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Agglutination of pseudomonad cells by plant products
Physiological
Plant P4fholog3
Agglutination A. J. ANDERSON
(1979)
15, 149-159
of Pseudomonad cells by plant products and C. JASALAVICH
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 (Acceptedfor
publication
January
U.S.A.
1979)
Cells of plant pathogenic and saprophytic species of pseudomonads were differentially agglutinated by preparations from Dark Red kidney bean leaves. Water homogenates of bean leaves caused agglutination of the saprophytic pseudomonads Pseudomonas juorescm and P. putida but no agglutination of the plant pathogens P. phasGolicola or P. gringae. Agglutination with P. tubaci was variable. The agglutinin was detected in both pellet and supernatant after centrifugation of the water homogenate at 6000 g for 10 min. This agglutinin was protease and trypsin stable, was insensitive to salt except above 0.15 M and required a divalent metal ion for activity. An agglutinin with apparently different properties was released by 0.3 M NaCl treatment of a crude preparation of isolated bean leaf cell walls. This wall-released agglutinin caused a more variable agglutination of both saprophytic and pathogenic pseudomonads. The wall agglutinin was protease and trypsin labile and was inhibited by salt or metal ions above 0.02 M. Agglutination of pseudomonad cells by characterized plant components was also demonstrated. Pectin and galacturonic acid but not other plant cell wall polysaccharides produced agglutination. The plant lectins concanavalin A, wheat germ agglutinin and phytohemagglutinin but not ricin caused agglutination of the bacterial cells.
INTRODUCTION Recent electron microscopy studies [S, 10, 1.5, 17, 201 have demonstrated that a physical binding of bacterial cells to the cell walls of a non-host plant may be a key event in a resistant interaction. It has been proposed that the bacterial cells become immobilized at their binding site through an envelopment process which involves an active restructuring of the plant cell wall surface [S, 91. This binding and encapsulating process has not been observed in a susceptible interaction, when the virulent bacteria remain free and multiply in the intercellular space. Consequently, a recognition process must exist which permits a plant to differentiate between an avirulent or virulent bacterial challenge. Sequeira et al. [16, 17, 181 have suggested that differential recognition of avirulent and virulent bacteria may involve a lectin component in the plant. A lectin isolated from potato tubers was observed to agglutinate only strains of pseudomonads that were avirulent in potato. The virulent strains were not agglutinated [18]. Thus, a lectin may be involved in establishing the binding of avirulent bacteria to the plant cell wall. Involvement of a lectin in regulating the specificity of binding bacteria has also been suggested for the interaction of species of nitrogen-fixing Rhizobia with their hosts [3, 51. However, here it is the compatible bacteria that are recognized and bound by the plant during the process of establishing the symbiotic relationship. 0048-4059/79/050149
+ 11$02.00/O
@ 1979 Academic
Press Inc.
(London)
Limited
150
A. J. Anderson
and
C. Jasalavich
The demonstration that surface interactions between plant and bacteria may play a key role in the specificity of the responses prompted these studies on the ability of plant products to agglutinate cells of pseudomonad species. Lectins and model plant cell wall polymers were examined for their potential to agglutinate cells of nonpathogenic saprophytes as well as cells of the plant pathogens. A search for agglutinins in extracts of bean leaves also was initiated since the immobilization response has been observed to occur with pseudomonads in this tissue [ 1.5, 201. MATERIALS
AND
METHODS
Culture of bacteria Bacterial isolates were obtained from the stock cultures of L. W. Moore and R. H. Cameron of Oregon State University. Pseudomonas tabaci, P. syringae and P. phaseolicola were used to represent plant pathogen species. Pseudomonas putida and P. juorescens were selected as examples of saprophytic species. The virulence of P. tabaci and of P. phaseolicola were confirmed periodically by inoculation into their hosts, tobacco and bean (Phaseolus vulgaris) respectively. The cultures were maintained at 22 “C by monthly transfer of cells onto a rich medium containing in 1 1: 10.5 g K,HPO,, 4.5 g KH,PO,, O-5 g sodium citrate, 1-O g (NH,)sSO,, 10 g nutrient broth (Difco Laboratories, Detroit, Michigan 48232, U.S.A.), 6 g yeast extract (Difco) 0.25 g MgSO,.7H,O, 2 g sucrose and 15 g agar (Difco). The cells used for agglutination assay were grown in the medium described above prepared without agar. The liquid medium was inoculated by loop transfer from the cultures growing on solid media and was shaken on a gyrating incubator at 24 “C. After 15, 36 or 40 h of growth, the cells were harvested by centrifugation at 10 000 g and the supernatants were discarded. The cell pellet was resuspended gently in distilled water and the suspension recentrifuged at 10 000 g. The washed pellet was resuspended in sterile distilled water at approximately 10’0 cells per ml. The cell concentration was determined by dilution plating and counting the viable cell colonies. Heat-killed bacteria were prepared by treatment of the cell suspensions at 121 “C for 20 min. Heated cells were pelleted by centrifugation and washed twice with water before suspension for bioassay. This procedure was an attempt to remove materials possibly solubilized by heating that may otherwise interfere in the agglutination response. Source of agglutinin
preparations
The lectin concanavalin A was obtained commercially from Sigma Chemical Company, P.O. Box 14508, St Louis, Missouri 63178, U.S.A., and phytohemaglutinin from Difco Laboratories. Ricin was isolated from castor bean and purified by following the procedures of Nicolson & Blaustein [I4]. Wheat germ agglutinin, purified to homogeneity, was a gift from B. Triplett of this department. The lectins were dissolved using distilled water to give a preparation of 0.5 mg protein per ml. Plant cell wall polymers, mannan, galactan, xylan, pectin and the uranic acid, galacturonic acid, were purchased from Sigma Chemical Company. Two methods were used to obtain bean (Phaseolus vulgaris) leaf extracts that were tested for agglutinin activity. One method involved water extraction and the second
Agglutination
of pseudomonad
cells
151
required salt treatment of a crude preparation of bean leaf cell walls. Dark Red Kidney beans were grown in vermiculite for 14 days at 24 “C under a light regime involving 10 h dark and 14 h of illumination. The leaves were homogenized in 100 g quantities in 200 ml 4 “C distilled water for 1 min in a Waring blender. The homogenate was filtered through a coarse sintered glass funnel to separate the insoluble material from the water extract. The filtrate was centrifuged first at 6000 g and then at 20 000 g for 10 min. The 6000 g and 20 000 g pellets were resuspended in distilled water (30 ml) and stored at 4 “C, as was the final 20 000 g supernatant. The dispersed pellets and the 20 000 g supernatant were used as crude extracts in agglutination assays. The second type of agglutinin preparation involved salt treatment of the insoluble material prepared by the above procedures. The insoluble material retained on the sintered glass funnels was washed extensively with 4 “C water until the washings were no longer green. The material was then resuspended in 0.3 M NaCl (200 ml per 100 g original leaf material) for 10 min at 22 “C. The suspension was filtered on a sintered glass funnel, and the filtrate was centrifuged at 20 000 g. The 20 000 g supernatant was concentrated by one of two procedures. One method involved an ultrafilter apparatus equipped with a Pellicon filter (Millipore Corporation, Bedford, Massachusetts 01730) that restricted flow of macromolecules of lo4 mol. wt. The alternate procedure involved rotary evaporation at 38 “C. The concentrated preparations were desalted by passage through a Bio-Gel P-10 (mesh 50-100) (Bio-Rad Laboratories, 2200 Wright Avenue, Richmond, CA 94804, U.S.A.) molecular sizing column (23 x 2.1 cm) using distilled water as the eluent, or by dialysis at 4 “C against distilled water. These desalted preparations were used as the source of the “salt extracted” agglutinin. Calorimetric analyses were used to determine the quantity of protein, hexose and uranic acid in the leaf agglutinin preparations [Z, S, 121. The analyses were standardized by using glucose (Sigma Chemical Company) for hexose, galacturonic acid (Sigma Chemical Company) for uranic acid and bovine serum albumen (Sigma Chemical Company) for protein. Assay for agglutinin
actiuity
Agglutinin activity was assayed by the addition of 100 ~1 of the bacterial suspension to 0.5 ml of a solution containing potential agglutinin activity or, for controls, 0.5 ml water. Assays of the water-extracted bean leaf agglutinin were supplemented with lo4 M MgCl,. Agglutination was judged by visual observation after 15 min at 22 “C. The activity was scored using a scale ranging from + + + + + for a flocculent precipitation of the bacterial cells to + where only faint granulation in the bacterial suspension was visible. One unit of agglutinin activity is defined as the amount in 500 ~1 required to give a + granulation of the appropriate pseudomonad cells under the assay conditions described above. Various sugars,used to attempt to prevent agglutination activity were obtained from the Sigma Chemical Company. Purification
of th,ewater-extracted
agglutinin
The agglutinin activity present in the water extract of bean leaves was partially purified by utilizing the ability of the factor to bind to bacterial cells only in the
A. J. Anderson
152
and
C. Jasalavich
were presence of Mgs+. Ion exchange and molecular sizing gel chromatography subsequently used for further purification. Two hundred ml of the 6000 g supernatant was mixed with O-5 g (wet wt) of 15 h harvested P. putidu cells at 24 “C for 15 min in the presence of lOa M MgCl,. The suspension was centrifuged at 6000 g for 10 min and the supernatant discarded. The pellet was resuspended in 200 ml distilled water, the suspension recentrifuged and the supernatant collected. The pellet was resuspended in distilled water and the resulting suspension recentrifuged at 6000 g to yield a second supernatant fraction, which was combined with the first. The combined supernatants were mixed with 20 ml of DEAE A-50-120 Sephadex resin (Sigma Chemical Company), equilibrated at pH 5.2 with 10 mM sodium acetate, and the slurry filtered through a sintered glass funnel. The resin was washed with 50 ml distilled water and the washings combined with the first filtrate. The volume of these combined filtrates was reduced to 10 ml in an ultrafilter apparatus containing a Pellicon membrane which restricted molecules of size greater than 104. A 2 ml sample of the concentrated preparation was applied to a Bio-Gel P-60 column (36 x 2 cm) equilibrated with O-1 M NaCI. Fractions of 5 ml were collected by eluting the column with 0.1 M NaCl and each fraction tested for agglutinin activity. The void volume of the column was determined by the position of elution of Blue Dextran (Sigma Chemical Company) and the included volume by the elution position of glucose. The glucose was detected by the anthrone calorimetric method
Determination
of the stability
of agglutinin
preparations
to proteolytic
activity
The stability of agglutinin preparations to proteolytic activity was determined using immobilized derivatives of trypsin and protease obtained from the Sigma Chemical Company. Fifty mg of distilled water-washed resins onto which the proteases were attached were added to 1.0 ml samples of the agglutinin preparations. The mixture was incubated for 12 to 48 h at 30 “C, filtered and the filtrate assayed for agglutinin activity. Control samples of agglutinin containing no protease were incubated to verify that the agglutinin alone was stable to these conditions. RESULTS
Agglutination
of pseudomonad cells by lectins
The characterized lectins [19] wheat germ agglutinin, concanavalin A, and phytohemagglutinin caused differential agglutination of the pathogenic and non-pathogenic species of pseudomonads examined (Table 1). Agglutination was prevented when O-075 M concentrations of N acetylglucosamine for wheat germ agglutinin, x-methyl glucoside for concanavalin A and galactosamine for phytohemagglutinin were included in the assay. Using cells harvested from 36 h cultures, wheat germ agglutinin agglutinated all the pseudomonad species and with phytohemagglutinin all species except P. putida or P. tabaci showed agglutination. A different agglutination pattern was observed with concanavalin A when cells from 15 or 36 h cultures were used. With 36 h cultured cells all cell types showed agglutination, although agglutination of P. putida and P. tabaci was weak. When 15 h cultures were used, cells of P. putida and P. phaselicola agglutinated strongly, P. fluorescent was not agglutinated
Agglutination
of pseudomonad
cells
153
and only weak agglutination of P. syringae was observed. No agglutination was observed with cells of any species using ricin as the lectin, or in the controls where no lectin was added. TABLE Agglutination
1
of @mdomonad Agglutinationa
Lectin
P. jluorescens
Ricin Wheat germ agglutinin Phytohemagglutinin Concanavalin A Concanavalin A
+++ ++++ +++ -0
cells by lectins of cell9
of the pseudomonad
P. putida
P. phaseolicola
-
-
-I-++ -I-+/++++c
+++++ +I++++ ++++O
species
P. syringae
P. tabaci
+++ ++ ++++ +c
+++ ++/+fC
a The conditions for the agglutination assay were as described in Materials and Methods. The absence of agglutination is denoted by a - symbol. When varying degrees of agglutination were observed using different preparations of cells of the same species the range is shown by including the symbol / in the table. Data are with pseudomonad cells harvested at five different cultures. b Pseudomonad cells were harvested from 36 h cultures except for those marked with footnote G. c Fifteen h cultures used.
Agglutination of cells by plant cell wall polysaccharides Mannan, xylan and galactan solutions at 0.05% (w/v) gave no agglutination of any pseudomonad cells. However, pectin (0.05% w/v) and galacturonic acid solutions caused the agglutination of both the pathogenic and saprophytic pseudomonad species (Table 2). Variation in the species agglutinated was observed between 15 and 40 h cultured cells, especially for P. phaseolicola and P. syringae. Generally the TABLIZ Agglutination
of fiseudomanad
2
cells by a range of concentrations Agglutination5
Pseudomonad
species
15 h cells P. j¶uorcscens P. putida P. phaseolicola P. syringae P. tabaci 40 h cells P. jluorescens P. jmtida P. $aseolicola P. syringae P. tabaci a The agglutination Table 1. b Final concentration
assay
by galacturonic
10-l
10-a
++++ ++ I/+++ +++ -I++++ ++ +++ ++++ +++ was conducted
of galacturonic
acid
of galacturonic
in reaction
(M) b
10-s
+++ + I,++ ++, +/+++ ++++ +++ +
as described
acid
mixture.
lo-’
-Iz -/+ -++
in Materials
aicd
z -and
Methods
and
A. J. Anderson
154
and C. Jasalavich
older cells were more agglutinable, although in the course of replications of these studies exceptions to the pattern in Table 2 have been observed. For example, contrary to the usual results, in one preparation out of five, 40 h P.fluorescem cells and 15 h P. @gae preparations failed to agglutinate in 10-l and 10e2 M galacturonic acid. Agglutination
of cells by bean leaf extracts
by water-extracted preparations. Agglutinin activity for pseudomonad cells was detected in the 6000 g and 20 000 g supernatants from the water homogenate of the bean leaves. Activity was also detected in the 20 000 g pellet and in the 6000 g pellet. Each of these supernatants and suspensions of pelleted material caused agglutination of 15 h cultured cells of P.Juorescens and P. putida, whereas cells of the plant pathogenic species were not agglutinated (Table 3). With 40 h cultures, cells
Agglutination
TABLE
Agglutination
of @mhmad
Agglutinationa Pseudomonad P. P. P. P. P.
fhoremns putida &aseolicola Syrngae taban
species
3
cells by mule water extracts
of bean &af tissue
of cells harvested 15h
++++ +++++
from 40h
cultures
at
+++ +-I-++ ++
o. The agglutination assays were conducted and scored as described in Materials and Methods and Table 1. Data are compiled from at least five replicates. The water extracts were prepared from bean leaves by the procedure described in Materials and Methods and consisted of suspensions of 6000 g and 20 000 g pelleted material as well as 6000 g and 20 000 g supernatants.
of P.juorescens and P. putida were agglutinated and there was also a weak agglutination of some preparations of P. tabaci. No reaction with P. syringae or P. phaseolicola (Table 3) was observed. Bacterial cells that were treated at 121 “C for 20 min were not agglutinated. The agglutination response was not prevented by the inclusion in the bioassay of 0.05 M concentrations of rhamnose, fucose, xylose, arabinose, ribose, deoxyribose, deoxyglucose, glucose, mannose, galactose, galactosamine, glucosamine, r+acetyl glucosamine, and a- or P-methyl glucoside. The activity present in the 6000 g supernatant was selected to conduct further studies. The agglutination of P. fluorescens and P. putida was dependent on the presence of a divalent metal ion. Passage of the crude agglutinin preparation through CM Sephadex caused a loss in activity which was restored by the addition of MgCl,, MgSO,, CaCl,, MnSO, or MnCl, at a concentration lOA M. Consequently lOA MgCl, was routinely added to all assays of this agglutinin preparation. The water-extracted agglutinin was partially purified by using a technique that depended upon absorption of the agglutinin to P. putida cells in the presence of MgCl, and its release into solution in the absence of MgCl,. The supernatant obtained from a control where P. putida cells were treated identically except for using
Agglutination
of pseudomonad
cells
155
water instead of the leaf extract, did not possessany agglutinin activity. The agglutinin obtained by the bacterial cell affinity technique was purified further by passage without absorption through DEAE Sephadex equilibrated in 10 mM sodium acetate, pH 5.2. The preparation partially purified by this procedure required (2 & 1) pg protein for one unit of agglutinin activity with P. putida cells. These preparations contained protein (220 pg ml-l) as well as hexose (23 pg ml-l) and uranic acid (2 [*g ml-l). The agglutinin activity in this partially purified preparation was stable to treatment with protease or trypsin and to heating at 121 “C for 15 min. The agglutinin was inactivated if the concentration of NaCl in the reaction mixture exceeded 0.15 M. The agglutinin eluted in the void fractions of Bio-Gel P-10 and Bio-Gel P-60 columns. Agglutination by salt extracts of crude bean leaf walls. Extracts prepared by salt treatment of the insoluble material from leaf homogenates possessed an agglutinin activity for both saprophytic and plant pathogenic pseudomonads (Table 4). A minimum TABLET Agglutination
of @uahonad
ccl12 by a 0.3 M .NaCl extra& Agglutination0
Pseudomonad P. P. P. P. P.
J7uorescens put&l &zscolicola syringae kzbaci
of crwh isolated bean leaf cell walls
of cells harvested 15h
species
-I++ -I-l-I+ ++
from 4Qh
cultures
at
++ ++ -I+++ +++ ++++
‘The agglutination assays were conducted and scored as described in Materials and Methods and Table 1. The salt extract of crude isolated bean leaf cell walls was prepared as described in Materials and Methods. Data are compiled from at least five replicated experiments.
concentration of O-3 M NaCl was required to extract the agglutinin from the crude bean leaf cell wall material. The agglutinin activity was detected only after the extracts were desalted by dialysis or by elution through a B&Gel P-10 column, when the activity was detected in the void fractions. Concentrations of NaCl greater than O-02 M or metal ions, Mg s+, Gas+ or Mns+ at a concentration of 10”’ M in the reaction mixture, inhibited agglutination. The agglutination activity in the B&Gel P-10 purified material was eliminated by protease or trypsin treatment. Agglutination was not inhibited by inclusion in the bioassay of 0.05 M concentrations of the sugars previously listed for the water-agglutinin assays. The species of pseudomonads agglutinated by this salt-extracted agglutinin showed variation between cells grown at different times. The pattern of agglutination genertilly obtained is summarized in Table 4 but as with agglutinatidn by galacturonic acid exceptions have been observed. Agglutination of 40 h cells of all cell types has been observed although in one preparation among five, the P. phaseolicola, cells were non-agglutinable. With 15 h cultures P. tuba& cells were routinely agglutinated and a weaker and more variable agglutination of P. syringae, P. .@mmm and P. putida occurred. The preparations of agglutinin present in the void fractions
156
A. J. Anderson
and
from the Bio-Gel P-10 column possessed protein (65 pg protein per ml) hexose (38 pg ml-l) and uranic acid (5 pg ml-l). These preparations 15( + 5) pg protein for 1 unit of agglutinin activity with P. tabuci cells.
C. Jasalavich
as well as required
CONCLUSIONS
Differential agglutination of both plant pathogenic and saprophytic pseudomonad cells occurred upon treatment with the characterized plant proteins, lectins, as well as with the plant cell wall components, pectin and galacturonic acid. Agglutination was also observed with as yet uncharacterized components present in extracts of bean leaves, a tissue in which immobilization of avirulent pseudomonads has been demonstrated [20]. Variation in the agglutinability of bacterial cells of the same species between different cell preparations was observed with each of the agglutinin preparations. This variation suggests that the surface physiology of the bacteria is changeable and sensitive to culture conditions. The age at which the cells were harvested and the treatment of the cells between the time of harvest and the agglutination assay are areas where we have observed variability. Also, our agglutination studies with the bean lectin phytohemagglutinin differ from those of Sing & Schroth [20]. Phytohemagglutinin agglutinated cells of our isolates of both saprophytic and plant pathogenic pseudomonads including P. phaseolicola, whereas Sing and Schroth [20] report only agglutination of the saprophyte P. putida and not the plant pathogens P. tomato or P. phareolicola. A possible explanation could be differences in the techniques for culturing the bacteria and in the conditions for agglutinin assay. The importance of culture conditions in the phenomenon of bacterial agglutination has also been reported by Bauer et al. [I]. These workers showed that culture age caused variability of the agglutination response of Rhizobia with soybean lectin. The agglutination of the pseudomonad cells by plant lectins can be explained by the ability of the lectin to recognize specific carbohydrate residues on the bacterial cell surface. Concanavalin A is known to bind to ol-mannosyl or a-glucosyl units [19], wheat germ agglutinin to N-acetylglucosamine oligomers [29] and phytohemagglutinin to galactosamine [19]. The observed inhibition of pseudomonad cell agglutination when the appropriate sugar was present along with the lectin, supports the idea that the agglutination involved the lectin recognition of bacterial cell surface carbohydrates. Indeed, the presence of amino sugars and glucose residues on cellurs face structures of pathogenic and non-pathogenic pseudomonad species has been demonstrated in our laboratory (unpublished observations). A lack of detectable galactose on the pseudomonad surface structures (unpublished observations) is consistent with the failure of ricin, a lectin that binds to galactose [19], to agglutinate any of the pseudomonad cells tested. Although these lectins are demonstrated to recognize the bacterial cells, their significance to a plant-pathogen relationship is uncertain. The lectins used in these studies were extracted from the seed of the appropriate plant: jack bean for concanavalin A, red kidney bean for phytohemagglutinin, wheat for wheat germ agglutinin and castor bean for ricin. Thus the lectins may not be present in the tissues that are challenged by the bacteria. Also, concanavalin A and phytohemagglutinin are believed to be located in the cytoplasm and not extracellularly [4]. Consequently, they would not be available to recognize the invading bacteria
Agglutination
of pseudomonad
cells
157
in the intercellular space unless plant cell damage occurs that allows lectin movement. Another possibility is that the leaf cell wall possesses its own lectin components. Sequeira et al. [16, 181 have cited preliminary evidence for such existence of lectin activity associated with cell walls of tobacco leaf tissue. The demonstration (Table 2) that the plant cell wall component, pectin, and its monomer, galacturonic acid, have agglutinin activity suggests that plant cell walls could bind both pathogenic and non-pathogenic pseudomonad species. Indeed, Lippincott &Lippincot have stated that thepecticcomponentisresponsibleforbindingt Agrobacterium cells to plant cell walls [11]. It is possible that preliminary binding of bacterial cells to plant cell walls is initiated by the pectin component and that secondary events, such as lectin interaction, then occur which determine whether or not the bacteria remain bound. The failure of virulent bacteria to bind to the plant cell wall could lie in their production of acidic components that negate the possible attractant effect of pectin. The existence of acidic structures in extracellular polysaccharides has been documented [13] for other bacterial species. Also, some pathogens such as P. phaseolicola [7] produce toxins that are negatively charged. Thus the secretion of a charged toxin may prevent the bacteria from adhering to the plant cell wall in the susceptible interaction. The possibility that recognition and binding of bacterial cells could involve factors in addition to pectin is supported by our observations of agglutinin activities detected in bean leaf extracts. The properties of the bean leaf agglutinin preparations are distinct from agglutination caused by pectin (Table 5). Agglutination by leaf TABLE A comparison
of the properties
5
of the agglutination
of pseuahonad cells by galacturonic water- and salt-extracted bean leaf agglutinins Agglutinin
Property (1) Effect of MgCl,, 10-s r.sb (2) Inhibition by O-2 Y NaClb Inhibition by 0.02 M NaClb (3) Effect of proteasec or trypsin (4) Agglutination of heat-killedd bacterial cells
Galacturonic
acid None None None None Active
(lOma Mu)
Water Required Inhibited None None None
acid, and the
source” extract
(bean
leaf)
Salt extract Inhibited Inhibited Inhibited Labile None
0 The agglutinins were assayed and prepared as described in Materials and Methods. The 6000 g supernatant was used as the water-extracted agglutinin. Cells from 40 h cultures of P. tabaci were used for the assays of the salt-extracted agglutinin and galacturonic acid. Cells from 40 h cultures of P. putida were used for assay of the water-extracted agglutinin and galacturonic acid. 6 The final concentration in the reaction mixture. E Treatment with enzymes followed the procedures given in Materials and Methods. d Cells of P. tab& and P. putida were treated at 121 “C for 20 min as described in Materials and Methods.
preparations and by galacturonic acid differ in sensitivity to salt and metal ions, in proteolytic stability, and in the agglutination of heat-killed bacterial cells (Table 5). Also the concentration of uranic acid in the leaf preparations (less than IO4 M) is insufficient to account for the agglutinin activities. The agglutinins present in these
158
A. J. Anderson
and
C. Jasalavich
water and salt extracts also appear to be distinct factors. The agglutinins differ in proteolytic stability and sensitivity to inorganic ions (Table 5) as well as in the species of pseudomonad cells agglutinated (Tables 3 and 4). Both of the leaf agglutinins appear to be macromolecular because they eluted in the void of a Bio-Gel P-10 column and failed to pass through an ultrafilter which restricted molecules of size greater than lo* daltons. The mode of action of the leaf agglutinins is speculative. It is possible that like lectins the leaf agglutinins are recognizing specific surface polysaccharides. The fact that no simple sugar has yet been shown to prevent the activity of the leaf agglutinins could be due to their requirement of a carbohydrate structure more complex than a monomer for recognition. Although the water-extracted agglutinin is heat and protease stable, these properties are in common with plant “3-1ectin” glycopeptide complexes that have an affinity for p-linked sugar residues [4]. The differential agglutination of the pseudomonad species observed with both leaf preparations suggests that some type of specific recognition event is occurring. The failure of the heatkilled bacterial cells to agglutinate with either leaf preparation also suggests that there is a critical recognition site. The variability of agglutination with age of the bacterial cells could be explained by a transient nature of the recognition site. The differential agglutination of pseudomonad cells with the leaf agglutinins is interesting. The preferential agglutination of saprophytic pseudomonads by the water-extracted material complements the immobilization ofP.pzctidu but not P. tomato reported by Sing & Schroth [ZO]. Other agglutinins, perhaps the salt-extracted agglutinin, could recognize plant pathogenic pseudomonads. Indeed, P. phaseolicola has been observed to bind to cell walls of a resistant bean variety [15]. Thus, although the agglutinins we have extracted have not been demonstrated to be lectins, it is intriguing that these activities are present in bean leaf tissue which displays an immobilization response.
REFERENCES 1. BHWANESWARI, T. V., PUEPPKE, S. G. & BAUER, W. D. (1977). The role of lectins in plantmicroorganism interactions 1. Binding of soybean lectin to Rhizobia. Plant Physioloo 60,486-491. 2. BITTER, T. & Mum, H. M. (1962). A modified uranic acid carbazole reaction. Analytical Bioch8mistry 4, 330-334. 3. BOHLOOL, B. B. & SCHMIDT, E. L. (1974). Lectins: a possible basis for specificity in the Rhizobium legume root nodule symbiosis. Science 185, 269-27 1. 4. CLARKE, A. E., KNOX, R. B. & JERMYN, M. A. (1975). Localization of lectins in legume cotyledons. Journal of Cell Science 19, 157-167. 5. DAZZO, F. B. & HUBBELL, D. H. (1975). Cross reactive antigens and lectins as determinants of symbiotic specificity in the Rhizobiam-clover association. Applied Microbiology 30, 1017-1033. 6. DISCHE, 2. (1962). Color reactions of carbohydrates. In Methods in Carbohydrate Chemistry, Vol. 1, Ed. by R. L. Whistler & M. L. Wolfrom, p. 479. Academic Press, New York. 7. GNANAMANICKAM, S. S. & PAT~L, S. S. (1976). Bacterial growth, toxin production and levels of ornithine carbamoyl transferase in resistant and susceptible cuhivars of bean inoculated with Pseudomonas phaseolicola. Phytopathology 66, 290-294. 8. (HEADMAN, R. N., HUANC, P. Y. & WHITE, J. A. (1976). Ultrastructural evidence for immobilization of an incompatible bacterium, Pseudomonaspi.ti, in tobacco leaf tissue. Phytopathology 66,754764. 9. GRAHAM, T. L., SEQUZIRA, L. & HUANC, T. S. R. (1977). Bacterial lipopolysaccharides as inducers of disease resistance in tobacco. A&.&d and Enuironmmtal Microbiology 34, 424-432. 10. HUANG, P. Y., HUANG, J. S. & GOODMAN, R. N. (1975). Resistance mechanism of apple shoots to an avirulent strain of Enuinia amylowa. Physiological Plant Pathology 6, 283-287.
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cells
159
11. LIPPINCOTI-, B. B. & LIPPINCOTT, J. A. (1977). Nature and specificity of the bacterium-host attachment in Agrobacterium infection. In Cell Wall Biochemistry Related tu S@$icity in Host-Plant Pathogen Interactions, Ed. by B. Solheim &J. Raa, pp. 439-51. Oslo Universitetsforlaget. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. 3ouwzal of Biological Chemistry 193, 265-275. 13. LUDERITZ, O., STAUB, A. M. & WESTPHAL, 0. (1966). Immunochemistry of 0 and R antigens of Salmonella and related Enterobacteriaceae. Bacteriological Reviews 30, 192-249. 14. NICOLSON, G. L. & BLAUSTEIN, J. (1972). The interaction of Ricks comma& agglutinin with normal and tumor cell surfaces. Biochimica et Biophysics Acta 266, 543-547. 15. ROEBUCK, P., SEXTON, R. & MANSFIELD, J. W. (1978). Ultrastructural observations on the development of the hypersensitive reaction in leaves of Phaseolus vulgaris C.V. Red Mexican inoculated with Pseudomonas phaseolicola (race 1). Physiological Plant Patholou 12, 151-157. 16. SEQUEIRA, L. (1978). Lectins and their role in host-pathogen specificity. Annual Review of Phytopathology 16, 453-481. 17. SEQUEIRA, L., GAARD, G. & DEZOETEN, G. A. (1977). Interaction of bacteria and host cell walls: its relationship to mechanisms of induced resistance. Physiological Plant Pathology 10, 43-50. 18. SEQUEIRA, L. & GRAHAM, T. L. (1977). Agglutination of avirulent strains of Pseudomonas solanacearum by potato lectin. Physiological Plant Pathology 11, 43-54. 19. SHARON, N. & LIS, H. (1972). Lectins: cell agglutinating and sugar specific proteins. Science 177, 949-959. 20. SING, V. 0. & SCHROTH, M. N. (1977). Bacteria-plant surface interactions: active immobilization of saprophytic bacteria in plant leaves. Sci.eme 197, 759-716.
Plant P4fholog3
Agglutination A. J. ANDERSON
(1979)
15, 149-159
of Pseudomonad cells by plant products and C. JASALAVICH
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 (Acceptedfor
publication
January
U.S.A.
1979)
Cells of plant pathogenic and saprophytic species of pseudomonads were differentially agglutinated by preparations from Dark Red kidney bean leaves. Water homogenates of bean leaves caused agglutination of the saprophytic pseudomonads Pseudomonas juorescm and P. putida but no agglutination of the plant pathogens P. phasGolicola or P. gringae. Agglutination with P. tubaci was variable. The agglutinin was detected in both pellet and supernatant after centrifugation of the water homogenate at 6000 g for 10 min. This agglutinin was protease and trypsin stable, was insensitive to salt except above 0.15 M and required a divalent metal ion for activity. An agglutinin with apparently different properties was released by 0.3 M NaCl treatment of a crude preparation of isolated bean leaf cell walls. This wall-released agglutinin caused a more variable agglutination of both saprophytic and pathogenic pseudomonads. The wall agglutinin was protease and trypsin labile and was inhibited by salt or metal ions above 0.02 M. Agglutination of pseudomonad cells by characterized plant components was also demonstrated. Pectin and galacturonic acid but not other plant cell wall polysaccharides produced agglutination. The plant lectins concanavalin A, wheat germ agglutinin and phytohemagglutinin but not ricin caused agglutination of the bacterial cells.
INTRODUCTION Recent electron microscopy studies [S, 10, 1.5, 17, 201 have demonstrated that a physical binding of bacterial cells to the cell walls of a non-host plant may be a key event in a resistant interaction. It has been proposed that the bacterial cells become immobilized at their binding site through an envelopment process which involves an active restructuring of the plant cell wall surface [S, 91. This binding and encapsulating process has not been observed in a susceptible interaction, when the virulent bacteria remain free and multiply in the intercellular space. Consequently, a recognition process must exist which permits a plant to differentiate between an avirulent or virulent bacterial challenge. Sequeira et al. [16, 17, 181 have suggested that differential recognition of avirulent and virulent bacteria may involve a lectin component in the plant. A lectin isolated from potato tubers was observed to agglutinate only strains of pseudomonads that were avirulent in potato. The virulent strains were not agglutinated [18]. Thus, a lectin may be involved in establishing the binding of avirulent bacteria to the plant cell wall. Involvement of a lectin in regulating the specificity of binding bacteria has also been suggested for the interaction of species of nitrogen-fixing Rhizobia with their hosts [3, 51. However, here it is the compatible bacteria that are recognized and bound by the plant during the process of establishing the symbiotic relationship. 0048-4059/79/050149
+ 11$02.00/O
@ 1979 Academic
Press Inc.
(London)
Limited
150
A. J. Anderson
and
C. Jasalavich
The demonstration that surface interactions between plant and bacteria may play a key role in the specificity of the responses prompted these studies on the ability of plant products to agglutinate cells of pseudomonad species. Lectins and model plant cell wall polymers were examined for their potential to agglutinate cells of nonpathogenic saprophytes as well as cells of the plant pathogens. A search for agglutinins in extracts of bean leaves also was initiated since the immobilization response has been observed to occur with pseudomonads in this tissue [ 1.5, 201. MATERIALS
AND
METHODS
Culture of bacteria Bacterial isolates were obtained from the stock cultures of L. W. Moore and R. H. Cameron of Oregon State University. Pseudomonas tabaci, P. syringae and P. phaseolicola were used to represent plant pathogen species. Pseudomonas putida and P. juorescens were selected as examples of saprophytic species. The virulence of P. tabaci and of P. phaseolicola were confirmed periodically by inoculation into their hosts, tobacco and bean (Phaseolus vulgaris) respectively. The cultures were maintained at 22 “C by monthly transfer of cells onto a rich medium containing in 1 1: 10.5 g K,HPO,, 4.5 g KH,PO,, O-5 g sodium citrate, 1-O g (NH,)sSO,, 10 g nutrient broth (Difco Laboratories, Detroit, Michigan 48232, U.S.A.), 6 g yeast extract (Difco) 0.25 g MgSO,.7H,O, 2 g sucrose and 15 g agar (Difco). The cells used for agglutination assay were grown in the medium described above prepared without agar. The liquid medium was inoculated by loop transfer from the cultures growing on solid media and was shaken on a gyrating incubator at 24 “C. After 15, 36 or 40 h of growth, the cells were harvested by centrifugation at 10 000 g and the supernatants were discarded. The cell pellet was resuspended gently in distilled water and the suspension recentrifuged at 10 000 g. The washed pellet was resuspended in sterile distilled water at approximately 10’0 cells per ml. The cell concentration was determined by dilution plating and counting the viable cell colonies. Heat-killed bacteria were prepared by treatment of the cell suspensions at 121 “C for 20 min. Heated cells were pelleted by centrifugation and washed twice with water before suspension for bioassay. This procedure was an attempt to remove materials possibly solubilized by heating that may otherwise interfere in the agglutination response. Source of agglutinin
preparations
The lectin concanavalin A was obtained commercially from Sigma Chemical Company, P.O. Box 14508, St Louis, Missouri 63178, U.S.A., and phytohemaglutinin from Difco Laboratories. Ricin was isolated from castor bean and purified by following the procedures of Nicolson & Blaustein [I4]. Wheat germ agglutinin, purified to homogeneity, was a gift from B. Triplett of this department. The lectins were dissolved using distilled water to give a preparation of 0.5 mg protein per ml. Plant cell wall polymers, mannan, galactan, xylan, pectin and the uranic acid, galacturonic acid, were purchased from Sigma Chemical Company. Two methods were used to obtain bean (Phaseolus vulgaris) leaf extracts that were tested for agglutinin activity. One method involved water extraction and the second
Agglutination
of pseudomonad
cells
151
required salt treatment of a crude preparation of bean leaf cell walls. Dark Red Kidney beans were grown in vermiculite for 14 days at 24 “C under a light regime involving 10 h dark and 14 h of illumination. The leaves were homogenized in 100 g quantities in 200 ml 4 “C distilled water for 1 min in a Waring blender. The homogenate was filtered through a coarse sintered glass funnel to separate the insoluble material from the water extract. The filtrate was centrifuged first at 6000 g and then at 20 000 g for 10 min. The 6000 g and 20 000 g pellets were resuspended in distilled water (30 ml) and stored at 4 “C, as was the final 20 000 g supernatant. The dispersed pellets and the 20 000 g supernatant were used as crude extracts in agglutination assays. The second type of agglutinin preparation involved salt treatment of the insoluble material prepared by the above procedures. The insoluble material retained on the sintered glass funnels was washed extensively with 4 “C water until the washings were no longer green. The material was then resuspended in 0.3 M NaCl (200 ml per 100 g original leaf material) for 10 min at 22 “C. The suspension was filtered on a sintered glass funnel, and the filtrate was centrifuged at 20 000 g. The 20 000 g supernatant was concentrated by one of two procedures. One method involved an ultrafilter apparatus equipped with a Pellicon filter (Millipore Corporation, Bedford, Massachusetts 01730) that restricted flow of macromolecules of lo4 mol. wt. The alternate procedure involved rotary evaporation at 38 “C. The concentrated preparations were desalted by passage through a Bio-Gel P-10 (mesh 50-100) (Bio-Rad Laboratories, 2200 Wright Avenue, Richmond, CA 94804, U.S.A.) molecular sizing column (23 x 2.1 cm) using distilled water as the eluent, or by dialysis at 4 “C against distilled water. These desalted preparations were used as the source of the “salt extracted” agglutinin. Calorimetric analyses were used to determine the quantity of protein, hexose and uranic acid in the leaf agglutinin preparations [Z, S, 121. The analyses were standardized by using glucose (Sigma Chemical Company) for hexose, galacturonic acid (Sigma Chemical Company) for uranic acid and bovine serum albumen (Sigma Chemical Company) for protein. Assay for agglutinin
actiuity
Agglutinin activity was assayed by the addition of 100 ~1 of the bacterial suspension to 0.5 ml of a solution containing potential agglutinin activity or, for controls, 0.5 ml water. Assays of the water-extracted bean leaf agglutinin were supplemented with lo4 M MgCl,. Agglutination was judged by visual observation after 15 min at 22 “C. The activity was scored using a scale ranging from + + + + + for a flocculent precipitation of the bacterial cells to + where only faint granulation in the bacterial suspension was visible. One unit of agglutinin activity is defined as the amount in 500 ~1 required to give a + granulation of the appropriate pseudomonad cells under the assay conditions described above. Various sugars,used to attempt to prevent agglutination activity were obtained from the Sigma Chemical Company. Purification
of th,ewater-extracted
agglutinin
The agglutinin activity present in the water extract of bean leaves was partially purified by utilizing the ability of the factor to bind to bacterial cells only in the
A. J. Anderson
152
and
C. Jasalavich
were presence of Mgs+. Ion exchange and molecular sizing gel chromatography subsequently used for further purification. Two hundred ml of the 6000 g supernatant was mixed with O-5 g (wet wt) of 15 h harvested P. putidu cells at 24 “C for 15 min in the presence of lOa M MgCl,. The suspension was centrifuged at 6000 g for 10 min and the supernatant discarded. The pellet was resuspended in 200 ml distilled water, the suspension recentrifuged and the supernatant collected. The pellet was resuspended in distilled water and the resulting suspension recentrifuged at 6000 g to yield a second supernatant fraction, which was combined with the first. The combined supernatants were mixed with 20 ml of DEAE A-50-120 Sephadex resin (Sigma Chemical Company), equilibrated at pH 5.2 with 10 mM sodium acetate, and the slurry filtered through a sintered glass funnel. The resin was washed with 50 ml distilled water and the washings combined with the first filtrate. The volume of these combined filtrates was reduced to 10 ml in an ultrafilter apparatus containing a Pellicon membrane which restricted molecules of size greater than 104. A 2 ml sample of the concentrated preparation was applied to a Bio-Gel P-60 column (36 x 2 cm) equilibrated with O-1 M NaCI. Fractions of 5 ml were collected by eluting the column with 0.1 M NaCl and each fraction tested for agglutinin activity. The void volume of the column was determined by the position of elution of Blue Dextran (Sigma Chemical Company) and the included volume by the elution position of glucose. The glucose was detected by the anthrone calorimetric method
Determination
of the stability
of agglutinin
preparations
to proteolytic
activity
The stability of agglutinin preparations to proteolytic activity was determined using immobilized derivatives of trypsin and protease obtained from the Sigma Chemical Company. Fifty mg of distilled water-washed resins onto which the proteases were attached were added to 1.0 ml samples of the agglutinin preparations. The mixture was incubated for 12 to 48 h at 30 “C, filtered and the filtrate assayed for agglutinin activity. Control samples of agglutinin containing no protease were incubated to verify that the agglutinin alone was stable to these conditions. RESULTS
Agglutination
of pseudomonad cells by lectins
The characterized lectins [19] wheat germ agglutinin, concanavalin A, and phytohemagglutinin caused differential agglutination of the pathogenic and non-pathogenic species of pseudomonads examined (Table 1). Agglutination was prevented when O-075 M concentrations of N acetylglucosamine for wheat germ agglutinin, x-methyl glucoside for concanavalin A and galactosamine for phytohemagglutinin were included in the assay. Using cells harvested from 36 h cultures, wheat germ agglutinin agglutinated all the pseudomonad species and with phytohemagglutinin all species except P. putida or P. tabaci showed agglutination. A different agglutination pattern was observed with concanavalin A when cells from 15 or 36 h cultures were used. With 36 h cultured cells all cell types showed agglutination, although agglutination of P. putida and P. tabaci was weak. When 15 h cultures were used, cells of P. putida and P. phaselicola agglutinated strongly, P. fluorescent was not agglutinated
Agglutination
of pseudomonad
cells
153
and only weak agglutination of P. syringae was observed. No agglutination was observed with cells of any species using ricin as the lectin, or in the controls where no lectin was added. TABLE Agglutination
1
of @mdomonad Agglutinationa
Lectin
P. jluorescens
Ricin Wheat germ agglutinin Phytohemagglutinin Concanavalin A Concanavalin A
+++ ++++ +++ -0
cells by lectins of cell9
of the pseudomonad
P. putida
P. phaseolicola
-
-
-I-++ -I-+/++++c
+++++ +I++++ ++++O
species
P. syringae
P. tabaci
+++ ++ ++++ +c
+++ ++/+fC
a The conditions for the agglutination assay were as described in Materials and Methods. The absence of agglutination is denoted by a - symbol. When varying degrees of agglutination were observed using different preparations of cells of the same species the range is shown by including the symbol / in the table. Data are with pseudomonad cells harvested at five different cultures. b Pseudomonad cells were harvested from 36 h cultures except for those marked with footnote G. c Fifteen h cultures used.
Agglutination of cells by plant cell wall polysaccharides Mannan, xylan and galactan solutions at 0.05% (w/v) gave no agglutination of any pseudomonad cells. However, pectin (0.05% w/v) and galacturonic acid solutions caused the agglutination of both the pathogenic and saprophytic pseudomonad species (Table 2). Variation in the species agglutinated was observed between 15 and 40 h cultured cells, especially for P. phaseolicola and P. syringae. Generally the TABLIZ Agglutination
of fiseudomanad
2
cells by a range of concentrations Agglutination5
Pseudomonad
species
15 h cells P. j¶uorcscens P. putida P. phaseolicola P. syringae P. tabaci 40 h cells P. jluorescens P. jmtida P. $aseolicola P. syringae P. tabaci a The agglutination Table 1. b Final concentration
assay
by galacturonic
10-l
10-a
++++ ++ I/+++ +++ -I++++ ++ +++ ++++ +++ was conducted
of galacturonic
acid
of galacturonic
in reaction
(M) b
10-s
+++ + I,++ ++, +/+++ ++++ +++ +
as described
acid
mixture.
lo-’
-Iz -/+ -++
in Materials
aicd
z -and
Methods
and
A. J. Anderson
154
and C. Jasalavich
older cells were more agglutinable, although in the course of replications of these studies exceptions to the pattern in Table 2 have been observed. For example, contrary to the usual results, in one preparation out of five, 40 h P.fluorescem cells and 15 h P. @gae preparations failed to agglutinate in 10-l and 10e2 M galacturonic acid. Agglutination
of cells by bean leaf extracts
by water-extracted preparations. Agglutinin activity for pseudomonad cells was detected in the 6000 g and 20 000 g supernatants from the water homogenate of the bean leaves. Activity was also detected in the 20 000 g pellet and in the 6000 g pellet. Each of these supernatants and suspensions of pelleted material caused agglutination of 15 h cultured cells of P.Juorescens and P. putida, whereas cells of the plant pathogenic species were not agglutinated (Table 3). With 40 h cultures, cells
Agglutination
TABLE
Agglutination
of @mhmad
Agglutinationa Pseudomonad P. P. P. P. P.
fhoremns putida &aseolicola Syrngae taban
species
3
cells by mule water extracts
of bean &af tissue
of cells harvested 15h
++++ +++++
from 40h
cultures
at
+++ +-I-++ ++
o. The agglutination assays were conducted and scored as described in Materials and Methods and Table 1. Data are compiled from at least five replicates. The water extracts were prepared from bean leaves by the procedure described in Materials and Methods and consisted of suspensions of 6000 g and 20 000 g pelleted material as well as 6000 g and 20 000 g supernatants.
of P.juorescens and P. putida were agglutinated and there was also a weak agglutination of some preparations of P. tabaci. No reaction with P. syringae or P. phaseolicola (Table 3) was observed. Bacterial cells that were treated at 121 “C for 20 min were not agglutinated. The agglutination response was not prevented by the inclusion in the bioassay of 0.05 M concentrations of rhamnose, fucose, xylose, arabinose, ribose, deoxyribose, deoxyglucose, glucose, mannose, galactose, galactosamine, glucosamine, r+acetyl glucosamine, and a- or P-methyl glucoside. The activity present in the 6000 g supernatant was selected to conduct further studies. The agglutination of P. fluorescens and P. putida was dependent on the presence of a divalent metal ion. Passage of the crude agglutinin preparation through CM Sephadex caused a loss in activity which was restored by the addition of MgCl,, MgSO,, CaCl,, MnSO, or MnCl, at a concentration lOA M. Consequently lOA MgCl, was routinely added to all assays of this agglutinin preparation. The water-extracted agglutinin was partially purified by using a technique that depended upon absorption of the agglutinin to P. putida cells in the presence of MgCl, and its release into solution in the absence of MgCl,. The supernatant obtained from a control where P. putida cells were treated identically except for using
Agglutination
of pseudomonad
cells
155
water instead of the leaf extract, did not possessany agglutinin activity. The agglutinin obtained by the bacterial cell affinity technique was purified further by passage without absorption through DEAE Sephadex equilibrated in 10 mM sodium acetate, pH 5.2. The preparation partially purified by this procedure required (2 & 1) pg protein for one unit of agglutinin activity with P. putida cells. These preparations contained protein (220 pg ml-l) as well as hexose (23 pg ml-l) and uranic acid (2 [*g ml-l). The agglutinin activity in this partially purified preparation was stable to treatment with protease or trypsin and to heating at 121 “C for 15 min. The agglutinin was inactivated if the concentration of NaCl in the reaction mixture exceeded 0.15 M. The agglutinin eluted in the void fractions of Bio-Gel P-10 and Bio-Gel P-60 columns. Agglutination by salt extracts of crude bean leaf walls. Extracts prepared by salt treatment of the insoluble material from leaf homogenates possessed an agglutinin activity for both saprophytic and plant pathogenic pseudomonads (Table 4). A minimum TABLET Agglutination
of @uahonad
ccl12 by a 0.3 M .NaCl extra& Agglutination0
Pseudomonad P. P. P. P. P.
J7uorescens put&l &zscolicola syringae kzbaci
of crwh isolated bean leaf cell walls
of cells harvested 15h
species
-I++ -I-l-I+ ++
from 4Qh
cultures
at
++ ++ -I+++ +++ ++++
‘The agglutination assays were conducted and scored as described in Materials and Methods and Table 1. The salt extract of crude isolated bean leaf cell walls was prepared as described in Materials and Methods. Data are compiled from at least five replicated experiments.
concentration of O-3 M NaCl was required to extract the agglutinin from the crude bean leaf cell wall material. The agglutinin activity was detected only after the extracts were desalted by dialysis or by elution through a B&Gel P-10 column, when the activity was detected in the void fractions. Concentrations of NaCl greater than O-02 M or metal ions, Mg s+, Gas+ or Mns+ at a concentration of 10”’ M in the reaction mixture, inhibited agglutination. The agglutination activity in the B&Gel P-10 purified material was eliminated by protease or trypsin treatment. Agglutination was not inhibited by inclusion in the bioassay of 0.05 M concentrations of the sugars previously listed for the water-agglutinin assays. The species of pseudomonads agglutinated by this salt-extracted agglutinin showed variation between cells grown at different times. The pattern of agglutination genertilly obtained is summarized in Table 4 but as with agglutinatidn by galacturonic acid exceptions have been observed. Agglutination of 40 h cells of all cell types has been observed although in one preparation among five, the P. phaseolicola, cells were non-agglutinable. With 15 h cultures P. tuba& cells were routinely agglutinated and a weaker and more variable agglutination of P. syringae, P. .@mmm and P. putida occurred. The preparations of agglutinin present in the void fractions
156
A. J. Anderson
and
from the Bio-Gel P-10 column possessed protein (65 pg protein per ml) hexose (38 pg ml-l) and uranic acid (5 pg ml-l). These preparations 15( + 5) pg protein for 1 unit of agglutinin activity with P. tabuci cells.
C. Jasalavich
as well as required
CONCLUSIONS
Differential agglutination of both plant pathogenic and saprophytic pseudomonad cells occurred upon treatment with the characterized plant proteins, lectins, as well as with the plant cell wall components, pectin and galacturonic acid. Agglutination was also observed with as yet uncharacterized components present in extracts of bean leaves, a tissue in which immobilization of avirulent pseudomonads has been demonstrated [20]. Variation in the agglutinability of bacterial cells of the same species between different cell preparations was observed with each of the agglutinin preparations. This variation suggests that the surface physiology of the bacteria is changeable and sensitive to culture conditions. The age at which the cells were harvested and the treatment of the cells between the time of harvest and the agglutination assay are areas where we have observed variability. Also, our agglutination studies with the bean lectin phytohemagglutinin differ from those of Sing & Schroth [20]. Phytohemagglutinin agglutinated cells of our isolates of both saprophytic and plant pathogenic pseudomonads including P. phaseolicola, whereas Sing and Schroth [20] report only agglutination of the saprophyte P. putida and not the plant pathogens P. tomato or P. phareolicola. A possible explanation could be differences in the techniques for culturing the bacteria and in the conditions for agglutinin assay. The importance of culture conditions in the phenomenon of bacterial agglutination has also been reported by Bauer et al. [I]. These workers showed that culture age caused variability of the agglutination response of Rhizobia with soybean lectin. The agglutination of the pseudomonad cells by plant lectins can be explained by the ability of the lectin to recognize specific carbohydrate residues on the bacterial cell surface. Concanavalin A is known to bind to ol-mannosyl or a-glucosyl units [19], wheat germ agglutinin to N-acetylglucosamine oligomers [29] and phytohemagglutinin to galactosamine [19]. The observed inhibition of pseudomonad cell agglutination when the appropriate sugar was present along with the lectin, supports the idea that the agglutination involved the lectin recognition of bacterial cell surface carbohydrates. Indeed, the presence of amino sugars and glucose residues on cellurs face structures of pathogenic and non-pathogenic pseudomonad species has been demonstrated in our laboratory (unpublished observations). A lack of detectable galactose on the pseudomonad surface structures (unpublished observations) is consistent with the failure of ricin, a lectin that binds to galactose [19], to agglutinate any of the pseudomonad cells tested. Although these lectins are demonstrated to recognize the bacterial cells, their significance to a plant-pathogen relationship is uncertain. The lectins used in these studies were extracted from the seed of the appropriate plant: jack bean for concanavalin A, red kidney bean for phytohemagglutinin, wheat for wheat germ agglutinin and castor bean for ricin. Thus the lectins may not be present in the tissues that are challenged by the bacteria. Also, concanavalin A and phytohemagglutinin are believed to be located in the cytoplasm and not extracellularly [4]. Consequently, they would not be available to recognize the invading bacteria
Agglutination
of pseudomonad
cells
157
in the intercellular space unless plant cell damage occurs that allows lectin movement. Another possibility is that the leaf cell wall possesses its own lectin components. Sequeira et al. [16, 181 have cited preliminary evidence for such existence of lectin activity associated with cell walls of tobacco leaf tissue. The demonstration (Table 2) that the plant cell wall component, pectin, and its monomer, galacturonic acid, have agglutinin activity suggests that plant cell walls could bind both pathogenic and non-pathogenic pseudomonad species. Indeed, Lippincott &Lippincot have stated that thepecticcomponentisresponsibleforbindingt Agrobacterium cells to plant cell walls [11]. It is possible that preliminary binding of bacterial cells to plant cell walls is initiated by the pectin component and that secondary events, such as lectin interaction, then occur which determine whether or not the bacteria remain bound. The failure of virulent bacteria to bind to the plant cell wall could lie in their production of acidic components that negate the possible attractant effect of pectin. The existence of acidic structures in extracellular polysaccharides has been documented [13] for other bacterial species. Also, some pathogens such as P. phaseolicola [7] produce toxins that are negatively charged. Thus the secretion of a charged toxin may prevent the bacteria from adhering to the plant cell wall in the susceptible interaction. The possibility that recognition and binding of bacterial cells could involve factors in addition to pectin is supported by our observations of agglutinin activities detected in bean leaf extracts. The properties of the bean leaf agglutinin preparations are distinct from agglutination caused by pectin (Table 5). Agglutination by leaf TABLE A comparison
of the properties
5
of the agglutination
of pseuahonad cells by galacturonic water- and salt-extracted bean leaf agglutinins Agglutinin
Property (1) Effect of MgCl,, 10-s r.sb (2) Inhibition by O-2 Y NaClb Inhibition by 0.02 M NaClb (3) Effect of proteasec or trypsin (4) Agglutination of heat-killedd bacterial cells
Galacturonic
acid None None None None Active
(lOma Mu)
Water Required Inhibited None None None
acid, and the
source” extract
(bean
leaf)
Salt extract Inhibited Inhibited Inhibited Labile None
0 The agglutinins were assayed and prepared as described in Materials and Methods. The 6000 g supernatant was used as the water-extracted agglutinin. Cells from 40 h cultures of P. tabaci were used for the assays of the salt-extracted agglutinin and galacturonic acid. Cells from 40 h cultures of P. putida were used for assay of the water-extracted agglutinin and galacturonic acid. 6 The final concentration in the reaction mixture. E Treatment with enzymes followed the procedures given in Materials and Methods. d Cells of P. tab& and P. putida were treated at 121 “C for 20 min as described in Materials and Methods.
preparations and by galacturonic acid differ in sensitivity to salt and metal ions, in proteolytic stability, and in the agglutination of heat-killed bacterial cells (Table 5). Also the concentration of uranic acid in the leaf preparations (less than IO4 M) is insufficient to account for the agglutinin activities. The agglutinins present in these
158
A. J. Anderson
and
C. Jasalavich
water and salt extracts also appear to be distinct factors. The agglutinins differ in proteolytic stability and sensitivity to inorganic ions (Table 5) as well as in the species of pseudomonad cells agglutinated (Tables 3 and 4). Both of the leaf agglutinins appear to be macromolecular because they eluted in the void of a Bio-Gel P-10 column and failed to pass through an ultrafilter which restricted molecules of size greater than lo* daltons. The mode of action of the leaf agglutinins is speculative. It is possible that like lectins the leaf agglutinins are recognizing specific surface polysaccharides. The fact that no simple sugar has yet been shown to prevent the activity of the leaf agglutinins could be due to their requirement of a carbohydrate structure more complex than a monomer for recognition. Although the water-extracted agglutinin is heat and protease stable, these properties are in common with plant “3-1ectin” glycopeptide complexes that have an affinity for p-linked sugar residues [4]. The differential agglutination of the pseudomonad species observed with both leaf preparations suggests that some type of specific recognition event is occurring. The failure of the heatkilled bacterial cells to agglutinate with either leaf preparation also suggests that there is a critical recognition site. The variability of agglutination with age of the bacterial cells could be explained by a transient nature of the recognition site. The differential agglutination of pseudomonad cells with the leaf agglutinins is interesting. The preferential agglutination of saprophytic pseudomonads by the water-extracted material complements the immobilization ofP.pzctidu but not P. tomato reported by Sing & Schroth [ZO]. Other agglutinins, perhaps the salt-extracted agglutinin, could recognize plant pathogenic pseudomonads. Indeed, P. phaseolicola has been observed to bind to cell walls of a resistant bean variety [15]. Thus, although the agglutinins we have extracted have not been demonstrated to be lectins, it is intriguing that these activities are present in bean leaf tissue which displays an immobilization response.
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