Polar localization of a symbiosis-specific phosphate transporter is mediated by a transient reorientation of secretion

Abstract

The arbuscular mycorrhizal (AM) symbiosis, formed by land plants and AM fungi, evolved an estimated 400 million years ago and has been maintained in angiosperms, gymnosperms, pteridophytes, and some bryophytes as a strategy for enhancing phosphate acquisition. During AM symbiosis, the AM fungus colonizes the root cortical cells where it forms branched hyphae called arbuscules that function in nutrient exchange with the plant. Each arbuscule is enveloped in a plant membrane, the periarbuscular membrane, that contains a unique set of proteins including phosphate transporters such as Medicago truncatula MtPT4 [Javot et al., (2007) Proc Natl Acad Sci USA 104:1720-1725], which are essential for symbiotic phosphate transport. The periarbuscular membrane is physically continuous with the plasma membrane of the cortical cell, but MtPT4 and other periarbuscular membrane-resident proteins are located only in the domain around the arbuscule branches. Establishing the distinct protein composition of the periarbuscular membrane is critical for AM symbiosis, but currently the mechanism by which this composition is achieved is unknown. Here we investigate the targeting of MtPT4 to the periarbuscular membrane. By expressing MtPT4 and other plasma membrane proteins from promoters active at different phases of the symbiosis, we show that polar targeting of MtPT4 is mediated by precise temporal expression coupled with a transient reorientation of secretion and alterations in the protein cargo entering the secretory system of the colonized root cell. In addition, analysis of phosphate transporter mutants implicates the trans-Golgi network in phosphate transporter secretion.

Fig. 1.

Protein localization in colonized cells is altered when genes are expressed from promoters that differ in activity relative to arbuscule development. Live-cell fluorescence and bright-field DIC images of longitudinal root sections of M. truncatula roots colonized with an AM fungus, G. versiforme. (A) pMtPT4:MtPT4-GFP localizes in the periarbuscular membrane (arrowhead) of colonized cortical cells. (B) pMtPT1:MtPT1-GFP localizes in the plasma membrane of root epidermal cells. (C) pMtPT4:MtPT1-GFP localizes in the periarbuscular membrane of colonized cortical cells and not in the plasma membrane. (D) pMtPT4:AtPIP2a-GFP does not localize in the periarbuscular or plasma membrane; instead it colocalizes with the HDEL-mCherry ER marker. (E) p35S:MtPT4-GFP accumulates mostly in the vacuole and ER, as shown by the presence of perinuclear signal (Inset), and not in the periarbuscular membrane (arrowhead). The signal is particularly strong in the vascular tissue. (F) p35S:MtPT1-GFP localizes in the plasma membrane and in the membrane that surrounds intracellular hyphae but not in the periarbuscular membrane. p35S:MtPT4-GFP includes the MtPT4 5′ UTR, whereas p35S:MtPT1-GFP contains the 35S 5′ UTR. For each construct, images are representative examples of at least 10 independent transgenic root systems; localization patterns were consistent among these independent experiments. Arrowheads indicate arbuscule branches. N, plant nucleus; T, arbuscule trunk; V, vascular tissue. (Scale bars: 20 μm.)

Fig. 2.

The MtBcp1 promoter drives expression before and during arbuscule formation and confirms the importance of gene expression coordinated with arbuscule development for localization in the periarbuscular membrane. (A) In cells with intracellular hyphae (IH) but without arbuscule branches (cell 1), pMtBcp1:MtPT4-GFP localizes in the ER as indicated by the peripheral and perinuclear ER pattern. Upon arbuscule branching (cells 2 and 3), MtPT4 accumulates to high levels in the periarbuscular membrane (arrowhead). (B) pMtBcp1:MtPT1-GFP localizes in the plasma membrane of noncolonized cortical (NC) cells, in cells with intracellular hyphae, and in the periarbuscular membrane of cells with branched arbuscules. (C) Coexpression of pMtBcp1:MtPT1-GFP and pMtBcp1:AtPIP2a-mCherry in two cells with young arbuscules just beginning to branch illustrates plasma membrane localization of both proteins but contrasting perinuclear ER localization of AtPIP2a (N) and periarbuscular membrane localization of MtPT1 (arrowhead). (D) Coexpression of two AtPIP2a fusions reveals plasma membrane and ER localization for pMtBcp1:AtPIP2a-mCherry (red) but only ER localization for pMtPT4:AtPIP2a-GFP (green). (Scale bars: 20 μm.)

Fig. 3.

Localization of two carbohydrate transporters, MtSTP and MtPLT, when expressed from three promoters that differ in activity relative to arbuscule development. Live-cell fluorescence and bright-field DIC images of longitudinal root sections of M. truncatula roots (A and B) and M. truncatula roots colonized with the AM fungus, G. intraradices (C–F). (A and B) p35S:MtMST-GFP and p35S:MtPLT-GFP localize in the plasma membrane. (C and D) pMtPT4:MtMST-GFP and pMtPT4:MtPLT-GFP localize in the periarbuscular membrane around the branches of young arbuscules and not in the plasma membrane. (E and F) pMtBcp1:MtSTP-GFP and pMtBcp1:MtPLT-GFP are expressed before and during arbuscule formation and localize in the plasma membrane of noncolonized cortical cells and in the plasma membrane and periarbuscular membrane of cells containing arbuscules. Arrowheads indicate localization in the periarbuscular membrane. V, vascular tissue. (Scale bars: 75 μm in A, 50 μm in B, 10 μm in C and D, 25 μm in E, and 10 μm in F.)

Fig. 4.

Mutant MtPT4^S115F is retained in the endomembrane system and colocalizes with ER and TGN markers. Localization of MtPT4^S115F-GFP expressed under pMtPT4 and coexpressed with HDEL-mCherry ER marker (A), GmMAN1-mCherry Golgi marker (B), and AtSyp61-mRFP TGN marker (C). MtPT4^S115F signal overlaps with the ER and Syp61 signals. MtPT4^S115F signals often occur adjacent to GmMAN1 signals but do not colocalize. (Left ) GFP fluorescence. (Center Left) mCherry/mRFP fluorescence. (Center Right) Overlay of both channels. (Right) DIC. Arrowheads indicate the arbuscule branch; arrows indicate endosomal localization of MtPT4^S115F-GFP. In addition to TGN, AtSyp61-mRFP also produces signal in the vacuole (C), likely because of overexpression and turnover. ER, Golgi, and TGN markers are expressed from the 35S promoter. Localization of these markers has been verified and is consistent in colonized and noncolonized cells (10). (Scale bars: 20 μm.) N, nucleus.

Fig. 5.

MtPT1^S117F is retained in endomembranes. AtSyp61-mRFP TGN marker coexpressed with pMtPT1:MtPT1^S117F-GFP (A) and pMtPT4:MtPT1^S117F-GFP (B). Colocalization between markers is observed in epidermal cells (A) and cortical cells harboring arbuscules (B). Retention in the ER is evident by reticulate and perinuclear signals. (Scale bars: 20 μm.)

Fig. 6.

Summary and model for phosphate transporter secretion during arbuscule development. (A) Table summarizing membrane localizations and illustrating that proteins expressed from the MtBcp1 promoter show localization patterns that combine those obtained from MtPT4 and p35S promoters. (B) Proposed model derived from data obtained through analysis of protein-GFP fusions expressed either from their native promoters and/or expressed ectopically from promoters that show differential activity during arbuscule formation. The cartoon depicts an uninfected cell (Upper) and a cortical cell harboring a developing arbuscule (Lower). In an uninfected cell, MtPT1, MtSTP, MtPLT, and AtPIP2a are secreted to the plasma membrane (black dashed line), whereas MtPT4 is retained in the ER. The MtPT1^S117F mutant protein is retained in the ER and likely in the TGN. During arbuscule development (Lower), newly synthesized MtPT4, MtPT1, MtSTP, and MtPLT, expressed from an arbuscule-specific promoter, are secreted to the periarbuscular membrane (gray dotted line), whereas AtPIP2a is retained in the ER. The mutant protein MtPT4^S115F is retained in the ER and likely in the TGN.

Fig. P1.

(Upper) The arbuscule creates a large interface optimized for nutrient exchange. It is surrounded by the periarbuscular membrane, which contains a distinct complement of proteins. A phosphate transporter protein (tagged with GFP) resides in the periarbuscular membrane; an aquaporin protein (tagged with mCherry) resides in the plasma membrane. Note that in this cell the aquaporin was expressed before arbuscule formation. (Lower) Protein distribution patterns when expressed ectopically (i.e., in an unnatural location) during arbuscule formation. Arrows represent the reorientation of newly synthesized phosphate or carbohydrate transporters toward the arbuscule together with synthesis of the periarbuscular membrane. In contrast, the aquaporin is retained in the ER, and a mutated MtPT4 stays in a cell organelle called the “trans-Golgi network” (TGN). N, nucleus.