Abstract
Inorganic phosphorus (Pi) is essential for plant growth, and phosphate (P) deficiency is a primary limiting factor in Pinus tabulaeformis development in northern China. P acquisition in mycorrhizal plants is highly dependent on the activities of phosphate transporters of their root-associated fungi. In the current study, two phosphate transporter genes, RlPT and LbPT, were isolated from Rhizopogon luteolus and Leucocortinarius bulbiger, respectively, two ectomycorrhizal fungi forming symbiotic interactions with the P. tabulaeformis. Phylogenetic analysis suggested that the sequence of the phosphate transporter of L. bulbiger is most closely related to a phosphate transporter of Hebeloma cylindrosporum, whereas the phosphate transporter of R. luteolus is most closely related to that of Piloderma croceum. The subcellular localization indicated that RlPT and LbPT were expressed in the plasma membrane. The complementation assay in yeast indicated that both RlPT and LbPT partially compensated for the absence of phosphate transporter activity in the MB192 yeast strain, with a K m value of 57.90 μmol/L Pi for RlPT and 35.87 μmol/L Pi for LbPT. qPCR analysis revealed that RlPT and LbPT were significantly up-regulated at lower P availability, which may enhance P uptake and transport under Pi starvation. Our results suggest that RlPT and LbPT presumably play a key role in Pi acquisition by P. tabulaeformis via ectomycorrhizal fungi.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 31060110 and 31360125). We would like to express our appreciation to Prof. J. Hegemann (Institut fur Mikrobiologie, Heinrich-Heine-Universitat Dusseldorf, Germany) for generously providing the yeast mutants (MB192) and two types of vectors (pUG23 and pUG23+GFP).
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Rong Zheng and Jugang Wang contributed equally to this work.
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Fig. S1
Sporocarps and ectomycorrhizal (ECM) characteristics of the fungi used in the present study. (a) Rhizopogon luteolus; (b) Leucocortinarius bulbiger; (c) mycorrhizal characteristics of Rhizopogon luteolus–Pinus tabulaeformis; (d) mycorrhizal characteristics of Leucocortinarius bulbiger–Pinus tabulaeformis. (GIF 81 kb)
Fig. S2
The ExPASy online hydrophobic prediction of RlPT (a) and LbPT (b). (GIF 123 kb)
Fig. S3
The DNA structures of RlPT and LbPT. The lavender boxes represent the exons, the white boxes represent the introns, and the characters in the box indicate the number of bases. (GIF 28 kb)
Fig. S4
The transmembrane-spanning domains predication of RlPT (a) and LbPT (b) by using TMHMM software. (GIF 182 kb)
Fig. S5
Functional expression of RlPT and LbPT in the MB192 strains and YPD medium. A staining test was performed to determine PT activity in the MB192 mutant yeast transformed with the expression vector pUG23 harboring no insert (MB192+eV) and pUG23+PT (MB192+RlPT/LbPT), and a WT yeast strain transformed with empty expression vector pUG23 (WT+eV) was cultured as a positive control. All aliquots (a and b are the experiment for the RlPT, c and d are the experiment for the LbPT) were cultivated in dishes containing 60 μmol/L (a and c) and 200 μmol/L (b and d) phosphate in a YPD medium at 30 °C for 3 days. The green arrows indicate that all aliquot concentrations decreased along the arrow directions (10-fold dilution each time). (GIF 361 kb)
Fig. S6
The growth (OD 600 nm) curves of MB192+eV, MB192+RlPT, MB192+LbPT, and WT+eV at optimal pH (pH = 6). All of the yeast strains were cultured in YNB medium that contained 80 μmol/L of phosphate at 30 °C and at 180 rpm, and the OD (600) values were determined every 5 h. (GIF 23 kb)
Fig. S7
The functional expression of RlPT and LbPT in yeast. A staining test was applied to determine the acid phosphatase enzyme activities in the MB192 mutant yeast transformed with the empty vector pUG23 (MB192+eV), pUG23+RlPT (MB192+RlPT), and pUG23+LbPT (MB192+LbPT), and a WT yeast strain transformed with empty pUG23 vector (WT+eV) was cultured as a positive control. All aliquots were cultivated in tubes containing 20, 60, 100, and 200 μmol/L of phosphatase YNB medium (pH = 4.8) at 30 °C and at 180 rpm for 24 h. Bromocresol purple was added to the culture medium as a color indicator, and a color shift reflected medium acidification, which correlates with the growth of the yeast cells. (GIF 104 kb)
Table S1
Primers used in this study. In the primers for functional verification and subcellular localization, the cDNA complementary sequences are the underlined bases and the homologous sequences of vectors are the bases without underlining. (XLS 17 kb)
Table S2
The main properties, including ORF length, predicted molecular weight, predicted isoelectric point, hydrophobicity/hydrophilicity, transmembrane regions, optimal pH, and K m values of functionally characterized PTs from ECM fungi. “–” means no data at present time. (XLS 33 kb)
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Zheng, R., Wang, J., Liu, M. et al. Molecular cloning and functional analysis of two phosphate transporter genes from Rhizopogon luteolus and Leucocortinarius bulbiger, two ectomycorrhizal fungi of Pinus tabulaeformis . Mycorrhiza 26, 633–644 (2016). https://doi.org/10.1007/s00572-016-0702-7
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DOI: https://doi.org/10.1007/s00572-016-0702-7