How does a symbiotic fungus modulate ... - Wiley Online Library

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How does a symbiotic fungus modulate expression of its host-plant nitrite reductase? Blackwell Publishing Ltd

Julie Bailly1, Jean-Claude Debaud1, Marie-Christine Verner1, Claude Plassard2, Michel Chalot3, Roland Marmeisse1 and Laurence Fraissinet-Tachet1 1

Université de Lyon, Lyon, F-69003, France; Université Lyon1, Lyon, F-69003, France; IFR 41, Lyon, Villeurbanne, F-69622, France; Laboratoire CNRS,

UMR5557, USC INRA 1193, Ecologie Microbienne, Bâtiment A. Lwoff, 43 Boulevard du 11 novembre 1918, F-69622 Villeurbanne Cedex, France; 2

INRA, UMR 1222, Rhizosphère & Symbiose, 2 Place Viala, F-34060 Montpellier Cedex 01, France; 3Nancy-University, Research Unit 1136 INRA/UHP

‘Tree–microbe Interactions’, BP 239, F-54506 Vandoeuvre-les-Nancy Cedex, France

Summary Author for correspondence: L. Fraissinet-Tachet Tel: +33 4 72 44 83 02 Fax: +33 4 72 43 16 43 Email: [email protected] Received: 22 November 2006 Accepted: 12 February 2007

• In the mycorrhizal association, changes in the metabolic activities expressed by the plant and fungal partners could result from modulations in the quantity and nature of nutrients available at the plant–fungus interface. This hypothesis was tested for the nitrite reductase gene in the association Hebeloma cylindrosporum × Pinus pinaster. • Transcripts from plant and fungal nitrite reductases and a fungal ammonium transporter were quantified in control uninoculated roots, extraradical mycelia and mycorrhizas formed by either wild-type or nitrate reductase deficient fungal strains. • The fungal genes were downregulated in mycorrhizas compared with extraradical hyphae. The plant nitrite reductase was induced only transiently by NO3− in the association with a wild-type strain, but permanently expressed at a high level in mycorrhizas formed by the deficient mutant. • These results suggest that reduced nitrogen compounds transferred from the fungus to the root cortical cells repress the plant nitrite reductase, thus highlighting a plant gene regulation by the nutrients available in the Hartig net. Key words: Hebeloma cylindrosporum, mycorrhiza, nitrate assimilation, nitrite reductase regulation, Pinus pinaster, symbiosis. New Phytologist (2007) 175: 155–165 No claim to original French government works. Journal compilation © New Phytologist (2007) doi: 10.1111/j.1469-8137.2007.02066.x

Introduction The mycorrhizal association between fungi and plant roots leads to the bidirectional exchange of essential nutrients between the two symbionts, and necessitates tight coordination between the metabolisms of the two partners. This could result in changes in the expression levels of key metabolic genes by reference to their levels in the free-living stages of either plant or fungus. Such modifications have indeed been detected in studies that either focused on one metabolic pathway, or scanned the entire transcriptome. The most clearcut examples include ‘mycorrhiza-specific’ genes, transcripts of which are not detectable in nonsymbiotic plant tissues or fungal hyphae. This is the case for the plant genes encoding

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plasma membrane-associated inorganic phosphate transporters, which are expressed only in arbuscule-containing root cortical cells of endomycorrhizal plants such as potato (Rausch et al., 2001) and Medicago truncatula (Harrison et al., 2002). The role of these plant symbiosis-specific transporters could be specifically to take up phosphate delivered by the fungus into the plant–arbuscule apoplastic interface. In this respect, these transporters could efficiently relay other plant phosphate transporters that have a broader tissue distribution and that can be downregulated upon mycorrhiza association (Liu et al., 1998). In most cases, the symbiotic association leads to the up- or downregulation of key metabolic genes that are otherwise expressed in nonsymbiotic tissues. This has been documented in studies focusing on genes necessary for the primary absorp-

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tion and assimilation of inorganic nitrogen sources. In the case of hybrid poplar plants, Selle et al. (2005) showed that the transcription of three plant ammonium transporter genes was five to nine times higher in ectomycorrhizas formed by the basidiomycete Amanita muscaria compared with their levels in control nonmycorrhizal roots. Upregulation of plant ammonium transporter in mycorrhizas could signify ammonia excretion by the fungus at the Hartig net level. This hypothesis is supported by the observation that an Amanita ammonium transporter, Am-AMT2, is downregulated in poplar mycorrhizas (Willmann et al., 2007), thus minimizing the capacity of the fungus to compete with plant cells to assimilate ammonium. An opposite trend of regulation was reported for plant nitrate reductase genes that were downregulated in two different associations: endomycorrhizas formed between maize and Glomus intraradices (Kaldorf et al., 1998) and ectomycorrhizas formed between Tilia platyphyllos and the truffle Tuber borchii (Guescini et al., 2003). In the latter example, downregulation of the plant gene was paralleled by upregulation of the fungal nitrate transporter, nitrate reductase and nitrite reductase genes, which could possibly relay the plant gene to maintain a high or constant capacity of reducing nitrate (Guescini et al., 2003, 2007). Most plant and fungal metabolic genes participating in the primary assimilation of major soil nutrients are usually directly or indirectly regulated at the transcriptional level by nutrient availability in soil or in culture medium. This has been abundantly documented, for example in the nitrateassimilation pathway which, in almost all organisms studied, is induced by nitrate and repressed by ammonium or other reduced N sources that can be assimilated at a lower energy cost compared with nitrate (for a review on fungi see e.g. Marzluf, 1997; on plants, Solomonson & Barber, 1990; Crawford, 1995). Carbon availability could also play a central role, as the expression of Tuber nitrate assimilation genes has been shown to be repressed by C depletion (Guescini et al., 2007). Although C starvation is not likely to occur in the fungus in contact with the plant, the possibility of a complex regulation involving both symbiotic partners and N and C fluxes cannot be ruled out. In this respect, the observed changes in the patterns of transcription of metabolic genes on mycorrhiza formation can be attributed to opposite origins. On one hand, it can be hypothesized that these changes are fully part of (and cannot be dissociated from) the ontogenic process that leads to the formation of a functional mycorrhiza. In this case, mycorrhizaspecific internal signals, not related to external nutrient availability, act to repress or induce the genes. On the other hand, it can also be hypothesized that the intimate association between plant cells and fungal hyphae modifies their immediate environment in terms of the quality and quantity of available nutrients. This new environment is perceived by the same sensors and transmitted by the same transduction pathways that operate in the free-living stages of the two symbionts and respond according to changes in external nutrient status.

New Phytologist (2007) 175: 155–165

In this study, we tested these alternative hypotheses for plant and fungal genes participating in nitrite reductase activity in the symbiotic association between the ectomycorrhizal plant Pinus pinaster and the basidiomycete fungus Hebeloma cylindrosporum. The main objective of this work is to analyse the expression of a single gene (coding a unique function) in both plant and fungal partners, in order to reveal at the transcriptional level how one function expressed by both partners could be coordinately regulated or modulated during symbiotic association. We also analysed as a control an additional fungal gene encoding a high-affinity ammonium transporter known to have an expression pattern similar to that of the fungal nitrite reductase. The plant gene studied encodes a plastidial ferredoxin-dependent nitrite reductase gene (EC 1.7.7.1), the sequence of which was identified among expressed sequence tags (ESTs) from P. pinaster roots. Hebeloma cylindrosporum has been studied extensively with respect to the transcriptional regulation of genes coding for proteins involved in utilization of nitrate (Jargeat et al., 2000) and ammonium (Javelle et al., 2001). In the present work we studied Hc-nir1 and Hc-amt1 that code, respectively, for a cytosolic NADPH-nitrite reductase (EC1.6.6.4; Jargeat et al., 2003) and a high-affinity ammonium transporter ( Javelle et al., 2001). When the fungus is grown in vitro, transcription of these two fungal genes is repressed by ammonium and active under N deprivation or in the presence of a secondary N source such as nitrate. In this respect, the H. cylindrosporum nitrate-assimilation pathway does not need nitrate induction, and the observed ammonium repression is a dominant phenomenon as the different genes of the nitrate assimilation pathway, including Hc-nir1, are all downregulated on a medium supplemented with NH4NO3 as N source ( Jargeat et al., 2003). The aim of this study is to identify the mechanisms responsible in ectomycorrhizal symbiosis for differential expression of both plant and fungal nitrite reductase genes. In order to manipulate the N supplied by the fungus to P. pinaster mycorrhizal roots, we inoculated the plants with either wild-type fungal strains or a nitrate reductase-deficient mutant, the only such mutant available for a mycorrhizal fungus (Marmeisse et al., 1998). This mutant able to form mycorrhizas is unable to convert nitrate into ammonium, and is therefore unable to supply the plant with reduced N molecules in the presence of nitrate as sole external N source.

Materials and Methods Biological material and culture conditions Three dikaryotic strains of Hebeloma cylindrosporum Romagnesi were used. The GCA6 wild-type (wt) strain was isolated from a fruit body collected in the field (Gryta et al., 1997). The D2 strain resulted from a cross between the wild-type homokaryons h1 (mating type A1 B2) and h7 (A2 B1) (Debaud & Gay, 1987). The nitrate reductase-deficient D2NR– strain, homogenic to

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the D2 wt strain, resulted from a cross between homokaryons h1.7 (A1 B2, Hc-nar1–) and h7.12 (A2 B1, Hc-nar1–), both mutated in the Hc-nar1 gene coding the nitrate reductase apoenzyme (Marmeisse et al., 1998). Fungal mycelia were grown in the dark at 22°C on YMG medium (Rao & Niederpruem, 1969) solidified or not with 0.8% agar. Pinus pinaster (Ait.) Sol. seeds (CEMAGREF, batch, France) were surface-sterilized and germinated in water-saturated sterile vermiculite, as described by Bogeat-Triboulot et al. (2004). The soil used for the development of mycorrhizas was collected in autumn 2002 on an Atlantic coastal sand dune planted with P. pinaster trees (‘Le Truc Vert’ site, south-west France) (Gryta et al., 1997). This nutrient-poor arenosol was characterized by 99% non-calcareous sand and 28 000 sequences publicly available for this species) and from H. cylindrosporum (Wipf et al., 2003; Lambilliotte et al., 2004). Such models could also be tested by using the natural variation in utilization of different N sources that exists between ectomycorrhizal fungi such as A. muscaria, which is naturally unable to use nitrate (Sawyer et al., 2003).

Acknowledgements We would like to thank Patrick Pastuszka (INRA Pierroton) for providing Pinus pinaster seeds and the IFR 41 and DTAMB of the University Lyon 1 for access to the quantitative PCR equipment.

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