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Identification of genes differentially expressed in ectomycorrhizal roots during the Pinus pinaster–Laccaria bicolor interaction

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Abstract

Ectomycorrhizal associations are of major ecological importance in temperate and boreal forests. The development of a functional ectomycorrhiza requires many genetic and biochemical changes. In this study, suppressive subtraction hybridization was used to identify differentially expressed genes in the roots of maritime pine (Pinus pinaster Aiton) inoculated with Laccaria bicolor, a mycorrhizal fungus. A total number of 200 unigenes were identified as being differentially regulated in maritime pine roots during the development of mycorrhiza. These unigenes were classified into 10 categories according to the function of their homologues in the GenBank database. Approximately, 40 % of the differentially expressed transcripts were genes that coded for unknown proteins in the databases or that had no homology to known genes. A group of these differentially expressed genes was selected to validate the results using quantitative real-time PCR. The transcript levels of the representative genes were compared between the non-inoculated and inoculated plants at 1, 5, 15 and 30 days after inoculation. The observed expression patterns indicate (1) changes in the composition of the wall cell, (2) tight regulation of defence genes during the development of mycorrhiza and (3) changes in carbon and nitrogen metabolism. Ammonium excess or deficiency dramatically affected the stability of ectomycorrhiza and altered gene expression in maritime pine roots.

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Abbreviations

DAI:

Days after inoculation

ECM:

Ectomycorrhiza

SSH:

Suppressive subtraction hybridization

qPCR:

Real-time quantitative PCR

IAA:

Indole acetic acid

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Acknowledgments

We are indebted to the anonymous reviewers for their thorough evaluation and constructive recommendations that helped to improve this manuscript. We would like to thank Dr. Francis Martin from INRA-Nancy (France) for the generous gift of Laccaria bicolor strain S238N. We also like to thank Marina Rueda-López for helping with the culture and maintenance of the strain S238N, Josefa Gómez-Maldonado for her collaboration in the picking and rearrangement of the clones of the SSH library and Jacob Pérez-Tienda for valuable suggestions. This work was supported by grants from the Ministerio de Ciencia e innovación (BIO2009-07490; BIO2012-33797) and Junta de Andalucía (CVI-3739).

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Authors and Affiliations

  1. Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Instituto Andaluz de Biotecnología, Universidad de Málaga, Campus Universitario de Teatinos s/n, 29071, Malaga, Spain

    Aranzazu Flores-Monterroso, Javier Canales, Fernando de la Torre, Concepción Ávila & Francisco M. Cánovas

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  1. Aranzazu Flores-Monterroso
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  2. Javier Canales
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  3. Fernando de la Torre
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  4. Concepción Ávila
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  5. Francisco M. Cánovas
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Corresponding author

Correspondence to Francisco M. Cánovas.

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425_2013_1874_MOESM1_ESM.doc

Fig. S1 Schematic representation of the protocol used to obtain the SSH library and validation of the procedure. Four identical replicates with cDNAs of SSH libraries (unsubtracted tester cDNAs (UT), forward subtracted cDNAs (FS), unsubtracted driver cDNAs (UD) and reverse subtracted cDNAs (RS), were independently hybridised with complex probes derived from unsubtracted tester (filter A), forward subtracted (filter B), unsubtracted driver (filter C) and reverse subtracted (filter D). A stronger signal was observed following the subtraction in both, the forward and driver libraries (DOC 541 kb)

Table S1 Genes with putative functions identified in the SSH libraries (XLS 138 kb)

Table S2 Sequences of oligonucleotides used for qPCR analysis of gene expression (XLSX 51 kb)

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Flores-Monterroso, A., Canales, J., de la Torre, F. et al. Identification of genes differentially expressed in ectomycorrhizal roots during the Pinus pinaster–Laccaria bicolor interaction. Planta 237, 1637–1650 (2013). https://doi.org/10.1007/s00425-013-1874-4

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