Two Types of Nodules Induced on Trifolium pratense by Mutants of Rhizobium leguminosarum bv. trifolii deficient in Exopolysaccharide Production

]. Plant Physiol. Vol. 147. pp. 93 -100 (1995)

Two Types of Nodules Induced on Trifolium pratense by Mutants of Rhizobium leguminosarum by. trifolii deficient in Exopolysaccharide Production ANNA SKORUPSKA 1,*, Urszula BiaLEK 1,2, TERESA URBANIK-SYPNIEWSKA!, and ANDRE VAN LAMMEREN2 1

Department of General Microbiology, Maria Curie-Sldodowska University, Akademicka 19,20·033 Lublin, Poland

2

Department of Plant Cytology and Morphology, Wageningen Agricultural University, Arboretumlaan 4, NL-6703 BD Wageningen, The Netherlands

Received January 5, 1995 . Accepted May 22, 1995

Summary

Several Tn5 mutants of the symbiotic bacterium Rhizobium /eguminosarum bv. trifolii 24.1 that fail to synthesize acidic exopolysaccharide, are able to induce non-nitrogen-fixing nodules on Trifolium pratense. Hybridization analysis indicated that Tn5 insertions were located on megaplasmids pRtb and pRtc and on the chromosome of R. /eguminosarum bv. trifolii 24.1. Microscopic analysis of nodules induced by these non-mucoid mutants revealed two types of nodules: infected nodules with infection threads and bacteroids, and non-infected nodules which neither contained infection threads nor bacteroids. Ultrastructural data on bacteroid development and plant cell differentiation in nodules induced by the mutants indicate a decrease in bacteroid and plant metabolism. Starch accumulation in the plant plastids and presence of osmiophilic droplets in the plant cells of the peripheral tissue was observed.

Key words: Trifolium pratense, exopolysaccharide, Rhizobium leguminosarum bu.

trifoli~

Tn5 mutants.

Abbreviations: EPS - exopolysaccharide, LPS - lipopolysaccharide, Fix- - non-nitrogen-fixing phe-

notype. Introduction

The formation of nitrogen-fIxing nodules on the roots of leguminous plants is a complex interaction between gramnegative soil bacteria as Rhizobium and Bradyrhizobium and members of the plant family Leguminosae. The early development of Rhizobium-legume symbiosis resulting in normal nodule formation can be summarized as follows: attachment of the bacteria to the root surface, curling of root hairs, formation of infection threads and induction of cell divisions in the root cortex leading to development of the nodule meristem. This multistep development is controlled by a complex of signals which are produced by both the bacteria and the .. Corresponding author. © 1995 by Gustav Fischer Verlag, Stuttgart

plant (Brewin, 1991; Long, 1992). Up to now two essential signals have been recognized in Rhizobium-legume symbiosis i.e. the Nod factors and acidic exopolysaccharide (EPS). In response to plant-derived flavones, Nod D gene product activates the common nodulation genes and host specificity genes. As a result a lipo-oligosaccharide signal called the Nod factor, is synthesized (for review see Lerouge, 1994). The Nod factor is able to induce root hair deformation and nodule meristem formation. In the next stage of symbiosis (the infection of the root hairs) the synthesis of EPS is required. For Rhizobium melilo~ there is strong genetic evidence that surface, acidic polysaccharides are required for further nodule development (Leigh et al., 1985; Finan et al., 1986). Alfalfa seedlings inoculated with Exo - mutants formed non-nitrogen-fixing nodules that contained few, if any bacteria and

94

ANNA SKORUPSKA, URSZULA BIALEK, TERESA URBANIK-SYPNIEWSKA, and ANDRE VAN lAMMEREN

no bacteroids. Root hair curling was significantly delayed and infection threads aborted at an early stage of infection (Arnold et al., 1993/94). The identification and analysis of exo genes of R. meliloti and their role in the infection process with Medicago sativa have been described (Arnold et al., 1993/94; Leigh and Walker, 1994). The genes for the biosynthesis of EPS I were located on megaplasmid 2 of R. meliloti within a 24 kb DNA region. Beside this exa-region only a few additional genes have been located on the chromosome. They were involved in EPS I biosynthesis or regulation (Zhan et al., 1989; Glazebrook et al., 1990). The complete nucleotide sequence of the exa-region on megaplasmid 2 has recently been established and the sequence information and mutational analysis revealed a total of 19 exo genes (Buendia et al., 1991; Becker et al., 1993 a, b; Muller et al., 1993). Relatively little is known of the genetic control of synthesis and regulation of EPS in other Rhizobium species. Exomutants of R. leguminosarum bv. phaseoli and bv. viciae which displayed Nod- or Fix- phenotypes, have been described (Borthakur et al., 1985). In R. leguminosarum bv. phaseoli the genes which affect the synthesis of EPS (pss and psi) were located on the pSym plasmid, near nod and fix genes (Borthakur and Johnston, 1987). Exo- mutants of R. Ieguminosarum bv. trifolii and Rhizobium sp. NGR234 isolated by Tn5 mutagenesis and by other techniques, were able to infect host legumes but the nodules formed were unable to fix nitrogen (Chakravorty et al., 1982; Chen et al., 1985; 1988; Deryfo et al., 1986; Skorupska et al., 1991). Nodules induced on clover by Exo- mutants of R./eguminosarum bv. trifolii were poorly developed, but infection threads occurred and bacteria were released into plant cells (Chakravorty et al., 1982; Deryfo et al., 1986; Skorupska et al., 1991). The mutated bacteria differentiated into bacteroids which were defective in nitrogen fIxation and degenerated prematurely. The aim of this paper was to find the effect of mutation in genes which control EPS production on morphology and function of the nodules induced by Exo- mutants.

Materials and Methods

Strains, plasmids and growth conditions Rhizobial strains used in this work are listed in Table 1. Rhizobium strains were cultured and maintained on mannitol-yeast extract agar medium (Vincent, 1970). Clover plants were grown on nitrogen-free Jensen's medium (Vincent, 1970). E. coli strains were cultured and maintained on LB medium (Sambrook et al., 1989). Antibiotics used were: kanamycin 50 mg' l -I and streptomycin 250mg·l- l .

Tn5 mutagenesis Tn5 mutagenesis was performed by mating E. coli strain 17.1 carrying suicide plasmid pSUP5011 (pBR325::Tn5-Mob, Ap'Cm'Km') with R. leguminosarum bv. trifolii strain 24.1 (Simon et al., 1983). Selection was carried out on mannitol-yeast-extract agar medium supplemented with kanamycin and streptomycin. The non-mucoid KmrSm' transconjugants were purified by successive single colony isolation and passages through the selective medium.

Plant nodulation tests Clover (Trifolium pratense L. cv. Hruszowska) seeds were surface sterilized with 6 % sodium hypochloride and germinated overnight on Jensen's medium at 28°C. The clover seedlings were transferred onto nitrogen-free agar slants and 5-day-old seedlings were inoculated with cultures of Exo- mutants and wild-type bacteria (Vincent, 1970). Plants were grown in a phytotron with 16/8 h photoperiod at 24/19°C. Four-week-old plants were assayed for nitrogen fixation ability by the acetylene reduction technique.

DNA manipulation 211g of total DNA from wild type and mutant bacteria was digested with EcoRI, electrophorezed in 1 % agarose and blotted onto a Hybond N filter. The filter was hybridized to pSUP2021 (pBR325::Tn5, AprCm'Km') (Simon et al., 1983) probe, labeled with 32p by nick translation (Sambrook et al., 1989) and was washed as follows: 3 x SSC, 0.1 % SDS; 1x SSC, 0.1 % SDS; 0.1 x SSC, 0.1 % SDS and 0.1 x SSC. All washings were performed for 15 min at 55°C. After washing steps the filter was exposed to X-ray film for 3 days at -70°C. Plasmids of R. leguminosarum bv. trifolii and its Tn5 derivatives were separated from chromosomal DNA by modified Eckhardt (1978) agarose gel electrophoresis (Hynes et al., 1985). DNA was blotted onto a Hybond N filter and hybridized to 32P-pSUP2021 DNA probe (Sambrook et al., 1989).

DOC-PAGE profiles oflipopolysaccharides (LPS) Bacterial mass was produced by growing the strains on mannitol yeast-extract agar plates (Vincent, 1970) for 48 h at 28°C. lPS was isolated using a mini-prep version of the phenol-water extraction (Bahrani and Oliver, 1991). The profiles of lPS were revealed by polyacrylamide gel electrophoresis in the presence of sodium deoxycholate (DOCPAGE) by silver staining according to Krauss et al. (1988). Electrophoresis was performed in a vertical gel apparatus (Hoeffer Scientific Instruments, model SE 250).

Light and electron microscopy Clover nodules harvested four weeks after inoculation were fixed with 3 % glutaraldehyde in 0.05 M sodium cacodylate buffer pH 7.2 (24 h, 22 0e), post-fixed in 2 % osmium tetroxide in sodium cacodylate buffer (12 h, 22°e), dehydrated in graded ethanol and acetone series, and embedded in the mixture of Epon and Spurr (1: 1) for 48 h at 60 °C. For light microscopy 1.4 11m sections were cut and stained with 1 % toluidine blue. Ultrathin sections (70 nm) were stained with uranyl acetate for 10 min and lead citrate for 15 min and examined under a JEOl JEM-1200 transmission electron microscope operating at 80 kV.

Results

Isolation andphysical analysis o/Exo- mutants Random Tn5 mutagenesis was carried out by mating E.

coli pSUP5011 with R. leguminosarum bv. tn/olii 24.1 to obtain mutants deficient in exopolysaccharide synthesis (Exo-). From the Km'Sm' transconjugants, we isolated several non-mucoid, rough colonies. Rough morphology of these mutants appeared on mannitol-yeast-extract and minimal agar medium. Thirteen Tn5 mutants revealed the same

Two types of nodules of Rhizobium leguminosarum bv. tn/olii Exo- mutants

pattern of bacteriophage sensitivity as the parental strain 24.1. They were sensitive to bacteriophages 3H, 411 and 412 and resistant to 1P. To confirm the presence of the Tn5 sequence in each Exomutant, a Southern blot of EcoRI-digested total DNA was probed with 32P-Iabeled pSUP2021 DNA which carries the Tn5 transposon. For all but one (Rt76), a single hybridizing band was observed (Table 1). To identify the location (plasmid or chromosome) of Tn5 insertion in each Exo- mutant, the Eckhardt procedure (Hynes et al., 1985) was used. The wild type parental strain exhibits 3 plasmid bands, pRta, b and c, at molecular weights approximately 180 kb, 300 kb and 500 kb (Fig. 1a). The lower band, pRta, has homology to the nifKDH genes (Skorupska et al., 1991). The Southern blot of the «Eckhardt,. gel of the Exo- mutants of R. leguminosarum bv. tn/alii 24.1 was hybridized to 32P-Iabeled pSUP2021 (Fig. 1b). The mutants Rt55 and Rt66, had Tn5 inserted into the largest plasmid, pRtc (- 500 kb), whereas the mutants Rt69, Rt74, Rt76 and Rt79 had Tn5 inserted in plasmid pRtb (- 300 kb). In the other mutants strains, Tn5 was not detected in the plasmid bands; therefore it was assumed to be inserted in chromosome (Fig. 1, Table 1).

1

2

3

4

5 6

7

8

9 10 11

95

Table 1: Characteristic of Exo- derivatives of R.leguminosarum bv. tn/olii strain 24.1. Mutant strains

Location of Tn5

Mol. weight of EcoRI::Tn5 DNA fragment (kb)

chromosom pRtc chromosom chromosom chromosom pRtc pRtb chromosom pRtb pRtb chromosom chromosom pRtb

11.8

infected

8.9 19.5

empty

Type of nodules

R. /epminosarum bv. tri/oJii Rt53 Rt55 Rt56 Rt62 Rt65 Rt66 Rt69 Rt71 Rt74 Rt76 Rt77 Rt78 Rt79

11.5

10.6 21,4 19.0 17.9 9.0 10.6; 5.7 17.9 10.6 12.7

infected infected empty empty empty

infected infected empty

infected infected infected

23456789

lPS

I

l PS II

Fig. 2: DOC-PAGE analysis of Exo- mutants and parental strain R. leguminosarum bv. tri/oli. Wild-type strain 24.1 (line 1), Rt76 (lane

1 2

3

4

5

6

7 8

9 10 11

2), Rt66 (lane 3), Rt55 (lane 4), Rt69 (lane 5), Rt79 (lane 6), Rt71 (lane 7), Rt62 (lane 8), Rt56 (lane 9). LPSII represents the lower molecular weight forms containing lipid A and core oligosaccharides. LPSI represents the higher molecular weight molecules consisting of lipid A. core oligosaccharides and 0 antigen oligosaccharides of different degrees of polymerization.

Charaeteristia 0/LPS 0/ the exopolysaccharide-deficient

mutants Fig. 1: Eckhardt plasmid gel electrophoresis and Southern blot analysis of R.leguminosarum bv. tn/olii 24.1 and its Exo- derivatives. a. Plasmid profile of R. leguminosarum bv. tn/olii strain 24.1 (line 6) and its Tn5 derivatives: Rt53 (line 1), Rt55 (line 2), Rt62 (line 3), Rt66 (line 4), Rt69 (line 5), Rt74 (line 8), Rt77 (line 9), Rt78 (line 10), Rt79 (line 11). Every Exo- mutant but Rt71 displayed the presence of three megaplasmids typical for parental strain Rt24.1: pRta (180 kb), pRtb (300 kb), pRtc (500 kb). b. Transposon Tn5 localization on Exo - mutants megaplasmids. Autoradiography showed Tn5 insertion either on plasmid pRtb for mutants Rt69 (line 5), Rt74 (line 8), Rt79 (line 11) or on plasmid pRtc for mutants Rt55 (line 1), Rt66 (line 4).

Acidic EPS was obtained from strain 24.1 by cetrimide precipitation. This procedure gave no precipitation of EPS when applied to the non-mucoid mutants. It as conceivable that alterations in polysaccharides other than EPS, e.g. LPS, could also have occurred. To investigate this possibility, the LPS profiles of the parental strain and the mutant strains were compared after electrophoresis and subsequent silver staining. All samples exhibited very similar migration patterns (Fig. 2). In all preparations tested, two banding regions were visible; one with a faster mobility containing, a series of regularly spaced prominent bands and a second with a slower mobility, containing a number of faint bands.

96

ANNA SKOllUPSKA, UIlSZULA BIALEK, TEllESA UIlBANIK-SYPNIEWSKA, and ANDRE VAN LAMMI!llEN

Table 2: Symbiotic properties of R. leguminosarum bv. tn/olii Exo- mutants.

R.legumin. bv. tn/olii 24.1 Rt53 Rt55 Rt56 Rt62 Rt65 Rt66 Rt69 Rt71 Rt74 Rt76 Rt77 Rt78 Rt79

Average no. of nodules/plant on primary root 14d. 28d.

on lateral root 14d. 28d.

2.6

3.5 0.1 0.4

3.2 0 0.4

14.4 2.3 5.0

~2

~1

3~

o o o o o o o o o

0.3 0.1 0.4 0.1

~1

0.2 0.8 0.1 0.5 0.2 0.9 0.5 0.9 0.8

M

1.0 0.4 0.8 0.1 0.5 2.8 0.1 1.1

0.5

~

4.0 5.2 3.8 8.0 5.6 6.4 5.5 6.9 7.9

14d.

Total 28d.

5.8

o

0.4 0.1 0.6 1.0 0.4 0.8 0.1 0.5 3.1 0.2 1.5 0.6

Green wet mass/plant

Reduction of acetylene nM ethylene/plant/h

127.5 42.9 47.5 39.6 39.9 40.8 40.4 44.7 48.0 50.0 36.0 48.8 44.2 43.3

120,4 2.1

(mg) 17.9 2.4 5.4 3.8 6.0 4.2 6.0 3.9 8.5 5.8 7.3 6.0 7.8 8.7

o

4.36 9.44 1.8 1.94 4.86 6.9 8.8 6.4 1.48 1.6 3.75

Given values were obtained from three experiments; 20 plants were used at each one.

Symbiotic properties ofexopolysaccharide-deficient mutants Exo- mutants were tested on nodule formation and nitrogen-fIxation abilities on clover. Inoculation of 5-day-old clover seedlings with wild-type strain 24.1 led to visible nodules within 5-7 days and the nodules were formed on primary and lateral roots. After 3- 4 weeks the nodules were pink, caused by the presence of leghemoglobin. All Exo - mutants tested induced non-nitrogen-fIxing nodules (Fix-) on clover. This was proved by the acetylene reduction test and measurement of green mass of clover plants. White, small nodules appeared 10-14days after inoculation, mainly on the lateral roots and occasionally on the primary roots (Table 2). The efficacy of nodulation was 40 - 60 % of the infected plants. We have never observed reversion to Fix+ phenotype. Bacteria reisolated from mutant-induced nodules were Kmr and non-mucoid.

Light and electron microscopy Based upon microscopic studies on four-week-old nodules elicited by Exo- mutants, two types of phenotypically different nodules were distinguished. Nodules were infected with baeteroids in the central tissue or were not infected and called the empty nodules. Infected nodules were induced by Exo- mutants Rt53, Rt56, Rt62, Rt71, Rt74, Rt77, Rt78 and Rt79. They were characterized by infection threads and very large plant cells packed with numerous baeteroids (Fig. 3 b, short arrows). In infected nodules we distinguished three developmental zones (Fig. 3 b) instead of the five zones found in nodules induced by wild-type bacteria (Fig. 3 a for zones I-III). Only the meristematic zone, the prefixation zone and interzone ll-ill were formed (Fig. 3 b). In the inner part of the peripheral tissue, surrounding the meristematic zone, heavily stained, grainy structures were found (Fig. 3 b, black arrows). Their location was restricted to 2-3 cell layers of the

peripheral tissue (Fig. 3 d). Electron microscopy studies revealed that these structures were highly osmiophilic and either surrounded by the cytoplasm or arranged in a string-ofbeads-like pattern along the tonoplast (Figs. 3 d, e). In the pre-fixation zone the infection threads penetrating plant cells were much larger (Fig. 4 b) than typical wild-type infection threads (Fig. 4 a). They were packed with degenerating bacteria (Fig. 4 b, white arrows). Ultrastructural analysis of the nodules induced by Exo- mutants of R. leguminosarum bv. tnfolii showed pronounced differences in the bacteroid appearance in comparison to wild-type baeteroids. Ultrastructural data on baeteroids are to be published elsewhere (Bialek et al., submitted). The morphology of infected and non-infected plant cells and bacteroids in the interzone llill are shown in Figs. 4 c, d. The bacteroids were smaller than in wild-type nodules, irregular in shape (Fig. 4 d) and often prematurely degenerated. Starch accumulation was more pronounced in mutant than in wild-type nodules (d. Figs. 4 c, d). Very large plastids harboring flattened starch grains were typical for the infected cells of the interzone llill, while in non-infected cells starch grains displayed ovoid appearance (Fig. 4 d). Non.infected nodules were induced by the Exo- mutants Rt55, Rt65, Rt69, and Rt76. The central tissue of these nodules was occupied by large, highly vacuolate plant cells (Fig. 3 c, short arrows) often containing a few plastids harbouring ovoid starch grains (Fig. 3 c, long arrows). Generally, starch accumulation in this type of nodules was much lower than in nodules induced by the other Exo- mutants (d. Figs. 3 b, c). Occasionally, in these nodules some infection threads and some infected plant cells were observed (not shown). When such traces of the infection were found, the nodules exhibited heavily stained, grainy structures in the plant cells in the inner part of the peripheral tissue surrounding the meristematic area. They were similar to the structures also found in the infected nodules induced by the other Exomutants.

Two types of nodules of Rhizobium leguminosarum bv. tnlolii Exo- mutants

97

Fig. 3: Light and electron micrographs of nodules induced by R. leguminosarum bv. tnlolii strain 24.1 and Exo- mutants. Bars represent 200 11m for a, b and c; 10 11m for d; 111m for e. a. Longitudinal section of a nodule induced by strain 24.1 showing four developmental zones: the meristematic zone (1), the pre-fixation zone (II), the interzone (II - ill) and the nitrogen-fixing zone (ill). b. Longitudinal section of a nodule induced by Exo - mutant RtS3. Note the lack of the nitrogen-fixing zone (ill). Long white arrows point to starch grains, short white arrows point to infected cells, black arrows point to grainy structures in the peripheral tissue. c. Longitudinal section of a nodule induced by Exo- mutant Rt69. Long white arrows point to starch grains, short white arrows point to highly vaCuolate plant cells. d. Electron micrograph showing the accumulation of the osmiophilic structures in the peripheral tissue (PT) and their absence in the meristematic zone (1). e. High magnification electron micrograph of the electron-dense structures shown on Fig. 3 d.

DI8CU881on

There is genetical evidence that plasmid and chromosomal genes affect EPS synthesis in fast growing rhizobia i.e. R. melilati (for review see Arnold et al., 1993/94). Recently Chen et al. (1993) reported that the second largest megaplasmid (300Mdal) of R. leguminosarum bv. tri/olii ANU 1173 carries genes whose products are involved in both EPS and LPS synthesis. Our studies clearly show that TnS insertions re-

suIt in bacterial disability to produce EPS, were located on the two largest megaplasmids and on the chromosome. Additionally, LPS of mutated rhizobia, analyzed by DOCPAGE, was not visibly changed in comparison to LPS of the parental strain. These data confirm that the plasmids pRtb and pRtc, which were considered to be cryptic in R. leguminosarum bv. tri/olii 24.1, are involved in the production of EPS. All Exo - mutants isolated so far were less efficient in infecting clover than the parental strain 24.1, and induced

Fig. 4: Electron micrographs of nodules induced by strain 24.1 and by Exo- mutant RtS3; bars represent Silm. a, b. Infection threads (IT) and baeteroids (B) in a wild-type (a) and a mutant nodule (b). c. d. Bacteroids and starch (S) appearance at the transition from the pre-fixation zone to the interzone ll-ill in a wild-type (c) and a mutant nodule (d). Note the differences in the shape of starch grains in infected (IC) and uninfected (DC) plant cells in both types of nodules.

Two types of nodules of Rhizobium leguminosarum bv. trifolii Exo- mutants nodules finally defective in nitrogen fixation. Although all Exo- mutants displayed the same non-mucoid phenotype, they elicited two types of nodules on clover roots i.e. infected and non-infected ones. Based on these nodule phenotypes we distinguish two groups of Exo - mutants. Infected nodules were elicited by the group of rhizobia appearantly mutated in genes controlling later stages of infection. In these nodules, the infection process was far in progress. We, however, observed enlarged infection threads which harboured degenerated bacteroids but plant cells were also invaded by numerous bacteroids. The second group of R. legu· minosarum bv. tn/olii Exo- mutants had to be mutated in genes controlling early stages of clover infection, and induced non-infected nodules devoided infection threads and bacteroids. The empty nodules induced by these mutants essentially resembled the nodules induced on alfalfa, by noninfecting Exo - mutants of R. meliloti described by Leigh et al. (1985) and Finan et al. (1986). Detailed genetical analysis of Exo- mutants is needed to explain the differences in the morphology and structure of the nodules. Niehaus et al. (1993) recently found indications that R. meliloti EPSI or a related compound might act as a suppressor of plant defence. EPSI-deficient mutants failed in normal nodule development, although they were able to penetrate into intercellular spaces of the root cortex and to induce the infection thread-like structures. In clover - R. leguminosarum bv. tn/olii interactions, EPS seems to be more important at later stages of the symbiosis since plant defence reactions occurred after the release of the first bacteroids. The change in surface structures of Exo - mutants might lead to the release of plant defence reactions. These signals could prevent the development of new infection threads, act against bacteria in already existing infection threads, as they seem to contain no protective matrix, and finally they could stop the development of the bacteroids released into plant cells. Such a block in development, both in shape as well as in succession of gene expression in released bacteroids was recently demonstrated in clover - Exo - mutant interaction (Bialek et al., submitted). Furthermore, the nodules induced by Exo- mutants exhibited electron-dense grainy structures in the cytoplasm of the cortical cells surrounding the meristematic area. The appearance and subcellular localisation of these osmiophilic structures shows striking similarity to storage protein deposits found in developing protein storage vacuoles of young pea cotyledons (Hoh et al., 1995). Similar structures were localized in 2-3 layers of specialized cortical cells surrounding the infected zone of the nitrogen-fixing peanut root nodules (Bal, 1993). They were described as localized pockets of electron-dense material at the plasma membrane-cell wall interface and were shown to be proteinaceous and most likely complexed with suberin or phenolics. Although the chemical composition of electrondense structures found in clover nodules elicited by Exomutants remains to be elucidated, their exclusive occurrance in mutant-induced nodules indicated that their accumulation could be a consequence of a deposition of plant defence related metabolites. On the other hand, the presence of these osmiophilic granules could be a result of the accumulation of metabolites synthesized by plant symbiont and not metabolized further due to bacterial deficiency in nitrogen reduc-

99

tion. Additionally, the pronounced starch accumulation in both infected and uninfected plant cells indicates slower metabolism of nodules. In R. meliloti - alfafa interactions EPSI deficient mutants were capable to induce infected nodules but nodulation was delayed up to 5 weeks after inoculation (Niehaus et al., 1993). That delay was needed for bacteria to overcome plant defence response. We suggest that in clover - R. leguminosarum bv. tn/olii interactions the absence of exopolysaccharides might trigger a comparable plant defence response after release of first bacteria from infection threads. As a result of incompatible interactions, nodule development and especially bacteroid maturation are stopped and no nitrogen fixation takes place. Acknowledgements

The authors are indebted to Sybout Massalt, Paul van Snippenburg and Allex Haasdijk for the photographic work and Mrs. Maria Maiek for excellent technical assistance.

References ARNOLD, W., A. BECKER, M. KELLER, A. RoXLAu, and A. POHLEll: The role of Rhizobium meliloti surface polysaccharides in the infection of Medicago sativa. Endocytobiosis & Cell Res. 10, 1728 (1993/94). BAHRANI, K. F. andJ. D. OUVER: Electrophoretic analysis of lipopolysaccharide isolated from opaque and translucent colony variants of Vibrio vulnificus using various extraction methods. Microbios 66,83-93 (1991). BAL, A. K.: Electron-dense material in the cell wall/plasma membrane interface of specialized cortical cells of peanut nodules. Cell Biology International, 17, 227 - 233 (1993). BECKER, A., A. KLE!CItMANN, W. ARNOLD, and A. POHLEll: Analysis of the Rhizobium meliloti exoH/exoKiexoL fragment: ExoK shows homology to excreted endo-p-1,4-glucanases and ExoH resembles membrane proteins. Mol. Gen. Genet. 238,145-154 (1993 a). BECKER, A., A. KLE!CItMANN, M. KELLER, W. ARNOLD, and A. POHLER: Identification and analysis of the Rhizobium meliloti exoAMONP genes involved in exopolysaccharide biosynthesis and mapping of promoters located on the exoHKLAMONOP fragment. Mol. Gen. Genet. 241, 367-379 (1993 b). BIALEK, U., A. SKORUPSKA, W.-c. YANG, T. BISSELING, and A. A. M. VAN LAMMEREN: Disturbed gene expression and bacteroid development in Trifolium pratense root nodules induced by Tn5 mutants of Rhizobium leguminosarum bv. trifoli defective in polysaccharide synthesis. Submitted. BORTHAlWR, D., J A. DOWNIE, A. W. B. JOHNSTON, and J LAMB: ps~ a plasmid-linked Rhizobium phaseoli gene that inhibits exopolysaccharide production, and which is required for symbiotic nitrogen fixation. Mol. Gen. Genet. 200, 278-282 (1985). BORTHAKUR, D. and A. W. B. JOHNSTON: Sequence of ps~ a gene on the symbiotic plasmid of Rhizobium phaseoli which inhibits exopolysaccharide synthesis and nodulation and demonstration that its transcription is inhibited by psr, another gene on the symbiotic plasmid. Mol. Gen. Genet. 207, 149-154 (1987). BREWIN, N. J: Development of the legume root nodule. Annu. Rev. Cell BioI. 7, 191-226 (1991). BUENDIA, A. M., B. ENENKEL, R. KOPLIN, K. NIEHAUS, W. ARNOLD, and A. POHLEll: The Rhizobium meliloti exoZ/exoB fragment of megaplasmid 2: ExoB functions as an UDP-glucose 4-epimerase and ExoZ shows homology to NodX of Rhizobium leguminosa· rum biovar viciae strain TOM. Mol. Microbiol. 5 (6), 15191530 (1991).

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ANNA SKOIlUPSKA, UIlSZULA BIALEI., TERESA UIlBANlK-SYPNIEWSKA, and ANDRE VAN LAMMEIlEN

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