Functional Characterization of a Magnesium Transporter of Root Endophytic Fungus Piriformospora indica

Abstract

Magnesium (Mg) is a crucial macronutrient required for the regular growth of plants. Here we report the identification, isolation and functional characterization of Mg-transporter PiMgT1 in root endophytic fungus Piriformospora indica. We also report the role of P. indica in the improvement of the Mg nutrition of the plant particularly under Mg deficiency condition. Protein BLAST (BLASTp) for conserved domains analysis showed that PiMgT1 belong to CorA like protein family of bacteria. We have also observed the presence of conserved 'GMN' signature sequence which suggests that PiMgT1 belongs to Mg transporter family. Phylogenetic analysis revealed that PiMgT1 clustered among fungal CorA family members nearer to basidiomycetes. Functionality of PiMgT1 was confirmed by complementation of a yeast magnesium transporter mutant CM66. We have observed that PiMgT1 restored the growth of mutant and showed comparable growth with that of WT. We found statistically significant (p < 0.05) two fold increase in the total intracellular Mg content of mutant complemented with PiMgT1 as compared to the mutant. These observations suggest that PiMgT1 is actively involved in Mg uptake by the fungus and may be helping in the nutritional status of the host plant.

Keywords: Arabidopsis thaliana; Piriformospora indica; high affinity transporter; magnesium transporter; plant growth activity.

FIGURE 1

Interaction of plant with Piriformospora indica. Plants were cultivated with or without P. indica in PNM media supplemented with different concentration of Mg for 15 days under long day light conditions.

FIGURE 2

Impact of P. indica colonization on the growth of the plant. Growth parameters of Arabidopsis seedlings after 15 days of co-cultivation under different Mg concentration (A) Biomass (B) Root length (C) Number of leaves. Each data set represents the means of 3 independent measurements ± SE ^∗indicates not significant as compared with the control (non-colonized); all other data are found significant at P < 0.05.

FIGURE 3

Measurement of chlorophyll contents of the plant during colonization with P. indica and during non-colonized stage. A. thaliana seedlings after 15 days of co-cultivation under different Mg concentration (A) chlorophyll a (B) chlorophyll b (C) total chlorophyll. Each data set represents the means of three independent measurements; all data are found significant at P < 0.05.

FIGURE 4

The phylogeny tree was inferred by using the maximum likelihood method based on the JTT matrix-based model. The tree with the highest log likelihood (–6494.5969) is shown. Boot strap value of 500 was taken in the tree construction. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pair wise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 44 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 99 positions in the final dataset. Evolutionary analyses were conducted using MEGA7.

FIGURE 5

Multiple alignment analysis. Multiple alignments analysis of PiMgT1 and PiMgT2 was performed using MultAlign tool with six homologs from closely related fungi. The analysis revealed conserved ‘GMN’ signature sequence (highlighted in rectangular blue color box) which is required to be designated as an Mg transporter.

FIGURE 6

Complementation assay. Growth complementation assay in comparison of WT of the mutant S. cerevisiae strain CM66 transformed individually with plasmid vector only (V), PiMgT1 and PiMgT2 indicated and serially dilution of each strain was spotted on SC^-URA medium containing different concentration of Mg (A) 10 μM (B) 100 μM (C) 1 mM (D) 4 mM (E) 10 mM (F) 100 mM (G) Control on YPD. Cobalt resistance assay: cobalt resistance assay in comparison of WT of the mutants S. cerevisiae strain CM66 which lacks Mg transport systems was transformed with plasmids vector only (V) PiMgT1 and PiMgT2 indicated and serially dilution of each strain was spotted on SC^-URA medium containing different concentration of CoCl₂ (H) 100 μM CoCl₂ (I) 500 μM CoCl₂ and fixed amount of 100 mM MgCl₂.

FIGURE 7

Analysis of growth. (A) Mutant transformed with PiMgT1 (B) WT (C) mutant transformed with the PiMgT2 (D) Mutant only.

FIGURE 8

Uptake of Mg. Uptake of Mg by mutant, WT, and mutant complemented with PiMgT1. Significant differences (p-value) were calculated with respect to mutant using two way ANOVA ^dp ≤ 0.05, ^cp ≤ 0.01, ^bp ≤ 0.005, ^ap ≤ 0.001.