A Phosphate Transporter from the Root Endophytic Fungus Piriformospora indica Plays a Role in Phosphate Transport to the Host Plant

Vikas Yadav,Manoj Kumar,Deepak Kumar Deep,Hemant Kumar,Ruby Sharma,Takshashila Tripathi,Narendra Tuteja,Ajay Kumar Saxena,Atul Kumar Johri

doi:10.1074/jbc.m110.111021

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.

Highlights

Nutrition—To know whether phosphate has a effect on plant growth, we have determined the total phosphate content in 1) maize plant colonized with wild-type P. indica, 2)
An unrooted phylogenetic tree demonstrates the close relationship between the PiPT protein and members of the plant Pht1 family and high affinity phosphate transporters of fungi (Fig. 1)
Southern blot analysis suggests the presence of a single PiPT gene in the genome of P. indica (Fig. 2)

Summary

EXPERIMENTAL PROCEDURES

Fungi, Bacteria, and Yeast Strains—Zea mays (var. pro33) plant and fungus P. indica [15] were used throughout the study, and Escherichia coli XL1-Blue was used for cloning purposes [20]. After obtaining homologous probe (928 bp), cDNA library was screened to get full-length PiPT cDNA by using Hybond-N nylon membranes (Amersham Biosciences) harboring recombinant ␭ phages, which were hybridized overnight at 60 °C in hybridization buffer containing 7% SDS, 0.5 M phosphate buffer (pH 7.5), 1 mM EDTA (pH 8), and 1% bovine serum albumin. For Southern blot, genomic DNA (25 ␮g) was digested with restriction enzymes and separated on 0.8% agarose gel, denatured, and transferred to a nylon membrane For both analyses, hybridization and washing conditions were kept the same as mentioned in the previous text. Summary of amino acids identity (%) between P. indica PiPT and other fungal and plant phosphate transporters

Organism name

Effect of PiPT on Phosphate

RESULTS

Hydrophobicity analysis and sequence alignment suggests that

DISCUSSION