attachment of azospirillum to isolated plant cells

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FEMS Microbiology Letters 49 (1988) 435-439 435 Published by Elsevier FEM 03108 Attachment of Azospirillum to isolated plant cells Mark Eyers, Jos Vanderleyden and August Van Gool F.A. Janssens Memorial Laboratory of Genetics, University of Leuven, Heverlee, Belgium Received 19 November 1987 Accepted 20 November 1987 Key words: Azospirillum brasilense; Agrobacterium tumefaciens; Zinnia elegans; Triticum monococcum; Attachment 1. SUMMARY Azospirillum brasilense cells were shown to at- tach to isolated plant cells. A rapid semi-quantita- tive assay to measure plant cell attachment, as was described previously for Agrobacterium, was found to work especially well for Azospirillum. Attach- ment was measured in an indirect way and con- firmed by direct observations through scanning electron microscopy. 2. INTRODUCTION Bacteria of the genus Azospirillum are free-riv- ing diazotrophs that can readily be isolated from the rhizosphere and roots of grasses and cereals [1]. These bacteria have potential plant growth- promoting activity in gnotobiotic conditions and in the field [2-7]. So far, the plant growth-promo- ting principle has not been identified. It has been postulated that biological nitrogen fixation by Azospirillum spp. in association with roots might contribute significant amounts of nitrogen to the Correspondence to: J. Vanderleyden, F.A. Janssens Memorial Laboratory of Genetics, University of Leuven, Willem de Croylaan 42, B-3030 Heverlee, Belgium. plant [4,8]. Using the 15N-isotope dilution tech- nique [9] or the N-balance technique [7], it is clear that no or very tittle N2, fixed by Azospirillum, is of benefit to the plant, except for A. halopraeferens in association with Kallar grass [10]. The second characteristic of the genus Azospirillum that has been related to plant growth-promoting activity is the synthesis of phytohormones [11]. It can be speculated that the observed changes in root de- velopment, i.e., decrease of the root elongation zone and increase in the number of root hairs, as a result of Azospirillum inoculaton, are due to hormonal effects. These changes might then be responsible for the increase in the rate of water and mineral uptake from the soil by the roots [12,13]. In order to obtain these plant growth-pro- moting effects, good colonization of the plant roots by Azospirillum appears to be a decisive factor [14]. It has been shown that bacteria of the genus Azospirillum can attach to root hairs [15-18]. In order to study the process of attachment and its role on the plant growth-promoting activity of Azospirillum, mutants deficient in attachment need to be isolated. Random transposon mutagenesis is a powerful technique to inactivate and tag bacterial genes, and has recently been demonstrated to be feasible for analysis of the genome of Azospirillum [19,20]. In order to isolate attachment-deficient 0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies

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Page 1: Attachment of Azospirillum to isolated plant cells

FEMS Microbiology Letters 49 (1988) 435-439 435 Published by Elsevier

FEM 03108

Attachment of Azospirillum to isolated plant cells

Mark Eyers, Jos Vanderleyden and August Van Gool

F.A. Janssens Memorial Laboratory of Genetics, University of Leuven, Heverlee, Belgium

Received 19 November 1987 Accepted 20 November 1987

Key words: Azospirillum brasilense; Agrobacterium tumefaciens; Zinnia elegans; Triticum monococcum; Attachment

1. SUMMARY

Azospirillum brasilense cells were shown to at- tach to isolated plant cells. A rapid semi-quantita- tive assay to measure plant cell attachment, as was described previously for Agrobacterium, was found to work especially well for Azospirillum. Attach- ment was measured in an indirect way and con- firmed by direct observations through scanning electron microscopy.

2. INTRODUCTION

Bacteria of the genus Azospirillum are free-riv- ing diazotrophs that can readily be isolated from the rhizosphere and roots of grasses and cereals [1]. These bacteria have potential plant growth- promoting activity in gnotobiotic conditions and in the field [2-7]. So far, the plant growth-promo- ting principle has not been identified. It has been postulated that biological nitrogen fixation by Azospirillum spp. in association with roots might contribute significant amounts of nitrogen to the

Correspondence to: J. Vanderleyden, F.A. Janssens Memorial Laboratory of Genetics, University of Leuven, Willem de Croylaan 42, B-3030 Heverlee, Belgium.

plant [4,8]. Using the 15N-isotope dilution tech- nique [9] or the N-balance technique [7], it is clear that no or very tittle N2, fixed by Azospirillum, is of benefit to the plant, except for A. halopraeferens in association with Kallar grass [10]. The second characteristic of the genus Azospirillum that has been related to plant growth-promoting activity is the synthesis of phytohormones [11]. It can be speculated that the observed changes in root de- velopment, i.e., decrease of the root elongation zone and increase in the number of root hairs, as a result of Azospirillum inoculaton, are due to hormonal effects. These changes might then be

responsible for the increase in the rate of water and mineral uptake from the soil by the roots [12,13]. In order to obtain these plant growth-pro- moting effects, good colonization of the plant roots by Azospirillum appears to be a decisive factor [14].

It has been shown that bacteria of the genus Azospirillum can attach to root hairs [15-18]. In order to study the process of attachment and its role on the plant growth-promoting activity of Azospirillum, mutants deficient in attachment need to be isolated. Random transposon mutagenesis is a powerful technique to inactivate and tag bacterial genes, and has recently been demonstrated to be feasible for analysis of the genome of Azospirillum [19,20]. In order to isolate attachment-deficient

0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies

Page 2: Attachment of Azospirillum to isolated plant cells

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mutants out of a large collection of transposon mutants, a rapid screening method needs to be available. In this paper we demonstrate that Azospirillum cells are able to attach to plant cells, either freshly prepared or derived from cell sus- pension cultures. Attachment to isolated plant cells can be easily monitored on a large number of bacterial isolates or mutants. (A preliminary account of part of this work was presented at the 4th Workshop on Azospirillum, held in Bayreuth, F.R.G., June 17-18, 1987).

3. MATERIALS AND METHODS

3.1. Bacterial strains and media The bacterial strains used in this study are

listed in Table 1. Escherichia coli HB101 was maintained on LB agar and grown at 37 °C in LB broth. Azospirillum and Agrobacterium strains were maintained on yeast extract-peptone (YEP) agar and grown at 28 ° C in YEP broth.

3.2. Plant cells Zinnia elegans Jacq. was grown from seed (Na-

tionale plantentuin, Meise). Seeds were ger- minated, and seedlings were grown in vermiculite. Leaves were rinsed in distilled water and then homogenized in Murashige-Skoog salts with a pes- tle and mortar. The homogenate was passed through a 250 #M screen (Bellco Glass, Inc.) and the cells were collected on a Miracloth filter

Table 1

Bacterial strains

Strains Relevant Reference and/ properties or source

E. coli HB101 E. coli, K12 [21]

Agrobacterium strains A. tumefaciens A208 Virulent M.-D. Chilton A. tumefaciens A1011 Avirulent,

chv::Tn5 (Km r) [22]

Azospirillum strains A. brasilense Sp7 Apr ATCC29145 A. brasilense 7030 Ap r, NG

induced Sm r, C. Elmerich derivative of Sp7

A. brasilense Sp245 Sp r J. Drbereiner

(Calbiochem). Cells were suspended in Murashige- Skoog salts at a concentration of 5 x 105 to 6 x 105 cells/ml. Suspensions obtained in this manner consisted mostly of single cells. Suspension cul- tures of Triticum monococcum were obtained from Dr. Hadlaczky (Institute of Genetics, Biological Research Center, Hungarian Academy of Scien- ces, Szeged, Hungary). They were maintained in C8I medium [23] with weekly transfers.

3.3. Assay of bacterial attachment to plant cells Radiolabeled bacteria were added to Zinnia

elegans leaf mesophyl cells or Triticum monococ- cure cells and their attachment was monitored. Zinnia leaf mesophyl cells were prepared im- mediately before use as described above. Bacteria were labeled by inoculating a single colony into 0.5 ml of YEP or L medium supplemented with 2 #Ci of t-[u-lnC]leucine (342 mCi/mmol; Amers- ham International plc) and grown overnight. The bacteria were washed twice in 10 mM MgSO 4. Specific activities of 10 -5 cpm per cell were ob- tained. Labeled bacteria were added to the plant cells at a final concentration of approximately 10 6

bacteria per ml, corresponding with a bacterium- to-plant cell ratio of approximately 3 : 1. The mix- ture was incubated at 25 °C on a rotary shaker at 40 rpm for 2 h. The number of bacteria attached to plant cells was determined after filtering the bacteria-plant cell mixture either through Nylon 20-/tm-pore filters (Bureau Technique Wintgens, S.A., Eupen, Belgium) when Zinnia elegans cells were used or through Miracloth filters when Tri- ticum monococcum culture cells were used. Only non-attached bacteria passed through these filters. After a wash with 10 mM MgSO4, the filters were placed in scintillation vials with 5 ml of scintil- lation liquid and counted in a liquid Tri-carb scintillation counter.

3. 4. Scanning electron microscopy After an incubation time of 2 h, unattached

and attached bacteria were separated by filtration as described above. Zinnia elegans cells with at- tached bacteria were fixed in 1% glutaraldehyde for 1 h. After a wash with bidistilled H20 the cells were collected on a 0.22-/tm-pore membrane filter (Millipore).

Page 3: Attachment of Azospirillum to isolated plant cells

Cells were sequentially dehydrated in aqueous solutions of 30, 50, 70, 90 and 100% ethanol and dried in a critical point drying apparatus. The samples were coated for 2 min with 60% gold-40% paladium and examined in a scanning electron microscope.

4. RESULTS

When radiolabeled Azospirillum cells (see sec- tion 3.3) are incubated with isolated plant cells for 2 h, followed by a separation of free bacteria and plant cells, radioactivity is detected in the plant cell fraction. This indicates that bacteria attach to the plant cells. By counting the number of bacteria added and the amount of radioactivity (cpm) per bacterial cell, the total cpm detected in the plant cell fraction is a measure for the number of bacteria attached to the plant cells. The results of these experiments are shown in Table 2. Taking into account the data of Douglas et al. [22] on A.

437

Table 2

At tachment of Agrobacterium tumefaciens or Azospirillum brasilense cells to Zinnia elegans mesophyl cells and Triticum monococcum suspension cells

Bacterial strains Percentage of added bacterial cells that remain attached to the plant cells b

Zinnia elegans Triticum monococcum

A. tumefaciens A208 2 4 / 2 7 / 2 2 / 2 6 2 3 / 2 4 / 2 5 / 2 3 A. tumefaciens A1011 5 / 4 / 7 / 8 9 / 1 0 / 1 0 / 1 2 A. brasilense Sp7 1 9 / 2 0 / 1 7 / 1 9 3 0 / 3 1 / 2 9 / 3 3 A. brasilense Sp7;

heat killed a 5 N.D. c A. brasilense Sp245 17/18 2 9 / 3 0 / 2 8 / 3 2 A. brasilense 7030 n.d. 3 1 / 3 2 / 3 0 / 3 0 E. coli HB101 3 / 2 / 2 / 2 3 / 3 / 4 / 2

a 20 rain at 60 o C. b Bacteria and plant cells in a 3 to 1 ratio were co-incubated

for 120 minutes at 2 8 ° C on a rotary shaker at 40 rpm. c Not determined.

tumefaciens attachment, cells of A. tumefaciens A208 were included as a positive control and cells of A. tumefaciens A1011 as a negative control. E.

% a t t a c h m e n t

3C) gO 9() l i 0 150 180 t i m e ( m i n u t e s )

Fig. 1. Time course of attachment of Agrobacterium and Azospiri/lum cells to plant cells. Radiolabded cells of A. tumefaciens A208 or A. brasilense Sp7 were mixed in a 3 :1 ratio with Zinnia elegans cells or Triticum monococcum cells. At various time points, samples were taken and treated as indicated in section 3.3. The data for at tachment of A208 to bamboo cells and Z. elegans cells were taken from Douglas et al. [22,24]. Bars indicate s tandard deviations of a min imum of three experiments. Symbols: O, Sp 7 / Tri ticum monococcum ; ~ , Sp 7 /Z inn ia elegans ; I , A208 /Z inn ia e legans ; 0, A 2 0 8 / b a m b o o cells; O, A208 / Triticum monococcum .

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438

coli cells were included as a control for non-rhizo- sphere bacteria. When A. brasilense Sp7 cells were killed by heat, only a residual attachment was observed. Whether this means that live bacteria are required for attachment or whether it reflects changes in the cell surface caused by the treat- ment, remains to be determined.

To reveal the time course of bacterial attach- ment the same experiment was repeated, taking different time points after starting the incubation of bacteria with plant cells. These results are sum- marized in Fig. 1. It can be seen that attachment increases during the first hour, and reaches a plateau after about 2 h. Interestingly, similar kinetics were observed for attachment of A. tume- faciens to plant cells [22,24]. Attachment of A. tumefaciens to plant cells has been shown to be an

active process, requiring the activity of specific genes [22,25]. For A. tumefaciens it has also been shown that attachment is not host specific, since they attach to cells of monocots and dicots equally well [24]. For Azospirillum, we observe (see also Table 2) higher values for T. monococcum (mono- cot) than for Z. elegans (dicot). Whether or not this reflects host specificity requires further inves- tigation (see also section 5).

The binding of Azospirillum cells to isolated Z. elegans cells was also examined with scanning electron microscopy (Fig. 2). Individual cells as well as clusters of cells are observed on the surface of a plant cell. Clustering of bacterial cells on the plant cell surface was repeatedly observed and could reflect an anchoring process by which bacterial cells help each other to bind to the plant cell as observed for Agrobacterium and Rhizobium [26,27].

Fig. 2. Scanning electron microscopy of a Z. elegans cell with attached A. brasilense Sp7 cells. Samples were prepared a s

explained in sections 3.3 and 3.4, except that an excess of bacterial cells over plant ce l l s w a s used.

5. DISCUSSION

Today's genetic research on plant growth-pro- moting rhizosphere bacteria is focused on the identification of the plant growth-promoting principles and on the characterization of plant root colonization genes. It can be postulated that plant root colonization is a multistep process that involves chemotaxis, recognition, attachment, pro- liferation, and possibly invasion of the root inter- ior.

For Azospirillum plant rhizocoenoses, we choose to study the role of attachment, because attachment genes have been cloned from other phytobacteria [22,28,30]. Recently we were able to demonstrate structural homology between A. tumefaciens chromosomal virulence (chv) genes and Azospirillum DNA [31]. Douglas et al. [22] developed a simple assay to monitor attachment of A. tumefaciens ceils to isolated plant cells. Using this assay, we could demonstrate that A. brasilense cells also attach to individual plant cells. Although we noticed different attachment values for Z. elegans ceils and T. monococcum cells, at this stage we do not like to speculate on the significance of these differences in terms of host range specificity, as we have not determined yet

Page 5: Attachment of Azospirillum to isolated plant cells

the optimal conditions of attachment for each plant cell system. Moreover, in order to do so, the described assay needs to be extended with a broader spectrum of plant cells. This system nev- ertheless allows us now to screen for attachment- deficient mutants of a particular Azospirillum strain. Mutations in the chv loci of A. tumefaciens cause deficiency in attachment [22]. The fact that in Azospirillum sequences homologous to the A. tumefaciens cho loci have been detected [31] and the observation that attachment of Agrobacterium and Azospirillum to isolated plant cells follow the same kinetics, prompts us to investigate the role of these sequences in the attachment of Azospirillum to plant cells.

ACKNOWLEDGEMENTS

This research was supported by grants from the 'Fonds voor Kollectief Fundamenteel Onderzoek' (F.K.F.O.-2.0013.85) and the 'CEC-contract no. TSD-A-255-B (RS)'. M.E. is the recipient of a fellowship of the I.W.O.N.L.

We are grateful to Dr. C. Douglas and Dr. G. Hadlaczky for the gift of bacterial strains and the plant cell line.

We thank the Department of Electron Mi- croscopy and Molecular Cytology of the Univer- sity of Amsterdam for the helpful suggestions and the use of the scanning electron microscope, J. Vloeberghs for technical assistance and P. Bosch- mans for typing the manuscript.

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