Fig 1.
In silico prediction of a Shaker-like channel in the fungus Hebeloma cylindrosporum.
The hydrophobicity profiles of the Shaker subunits from H. cylindrosporum (HcSKC), L. bicolor (LbSKC), and X. laevis (XlKV2.1) (a) and their partial amino acid sequence alignment (b) predicted six transmembrane domains (S1 to S6). One pore domain in HcSKC and LbSKC subunits containing the conserved G[YF]G[DE] motif of the Shaker channel selectivity filter was also detected. The S4 transmembrane domain corresponds to the predicted voltage-sensor domain. (c) The plausible 3-D model of the fungal HcSKC subunit showing the transmembrane (S1 to S6) and pore (P) domains was generated by homology with the 3-D structure of RnKV2.1 K+ channel from R. norvegicus (NP_037318.1; S2 Table) as a template using Swiss-Model server [33] (http://swissmodel.expasy.org/). The GYGE signature motif of the pore region was highlighted in green. The sequence alignment of HcSKC and RnKV2.1 polypeptides is displayed in S1 Fig.
Fig 2.
Phylogenetic tree of KV channels in fungi, and representative animal and plant species.
The Shaker-like K+ channel tree of publicly available sequenced fungi from the Basidiomycota phylum and from basal fungi (Cryptomycota, Chytridiomycota, Blastocladiomycota, Zoopagomycota and Mucoromycota) was calculated by the maximum likelihood method (1000 bootstraps). Shaker-like proteins were found only in one putative Ascomycota species (Saitoella complicata) and not in the Pucciniomycotina and Ustilaginomycotina subphyla of Basidiomycota. Ectomycorrhizal (ECM), arbuscular mycorrhizal (AM), and orchid mycorrhizal (OM) fungi are indicated by yellow, green, and blue dots, respectively. KV channels from representative land plant and animal species were included in the tree. All species displayed in this tree are listed in the S2 Table. Shaker-like proteins from H. cylindrosporum (HcSKC) and L. bicolor (LbSKC) used in this study are indicated by black arrows.
Fig 3.
HcSKC transcript localization in Hebeloma cylindrosporum—Pinus pinaster ectomycorrhizas.
In situ hybridization in ectomycorrhizas from 2-month-old co-cultures of P pinaster–H. cylindrosporum with HcSKC-specific probes. (a) Control with a sense probe did not show any signal. (b) Using the antisense probe, HcSKC transcripts were detected in extra-radical hyphae, the fungal sheath and the Hartig net of ectomycorrhizas. Bars, 50 μm. c.c., cortical cell; exh, extraradical hyphae; Hn, Hartig net; sh, fungal sheath.
Fig 4.
Potassium content in Hebeloma cylindrosporum HcSKC downregulated lines.
K+ content was measured in fungal lines transformed with the empty (Ctrl) or HcSKC-silencing vectors (RNAi-SKC-5 and RNAi-SKC-7) using an atomic absorption flame spectrophotometer. Statistical differences were evaluated using the Student’s test with respect to the Ctrl strain (*, P < 0.05; **, P < 0.01). n = 6.
Fig 5.
Relative expression of putative K+ channels and transporters in Hebeloma cylindrosporum control and HcSKC-silencing lines.
Relative expressions of the K+ transporters HcTrk1 (a), HcTrk2 (b), HcHAK (c) and the K+ channels HcTOK1 (d), HcTOK2.1 (e) and HcTOK2.2 (f) were analyzed in dependence on K+ shortage in control and RNAi fungal lines. The relative expressions of the K+ transporter HcTrk2 (b) and the K+ channels HcTOK1 (d) and HcTOK2.1 (e) were altered in RNAi-SKC-5 and RNAi-SKC-7 lines in comparison to the transgenic control line (Ctrl) under K+ limiting conditions. The relative expressions of HcTrk1 (a) and HcHAK (c) K+ transporters, and of the K+ channel HcTOK2.2 (f) were not affected by K+ shortage in RNAi-SKC-5 and RNAi-SKC-7 lines in comparison to the transgenic control line (Ctrl). Mean values are provided with the standard error (n = 6). Statistical differences were determined using a one-way ANOVA followed by Dunnett’s test relative to Ctrl strain for each data point P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).
Fig 6.
Potassium content in 2-month-old Pinus pinaster plants growing alone or in co-culture with Hebeloma cylindrosporum under K+-sufficient or -deficient conditions.
Root and shoot contents were measured in non-mycorrhizal plants (NM) and plants inoculated with fungi transformed with the empty (Ctrl) or HcSKC-silencing vectors (RNAi-SKC-5 and RNAi-SKC-7), and growing at standard K+ (SK medium, 1 mM K+, a) or low K+ (LK medium, 0.05 mM K+, b). Mean values are provided with the standard error (n = 5–6). Different letters indicate significant differences between treatments according to one-way ANOVA followed by Tukey HSD post-hoc tests (P < 0.05).