Plant Molecular Biology 12: 307-315, 1989 © 1989 Kluwer Academic Publishers. Printed in Belgium
307
Peribacteroid membrane nodulin gene induction by japonicum mutants
Bradyrhizobium
Robert B. Mellor, ~ Christine Garbers 2 and Dietrich Werner*
Botanisches Institut, Fachbereich Biologic der Philipps-UniversitiitMarburg, D-3550 Marburg, Federal Republic of Germany; ~Present address: Botanisches Institut der Universitat Basel CH-4056 Basel Switzerland; 2present address: Max-Planck-Institut far Ziichtungsforschung, D-5000 K6ln, Federal Republic of Germany (*authorfor correspondance) Received 4 May 1988; accepted in revised form 13 December 1988
Key words: biological signalling, Bradyrhizobiumjaponicum, Glycine max, peribacteroid membrane, root nodules, symbiosis
Abstract
Seventeen translation products from Glycine max root mRNA precipitated with antiserum prepared against a peribacteroid membrane preparation from effective root nodules. Messenger RNA fromfix + nodules coded for these 17 products plus 7 other nodule-specific polypeptides which bound to the antiserum. Of these 7 nodulins only 4 were present when nodules were infected with Bradyrhizobium japonicum 110 r/f 15 2960, which induces the plant to produce 'empty' peribacteroid membranes. In nodules infected with B. japonicum strains inducing either very short-lived or defective peribacteroid membrane, only 5 or 6, respectively, of these nodulins could be detected. From these results we hypothesize that the microsymbiont is responsible for the production of at least 4 different signals leading to peribacteriod membrane formation by the plant.
Introduction
The symbiosis of leguminous plants with bacteria of the family Rhizobiaceae involves a chain of events culminating in the formation of a new plant organ, the nodule. Fully mutualistic (fix + ) symbioses are characterized by the presence of over 20 'nodule-specific' proteins, the nodulins [13, 20]. Nodulins appear to be induced by a process involving at least two steps in nodules of Medicago [24], lupin [12], pea [9] and soybean [8]. All fi x+ and many fix- bacteroids are surrounded in the nodule by a host membrane, called the peribacteroid membrane (PBM). PBM is derived from the biosynthetic part of the plant
endomembrane system [ 18]. How the prokaryote induces the eukaryotic partner to produce the peribacteroid membrane is unknown. However PBM contains several nodulins [4, 5, 29]. One of these, nodulin 26, is expressed independently of PBM formation whereas other PBM nodulins are dependent on the appearance of PBM [21]. On the other hand, PBM synthesis ean proceed independently of bacteroid proliferation [26]. In order to understand this process, we have used bacterial mutants provoking a range of symbiotic phenotypes and analysed the expression of all host genes coding for PBM polypeptides by studying nodule mRNA composition.
308 Materials and methods
Growth of plants The axenic titre-dependent infection of 10-dayold sterile phytotron-grown seedlings was as before [31]. Nodule tissue 28days old was harvested on ice and used immediately. Plants infected with f i x - bacteria were supplied with exogenous N from the 17th day [32].
trols are presented by MeUor etaL [14]. Pure membrane contained no leghaemoglobin, no G D P - D M P mannosyltransferase, glucan synthetases II or I, latent IDPase, galactosyldiacylglyceride transferase or fumarase [16]. Thus, the preparations were not contaminated with either host cell cytoplasm or host ER, plasma membrane, Golgi or plastid-membranes, or mitochondria.
Preparation of anti-PBM serum Bacteria Bacteria were designated according to their ability to fix N 2 gas in a derepressed free-living form (ni39 and in symbiotic nodules Oqx). Bradyrhizobium japonicum 61-A-101 is a mf + fix + wild type which differentiates into bacteroids surrounded by a stable peribacteroid membrane. For characterization, see Werner and Mrrschel
[31]. B. japonicum RH-31 Marburg is an UVinduced mutant of the above, nif- f i x - , differentiates into bacteroids surrounded by a peribacteroid membrane. Bacteroids are digested inside membrane envelopes in the vicinity of the host cell nucleus [33]. B.japonicum 61-A-24 is a nif- f i x - field isolate. Differentiated bacteroids lie naked in the host cell cytoplasm due to early loss of peribacteroid membrane [32]. B.japonicum 110, nif + fix + is a field isolate similar to 6 l-A- 101. B. japonicum 110 spc 4 A3 is a nif E deletion mutant of the strain above, nif- f i x - , differentiating into bacteroids surrounded by stable peribacteroid membrane [25]. B.japonicum 110 rifl5 2960 is nif ÷ f i x - , does not differentiate into bacteroids but induces 'empty' peribacteroid membrane envelope formation [26].
Pooled pure PBM was subjected first to Smith degradation [ 11 ], then sulphitolysed [23], a process which unfolds proteins using antigenically neutral SO4 z- groups and destroys antigenic glycolipids associated with membranes [10]. Proteins were deglycosylated with E n d o D and Endo H as described by Rosenfeld etal. [27], then freeze-dried, resuspended in 50 m M pyridine acetate buffer, pH 5.5 and polypeptides were purified from sugars and saccharide fragments by passage over a G200 column. Proteins were portioned into 200/~g aliquots, freeze-dried, and stored at - 70 °C until use. Aliquots, resuspended in 750 #1 complete Freund's adjuvant, were injected intra muscularly into alternatively the right and left thighs of Klein X Chincilla F1 hybrid rabbits 2, 3, 4, 5, 7, 10, 14, 17, 21 and 28 days after the first injection (day 1). Rabbits were bled on day 38. After clotting, serum was brought to 50 ~o (NH4)2SO 4 saturation and the precipitated proteins dialysed overnight against physiologically buffered phosphate saline. This solution was treated with insolubilized Endo D and Endo H to remove contaminating antibodies against these proteins, whereupon the IgG fraction was purified by F P L C on an alkylsepharose H R 5/5 column (Pharmacia).
Gold labelling
Tissue fractionation
0.01~o AuC14 in citrate buffer was heated at
Pure PBM was isolated as described by Mellor and Werner [16]. Electron microscopical con-
100 °C as before [22] to give gold grains of 10 nm diameter. 10 ml of this solution bound 400 #g protein. We routinely bound antiserum to gold at a
309 ratio of 80/~g per ml. After centrifugation at 50000 x gfor 30 min, gold conjugate was washed once in fresh buffer, then treated with 800/~g per ml original volume bovine serum albumin (BSA) to block any remaining binding sites, whereupon the washing 15rocedure was repeated to remove excess protein.
Electrophoresis One-dimensional SDS-PAGE (intact membranes) was performed in Tris-glycine buffer as before [17], using 1 mm thick 10-15~o acrylamide gradient gels (c--0.27 - 0.4%). Twodimensional urea-IEF X SDS-PAGE (translation products) was as described [34]. SDSPAGE gels were 2 mm thick and the gradient was t = 12-12~, c---0.52-1.5~. Gels were either stained with Coomassie brillant blue R250 or western-blotted onto nitrocellulose paper at 10 Vcm- ~ for 2 h (1 mm gels) or 4 h (2 mm gels). Blots were either stained with immunogold (10 ml concentrated gold conjugate containing 1 mg antiserum for 4-6 h) or with 10-3~o amido black. Blots of separated translation products were dried and layed on Dupont Cronex 4 film for 3 weeks in an intensifier box.
RNA used for translation experiments. RNA translations were run for 60 min at 30 ° C in 35 #1 ready-to-use rabbit reticulocyte lysate system with supplementary amino acids (Serva) and 50 #Ci (35S) methionine (Amersham, translation grade). 0.3-0.1/~g poly(A) + was used, resulting in ca. 20 000 cpm/~1 - 1incorporation into protein. 25/21 of antiserum was added and followed by incubation at 4 °C for 24 h. Immuno complexes were insolubilized by incubation (4 ° C, 24 h) with 40 #1 protein A-Sepharose 6B (Sigma). Sepharose was recovered by centrifugation and washed in phosphate-buffered saline. The pellet, which contained around 14000 cpm, was finally resuspended in 50 #1 8.5 M urea, containing 2% NP40, 0.5~o mercaptoethanol and 5 ~ ampholine (pH 3-10) prior to isoelectric focusing.
Results
Figure 1 shows that conjugated antiserum against PBM bound to polypeptides present in the peribacteroid membrane. Several peptides only weakly visible on the Coomassie-stained gel were heavily stained with antiserum and all other major polypeptides showed reaction with the antiserum.
RNA isolation and translation Tissue was pulverized in liquid nitrogen, then extracted at a iatio of 1 g to 2.5 ml in 0.2 M sodium acetate, pH 5.0, 1% SDS, 10 mM EDTA, whereupon 2.5 ml of phenol, containing 0.1 ~o 8-hydroxyquinoline, was added. After constant agigation for 5min, 2.5ml ehloroform:isoamyl alcohol (24:1) was added. After mixing and low-speed centrifugation the aqueous phase was washed first with phenol :chloroform (1:1), then with chloroform. LiC1 was added to an end concentration o f 2 M and precipitated RNA allowed to sediment overnight at 4 oC. RNA pellets were washed first in 2 M LiCI, then twice in 60% ethanol. RNA was then subjected to two cycles of oligo(dT) cellulose chromatography and released poly(A) +
Fig. 1. Proteins separated by SDS-PAGE. Lanes 1, 4 and 5, marker proteins; lanes 2 and 3, peribacteroid membrane proteins. Lanes 1 and 2, Coomassie blue-stained; lanes 3-5, blotted and stained with either immunogold (lanes 3 and 4) or amido schwarz (lane 5).
310
Fig. 2. Autoradiogramof two-dimensionalIEF/SDS-PAGE separation of anti-PBM immunoprecipitatedtranslation products coded by mRNA from (a) uninfectedroot tissue nst proteins 14-17 are circled, or (b) nodules infected with Bradyrhizobium japonicum 61-A-101. In b, nst proteins 14-17 are marked with black stars and nodulins A-G are encircled.
The antiserum did not react with marker proteins and no longer bound to PBM proteins when the pH was lowered to 2.5. No staining was evident when conjugated null serum was used (data not shown), showing non-specific binding was negligable. Cross reaction was not found using up to 100/~g plant cytoplasm in Laurell immunoelectrophoresis gels containing up to 63 #g antiserum ml gel- 1 (data not shown). Cross reactivity with other subcellular fractions has not yet been explored. Since the antigen used contained no other membrane type, such reactivity would indicate biogenetic relationships, which we intend to investigate in the future. Membrane antigen was deglycosylated before immunization proceeded and contained practically no bound saccharide, which has previously been found to be very antigenic [3]. Thus, our antiserum contained antibodies recognising the polypeptide parts of peribacteroid membrane proteins.
Translation products from root RNA binding to anti-PBM were separated electrophoretically. Seventeen proteins were detected after autoradiography, 4 of which were present in only trace quantities (Fig. 2a, 1-13 and 14-17). Thus, RNA coding for 17 PBM proteins is present in root tissue. Immunoprecipitated translation products from effective nodules showed not only these 17 proteins but also 7 other ones (Fig. 2b, A-G). These are presumed to be membrane (PBM) nodulins. This assumption is supported by the data of Legocki and Verma [ 13 ] who detected nodulespecific translation products in similar positions which did not react with nodule-specific antisera prepared from soluble nodule fractions. The number of 7 nodulins is a minimum number. Due to possible RNA degradation we cannot rule out that others, especially of higher molecular weight, were not detected in our system. Four proteins found in gels using RNA from
311 root tissue (Nos 14-17) were strongly stimulated in nodules. These products (42-48 kDa) hence appear to belong to the class of nodule-stimulated (nst) proteins. RNA was also analysed from nodules where 'empty' PBM is made [26]. Figure 3 shows that these bacteria (r/f 15 2960) stimulated the plant to produce RNA coding for the 17 proteins as in roots, but also for 4 nodulins (A-D), indicating that the amounts of translatable RNA coding for the other 3 (nodulins E - G ) was present at unde-
tectable levels. The 4 nst proteins were detected at levels similar to those found in uninfected root tissue. In nodules infected with the parent type of this strain or with the mutant spc4 A3, the same immunoprecipitated polypeptide pattern as in the effective 61-A-101 strain was found (compare Fig. 3b with Fig. 2b). Thus, the changed polypeptide pattern with n f l 5 2960-infected tissue cannot be traced back to trivial factors such as nitrogen starvation. The formation of either normal morphology or
Fig. 3. As in Figure 2, with mRNA from nodules infected with (a) Bradyrhizobiumjaponicum rif 15 2960 or (b) Bradyrhizobium japonieum 110, the parental wild type. Missing nodulins E, F and G are encircled and black stars mark the position ofnst proteins 14-17,
312 'empty' PBMs are not, however, the only responses of the plant to symbiotic infection. Figure 4a shows that after infection with strain RH-31 Marburg, lyric vacuoles are formed in the vicinity of the host cell nucleus where bacteria are digested [33]. In nodules infected with strain 61-A-24, P B M in unstable and breaks down early in the colonization process [32], (Fig. 4b). This leads to elevated phytoalexin levels in such nodules [30]. Translation products from plant R N A extracted from such nodules, immunoprecipitated with anti-PBM, are shown in Fig. 4. Both bacterial strains stimulated the plant to produce R N A coding for the proteins also found on gels using rif 15 2960 nodules (i.e. 17 non-nodulins and nodulins A - D ) but showed differences in the expression of the remaining nodulin genes
(encoding nodulins E, G and F; Fig. 2). Mutant RH-31 failed to stimulate the plant to produce detectable levels of one nodulin (nodulin F). Using nodules infected with strain 61-A-24, not only could nodulin F not be detected, but the gel was also clear at the position ofnodulin G and the expression of genes coding for the nst-proteins 14 and 15 sunk to root levels.
Discussion This in vitro translation o f m R N A and subsequent analysis of radioactive polypeptides by twodimensional electrophoresis has been used to study gene expression in response to symbiotic infection in peas [1] and soybeans [8, 13]. In the
Fig. 4. Ultrastructural morphologyof peribacteroid membranes correlated with autoradiogram of two-dimensional IEF/SDSPAGE separatation ofimmunoprecipitated translation products coded by mRNA from nodules infected with a) Bradyrhizobium japonicum RH 31 (lysis of bacteroids in the vicinity of the host cell nuclesu) or b)B.japonicum 61-A-24 (very early loss of peribacteroid membrane), a) n = host cell nucleus, v = vacuole. The circle marks the position of missing nodulin F. b) c = cell wall, bar = 1/~m.Circles indicate the position of missing nodulins F and G. Black stars represent the weaklyexpressed proteins 14 and 15.
313 Table I.
Plant response to bacterial signals. 1-13
Root
+
14-15
16-17
nodulins
nodulin
nodulin
nodulin
stimulated
stimulated
A-D
E
F
G
-
-
Bradyrhizobium japonicum 61-A-101
+
+
+
+
+
+
+
110
+
+
+
+
+
+
+
110 r i f 15 2 9 6 0
+
-
-
+
-
-
-
R H 31
+
+
+
+
+
-
+
61-A-24
+
-
+
+
+
-
-
110 s p c 4 A 3
+
+
+
+
+
+
+
Summary of plant responses to bacterial signals expressed as introduction or stimulation of groups of peribacteroid membrane protein component
genes.
studies reported here translation products from uninfected root RNA were precipitated by antiserum directed against peribacteroid membrane polypeptides. These consitutively synthesized proteins are presumably, in uninfected root, sequestered in other membranes [3]. The factors inducing these proteins to redistribute themselves, at least partly, into the PBM in root nodules, are unknown. However Verma et aL [29] have speculated that relevant factors may include the distinctive lipid composition of the PBM [ 15]. The nodulins detected in these experiments fall into two size groups, one around 24-25 kDa and the second between 26 and 29 kDa. Until the exact molecular weights are precisely determined we have chosen to call them nodulins A - G (Fig. 2b). In effective symbioses PBM nodulins have already been described at 24 kDa [4] and 26-29 kDa [5]. The nodulins described here are clearly different from other structural proteins such as nodulin 75, which has a function in nodule morphogenesis [6]. Table 1 summarizes the anti-PBM-binding translation products from RNA of nodules formed by various strains of Bradyrhizobium japonicum. From this the following points can be made. 1. The presence ofnodulins A - D was always correlated with nodule induction (it must be stressed here that the 'empty' PBM structures formed by
r/f 15 2960 may be energetically and metabolically inert). 2. Nodulins A - G and relatively large amounts of nst-proteins 14-17 are synthesised by nodules producing an effective symbiotic PBM phenotype. 3. Bacteroids normally exist in a lyric environment [ 14, 18] from which they can defend themselves [ 7 ], but the failure of the bacteria to stimulate the plant to express the nodulin F gene results in the fusion of PBMs and the digestion of bacteroids. 4. The lack of nodulins F and G together with the failure of the bacterium to stimulate production of nst proteins 14 and 15 is associated with an unstable situation where peribacteroid membrane (but not the bacteroid) break down. The analyses presented in this report support the theory that upon commencement of the colonization process the microsymbiont sends a signal to the plant host ordering the production of basic peribacteroid membrane [26]. This PBM may not, however, be symbiotically effective and contains nodulins A - D only. The bacterial signals leading to the expression of nodulins E - F require the physical presence of the bacteroid in the host cell. This interpretation is in good agreement with that of Morrison and Verma [21 ] who found PBM nodulin 26 expressed at normal levels infix- nodules without PBM, whereas PBM no-
314 dulin 27 (and others) was only found after the endocytosis of the microsymbiont. Earlier studies on phospholipid biosynthesis [15, 19] and glycosyltransferase activities [17] in nodules infected with various mutant bacteria also indicated a twostep process of P B M biogenesis. It has recently been reported that once the peribacteroid compartment is inhabited by endocytosed bacteria, bacteroids can influence the plant to change the protein composition of the P B M [34], indicating that this second step also consists of at least two parts. Judging by the nodulin and nst protein response documented here, this second step, the expression of genes coding for P B M nodulins E, F and G, can be regarded as consisting of at least 3 separate and independent parts. Early P B M nodulin gene induction (expression before endocystis) appears to be the result of at least one bacterial signal. The genes responsible for signal production are not known, but they do not appear to be mfgenes. Interestingly, the transfer of bacterial polypeptides, but not nucleic acids, to the host cell at this stage has been postulated by various groups [2, 28]. Bacterial genes responsible for the further 3 biological signals produced by internalized bacteria are also unknown. Since n f l 5 2960 possesses full sets of functional m f a n d nod genes and yet does not provoke the late plant responses, the involvement of these sets of genes can be ruled out. Four new fix loci influencing P B M development have recently been identified in Rhizobium meliloti [24] and our future studies will be aimed at characterizing such loci in Bradyrhizobium japonicum.
Acknowledgements We thank Prof. H. Hennecke (Ztirich) for the gift of m a n y of the bacterial mutants used. Finance was provided from the Sonderforschungsbereich 305, project 'Ecophysiology - processing of environmental signals'. Ms H. Thierfelder routinely isolated R N A and Ms L. Karner typed the manuscript.
References 1. BisselingT, Franssen H., Govers F, Goudemans T, Louwerse J, Moerman M, Nap JP, van Kammen A: Nodulin gene expression in Pisumsativum. In: Evans HJ, Bottomley PJ, Newton WE (eds) Nitrogen Fixation Research Progress, pp53-59. Martinus Nijhoff, Dordrecht/Boston/Lancaster (1985). 2. Bruening ML, Wullstein LH: Evidencefor the transfer of rhizobial metabolites to the polyploid nuclei of young clover nodules. Physiol Plant 27:244-252 (1972). 3. Brewin NJ, Robertson JG, Wood EA, WellsB, Larkin AP, Galfre G, Butcher GW: Monoelonal antibodies to antigens in the peribacteroid membrane from Rhizobium induced root nodules of pea cross-react with plasma membranes and Golgi bodies. EMBOJ 4: 605-611 (1985). 4. Fortin MG, Zelechowska M, Verma DPS: Specific targetting of membrane nodulins to the peribacteroidenclosing compartment in soybean nodules. EMBO J 4: 3041-3046 (1985). 5. Fortin MG, Morrison NA, Verma DPS: Nodulin-26a, a peribacteroid membrane nodulin expressed independently of the peribacteroid compartment. Nucleic Acids Res 15:813-824 (1987). 6. Franssen HP, Nap JP, Goudemans T, Stiekema W, van Dam H, Govers F, LouwerseJ, van Kammen A, Bisseling T: Characterization of eDNA for nodulin-75 of soybean: A gene product involvedin early of root nodule development. Proc Natl Acad. Sci USA 84:4495-4499 (1987). 7. Garbers C, Meckbach, R, Mellor RB, Werner D: Protease (thermolysin) inhibition activity in the peribacteroid space of Glycinemax root nodules. J Plant Physiol 132:442-445 (1988). 8. Gloudemans T, de Vries S, Bussink HJ, Malik NSA, Franssen HJ, Louwerse J, BisselingT: Nodulin gene expression during soybean (Glycine max) nodule development. Plant Mol Biol 8:395-403 (1987). 9. Govers F, Nap JP, MoermanM, FranssenHJ, van Kammen A, BisselingT: cDNA cloning and developmental expression of pea nodulin genes. Plant Mol Biol 8:425-435 (1987). 10. Herrlinger JD: Antigenspezifische Unterdrickung der AntikOrperbildung sensibilisierterTiere. Fischer Verlag, Stuttgart (1979). 11. Kim YS, Perdomo J, Nordberg: Glycoprotein biosynthesis in small intestinal mucosa. J Biol Chem 246: 5466-5476 (1971). 12. Konieczny A., SzczyglowskiK, Boron L, Przybylska M, Legocki AB: Expression of lupin nodulin genes during root nodule development. Plant Sci 55:145-149 (1988). 13. Legocki RP, Verma DPS: Identification of'nodule specific' host proteins (nodulins) involvedin the development of Rhizobium-legume symbiosis.Cell 20:153-163 (1980).
315 14. MeUor RB, Mtirschel E, Werner D: Legume root response to symbiotic infection. Enzymes of the peribacteroid space. Z Naturforsch 39c: 123-125 (1984). 15. Mellor RB, Christensen TMIE, Bassarab S, Werner D: Phospholipid transfer from ER to the peribacteroid membrane in soybean nodules. Z Naturforsch 40c: 73-79 (1985). 16. Mellor RB, Werner D: The fractionation of Glycine max root nodule cells: A methodological overview. Endocyt C Res 3:317-336 (1986). 17. Mellor RB, ChristensenTMIE, Werner D: Choline kinase II is present only in nodules that synthesize stable peribacteroid membranes. Proc Natl Acad Sci USA 83: 659-663 (1986). 18. Mellor RB, Werner D: Peribacteroid membrane biogenesis in mature legume root nodules. Symbiosis 3: 75-100 (1987). 19. Mellor RB, Thierfelder H, Pausch G, Werner D: The occurrence of choline kinase II in the cytoplasm of soybean root nodules infected with various strains of Bradyrhizobium japonicum. J Plant Physiol 128:169-172 (1987). 20. Morrison NA, Bisseling T, Verma DPS: Development and differentiation of the root nodule: Involvement of plant and bacterial genes. In: Browder W (ed) Developmental Biology, Vol. 5, pp. 405-425. Plenum, New York (1988). 21. Morrison NA, Verma DPS: A block in the endocytosis of Rhizobium allows cellular differentiation in nodules but affects the expression of some peribacteroid membrane nodulins. Plant Mol Biol 9:185-196 (1987). 22. Ostrowski E, Mellor RB, Werner D: The use of colloid gold labelling in the detection of plasma membrane from symbiotic and non-symbiotic Glyeine max root cells. Physiol Plant 66:270-276 (1986). 23. Pechere JF, Dixon GH, Maybury RH, Neurath H: Cleavage of disulfide bonds in trypsinogen and ~-chymotrypsinogen. J Biol Chem 233:1364-1372 (1958). 24. PutnokyP, GrosskopfE, Cam Ha DT, Kiss GB, Kondorosi A: Rhizobiumfix genes mediate at least two communication steps in symbiotic nodule development. J Cell Biol 106:597-607 (1988).
25. Regensburger B, Hennecke H: Free-living and symbiotic nitrogen fixing ability of Rhizobiumjaponieum is unaffected by rifampicin resistance mutations. FEMS Microbiol Lett 21:77-81 (1984). 26. Regensburger B, Meyer L, FilserM, Weber J, Studer D, Lamb JW, Fischer HM, Hahn M, Hennecke H: Bradyrhizobiumjaponicum mutants defective in root-nodule bacteroid development and nitrogen fixation. Arch Microbial 144:355-366 (1986). 27. Rosenfeld MG, Kreibich G, Porov D, Kato K, SabatiniDD: Biosynthesis of lysosomal hydrolases: Their synthesis in bound polysomes and the role of coand post-translational processing in determining their subcellular distribution. J Cell Biol 93:135-143 (1982). 28. Truchet G, Michel M, Denarie J: Sequential analysis of the organogenesis of lucerne root nodules using symbiotically defective mutants of Rhizobium meliloti. Differentiation 16:163-172 (1980). 29. Verma DPS, Fortin MG, Stanley J, Mauro VP, Purohit S, MorrisonN: Nodulins and nodulin genes of Glycine max: A perspective. Plant Mol Biol 7:51-61 (1986). 30. Werner D, Mellor RB, Hahn M, Grisebach H: Soybean root response to symbiotic infection: Glyceollin I accumulation in an ineffective type of soybean nodule with an early loss of the peribacteroid membrane. Z Naturforsch 40:179-181 (1985). 31. Werner D, M/Srschel E: Differentiation of nodules of Glycine max. Ultrastructural studies of plant cells and bacteroids. Planta 141:169-177 (1978). 32. Werner D, M6rschel E, StripfR, Winchenbach B: Development of nodules of Glycine max infected with an ineffective strain of Rhizobium japonicum Planta 147: 320-329 (1980). 33. Werner D, M6rschel E, Kort R, Mellor RB, Bassarab S: Lysis of bacteroids in the vicinity of the host cell nucleus in an ineffective (fix - ) root nodule of soybean (Glycine max). Planta 162:8-16 (1984). 34. Wcrner D, MOrschel E, Garbers C, Bassarab S, MellorRB: Particle density and protein composition of the peribacteroid membrane from soybean root nodules is affected by mutation in the microsymbiont Bradyrhizobiumjaponicum. Planta 174:263-270 (1988).
307
Peribacteroid membrane nodulin gene induction by japonicum mutants
Bradyrhizobium
Robert B. Mellor, ~ Christine Garbers 2 and Dietrich Werner*
Botanisches Institut, Fachbereich Biologic der Philipps-UniversitiitMarburg, D-3550 Marburg, Federal Republic of Germany; ~Present address: Botanisches Institut der Universitat Basel CH-4056 Basel Switzerland; 2present address: Max-Planck-Institut far Ziichtungsforschung, D-5000 K6ln, Federal Republic of Germany (*authorfor correspondance) Received 4 May 1988; accepted in revised form 13 December 1988
Key words: biological signalling, Bradyrhizobiumjaponicum, Glycine max, peribacteroid membrane, root nodules, symbiosis
Abstract
Seventeen translation products from Glycine max root mRNA precipitated with antiserum prepared against a peribacteroid membrane preparation from effective root nodules. Messenger RNA fromfix + nodules coded for these 17 products plus 7 other nodule-specific polypeptides which bound to the antiserum. Of these 7 nodulins only 4 were present when nodules were infected with Bradyrhizobium japonicum 110 r/f 15 2960, which induces the plant to produce 'empty' peribacteroid membranes. In nodules infected with B. japonicum strains inducing either very short-lived or defective peribacteroid membrane, only 5 or 6, respectively, of these nodulins could be detected. From these results we hypothesize that the microsymbiont is responsible for the production of at least 4 different signals leading to peribacteriod membrane formation by the plant.
Introduction
The symbiosis of leguminous plants with bacteria of the family Rhizobiaceae involves a chain of events culminating in the formation of a new plant organ, the nodule. Fully mutualistic (fix + ) symbioses are characterized by the presence of over 20 'nodule-specific' proteins, the nodulins [13, 20]. Nodulins appear to be induced by a process involving at least two steps in nodules of Medicago [24], lupin [12], pea [9] and soybean [8]. All fi x+ and many fix- bacteroids are surrounded in the nodule by a host membrane, called the peribacteroid membrane (PBM). PBM is derived from the biosynthetic part of the plant
endomembrane system [ 18]. How the prokaryote induces the eukaryotic partner to produce the peribacteroid membrane is unknown. However PBM contains several nodulins [4, 5, 29]. One of these, nodulin 26, is expressed independently of PBM formation whereas other PBM nodulins are dependent on the appearance of PBM [21]. On the other hand, PBM synthesis ean proceed independently of bacteroid proliferation [26]. In order to understand this process, we have used bacterial mutants provoking a range of symbiotic phenotypes and analysed the expression of all host genes coding for PBM polypeptides by studying nodule mRNA composition.
308 Materials and methods
Growth of plants The axenic titre-dependent infection of 10-dayold sterile phytotron-grown seedlings was as before [31]. Nodule tissue 28days old was harvested on ice and used immediately. Plants infected with f i x - bacteria were supplied with exogenous N from the 17th day [32].
trols are presented by MeUor etaL [14]. Pure membrane contained no leghaemoglobin, no G D P - D M P mannosyltransferase, glucan synthetases II or I, latent IDPase, galactosyldiacylglyceride transferase or fumarase [16]. Thus, the preparations were not contaminated with either host cell cytoplasm or host ER, plasma membrane, Golgi or plastid-membranes, or mitochondria.
Preparation of anti-PBM serum Bacteria Bacteria were designated according to their ability to fix N 2 gas in a derepressed free-living form (ni39 and in symbiotic nodules Oqx). Bradyrhizobium japonicum 61-A-101 is a mf + fix + wild type which differentiates into bacteroids surrounded by a stable peribacteroid membrane. For characterization, see Werner and Mrrschel
[31]. B. japonicum RH-31 Marburg is an UVinduced mutant of the above, nif- f i x - , differentiates into bacteroids surrounded by a peribacteroid membrane. Bacteroids are digested inside membrane envelopes in the vicinity of the host cell nucleus [33]. B.japonicum 61-A-24 is a nif- f i x - field isolate. Differentiated bacteroids lie naked in the host cell cytoplasm due to early loss of peribacteroid membrane [32]. B.japonicum 110, nif + fix + is a field isolate similar to 6 l-A- 101. B. japonicum 110 spc 4 A3 is a nif E deletion mutant of the strain above, nif- f i x - , differentiating into bacteroids surrounded by stable peribacteroid membrane [25]. B.japonicum 110 rifl5 2960 is nif ÷ f i x - , does not differentiate into bacteroids but induces 'empty' peribacteroid membrane envelope formation [26].
Pooled pure PBM was subjected first to Smith degradation [ 11 ], then sulphitolysed [23], a process which unfolds proteins using antigenically neutral SO4 z- groups and destroys antigenic glycolipids associated with membranes [10]. Proteins were deglycosylated with E n d o D and Endo H as described by Rosenfeld etal. [27], then freeze-dried, resuspended in 50 m M pyridine acetate buffer, pH 5.5 and polypeptides were purified from sugars and saccharide fragments by passage over a G200 column. Proteins were portioned into 200/~g aliquots, freeze-dried, and stored at - 70 °C until use. Aliquots, resuspended in 750 #1 complete Freund's adjuvant, were injected intra muscularly into alternatively the right and left thighs of Klein X Chincilla F1 hybrid rabbits 2, 3, 4, 5, 7, 10, 14, 17, 21 and 28 days after the first injection (day 1). Rabbits were bled on day 38. After clotting, serum was brought to 50 ~o (NH4)2SO 4 saturation and the precipitated proteins dialysed overnight against physiologically buffered phosphate saline. This solution was treated with insolubilized Endo D and Endo H to remove contaminating antibodies against these proteins, whereupon the IgG fraction was purified by F P L C on an alkylsepharose H R 5/5 column (Pharmacia).
Gold labelling
Tissue fractionation
0.01~o AuC14 in citrate buffer was heated at
Pure PBM was isolated as described by Mellor and Werner [16]. Electron microscopical con-
100 °C as before [22] to give gold grains of 10 nm diameter. 10 ml of this solution bound 400 #g protein. We routinely bound antiserum to gold at a
309 ratio of 80/~g per ml. After centrifugation at 50000 x gfor 30 min, gold conjugate was washed once in fresh buffer, then treated with 800/~g per ml original volume bovine serum albumin (BSA) to block any remaining binding sites, whereupon the washing 15rocedure was repeated to remove excess protein.
Electrophoresis One-dimensional SDS-PAGE (intact membranes) was performed in Tris-glycine buffer as before [17], using 1 mm thick 10-15~o acrylamide gradient gels (c--0.27 - 0.4%). Twodimensional urea-IEF X SDS-PAGE (translation products) was as described [34]. SDSPAGE gels were 2 mm thick and the gradient was t = 12-12~, c---0.52-1.5~. Gels were either stained with Coomassie brillant blue R250 or western-blotted onto nitrocellulose paper at 10 Vcm- ~ for 2 h (1 mm gels) or 4 h (2 mm gels). Blots were either stained with immunogold (10 ml concentrated gold conjugate containing 1 mg antiserum for 4-6 h) or with 10-3~o amido black. Blots of separated translation products were dried and layed on Dupont Cronex 4 film for 3 weeks in an intensifier box.
RNA used for translation experiments. RNA translations were run for 60 min at 30 ° C in 35 #1 ready-to-use rabbit reticulocyte lysate system with supplementary amino acids (Serva) and 50 #Ci (35S) methionine (Amersham, translation grade). 0.3-0.1/~g poly(A) + was used, resulting in ca. 20 000 cpm/~1 - 1incorporation into protein. 25/21 of antiserum was added and followed by incubation at 4 °C for 24 h. Immuno complexes were insolubilized by incubation (4 ° C, 24 h) with 40 #1 protein A-Sepharose 6B (Sigma). Sepharose was recovered by centrifugation and washed in phosphate-buffered saline. The pellet, which contained around 14000 cpm, was finally resuspended in 50 #1 8.5 M urea, containing 2% NP40, 0.5~o mercaptoethanol and 5 ~ ampholine (pH 3-10) prior to isoelectric focusing.
Results
Figure 1 shows that conjugated antiserum against PBM bound to polypeptides present in the peribacteroid membrane. Several peptides only weakly visible on the Coomassie-stained gel were heavily stained with antiserum and all other major polypeptides showed reaction with the antiserum.
RNA isolation and translation Tissue was pulverized in liquid nitrogen, then extracted at a iatio of 1 g to 2.5 ml in 0.2 M sodium acetate, pH 5.0, 1% SDS, 10 mM EDTA, whereupon 2.5 ml of phenol, containing 0.1 ~o 8-hydroxyquinoline, was added. After constant agigation for 5min, 2.5ml ehloroform:isoamyl alcohol (24:1) was added. After mixing and low-speed centrifugation the aqueous phase was washed first with phenol :chloroform (1:1), then with chloroform. LiC1 was added to an end concentration o f 2 M and precipitated RNA allowed to sediment overnight at 4 oC. RNA pellets were washed first in 2 M LiCI, then twice in 60% ethanol. RNA was then subjected to two cycles of oligo(dT) cellulose chromatography and released poly(A) +
Fig. 1. Proteins separated by SDS-PAGE. Lanes 1, 4 and 5, marker proteins; lanes 2 and 3, peribacteroid membrane proteins. Lanes 1 and 2, Coomassie blue-stained; lanes 3-5, blotted and stained with either immunogold (lanes 3 and 4) or amido schwarz (lane 5).
310
Fig. 2. Autoradiogramof two-dimensionalIEF/SDS-PAGE separation of anti-PBM immunoprecipitatedtranslation products coded by mRNA from (a) uninfectedroot tissue nst proteins 14-17 are circled, or (b) nodules infected with Bradyrhizobium japonicum 61-A-101. In b, nst proteins 14-17 are marked with black stars and nodulins A-G are encircled.
The antiserum did not react with marker proteins and no longer bound to PBM proteins when the pH was lowered to 2.5. No staining was evident when conjugated null serum was used (data not shown), showing non-specific binding was negligable. Cross reaction was not found using up to 100/~g plant cytoplasm in Laurell immunoelectrophoresis gels containing up to 63 #g antiserum ml gel- 1 (data not shown). Cross reactivity with other subcellular fractions has not yet been explored. Since the antigen used contained no other membrane type, such reactivity would indicate biogenetic relationships, which we intend to investigate in the future. Membrane antigen was deglycosylated before immunization proceeded and contained practically no bound saccharide, which has previously been found to be very antigenic [3]. Thus, our antiserum contained antibodies recognising the polypeptide parts of peribacteroid membrane proteins.
Translation products from root RNA binding to anti-PBM were separated electrophoretically. Seventeen proteins were detected after autoradiography, 4 of which were present in only trace quantities (Fig. 2a, 1-13 and 14-17). Thus, RNA coding for 17 PBM proteins is present in root tissue. Immunoprecipitated translation products from effective nodules showed not only these 17 proteins but also 7 other ones (Fig. 2b, A-G). These are presumed to be membrane (PBM) nodulins. This assumption is supported by the data of Legocki and Verma [ 13 ] who detected nodulespecific translation products in similar positions which did not react with nodule-specific antisera prepared from soluble nodule fractions. The number of 7 nodulins is a minimum number. Due to possible RNA degradation we cannot rule out that others, especially of higher molecular weight, were not detected in our system. Four proteins found in gels using RNA from
311 root tissue (Nos 14-17) were strongly stimulated in nodules. These products (42-48 kDa) hence appear to belong to the class of nodule-stimulated (nst) proteins. RNA was also analysed from nodules where 'empty' PBM is made [26]. Figure 3 shows that these bacteria (r/f 15 2960) stimulated the plant to produce RNA coding for the 17 proteins as in roots, but also for 4 nodulins (A-D), indicating that the amounts of translatable RNA coding for the other 3 (nodulins E - G ) was present at unde-
tectable levels. The 4 nst proteins were detected at levels similar to those found in uninfected root tissue. In nodules infected with the parent type of this strain or with the mutant spc4 A3, the same immunoprecipitated polypeptide pattern as in the effective 61-A-101 strain was found (compare Fig. 3b with Fig. 2b). Thus, the changed polypeptide pattern with n f l 5 2960-infected tissue cannot be traced back to trivial factors such as nitrogen starvation. The formation of either normal morphology or
Fig. 3. As in Figure 2, with mRNA from nodules infected with (a) Bradyrhizobiumjaponicum rif 15 2960 or (b) Bradyrhizobium japonieum 110, the parental wild type. Missing nodulins E, F and G are encircled and black stars mark the position ofnst proteins 14-17,
312 'empty' PBMs are not, however, the only responses of the plant to symbiotic infection. Figure 4a shows that after infection with strain RH-31 Marburg, lyric vacuoles are formed in the vicinity of the host cell nucleus where bacteria are digested [33]. In nodules infected with strain 61-A-24, P B M in unstable and breaks down early in the colonization process [32], (Fig. 4b). This leads to elevated phytoalexin levels in such nodules [30]. Translation products from plant R N A extracted from such nodules, immunoprecipitated with anti-PBM, are shown in Fig. 4. Both bacterial strains stimulated the plant to produce R N A coding for the proteins also found on gels using rif 15 2960 nodules (i.e. 17 non-nodulins and nodulins A - D ) but showed differences in the expression of the remaining nodulin genes
(encoding nodulins E, G and F; Fig. 2). Mutant RH-31 failed to stimulate the plant to produce detectable levels of one nodulin (nodulin F). Using nodules infected with strain 61-A-24, not only could nodulin F not be detected, but the gel was also clear at the position ofnodulin G and the expression of genes coding for the nst-proteins 14 and 15 sunk to root levels.
Discussion This in vitro translation o f m R N A and subsequent analysis of radioactive polypeptides by twodimensional electrophoresis has been used to study gene expression in response to symbiotic infection in peas [1] and soybeans [8, 13]. In the
Fig. 4. Ultrastructural morphologyof peribacteroid membranes correlated with autoradiogram of two-dimensional IEF/SDSPAGE separatation ofimmunoprecipitated translation products coded by mRNA from nodules infected with a) Bradyrhizobium japonicum RH 31 (lysis of bacteroids in the vicinity of the host cell nuclesu) or b)B.japonicum 61-A-24 (very early loss of peribacteroid membrane), a) n = host cell nucleus, v = vacuole. The circle marks the position of missing nodulin F. b) c = cell wall, bar = 1/~m.Circles indicate the position of missing nodulins F and G. Black stars represent the weaklyexpressed proteins 14 and 15.
313 Table I.
Plant response to bacterial signals. 1-13
Root
+
14-15
16-17
nodulins
nodulin
nodulin
nodulin
stimulated
stimulated
A-D
E
F
G
-
-
Bradyrhizobium japonicum 61-A-101
+
+
+
+
+
+
+
110
+
+
+
+
+
+
+
110 r i f 15 2 9 6 0
+
-
-
+
-
-
-
R H 31
+
+
+
+
+
-
+
61-A-24
+
-
+
+
+
-
-
110 s p c 4 A 3
+
+
+
+
+
+
+
Summary of plant responses to bacterial signals expressed as introduction or stimulation of groups of peribacteroid membrane protein component
genes.
studies reported here translation products from uninfected root RNA were precipitated by antiserum directed against peribacteroid membrane polypeptides. These consitutively synthesized proteins are presumably, in uninfected root, sequestered in other membranes [3]. The factors inducing these proteins to redistribute themselves, at least partly, into the PBM in root nodules, are unknown. However Verma et aL [29] have speculated that relevant factors may include the distinctive lipid composition of the PBM [ 15]. The nodulins detected in these experiments fall into two size groups, one around 24-25 kDa and the second between 26 and 29 kDa. Until the exact molecular weights are precisely determined we have chosen to call them nodulins A - G (Fig. 2b). In effective symbioses PBM nodulins have already been described at 24 kDa [4] and 26-29 kDa [5]. The nodulins described here are clearly different from other structural proteins such as nodulin 75, which has a function in nodule morphogenesis [6]. Table 1 summarizes the anti-PBM-binding translation products from RNA of nodules formed by various strains of Bradyrhizobium japonicum. From this the following points can be made. 1. The presence ofnodulins A - D was always correlated with nodule induction (it must be stressed here that the 'empty' PBM structures formed by
r/f 15 2960 may be energetically and metabolically inert). 2. Nodulins A - G and relatively large amounts of nst-proteins 14-17 are synthesised by nodules producing an effective symbiotic PBM phenotype. 3. Bacteroids normally exist in a lyric environment [ 14, 18] from which they can defend themselves [ 7 ], but the failure of the bacteria to stimulate the plant to express the nodulin F gene results in the fusion of PBMs and the digestion of bacteroids. 4. The lack of nodulins F and G together with the failure of the bacterium to stimulate production of nst proteins 14 and 15 is associated with an unstable situation where peribacteroid membrane (but not the bacteroid) break down. The analyses presented in this report support the theory that upon commencement of the colonization process the microsymbiont sends a signal to the plant host ordering the production of basic peribacteroid membrane [26]. This PBM may not, however, be symbiotically effective and contains nodulins A - D only. The bacterial signals leading to the expression of nodulins E - F require the physical presence of the bacteroid in the host cell. This interpretation is in good agreement with that of Morrison and Verma [21 ] who found PBM nodulin 26 expressed at normal levels infix- nodules without PBM, whereas PBM no-
314 dulin 27 (and others) was only found after the endocytosis of the microsymbiont. Earlier studies on phospholipid biosynthesis [15, 19] and glycosyltransferase activities [17] in nodules infected with various mutant bacteria also indicated a twostep process of P B M biogenesis. It has recently been reported that once the peribacteroid compartment is inhabited by endocytosed bacteria, bacteroids can influence the plant to change the protein composition of the P B M [34], indicating that this second step also consists of at least two parts. Judging by the nodulin and nst protein response documented here, this second step, the expression of genes coding for P B M nodulins E, F and G, can be regarded as consisting of at least 3 separate and independent parts. Early P B M nodulin gene induction (expression before endocystis) appears to be the result of at least one bacterial signal. The genes responsible for signal production are not known, but they do not appear to be mfgenes. Interestingly, the transfer of bacterial polypeptides, but not nucleic acids, to the host cell at this stage has been postulated by various groups [2, 28]. Bacterial genes responsible for the further 3 biological signals produced by internalized bacteria are also unknown. Since n f l 5 2960 possesses full sets of functional m f a n d nod genes and yet does not provoke the late plant responses, the involvement of these sets of genes can be ruled out. Four new fix loci influencing P B M development have recently been identified in Rhizobium meliloti [24] and our future studies will be aimed at characterizing such loci in Bradyrhizobium japonicum.
Acknowledgements We thank Prof. H. Hennecke (Ztirich) for the gift of m a n y of the bacterial mutants used. Finance was provided from the Sonderforschungsbereich 305, project 'Ecophysiology - processing of environmental signals'. Ms H. Thierfelder routinely isolated R N A and Ms L. Karner typed the manuscript.
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