterile cell suspensions) showed light electrolyte loss or cell death, which became substantial after 20 h. This can be explained by the very low rate of shaking, not allowing the cells to remain in suspension. Higher rates of shaking (rotary shaker, 150 rev./min, in flasks) gave very stable controls after 48 h, even in the assay media, but did not result in any reaction of the plant cells when co-cultured with the bacteria (results not shown), thereby contrasting with previous reports
[5,18]. The correlation between cell death and electrolyte loss has been already described on tobacco cell suspensions during a hypersensitive reaction [18,32]. However, cell death assessment is the method generally used to monitor the reaction of plant cells to phytopathogenic bacteria. Our results confirmed that this method, which is time consuming and often subjective, can be successfully replaced by an objective, quick and non destructive method of measurement of extracellular conductivity, for incompatible situations as well as for compatible situations. Furthermore, the method described here showed that this conductivity can be easily determined from very small samples of cell cultures (0.5-1 ml). Co-cultures of cell suspensions with E. amylovora, associated with electrolyte leakage measurements can be considered as a fast, reliable and straightforward technique for the study of the first events associated with plant-bacteria interaction. In addition, they should provide a useful tool for the study of physiological, biochemical and molecular mechanisms of hypersensitive and disease reactions induced by this bacterium. Acknowledgements Most of this work took place at the University of Bath, UK The authors are grateful to Dr. R.M. Cooper for access to the laboratory facilities at the School of Biological Sciences (University of Bath). The authors are particularly grateful to Dr. S. Ochatt for very fruitful discussions at the origin
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