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To cite this article: Philippe Simoneau , Christine Juge , Jean-Yves Dupuis , Jean-. Daniel Viémont , Jean-Christophe Moreau & Désiré-Georges Strullu (1994) ...
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Protein biosynthesis changes during mycorrhiza formation in roots of micropropagated birch a

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Philippe Simoneau , Christine Juge , Jean-Yves a

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Dupuis , Jean-Daniel Viémont , Jean-Christophe a

Moreau & Désiré-Georges Strullu

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Laboratoire des Interactions PlantesMicroorganismes , Université d'Angers , 2 boukvard Lavoisier, F-49045 , Angers Published online: 27 Apr 2013.

To cite this article: Philippe Simoneau , Christine Juge , Jean-Yves Dupuis , JeanDaniel Viémont , Jean-Christophe Moreau & Désiré-Georges Strullu (1994) Protein biosynthesis changes during mycorrhiza formation in roots of micropropagated birch, Acta Botanica Gallica, 141:4, 429-435, DOI: 10.1080/12538078.1994.10515179 To link to this article: http://dx.doi.org/10.1080/12538078.1994.10515179

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;leta bot. GaUica, 1994, 141 (1-), 429-435.

Protein biosynthesis changes during mycorrhiza formation in roots of micropropagated birch

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by Philippe Simoneau, Christine Juge, Jean-Yves Dupuis, Jean-Daniel Viemont, Jean-Christophe Moreau, and Desire-Georges Strullu Laboratoire des lnteraction.s Plantes-iHicroorganismes, Universite d'Angers, 2 boulevard Lavoisier, F -49015 Angers

Summary.- Birch (Betula pendula Roth) micropropagated plantlets were inoculated with 7 different isolates of the mycorrhizal fungus Paxil/us involutus Baisch. Based on the level of fungal ergosterol measured in roots at the end of the "mycorrhiza formation stage", strain PO was chosen as the reference strain. Electrophoretic analysis of in vivo labeled proteins extracted from my· corrhizal roots 96 h post-inoculation with this strain, noninoculated roots. and free-living mycelium, revealed that specific polypeptides were synthesized during ectomycorrhiza formation. To examine hypothetical similarity between some of these polypeptides and defense proteins, parts of corresponding putative genes of birch were isolated. Partial sequencing of one clone have shown that it contained a portion of the gene for phenylalanine ammonialyase. Resume.- Des vitroplants de bouleau (Betula pendula Roth) ont ate inocules in vitro avec 7 isolats differents du champignon mycorhizien Paxil/us involutus Batsch. En utilisant comma critere de compatibil~e Ia quantile d'ergosterol fongique retrouvee au niveau des racines Ia fin du slade de formation des mycorhizes, Ia souche PO a ate retenue comma souche de reference. L'analyse electrophoretique des proteines marquees in vivo, et extraites de racines 96 h apres !'inoculation avec cette souche, de racines non-inoculees et du mycelium fongique, a permis de montrer que des polypeptides specrtiques etaient synthetises lors du developpement de l'organe symbiotique. Afin de verifier Ia similitude entre certains de ces polypeptides et des proteines induites lors des reactions de defense, des portions de genes de bouleau pouvant coder pour de telles proteines ont ate clonees. Le sequenctage partial de l'un de ces clones a montre qu'il contenait une partie d'un ADNc codant pour Ia phenylalanine ammonia-lyase.

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Key words : Birch - mycorrhiza - symbiosis-related proteins • phenylalanine ammonia-lyase -sequence.

©Societe botanique de France 1994. ISSN 1253-8078.

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During the development of ectomycorrhizas, a series of interactions between the host plant and the fungal symbiont is required (Martin & Hilbert, 1991). Chemotropic attraction of the fungus (Horan & Chilvers, 1990) and recognition of fungal lectins by receptor sites on the roots (Giollant et al., 1993) might represent the initial stage of mycorrhiza formation. Thereafter a sequence of morphological events, including sheath development and Hartig net formation, occurs in colonized roots and leads to the emergence of a new symbiotic organ, i.e. the mature mycorrhiza (Horan et al., 1988). Based on these observations the infection process has been divided in three stages called respectively "preinfection", "mycorrhiza-formation", and "mature mycorrhiza" (Hilbert et al., 1991 ; Simoneau et al., 1993). The relative length of each stage may be modulated by varying degrees of aggressiveness of the fungal isolates toward the host plant roots. The development of modern techniques for the synthesis of mycorrhizas, where large numbers of roots arc inoculated simultaneously (Chilvers et al., 1986 ; Malajczuk et al., 1990), has allowed the characterization of fungal isolates according to such criteria (Malajczuk et al., 1990). Using this model system, Hilbert et al. (1991) have shown that symbiosis-related polypeptides accumulate in eucalypt roots challenged with an aggressive isolate of Pisolithus tinctorius while changes in polypeptide patterns were not observed in the roots treated with a non-aggressive one. Recently a similar approach was used to demonstrate that an accumulation of few polypeptides also occurs in developing synthetic birch ectomycorrhizas obtained by associating the roots of micropropagated clonal plants with the cctomycorrhizal fungus, Paxillus mvolutus (Simoneau et al., 1993).

This symbiotic system, in which all the plants have exactly the same genetic background, represent a useful tool for the comparison of mycorrhizal efficiencies of various fungal isolates and subsequent analysis of the molecular events that accompany successful or unsuccessful root colonisation. In this study, we examined therefore, the dynamic of the infection of cloned birch plants inoculated with different isolates of P. involutus and, using the most efficient one, by in vivo protein synthesis during the "mycorrhiza-formation" stage. We also report on the cloning of birch genes for phenylalanine ammonialyase (PAL) and chalcone-synthase (CHS) that might be transiently induced in developing mycorrhizas.

MATERIALS AND METHODS Synthesis of mycorrhlzas Clonal plants of Betula pendula were micropropagated as previously described (Grellier et a/., 1984), except that glass beads were used as an alternative to agar in the medium used lor the last transfer before fungal inoculation. Isolates of P. involutus were isolated from sporocarps collected under birch (isolates Mi. PO). eucalypt (isolate COU), poplar (isolate ANU, MAl), oak (isolate NAU), and aspen (isolate PEP), and cultured on a cellophane membrane in Petri dishes containing MMN solid medium (Marx, 1969). The mycorrhizas were synthesized and sampled as previously described (Simoneau et al., 1993). For in vivo labeling experiments, before sampling, free-living mycelium and roots of ectomycorrhizal and non inoculated plants were immersed in micropropagation medium containing 50 IJCilml of 35S-methionine, incubated 4 h in the growth chamber at 23•c. and washed in medium containing SmM cold methionine. The proportion of the fungal biomass in the inoculated roots was estimated by measuring their fungal ergosterol content by reversephase high performance liquid chromatography (Martin eta/., 1990). Protein extraction and electrophoresis Protein were extracted, solubilized and separated by two-dimensional polyacrylamide gel electrophoresis as previously described (Simoneau et a/., 1993). Alter electrophoresis, gels were impregnated w~h Entensily (Du pont de Nemours S.A., Les Ulis, France), dried under vacuum, and autolluorographed w~h Kodak XAAS films at -7o•c.

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P. SIMONEAU ET AL. Nucleic acid manipulations Total cellular RNA was isolated from birch roots as described by Friemann eta/. (1992) except that the CsCI purification step was replaced by selective LiCI precipitation. Polymerase chain reaction (PCR) amplification was carried out using degenerate sense (5'-GAAGGTCT(G, C, T)GCC(A,C)T(C, T)GT(G,C) AATGG-3' and 5'-ATGATGTACCA(A,G)CA(A,G)GG(A,G,C,T)TGCTT-3' for PAL and CHS respectively) and antisense (5'-TCGGCGCT(C,T)TG(G,T)ACATGG(C,T)TGGT-3' and 5'-CC(A,G)AA(A,G,C,T) CC(A,G)AACA(A,G)AACACCCCA(A,T)TC-3' for PAL and CHS respectively) primers specific of portions of PAL and CHS consensus sequences. Templates for the amplification reactions were diluted samples of the first strand eDNA synthesized from total root RNA using an oligo dT primer. Thirty cycles of denaturation (92°C, 1.5 min), annealing (52°C, 1.5 min), and extension (72°C, 3 min) were used. PCR products were cloned into pCR-Script SK(+) (Stratagene, La Jolla, CA, USA) according to manufacturer's instructions. DNA sequences were determined by the dideoxy method. All other routine manipulations were carried out using standard procedures.

RESULTS Dynamics of the infection The relative aggressiveness of seven P. involutus isolates toward birch roots was estimated by measuring the fungal ergosterol content in inoculated roots at various times (Fig. 1). For each association, the curve shape was similar with three characteristic phases : a lag period of at least 24 h ("preinfection stage") where no ergosterol was detected, a regular increase of the fungal biomass in roots ("mycorrhiza formation stage"), and a plateau or a slight decrease. The main differences in the colonization process were : i) the length of the "mycorrhiza formation stage" that varied from 168 h to at least 336 h with the Mi strain, and ii) the maximum value of fungal biomass, from 12% with isolates COU and MAl to 22 % with the PO strain, in inoculated roots at the end of the "mycorrhiza formation stage".

In vivo protein synthesis Based on the content of fungal sterol in inoculated roots, isolate PO was con-

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sidered as the most efficient one and selected for studying polypeptide changes occurring in roots during the "mycorrhiza-formation stage". Proteins from 96-h-old ectomycorrhizas were in vivo labeled and analyzed by two-dimensional gel electrophoresis. Comparison of the resulting pattern (Fig. 2A) with relevant controls, i.e. non-inoculated roots, free-living mycelium, or both (Fig. 2B, co-analysis gel) indicated that two major modifications of in vivo protein synthesis occurred in developing mycorrhizas. First, there was a significant decrease in the synthesis of some polypeptides, particularly polypeptide D6 which synthesis was almost completely abolished in ectomycorrhizas. Second some polypeptides (E1 to E8) were synthesized at higher rates in symbiotic tissues and were nearly absent in controls. The molecular masses of these polypeptides ranged from 12 kD to 82 kD and their isoelectric point from 5.2 to 6.1. Cloning of birch homologous probes Preliminary investigations suggested that some of the newly synthesized symbiosis-related polypeptides might correspond to proteins involved in a plant response similar to a defense reaction, including induction of enzymes of the phenylpropanoid pathway (unpublished observations). In order to examine further this possibility, parts of putative genes for PAL and CHS from birch have been amplified from first-strand cDNAs using specific primers. Fragments of the expected sizes (680 and 660 bp respectively) have been obtained (data not shown) and cloned into an appropriate plasmid vector. The amino acid sequence (Fig. 3), deduced from partial analysis of the nucleotide sequence of recombinant plasmids carrying the 680 bp fragment, shows 85 % sequence identity with the corresponding portion of the bean PAL sequence (Edwards et al., 1985).

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Fig. 1.- Fungal biomass in birch roots inoculated with seven different Paxillus involutus isolates. The level of infection was estimated by measuring the ergosterol content of sampled roots. Each value is the mean of three replicates. (•): PO, (D): NAU, (e): ANU, (.t.): PEP, (0): COU, {.t.): MAl, (0): Mi. Fig. 1.- Biomasse fongiques dans des racines de bouleau inoculees avec sept isolats differents de Paxillus involutus. Le degre d'infection a ete estime en mesurant Ia quanme d'ergosterol racinaire. Chaque valeur correspond a Ia moyenne de trois repetitions. (•): PO, (D): NAU, (•): ANU, ("): PEP. (0): COU, {.t.) : MAl, (()) : Mi.

DISCUSSION

The model system for mycorrhiza formation used in this study provided a convenient method for the comparison of dynamics of infection of roots by various fungal isolates. Considering isolate PO as the reference strain, the 6 other isolates that have been tested could be divided in at least three groups: i) those that behave almost like the reference strain (ANU, PEP, NAU), ii) those with a reduced level of root colonization (COU, MAl), and iii) those that formed mycorrhizas at a very slow rate (Mi). The use of cloned micropropagated birch plants to select P. involutus isolates efficient for growth promotion has already been reported (Strullu et al., 1986). However, there is not necessarily a correlation between the growth promotion and the rates and levels of root colonization by the fungal symbiont. On

the basis of the latter criteria, P. involutus strain PO was selected as the most efficient isolate. When birch plants were inoculated with this strain, newly synthesized polypeptides could be detected in roots 48 h after the fungal attachment. These proteins arc probably equivalent to early ectomycorrhizins previously found in eucalypt (Hilbert et al., 1991). Moreover based on gel migration patterns, it is likely that some of the polypeptides characterized here (E6, E7, E8) and the low molecular weight symbiosis-related (SR) polypeptides detected on silver-stained gels of proteins from developing birch ectomycorrhizas (Simoneau et al., 1993) are identical. It has been suggested that some of the SRproteins that accumulate in ectomycorrhizal roots may be related to specific proteins synthesized in other plant-microbe interactions (Hilbert et al., 1991). Recent data reporting increased activi-

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Fig. 2.- Changes in protein biosynthesis in developing ectomycorrhizas. Ftuorographs of gels of in vivo labeled proteins extracted from inoculated roots at 96 h post inoculation (A) and from a mixture of noninoculated roots and free-living mycelia with a 9/1 ratio (8) Similar amounts of radioactivity were loaded onto the gels. Arrows indicate symbiosis-related polypeptides : Circled spots indicate major down-regulated polypeptides. Numbers on the top of the gels correspond to pH values ; molecular masses of size markers are indicated in kD. Fig. 2.- Modifications de Ia synthese proteique durant le developpement des ectomycorhizes. Fluorogrammes de gels de proteines marquees in vivo et extraites de racines a 96 h apres inoculation (A) au d'un melange de racines non-inoculees et de mycelium libre dans un rapport 9/1. Des quantites equivalentes de radioactivite ant ete deposees sur chaque gel. Les fleches indiquent les polypeptides induits Iars de Ia symbiose. Les taches encerclees correspondent aux polypeptides majeurs dont Ia synthese est diminuee. Les chiffres en haul des gels indiquent les pH . les masses moleculaires des proteines standards sont indiquees en kD.

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KLLFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIPMASYCSELQFLANPVTNHVQSP II

II 111111111 II IIIII II

IIIII I 1111 II II I 11111111 IIIII I II II

KUMFAQFSELVNDFYNNGLPSNLTASRNPSLDYGFKGAEIAMASYCSELQYLANPVTSHVQSA Fig. 3.- Amino acid sequence deduced from nucleotide sequence of the 3' end of the 680 bp PCR-derived eDNA clone (upper line) and comparison with corresponding part of the bean PAL protein (lower line; residues 202 to 267). Identities are marked by vertical bars. The single letter code for amino acids is used. Fig. 3.- sequence en acides amin6s dllduite de Ia s6quence nucl6otidique de l'extr6mit6 3' de I'ADNc de 680pb obtenu par PCR (llgne du haul) et comparaison avec Ia partie correspondante de Ia PAL de haricot (residus 202 267). Les identit6s de s6quence sent lndiqu6es par des lignes verticales. Les acides amin6s sent repr6sent6s selon 1e code une lettre.

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a

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ties of enzymes (chitinases, PAL), usually induced by pathogens, in plant cell cultures (Sauter & Hager, 1989 ; Campbell & Ellis, 1992) or in roots (see the article from Lapeyrie et al. in the same issue) challenged with ectomycorrhizal fungus-derived elicitors, support this contention. To further test this hypothesis and study regulation of the expression of these proteins at the transcriptional level, homologous DNA probes are required. Using reverse transcription followed by PCR with primers specific of conserved regions on genes coding for defense proteins it has already been possible to clone a portion of a birch root transcript for PAL.

In conclusion, our model system for the synthesis of mycorrhizas in micropropagated birch plants is well suited to study the molecular basis of ectomycorrhiza organogenesis since : i) all the plants have the same genetic background, ii) the dynamics of infection of roots by various fungal isolates is well characterized, and iii) homologous DNA probes for genes induced during plant defense reactions are available.

Acknowledgements.- We thank Dr. F.F. Lapeyrie (INRA, Nancy, France) lor providing us some of the P. invo/utus Isolates. This work was supported by a grant from the Eurosilva-Eureka programme No. 447.

BIBLIOGRAPHY Campbell M.M. and B.E. Ellis, 1992.- Fungal elicitormediated responses in pine cell culture. I. Induction of the phenylpropanold metabolism. Planta, 186, 409-417. Chilvers G.A., P.A. Douglass and F. F. Lapeyrie, 1986.A paper-sandwich technique for rapid synthesis of ectomycorrhizas. New Phytol., 103, 397-402. Edwards K.E., C.L. Cramer, G.P. Bolwell, R.A. Dixon, W. Schuch and C.J. Lamb, 1985.- Rapid transient induction of phenylalanine ammonia-lyase mANA in elicitor-treated bean cells. Proc. Nat/. Acad. Sci. USA. 82, 6731-6735. Friemann A., M. Lange, W. Hachtel and K. Brinkmann, 1992.-lnduction of nitrate assimilatory enzymes in the tree Betula pendula. Plant Physiol., 99, 837842. Giollant M., J. Guillot, M. Damez, M. Dusser, P. Didier and E. Didier, 1993.- Characterization of a lectin from Lactarius deterrimus. Plant Physic/, 101, 513-522. Grellier B., R. Letouze and D.G. Strullu, 1984.- Micropropagation of birch and mycorrhizal formation in

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lu, 1993.- Symbiosis-related polypeptides associated wijh the early stages of ectomycorrhiza organogenesis in birch (Betula pendula Roth). New Phytol., 124, 495-504. Strullu D.G., B. Grellier, C. Remand and R. Letouze, 1986.- Mycorhization in vitro: efficacite des souches fongiques selectionnees. In : Mycorrhizae : physiology and genetics. Gianinazzi-Pearson V. & S. Gianinazzi (eds), INRA, Paris, 531-534.