Gene expression studies in different genotypes of an ectomycorrhizal fungus require a 1 high number of reliable reference genes. 2 3

Abstract 13 Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the 14 expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-15 PCR measurements strongly depends on the normalization with appropriate endogenous 16 reference genes. In this study a set of candidate reference genes for the use in gene expression 17 studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or 18 cadmium was identified, evaluated and validated. Seven candidate genes were selected from 19 cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to 20 evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in 21 different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has 22 its own most stably expressed set of reference genes, regardless of the metal treatments, i

Micronutrient transport in mycorrhizal symbiosis; zinc steals the show.

International audienceMycorrhizal fungi provide their host plant with essential nutrients in exchange for sugars and/or lipids. Traditionally, transport and translocation of macronutrients, including nitrogen and phosphorus, throughout the fungal mycelium and towards the host plant are well studied. However, the regulation of nutrient exchange and their contribution in the morphogenesis and development of mycorrhizas remains unclear. In this Opinion, we argue that adding micronutrients in the current models of symbiotic transport is essential to fully understand the establishment, maintenance, and functioning of mycorrhizal associations. Homeostatic mechanisms at the cellular level and the first transport proteins involved have been recently documented for zinc (Zn) in arbuscular mycorrhizal, ectomycorrhizal, and ericoid mycorrhizal fungi. Mycorrhizal plants benefit from an improved Zn status in control conditions and are better protected when environmental Zn availability fluctuates. These recent progresses are paving the way for a better understanding of micronutrient allocation in mycorrhizas. Revising our vision on the role of micronutrients, particularly of Zn, in these interactions will allow a better use of mycorrhizal fungi in sustainable agriculture and forestry, and will increase management practices in waste land, as well as in agricultural and natural ecosystems

Gene expression profiling of a Zn-tolerant and a Zn-sensitive Suillus luteus isolate exposed to increased external zinc concentrations

Complementary DNA (cDNA)-amplified fragment-length polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in different classes of expression pattern, were isolated and sequenced. Thirty-six of the represented genes showed homology to function-known genes, whereas 6 matched unknown protein coding sequences, and 102 were possibly novel. Although relatively few TDFs were found to be responsive to the different zinc treatments, their modulated expression levels may suggest a different transcriptional response to zinc treatments in both isolates. Among the identified genes that could be related to heavy-metal detoxification or the tolerance trait were genes encoding for homologues of a heat-shock protein, a putative metal transporter, a hydrophobin, and several proteins involved in ubiquitin-dependent proteolysis. © 2007 Springer-Verlag.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification, evolution and functional characterization of two Zn CDF-family transporters of the ectomycorrhizal fungus Suillus luteus

Two genes, SlZnT1 and SlZnT2, encoding Cation Diffusion Facilitator (CDF) family transporters were isolated from Suillus luteus mycelium by genome walking. Both gene models are very similar and phylogenetic analysis indicates that they are most likely the result of a recent gene duplication event. Comparative sequence analysis of the deduced proteins predicts them to be Zn transporters. This function was confirmed by functional analysis in yeast for SlZnT1. SlZnT1 was able to restore growth of the highly Zn sensitive yeast mutant zrc1 and localized to the vacuolar membrane. Transformation of zrc1 yeast cells with SlZnT1 resulted in an increased accumulation of Zn compared to empty vector transformed zrc1 yeast cells and equals Zn accumulation in wild type yeast cells. We were not able to express functional SlZnT2 in yeast. In S. luteus, both SlZnT genes are constitutively expressed whatever the external Zn concentrations. A labile Zn pool was detected in the vacuoles of S. luteus free-living mycelium. Therefore we conclude that SlZnT1 is indispensable for maintenance of Zn homeostasis by transporting excess Zn into the vacuole

The SlZRT1 Gene Encodes a Plasma Membrane-Located ZIP (Zrt-, Irt-Like Protein) Transporter in the Ectomycorrhizal Fungus Suillus luteus.

Zinc (Zn) is an essential micronutrient but may become toxic when present in excess. In Zn-contaminated environments, trees can be protected from Zn toxicity by their root-associated micro-organisms, in particular ectomycorrhizal fungi. The mechanisms of cellular Zn homeostasis in ectomycorrhizal fungi and their contribution to the host tree's Zn status are however not yet fully understood. The aim of this study was to identify and characterize transporters involved in Zn uptake in the ectomycorrhizal fungus Suillus luteus, a cosmopolitan pine mycobiont. Zn uptake in fungi is known to be predominantly governed by members of the ZIP (Zrt/IrtT-like protein) family of Zn transporters. Four ZIP transporter encoding genes were identified in the S. luteus genome. By in silico and phylogenetic analysis, one of these proteins, SlZRT1, was predicted to be a plasma membrane located Zn importer. Heterologous expression in yeast confirmed the predicted function and localization of the protein. A gene expression analysis via RT-qPCR was performed in S. luteus to establish whether SlZRT1 expression is affected by external Zn concentrations. SlZRT1 transcripts accumulated almost immediately, though transiently upon growth in the absence of Zn. Exposure to elevated concentrations of Zn resulted in a significant reduction of SlZRT1 transcripts within the first hour after initiation of the exposure. Altogether, the data support a role as cellular Zn importer for SlZRT1 and indicate a key role in cellular Zn uptake of S. luteus. Further research is needed to understand the eventual contribution of SlZRT1 to the Zn status of the host plant

A Transcriptomic Atlas of the Ectomycorrhizal Fungus Laccaria bicolor

Trees are able to colonize, establish and survive in a wide range of soils through associations with ectomycorrhizal (EcM) fungi. Proper functioning of EcM fungi implies the differentiation of structures within the fungal colony. A symbiotic structure is dedicated to nutrient exchange and the extramatricular mycelium explores soil for nutrients. Eventually, basidiocarps develop to assure last stages of sexual reproduction. The aim of this study is to understand how an EcM fungus uses its gene set to support functional differentiation and development of specialized morphological structures. We examined the transcriptomes of Laccaria bicolor under a series of experimental setups, including the growth with Populus tremula x alba at different developmental stages, basidiocarps and free-living mycelium, under various conditions of N, P and C supply. In particular, N supply induced global transcriptional changes, whereas responses to P supply seemed to be independent from it. Symbiosis development with poplar is characterized by transcriptional waves. Basidiocarp development shares transcriptional signatures with other basidiomycetes. Overlaps in transcriptional responses of L. bicolor hyphae to a host plant and N/C supply next to co-regulation of genes in basidiocarps and mature mycorrhiza were detected. Few genes are induced in a single condition only, but functional and morphological differentiation rather involves fine tuning of larger gene sets. Overall, this transcriptomic atlas builds a reference to study the function and stability of EcM symbiosis in distinct conditions using L. bicolor as a model and indicates both similarities and differences with other ectomycorrhizal fungi, allowing researchers to distinguish conserved processes such as basidiocarp development from nutrient homeostasis

Laccaria bicolor MiSSP8 is a small-secreted protein decisive for the establishment of the ectomycorrhizal symbiosis

The ectomycorrhizal symbiosis is a predominant tree–microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long‐term and intimate relationship between the soil‐borne fungi and the roots of trees. Mycorrhiza‐induced Small‐Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8‐RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C‐terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small‐secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.Fil: Pellegrin, Clément. université de Lorraine; FranciaFil: Pellegrin, Clément.Fil: Ruytinx, Joske.Fil: Guinet, Frédéric.Fil: Kemppainen, Minna.Fil: Frei dit Frey, Nicolas.Fil: Puech Pagès, Virginie.Fil: Hecker, Arnaud.Fil: Pardo, Alejandro Guillermo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Micología Molecular; ArgentinaFil: Martin, Francis M..Fil: Veneault Fourrey, Claire

Comparative genomics and expression levels of hydrophobins from eight mycorrhizal genomes

Hydrophobins are small secreted proteins that are present as several gene copies in most fungal genomes. Their properties are now well understood: they are amphiphilic and assemble at hydrophilic/hydrophobic interfaces. However, their physiological functions remain largely unexplored, especially within mycorrhizal fungi. In this study, we identified hydrophobin genes and analysed their distribution in eight mycorrhizal genomes. We then measured their expression levels in three different biological conditions (mycorrhizal tissue vs. free-living mycelium, organic vs. mineral growth medium and aerial vs. submerged growth). Results confirmed that the size of the hydrophobin repertoire increased in the terminal orders of the fungal evolutionary tree. Reconciliation analysis predicted that in 41% of the cases, hydrophobins evolved from duplication events. Whatever the treatment and the fungal species, the pattern of expression of hydrophobins followed a reciprocal function, with one gene much more expressed than others from the same repertoire. These most-expressed hydrophobin genes were also among the most expressed of the whole genome, which suggests that they play a role as structural proteins. The fine-tuning of the expression of hydrophobin genes in each condition appeared complex because it differed considerably between species, in a way that could not be explained by simple ecological traits. Hydrophobin gene regulation in mycorrhizal tissue as compared with free-living mycelium, however, was significantly associated with a calculated high exposure of hydrophilic residues

Comparative genomics reveals dynamic genome evolution in host specialist ectomycorrhizal fungi.

While there has been significant progress characterizing the 'symbiotic toolkit' of ectomycorrhizal (ECM) fungi, how host specificity may be encoded into ECM fungal genomes remains poorly understood. We conducted a comparative genomic analysis of ECM fungal host specialists and generalists, focusing on the specialist genus Suillus. Global analyses of genome dynamics across 46 species were assessed, along with targeted analyses of three classes of molecules previously identified as important determinants of host specificity: small secreted proteins (SSPs), secondary metabolites (SMs) and G-protein coupled receptors (GPCRs). Relative to other ECM fungi, including other host specialists, Suillus had highly dynamic genomes including numerous rapidly evolving gene families and many domain expansions and contractions. Targeted analyses supported a role for SMs but not SSPs or GPCRs in Suillus host specificity. Phylogenomic-based ancestral state reconstruction identified Larix as the ancestral host of Suillus, with multiple independent switches between white and red pine hosts. These results suggest that like other defining characteristics of the ECM lifestyle, host specificity is a dynamic process at the genome level. In the case of Suillus, both SMs and pathways involved in the deactivation of reactive oxygen species appear to be strongly associated with enhanced host specificity