A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi

Abstract

Many plants have the capacity to obtain phosphate via a symbiotic association with arbuscular mycorrhizal (AM) fungi. In AM associations, the fungi release phosphate from differentiated hyphae called arbuscules, that develop within the cortical cells, and the plant transports the phosphate across a symbiotic membrane, called the periarbuscular membrane, into the cortical cell. In Medicago truncatula, a model legume used widely for studies of root symbioses, it is apparent that the phosphate transporters known to operate at the root-soil interface do not participate in symbiotic phosphate transport. EST database searches with short sequence motifs shared by known phosphate transporters enabled the identification of a novel phosphate transporter from M. truncatula, MtPT4. MtPT4 is significantly different from the plant root phosphate transporters cloned to date. Complementation of yeast phosphate transport mutants indicated that MtPT4 functions as a phosphate transporter, and estimates of the K(m) suggest a relatively low affinity for phosphate. MtPT4 is expressed only in mycorrhizal roots, and the MtPT4 promoter directs expression exclusively in cells containing arbuscules. MtPT4 is located in the membrane fraction of mycorrhizal roots, and immunolocalization revealed that MtPT4 colocalizes with the arbuscules, consistent with a location on the periarbuscular membrane. The transport properties and spatial expression patterns of MtPT4 are consistent with a role in the acquisition of phosphate released by the fungus in the AM symbiosis.

Figure 1.

Features of the Arbuscule–Cortical Cell Interface. The plant periarbuscular membrane (dotted line) and the plasma membrane of the cortical cell are contiguous. The apoplastic compartment resulting from the envelopment of the arbuscule is called the periarbuscular space.

Figure 2.

Unrooted Tree Based on a ClustalW Alignment Showing MtPT4 and Other Phosphate Transporters of the PHS Family. Plant phosphate transporters that have been characterized are shown in boldface.

Figure 3.

DNA Gel Blot of M. truncatula DNA Hybridized with a MtPT4 Probe Corresponding to the Coding Sequence of the MtPT4 Gene. The DNA in lanes 1 to 5 was digested with enzymes DraI, BamHI, EcoRV, HindIII, and EcoRI, respectively. The MtPT4 gene does not contain EcoRV and EcoRI sites.

Figure 4.

Phosphate Transport Properties of NS219 and PAM2 Expressing MtPT4. (A) Phosphate uptake in PAM2 expressing pWV3-MtPT4 (open squares) and pWV3 (closed squares). The external Pi concentration was 880 μM, and the pH was 4.7. Values are means of duplicate samples ± sd. (B) Phosphate uptake in NS219 expressing pWV3-MtPT4 (open circles) and pWV3 (closed circles). The external Pi concentration was 110 μM, and the pH was 4.7. Values are means of triplicate samples ± sd. (C) Phosphate uptake rates plotted as a function of phosphate concentration in PAM2 expressing pWV3-MtPT4 (open squares) and pWV3 (closed squares) and NS219 expressing pWV3-MtPT4 (open circles) and pWV3 (closed circles). The external pH was 4.7. Values are means of duplicate samples ± sd. Curves were fitted by nonlinear regression. K_m values were derived from three replicate experiments for each strain. Results from one representative experiment are shown. (D) Phosphate uptake in PAM2 expressing pWV3-MtPT4 (gray bars) and pWV3 (white bars) as a function of pH. Values are means of duplicate samples ± sd.

Figure 5.

Expression of MtPT4. RNA gel blot of RNA isolated from noncolonized M. truncatula roots (Mt) and M. truncatula roots colonized with G. versiforme (Mt/Gv). Roots were harvested at 1 to 5 weeks after inoculation. The blot was hybridized with an MtPT4 probe (top) and then stripped and rehybridized with an 18S rRNA probe (bottom).

Figure 6.

Expression of MtPT4. (A) Protein gel blot of microsomal proteins isolated from M. truncatula roots (Mt) and M. truncatula roots colonized with G. versiforme (Mt/Gv) harvested at 2, 3, and 4 weeks after inoculation. (B) M. truncatula roots (Mt) and M. truncatula roots colonized with G. gigantea (Mt/Gg) harvested at 5 weeks after inoculation. The blots were probed with affinity-purified MtPT4 (top) and MtPT1 (bottom) antibodies. Size markers (kD) are shown (right).

Figure 7.

Cell Type–Specific Expression of the MtPT4 Promoter. (A) and (B) Histochemical staining for GUS activity in M. truncatula roots carrying an MtPT4 promoter–UidA fusion. Roots were colonized with G. versiforme. Arrows indicate positive GUS staining in cells with arbuscules. The root shown in (B) was counterstained with acid fuschin. Bars = 200 μm (A) and 50 μm (B). (C) Epifluorescence micrograph of the root shown in (B) revealing arbuscules (a) and intraradical hyphae (ih). Acid fuschin also stained the vascular tissue.

Figure 8.

Immunolocalization of MtPT4. Laser scanning confocal microscopy images of mycorrhizal roots of M. truncatula/G. versiforme probed with MtPT4 antibodies. The MtPT4 antibodies were visualized with a secondary antibody conjugated with AlexaFluor 488. The roots were counterstained with WGA–Texas red to visualize G. versiforme. (A) and (D) WGA–Texas red staining revealing G. versiforme arbuscules. (B) and (E) Corresponding images showing green fluorescence from MtPT4 immunostaining. (C) and (F) Merged images showing both red and green fluorescence. The MtPT4 signal is visible surrounding the branches of the arbuscule (arrowheads). (A) to (C) show projections of 39 optical sections taken at 0.16-μm intervals; bar = 30 μm. (D) to (F) show projections of 43 optical sections taken at 0.3 μm intervals; bar = 10 μm.

Figure 9.

Immunolocalization of MtPT4. Laser scanning confocal microscopy images of mycorrhizal roots of M. truncatula/G. versiforme probed with MtPT4 antibodies. (A), (D), (G), and (J) Red fluorescence from WGA–Texas red staining. (B), (E), and (H) Corresponding images showing green fluorescence from MtPT4 immunostaining. (C), (F), and (I) Merged images showing both red and green fluorescence. (K) Immunostaining with MtPT4 preimmune serum. (A) to (C) show a single optical section through the branches of an arbuscule; bar = 10 μm. (D) to (F) show projections of 18 optical sections taken at 0.4-μm intervals. MtPT4 signals are not detected around the very young arbuscules (ya). The MtPT4 signal is just detectable around the developing arbuscule (da). Mature arbuscules (ma) in the same area of the root are associated with strong MtPT4 immunostaining. Bar = 50 μm. (G) to (I) show projections of 22 optical sections taken at 0.3-μm intervals. The older, collapsed arbuscule (ca) does not show MtPT4 immunostaining. Punctate staining is visible surrounding the degenerating arbuscules (dga). Strong MtPT4 immunostaining is visible surrounding the mature arbuscules. Bar = 30 μm. (J) and (K) show projections of 15 optical sections taken at 0.5-μm intervals. The MtPT4 preimmune serum did not stain the arbuscules. Bar = 10 μm.