Symbiosis-related polypeptides associated with the early stages of ectomycorrhiza organogenesis in birch {Betula pendula Roth)

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1 New Phytoi. (1993), 124, Symbiosis-related polypeptides associated with the early stages of ectomycorrhiza organogenesis in birch {Betula pendula Roth) BY P. SIMONEAU*, J. D. VIEMONT, J. C. MOREAU AND D. G. STRULLU Laboratoire de Phytonique, Universite' d'angers, 2 Bd Lavoisier, Angers Cedex, France {Received 3 February 1993; accepted 17 March 1993) SUMMARY As a preliminary step towards elucidation of the molecular basis of ectomycorrhiza differentiation, polypeptide changes at different stages of mycorrhiza development were analyzed in birch {Betula pendula Roth). Timesequencing of the stages in the infection process of clonal plants inoculated with a compatible isolate of Paxillus involutus Batsch revealed that by 8 d mature ectomycorrhizas were obtained. Total phenol-extracted proteins of roots during the ' mycorrhiza formation stage' (2-8 d post-inoculation) were separated by two-dimensional polyacrylamide gel electrophoresis and the resulting patterns compared with non-mycorrhizal roots and mycelium. Alteration in the concentration of polypeptides from the host plant roots was limited even after 8 d of contact between the symbionts. However, seven novel polypeptides were detected 4 d after inoculation, three of them being already present m 2-d-old ectomycorrhizas. These findings demonstrate that symbiosis-related polypeptides accumulate in ectomycorrhizal roots prior to any of the morphological changes characterizing the symbiotic state. Key words: Ectomycorrhiza, differentiation, symbiosis, polypeptides, Betula pendula, Paxillus involutus. the extension of the results obtained for the INTRODUCTION PisoHthus/Eucalyptus symbiosis to other symbiotic The ectomycorrhiza symbiosis induced by specific systems at least until more detailed analyses with soil fungi on tree roots are very frequent phenomena. different host plants have been performed. Whatever The morphological events that accompany the the situation in fully-developed ectomycorrhizas, establishment of this type of association have been developmental genetic data (Martin & Hilbert, 1991) extensively studied (for reviews see Harley& Smith, suggest that preceding the establishment of the 1983; Strullu, 1985). Ultimately, a compatible symbiotic state, i.e. the functioning mycorrhiza, a interaction leads to the differentiation of active series of interactions between the two symbionts, ectomycorrhizas. The formation and function of that probably begins with chemotropic attraction of these symbiotic organs which consist of both host the fungus (Horan & Chilvers, 1990), is required, and fungal tissues, probably result from complex Although it has recently been shown that specific interactions between products from develop- proteins are synthesized prior to any morphological mentally-critical genes (Martin & Hilbert, 1991). changes during the development of eucalypt Extensive changes in concentration of particular ectomycorrhiza (Hilbert, Costa & Martin, 1991), polypeptides were observed in fully-developed euca- our knowledge of the molecular events in the initial lypt mycorrhizas (Hilbert & Martin, 1988). How- stages of the plant-fungus association is still scarce, ever, the fact that such differential accumulation of It seems therefore of a prime importance to search proteins was not detected in ectomycorrhizas of for a possible accumulation of specific polypeptides Picea abies (Guttenberger & Hampp, 1992) precludes in other ectomycorrhizal systems. The feasibility of controlled in-vitro association * To whom correspondence should be addressed. between clonal plants of Betula pendula Roth and the

2 496 P. Simoneau and others Roots (short roots plus tap roots) in contact with the fungal mycelium were collected at various times post-inoculation and frozen in liquid N,^ after excision of excess extramatrical myceliuni. These samples, plus roots from control plants and residual mycelium on the cellophane discs, were stored at C until use. o o I 3 O O 10 - Estimation of the infection level of inoculated roots - 5- at c 3 Days Figure 1. Fungal biomass in birch roots inoculated with Paxilius involutus. Roots from birch (clone B747) were inoculated with mycelium from Paxillus involutus (isolate PO) using a sandwich technique. The level of infection was estimated by measuring the ergosterol content of sampled roots. The mean value of the ergosterol content oi Paxillus involutus mycelia (2/<g mg ' fresh weight) was used as conversion factor. Eacb value is the mean of three replicates (±SD). ectomycorrhizal fungus, Paxillus involutus (Batsch), has been previously demonstrated and the infection process cytologically studied (Grellier, Letouze & Strullu, 1984). This symbiotic system, in which all the plants have exactly the same genetic background, has therefore been used to examine changes in gene expression during the mycorrhizal infection process. Here, we present results concerning the study of the changes in polypeptide patterns that occur during the early steps of mycorrhizal infection. MATERIALS AND METHODS Synthesis of mycorrhizas Micropropagated clonal plants of Betula pendula were obtained as previously described (Grellier et ai., 1984, 1989), except that glass beads were used as an alternative to agar in the medium used for the last transfer before fungal inoculation. The isolate (PO) of Paxillus involutus, derived from a sporocarp collected under birch (western France), was cultured on a cellophane membrane in Petri dishes containing MMN solid medium (Marx, 1969). The symbionts were associated using a sandwich technique (Chilvers, Douglass & Lapeyrie, 1986). Briefly 8-wk-old plants were layered over the surface of a solid micropropagation medium (Grellier et al., 1984) in large Petri dishes and their root systems covered with cellophane membranes invaded by mycelia. Plants were then returned to the controlled environment chamber (16 h light at 23 C followed by 8 h dark at 17 C, with an irradiance of 1()OO//J cm"^ s""') and left until sampling. Control plants received the same treatment except that their roots were covered with sterile cellophane discs. The proportion of fungal biomass in the inoculated roots was estimated by measuring their fungal ergosterol content by reverse-phase HPLC (Martin, Delaruelle & Hilbert, 1990). The rate of ectomycorrhizal development was also determined by examining sections of roots prepared and stained as previously described (Grellier et al., 1984). Protein extraction and solubilization For every protein extraction, samples corresponding to the root tips in contact with the fungal mycelium {500 mg), non-inoculated control roots (500 mg) and free-living mycelium (250 mg) were powdered in liquid Ng with a mortar and a pestle. Total proteins were then phenol-extracted from this powder (Schuster & Davies, 1983); the dry pellet from the last precipitation step was resuspended in solubilization buffer (Laemmli 1970) where /?-mercaptoethanol had been replaced by 5 mm dithiothreitol, and placed for 3 min in a boiling water bath. Non-solubilized materials were removed by a short centrifugation in a bench microfuge. Protein concentration of the supernatant was determined using the micro-bc A kit (Pierce). For the two-dimensional analysis, proteins were precipitated from the solubilization buffer with four volumes of 0-07 % (v/v) /?-mercaptoethanol in cold acetone, collected by centrifugation (10000^, 30 min, 4 C), rinsed once with cold precipitation solution, dried under vacuum and resuspended for 1 h at 37 C in lysis buffer (O'Farrell, 1975) containing 2"o (v/v) preblended ampholytes (ph range 3-^10; Pharmacia). Electrophoretic analysis Two-dimensional polyacrylamide gel electrophoresis (2D~PAGE) was as described by O'Farrell (1975) with slight modifications. Proteins were loaded at the basic end of tube gels (diameter, 0-8 mm ; length, 12 mm) containing ampholytes : 3 % (v/v) ph range 3-10 (Pharmacia) and 1 % (v/v) ph range 4-8 (BDH), that had been prefocalized for 1 h at 1200 V (1 W for 6 gels). Gels were run for 2 h at 500 V, 18 h at 1200 V and 0-5 h at 1500 V, extruded from the glass tubes, soaked for 15 min in equilibration buffer (Tris-HCl 0125 mm ph 6 8, glycerol 10% v/v, SDS 2-3% w/v) and stored at C until use. Electrophoresis in the second dinaension

3 Early symbiosis-related polypeptides in birch ectomycorrhizas 497 was performed as described except that the equilibrated gels were placed directly on the top of 12-5% (w/v) acrylamide separating gels (137x 137x 1 mm) without any sealing (Garrels, 1979). Alternatively, 15% (w/v) acrylamide gels were used for better resolution of low-molecularweight (MW) polypeptides. Gels were run at 6 W per gel until the bromophenol blue had reached the bottom. After electrophoresis gels were stained with silver nitrate according to the procedure of Oakley, Kirsh & Morris (1980). The apparent MW and isoelectric point of polypeptides were estimated by comparing their migration with that of standard proteins with known molecular masses (phosphorylase b, 97-4 kda; bovine serum albumin, 66-0 kda; ovalbumin, 450 kda; chymotrypsinogen, 25-0 kda; cytochrome C, 12-4 kda) or isoelectric point (carbamylated carbonic anhydrase; Pharmacia). Changes in accumulation of specific polypeptides were recorded only when they occurred in all the gels under comparison (four successive inoculation experiments and two independent protein extractions, i.e. eight gels per type of sample). Polypeptides were named by a letter E, D, or F for ectomycorrhiza-specific, ectomycorrhiza-decreased, and fungal-specific polypeptides, respectively, followed by a number denoting their relative molecular masses. RESULTS Dynamics of the infection In order to study the changes that occur at the protein level during the early stages of colonization of the root by the fungal symbiont, it was first necessary to examine the rate of ectomycorrhizal development under our experimental conditions. This was performed both quantitatively by measuring the ergosterol content in inoculated roots and qualitatively by microscopic observations of root sections at various times post-inoculation. The rapid increase in ergosterol content of roots (Fig. 1), consecutive to the fungal colonization revealed that the interaction between the two partners was compatible. Based on the curve shape the following sequence of events was defined: (i) a ' preinfection stage' (from 0 to 2 d of contact) where ergosterol w as barely detectable, (ii) a 'mycorrhiza formation stage' (from 2 to 8 d) characterized by a continuous accumulation of ergosterol in roots and (iii) a 'mature mycorrhiza stage' (more than 8 d of contact) where Figure 2. Birch roots 2-8 d after inoculation with Paxillus involutus. Longitudinal sections through roots after 2 d (A) and 4 d (B), showing the attachment of the fungal mycelium (h) to the root surface (A) and further development of the fungal sheath (m) (B); x 300. Stereomicrograph of roots after 8 d (C) showing young mature mycorrhizas formed on second and third-order roots; x40. c. Cortical cells; e, epidermis.

4 498 P. Simoneau and others SDS Figure 3. Two-dimensional patterns of polypeptides from non-inoculated roots (A), 8 d-old-ectomycorrhizas (B) and free-living mycelia (C). Gels (12-5 "o acrylamide) were loaded with 300/<g (A), 450//g (B) and 150/ig (C) of proteins. Gels were silver-stained. Arrows indicate major fungal polypeptides; circled spots correspond to root polypeptides whose concentrations were significantly decreased during mycorrhizal development; squared spots indicate two major root polypeptides that were not affected by fungal inoculation with the fungus and therefore served as reference polypeptides. Triangles indicate the position of the symbiosis-related polypeptides detected in mature ectomycorrhizas. Numbers on the top of the gels correspond to ph values; molecular masses of size markers are indicated in kda.

5 Early symbiosis-related polypeptides in birch ectomycorrhizas lef ISDS 974 Figure 3(C). For legend see opposite. accumulation of ergosterol had stopped. This sequence was confirmed by microscopic observations which revealed the following events: (i) at 2 d after inoculation, contacts were already established between the root and the hyphae: attachment of the fungus to the root surface and initiation of the mantle by proliferating hyphae were evident (Fig. 2A); (ii) at 4 d, a fungal sheath developed by accumulation of several layers of hyphae was observed (Fig. 2B) and (iii) after 8 d of contact, about 75 % of the second-order roots examined had the anatomy of mature mycorrhizas (Fig. 2C). At this stage, these synthetic mycorrhizas were comparable to those analyzed in a previous study (Grellier et al., 1989) with an Hartig net beginning to extend from the deepest layers of the fungal mantle between cortical cells, elongating radially. By contrast, such typical organization was never observed in first-order roots, although the fungus had totally enveloped them after 8 d. Changes in polypeptide patterns of mature ectomycorrhizas Alterations in the relative accumulation of polypeptides in fully developed mycorrhizas were estimated by paired comparison of two-dimensional gels from 8-d-old mycorrhizas with those from control roots and mycelia (Fig. 3). Adjustment of the amount of proteins loaded onto the former gels was necessary since these samples contained proteins from both partners. This was performed using the following data: (i) 22% of the mass in the mature 8-d-old ectomycorrhizas was fungal based on the ergosterol content and, (ii) double the proteins were phenol-extracted from the fungus than from an equal amount of roots, i.e. 1-8 and 0-9 mg of protein were respectively extracted from 1 g of frozen material. Gels in Figure 3 were therefore loaded with 300, 450, and 150/ig of non-infected root, ectomycorrhiza, and fungal proteins, respectively. Highly resolved gels were obtained, more than 800 inoculated or non-inoculated root polypeptides and approximately 450 fungal polypeptides were resolved with good reproducibility, 95 % of them being consistently found at nearly the same relative position in eight independent experiments. Upon global inspection, the most striking difference between gels was the presence in inoculated roots (Fig. 3 B) of a number of proteins that were absent in control roots (Fig. 3 A). The great majority of them were easily identified as proteins constituting the fungal mycelium (Fig. 3C), e.g. spots Fj to F^^; note that, based on staining intensity, the multispot V^.^ ^^ represents the most abundant polypeptide in the mycelium from P. involutus (isolate PO). Concomitantly, a decreased accumulation of a small number of plant polypeptides was noticed during fungal colonization (e.g. spots Dj to Dg with MW ranging from 110 to 25 kda). A few novel polypeptides (Ej, Ej, and E5 with MW of 70, 47, and 27 kda, respectively) were also detected in roots after 8 d of close contact with the ectomycorrhizal fungus. Based on its position on the 2D-map, the

6 500 P. Simoneau and others 4-8 lef SDS » «4-8 I I 6-7 I B Figure 4. For part C and legend, see opposite.

7 501 Early symbiosis-related polypeptides in birch ectomycorrhizas SDS Figure 4. Changes in root polypeptides during ectomycorrhizal development. Gels (15 ' acrylamide) were loaded with 300 //g of proteins extracted from inoculated roots sampled 2 d (B) and 4 d (C) post-inoculation or a mixture of non-inoculated roots and free-living mycelia with a 9/1 ratio (A). Arrows indicate symbiosisspecific polypeptides. Other symbols are as in Figure 3. Numbers on the top of the gels correspond to ph values; molecular masses of size markers are indicated in kda. former might correspond to a modified form of the fungal polypeptide F^j that was found in free-living mycelia. Modifications of polypeptide patterns during the mycorrhizal development stage Based on the dynamic of root colonization, early stages of the infection process were considered to be situated between 2 and 8 d of contact. In order to check for the possible appearance of novel polypeptides during the mycorrhiza formation stage, twodimensional patterns of proteins from developing ectomycorrhizas were established 2 and 4 d postinoculation and compared with those from noninoculated roots and free-living mycelia. To facilitate the comparison, the latter controls were performed by running, on same co-analysis gels, proteins extracted from plant and fungal material mixed at appropriate ratios. Only polypeptides that were detected in gels from inoculated roots but not in the co-analysis gels were considered as putative symbiosis-related proteins. The three novel polypeptides found in 8-d-old ectomycorrhizas were never detected in roots 2 or 4 d after inoculation (Fig. 4B, C). At these stages of the mycorrhiza formation, most of the changes were recorded in a region defined by a ph range from 4-8 to 6-2 and MW from 12-4 to 30 kda. Thus at 4 d post-inoculation, polypeptides E3, E4, Eg, E-, Eg, Eg, and Em present in developing ectomycorrhizas (Fig. 4C) were never detected in control roots or mycelia (Fig. 4A). Sequential accumulation of these polypeptides in roots during the early stages of the infection is illustrated in Figure 5. At least three symbiosis-related polypeptides. Eg, Ey, and Ej,,, appeared as early as 2 d post-inoculation (Fig. 5 B) but were still present in 8d-old ectomycorrhizas (Fig. 5D). The four other polypeptides transiently accumulated in 4-d-old ectomycorrhizas and were barely detectable in roots after 8 d of contact with the fungus (Fig. 5C). DISCUSSION The results presented here show that, under our experimental conditions, 8 d of contact between the two partners were required to form fully-developed birch ectomycorrhizas. Based on the shape of the curve obtained by measuring the ergosterol content of roots during the infection process, the dynamic of root colonization with P. involutus was similar, even if slower, than that recorded for E. globulus with a

8 502 P, Simoneau and others Figure 5. Sequential accumulation of symbiosis-related polypeptides during ectomycorrhizal development. The region of silver-stained gels where early symbiosis-related polypeptides accumulate is shown. Gels were loaded with proteins extracted from inoculated roots at 2 (B), 4 (C), and 8 d (D) post-inoculation and a mixture of non-inoculated roots and free-living mycelia with a 9/1 ratio (A). Symbols are as in Figure 4. Numbers on the top ot the gels correspond to ph values; molecular masses of size markers are indicated in kda. compatible isolate of Pisolithus tinctorius (Hilbert et al., 1991); the same sequence of events was therefore defined: 'preinfection', 'mycorrhiza formation', and 'mature mycorrhiza' stages. Only slight changes in polypeptide contents of birch roots were recorded during the overall mycorrhizal development: accumulation of seven specific polypeptides during the initiation of the ectomycorrhizal symbiotic state and down-regulation of a small number of root polypeptides in mature ectomycorrhizas. The finding of new polypeptides in developing birch ectomycorrhizas is in line with results from Hilhevtet al. (1991) which were able to identify, using a similar approach, at least seven unique polypeptides that accumulated in eucalypt roots as early as 12h after the fungal attachment, i.e. well before the completion of the differentiation of the symbiotic organs. By contrast, the phenomenon of polypeptide 'cleansing', that was quantitatively predominant in eucalypt ectomycorrhizas exhibiting all the basic features of a typical symbiotic organ (Martin & Hilbert, 1991) was not observed in birch roots after 8 d of contact with the fungal symbiont (end of the mycorrhiza formation stage). Recently, it has been suggested that neither the phenomenon of polypeptide 'cleansing' nor the accumulation of symbiosis-related polypeptides occurred in fullydeveloped ectomycorrhizas from P. abies (Guttenberger & Hampp, 1992). These findings may indicate that different tree species do not behave in a similar way when challenged with various kinds of compatible ectomycorrhizal fungi. Considering this, the response of birch roots towards mycorrhizal infection would be situated between that observed in eucalypt and that observed in Norway spruce. However, it remains possible that this discrepancy also results from differences in: (i) the type of inoculated material, cloned birch plantlets with highly homogeneous genetic background vs young seedlings of E. globulus and P. abies, (ii) the inoculation procedure, allowing or not the obtention of mycorrhiza on the tap roots, (iii) the sampling

9 Early symbiosis-related polypeptides in birch ectomycorrhizas method, isolated ectomycorrhizas or whole root system, and (iv) the kind of electrophoretic analysis, highly resolutive gels vs highly sensitive micromethods. Interestingly, comparison of the MW of eucalypt early ectomycorrhizins and the symbiosis-related polypeptides detected in birch roots reveals some homologies. These polypeptides were clustered in two major groups with molecular masses in the regions of 32 kda and 11 to 17 kda. Besides this, in both eucalypt and birch, the symbiosis-related polypeptides that have been identified to date were acidic polypeptides. The most likely explanation for this finding is probably the fact that isoelectrofocusing only poorly resolves polypeptides with a basic isoelectric point (O'Farrell, Goodman & O'Farrell, 1977). Moreover, some of the differences recorded in the basic part of the 2-D patterns have been taken with caution due to weaker resolution and reproducibility in this region. Therefore, it is highly probable that basic symbiosis-related polypeptides also exist but were not identified with this type of analysis. The low molecular mass of some of the early SRpolypeptides detected in birch roots could be interpreted as the result of proteolysis occurring during the extraction procedure (Guttenberger & Hampp, 1992). Although it is not possible to completely rule out this hypothesis, it seems highly improbable that such a degradation could explain our results since only spots that were constantly found in similar but independent extracts were retained for comparison of the polypeptide patterns, which were in any event very reproducible. Moreover, the action of proteases was strongly minimized by quickly freezing the sampled tissues in liquid Ng, homogenization with phenol in presence of PMSF as protease inhibitor (Hurkman & Tanaka, 1986), and solubilization of the precipitated proteins in a boiling buffer containing SDS (Colas des Francs, Thiellement & De Vienne, 1985). Based on the present data, it is clear that only subtle qualitative and quantitative changes in protein content of birch roots occurred during the early fungal colonization. Among the novel polypeptides, which could result from differential protein synthesis or post-translational modifications of pre-existing polypeptides, some might correspond to hostdefense proteins. Indeed recent data have demonstrated significant increases in activities of chitinase (Sauter & Hager, 1989), and enzymes associated with lignin synthesis including phenylalanine ammonia-lyase (Campbell & Fllis, 1992 a, b) in plant cell cultures challenged with ectomycorrhizal fungus-derived elicitors. On the other hand it is likely that other polypeptides, of fungal or plant origin, are more specifically involved in the establishment of the symbiotic state, e.g. maintenance of the fungi in the developing symbiotic root organ. The molecular mechanisms that orchestrate this perfectly tuned infection process where the fungal symbiont is neither becoming pathogenic nor rejected by the host, while probably triggering a defense response, remains to be clarified. Therefore, a comparative analysis of the complexity of transcripts from birch ectomycorrhizas with those from free-living partners, using both hybridization experiments with specific probes and electrophoretic analysis of in vitro translation products, is currently underway. ACKNOWLEDGEMENTS We thank Dr P. Gadal (University of Paris VI, France) for critical reading of the manuscript. This work was supported by a grant from the Eurosilva-Eureka programme No REFERENCES Campbell MM, Ellis BE. 1992a. Fungal elicitor-mediated responses in pine cell culture. I. Induction of phenylpropanoid metabolism. Planta 186: Campbell MM, Ellis BE Fungal elicitor-mediated responses in pine cell culture. III. Purification and characterization of phenylalanine ammonia-lyase. Plant Physiology 9%: Chilvers GA, Douglass PA, Lapeyrie FF A papersandwich technique for rapid synthesis of ectomycorrhizas. New Phytologist 103; 397^02. 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10 504 P. Simoneau and others Oakley BR, Kirsh DR, Morris NR A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry 105: O'Farrell PH High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250: O'Farrell PZ, Goodman HM, O'Farrell PH High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12: Sauter M, Hager A The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspensioncultures cells of its host Picea abies. Planta 179: Schuster AM, Davies E Ribonucleic acid and protein metabolism in pea epicotyls. I. The aging process. Plant Physiology 73: Strullu DG Les mycorhizes. In: Handbiich der Plfanzenanatomie, vol. XIII, part 2. Berlin & Stuggart: Gebruder Borntraeger.

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