Fig. S1. A. Schematic representation of L. bicolor fHANT-AC structure. LbNr, nitrate reductase; LbNrt, nitrate transporter; LbNir, nitrite reductase genes. White arrows between the LbNrt and LbNir refer to detected ORFs separating the fHANT-AC genes. ID numbers presented are L. bicolor genome v2.0 assembly JGI protein ID numbers.

B. A schematic representation of LbNrt 1902 bp coding sequence and its upstream region. The black bars represent the location of seven introns and the yellow bar the minimal promoter detected with NNPP version 2.2. The putative transcription start site, TSS (+1), is marked with an arrow. The red bars in the LbNrt upstream sequence represent sites with similarity to NIT2/AreA-type global positive acting GATA factor binding sites detected by TESS search. The binding sites of primers used for constructing the ihpRNA expression silencing vector for LbNrt (LbNrt-R/LbNrt-F), and the amplicons' sizes produced from both gDNA and mRNA are also indicated.


Fig. S2. Phylogenetic analysis of Laccaria nitrate transporter with a set of fungal, algal, higher plant NRT2 and bacterial NNP proteins. The aligning of the predicted polypeptides (ClustalW), the neighbour-joining analysis (using Poisson correction and 1000 bootstrap replicas) and the visualization of the generated phylogenetic trees were performed with the MEGA 4.0 software package. For sequence sources, see experimental procedures (Appendix S1).


Fig. S3. A. Secondary structure predictions of LbNRT by using TMHH2, TopPred and TMpred. The numbers (1–503) refer to amino acid positions and the roman numbers (I–XII) to predicted transmembrane α-helices. The numbers within the N- and C-terminal tails and the trans-helical loops in the scheme of LbNRT refer to the range of amino acids proposed to be involved in each structure by the three secondary structure detection programs used.

B. Amino acid sequence of the long cytosolic loop between the VI and VII transmembrane helices and the putative posttranslational regulatory sequences in it. The amino acids in bold represent the conserved S/T-x-R/K protein kinase C recognition motives. The amino acids in bold with asterisks mark the potentially phosphorylated serine and threonine residues detected by NetPhos2. The underlined amino acids represent a weak PEST sequence detected by PESTfind.

C. MFS I, MFS II, NNPI and NNPII sequence signatures in LbNRT. The numbers refer to amino acids in the protein. The fully conserved amino acids of MFS and NNP signature sequences are marked in bold and underlined. The amino acids not present in the consensus sequence or the potentially additional ones are presented with small letters. The absence of an amino acid is marked with a dash.

D. A 3D model of LbNRT, amino acids 20–499, obtained via homology modelling based on E. coli glycerol-3-phosphate transporter crystal structure at the SWISS-MODEL server. N- and C-terminal ends of the protein are marked in the model.


Fig. S4. A. RT-PCR assay showing the relative LbNrt and LbNr transcript levels of nitrate-grown wild type and two strongly LbNr-silenced strains, 21 and 30 (for the description of the strains, see Kemppainen and Pardo, 2010). gDNA, amplicons produced from genomic DNA template, C−, no template PCR control.

B. Northern blot detection and confirmation of the RT-PCR results for the unaffected LbNrt transcript status in the LbNr-silenced fungus.


Appendix S1. Experimental procedures.


Appendix S2. pHg/pSγ/LbNrtLoop T-DNA sequence (LB[RIGHTWARDS ARROW]RB 7680 bp).

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