WITHDRAWN: A phosphate transporter from the root endophytic fungus Piriformospora indica plays a role in phosphate transport to the host plant

Abstract

Because pure cultures and a stable transformation system are not available for arbuscular mycorrhizal fungi, the role of their phosphate transporters for the symbiotic interaction with the plant up till now could not be studied. Here we report the cloning and the functional analysis of a gene encoding a phosphate transporter (PiPT) from the root endophytic fungus Piriformospora indica, which can be grown axenically. The PiPT polypeptide belongs to the major facilitator superfamily. Homology modeling reveals that PiPT exhibits twelve transmembrane helices divided into two halves connected by a large hydrophilic loop in the middle. The function of the protein encoded by PiPT was confirmed by complementation of a yeast phosphate transporter mutant. The kinetic analysis of PiPT (K(m) 25 mum) reveals that it belongs to the high affinity phosphate transporter family (Pht1). Expression of PiPT was localized to the external hyphae of P. indica colonized with maize plant root, which suggests that external hyphae are the initial site of phosphate uptake from the soil. To understand the physiological role of PiPT, knockdown transformants of the gene were prepared using electroporation and RNA interference. Knockdown transformants transported a significantly lower amount of phosphate to the host plant than wild-type P. indica. Higher amounts of phosphate were found in plants colonized with wild-type P. indica than that of non-colonized and plants colonized with knockdown PiPT P. indica. These observations suggest that PiPT is actively involved in the phosphate transportation and, in turn, P. indica helps improve the nutritional status of the host plant.

FIGURE 1.

Unrooted phylogenetic relationship of PiPT with other high affinity phosphate transporters from plants and fungi. Protein names are followed by GenBank^TM (GB) accession numbers: GmPT (DQ074452) from Glomus mosseae; GiPT (AF359112) from Glomus intraradices; GvPT (U38650) from Glomus verisforme; PHO84 (D90346) from S. cerevisiae; LePT1 (AF022873), LePT2 (AF022874), and LePT4 (AY885651) from Lycopersicon esculentum; AtPT1 (U62330) and AtPT2 (U62331) from A. thaliana; StPT1 (X98890), StPT2 (X98891), StPT4 (AY793559), and StPT5 (AY885654) from Solanum tuberosum; MtPT1 (AF000354), MtPT2 (AF000355), and MtPT4 (AY116210) from Medicago truncatula; SrPT1 (AJ286743) and SrPT2 (AJ286744) from Sesbania rostrata; NtPT1 (AF156696), NtPT2 (AB042950), NtPT3 (AB042951), and NtPT4 (AB042956) from Nicotiana tobacum. The evolutionary history was inferred using the Neighbor-Joining method (72). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (73) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete Deletion option). Phylogenetic analyses were done by using MEGA4 (74).

FIGURE 2.

Southern blot analysis of P. indica genomic DNA digested with BamHI (lane 1), HindIII (lane 2), or EcoRI (lane 3). The blots were hybridized with labeled PiPT cDNA. The PiPT gene does not contain EcoRI and HindIII sites.

FIGURE 3.

Alignment of the deduced amino acid sequence of PiPT with G. versiforme (GvPT), yeast PHO84, Pholiota nameko (PnPT), and Magnaporthe grisea (MgPT) by using MULTIALIN (45). The degree of sequence conservation at each position amino acids is shown in red, and low consensus amino acids are shown in blue. Membrane-spanning domains (TM) of PiPT as predicted by TopPred (75) are shown as helices over the corresponding amino acid sequences indicated by numerals (TM1–TM12). Prediction of functional motifs in PiPT polypeptide was performed with PROSITE data base (available on-line) (52). *, signature tag of the MFS transporter. (Consensus symbols: “!” is any sequence of IV, “$” is any sequence of LM, “%” is any sequence of FY, and “#” is any sequence of NDQEBZ.) Green boxed sequences are potential phosphorylation sites for cAMP- and cGMP-dependent protein kinase.

FIGURE 4.

Ribbon representation of PiPT model based on homology modeling using GlPT as template. The predicted PiPT shape is trapezoidal with dimensions of ∼46 × 27 Å from the bottom and ∼33 × 26 Å from the top, and its height is ∼42 Å.

FIGURE 5.

a, effect of different concentrations of phosphate on the expression of PiPT transcripts. Northern blots show total RNA isolated from P. indica grown in MN media containing the indicated different phosphate concentrations for 1, 5, 10, and 15 days. A picture of the gel shows uniform loading of RNA. b, RT-PCR was used to assess abundance of PiPT transcript at 5, 10, 15, and 20 days showed 680-bp-amplified PiPT DNA fragment from colonized maize plant roots (+PI). As a negative control, 5- and 20-day plants without P. indica (−PI) were taken. DNA size markers are shown at the extreme left as M. PiTef was used as a reference gene. c, Trypan blue staining of maize plant roots showing the presence of intracellular chlamydospores of P. indica in the cortical cells showing the colonization at 5 days or onward (black arrow). A high degree of colonization was observed with time, and no colonization was observed in control roots.

FIGURE 6.

Complementation of PiPT gene using yeast phosphate uptake-mutant (MB192) and kinetics of phosphate uptake. a, acid phosphatase activity was checked in yeast, pho84 mutant containing only vector (p112A1NE), MB192 mutant cells with vector plus PHO84 insert, MB192 mutant cells with vector plus PiPT insert grown on high phosphate medium for 36 h. b, phosphate uptake into yeast MB192 mutant cells transformed with vector (no phosphate transporter gene) (black), PHO84 (line), and with PiPT cDNA (gray). Means and standard errors of means of three replicate determinations consisting of three measurements each are shown in a and b. The time period for the phosphate accumulation was 3 min. c, nonlinear regression of phosphate uptake of MB192 transformed with PiPT versus external phosphate concentration at pH 4.5. *, PiPT and PHO84 (positive control) indicates that the data are statistically significant (p < 0.001) as compared with control; i.e. MB192 mutant cells only with vector (no insert). Significance has been calculated using a t test (SigmaStat, version 2.0).

FIGURE 7.

Localization of PiPT expression in external (EH) and internal hyphae (IH) from maize plant root colonized with P. indica. For the determination of relative expression of PiPT level, cDNA was synthesized from RNA isolated from EH and IH and subjected to real-time PCR using specific primers and SYBR green I. The comparative C_t method was applied to analyze the data. For experimental samples, targeted (PiPT) quantity was determined and divided by the target quantity of the calibrator (Tef). Thus, the calibrator becomes the 1× sample, and all other quantities are expressed as an n-fold difference relative to the Tef. The values obtained for PiPT expression for EH and IH were 1- and 0.055-fold, respectively, relative to Tef. The means ± S.D. of three independent determinations are presented. Asterisks indicate significant differences from EH at p < 0.01.

FIGURE 8.

Characterization of KD-PiPT P. indica. a, the transcript levels of the PiPT gene in KD and wild-type P. indica. Wild-type control (WTC); transformed colony 2 (TC2) and TC5, with an RNAi construct. The P. indica colonies were first grown in AMM, at day 4 AMM was replaced with fungal minimal media with a 10 μm supplement of phosphate, and RNA was extracted from P. indica colonies at 3 days after transfer of media. Expression levels of PiPT were determined by real-time PCR as described in the legend of Fig. 7. The values obtained for PiPT expression for WTC, TC2, and TC5 were 1-, 0.4-, and 0.39-fold, respectively, relative to Tef. The means ± S.D. of three independent determinations are presented. Asterisks indicate significant differences from WTC at p < 0.01. b, Northern blot analysis of siRNAs of the PiPT in P. indica transformants. Blot was exposed overnight to show detectable siRNA accumulation in the KD-PiPT P. indica (TC2 and TC5), C, wild-type P. indica. DNA oligonucleotides (16 and 22 nucleotides (nt)) were used as molecular size markers for siRNA analysis. Equal loading of total RNA was estimated by ethidium bromide staining of rRNAs (predominant RNA).

FIGURE 9.

Bi-compartment Petri dish culture system to study the transport of radiolabeled (³²P) orthophosphoric acid to maize plants via P. indica. A, lateral view. B and C, top view showing both compartments separated by a small glass Petri plate (used as compartment 2). The black arrow indicates the P. indica growth in compartment 1, and the white arrow indicates the growth of P. indica in compartment 2 (D). Colonization of the maize roots was by P. indica.

FIGURE 10.

In A: transport of phosphorus to maize plants by P. indica carried out in the bi-compartment Petri dish culture system. Radioactivity incorporated in plants was demonstrated by autoradiography. Radioactivity count intensities are shown in false color code (vertical bar, low to high). Panels: i, whole maize plant before autoradiography; ii, false-color autoradiograph of the maize plant obtained after 3 h of exposure of the maize plant; and iii, microscopic view of a sample of plant root before autoradiography. a, maize plants were colonized with wild-type P. indica (WT); b, maize plants were colonized with KD-P. indica (KD); c, maize plants were grown alone without P. indica (C). In B: amount of ³²P transferred to the maize plant components by P. indica. Radioactivity was measured three times independently (number of transformants used was n = 2). The mean ± S.D. of three independent measurements are shown. The bar labeled with the asterisk represents significance as compared with the wild-type P. indica (p < 0.05).

FIGURE 11.

Effect of PiPT on phosphate nutrition and plant growth. Shown are biomass (a) and total phosphate (b) content. Error bars denote the ±S.D. of the mean from plants of three replicate plates. *, significant difference from the maize plant colonized with wild-type P. indica (taken as a control). c, growth-promoting performance of P. indica at low (10 μm) and high (1 mm) phosphate concentrations. The mean ± S.D. of three independent measurements are shown. The bar labeled with the asterisk represents significant (p < 0.05) as compared with their respective control.