Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants Is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3

Analyses of phosphate and ammonium transporter mutants reveal differential requirements for these proteins during arbuscular mycorrhizal symbiosis depending on the plant nutrient status.

Abstract

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.

INTRODUCTION

Most vascular flowering plants are capable of forming symbioses with obligate biotrophic fungi of the phylum Glomeromycota, generally referred to as arbuscular mycorrhizal (AM) fungi (Lanfranco and Young, 2012). These mutualistic associations develop in the plant roots where the AM fungi colonize the cortex to access carbon from the plant. In addition, the fungi develop an extraradical mycelium that captures mineral nutrients from the soil. Subsequently, some of these nutrients are transferred to the plant (Smith and Read, 2008).

Phosphorus (P) and nitrogen (N), two major mineral nutrients required by plants, can both be acquired through the symbiosis (reviewed in Smith and Smith, 2011). AM fungi can transfer substantial amounts of P, as phosphate (Pi), to their plant hosts and as Pi is relatively immobile in the soil, this usually has a major impact on plant growth (Sanders and Tinker, 1971). The effects of N delivery through the symbiosis are less apparent, likely because nitrate and ammonium ions are mobile in the soil and therefore more readily accessible to the root. However, tracer studies demonstrate that N transfer between fungal symbionts and their hosts does occur (Johansen et al., 1993; Hodge et al., 2001) and in certain circumstances the quantities can be significant (Tanaka and Yano, 2005; Leigh et al., 2009; Hodge and Fitter, 2010). The symbiosis is also regulated in response to these mineral nutrients, and colonization levels decrease when shoot P or N levels are high (Koide and Li, 1990; Blanke et al., 2005; Breuillin et al., 2010).

During AM symbiosis, mineral nutrients are delivered to the plant through arbuscules, highly branched hyphae that develop in the cortical cells. Arbuscules are surrounded by the periarbuscular membrane, which is continuous with the plasma membrane of the root cell but is 4- to 10-fold larger in surface area; thus, the arbuscule and periarbuscular membrane provide an extensive interface for nutrient exchange (reviewed in Harrison, 2012). Despite their beneficial function, arbuscules are terminally differentiated structures with a relatively short life span and they degenerate after ∼4 to 8 d depending on the species involved (Alexander et al., 1988, 1989). In an active symbiosis, development of new arbuscules and degeneration of old arbuscules occur continuously in the root cortex.

Symbiotic P and N transfer begins in the extraradical hyphae where uptake from the soil is followed by long-distance transfer to the arbuscules. The current models suggest that Pi and N move as polyphosphate (polyP) and arginine, respectively, and that translocation through the hyphae occurs in vacuoles (Cox et al., 1980; Govindarajulu et al., 2005; Cruz et al., 2007; Hijikata et al., 2010). In yeast, polyP and arginine are physically associated and a similar suggestion has been made for AM fungi (Durr et al., 1979; Govindarajulu et al., 2005). Once the polyP and arginine reach the arbuscule, they are catabolized. Pi and ammonium are released from the arbuscule to the periarbuscular apoplast where they can be accessed by plant transporters on the periarbuscular membrane (Ezawa et al., 2001; Govindarajulu et al., 2005; Cruz et al., 2007). Currently, transporters or channels involved in these efflux processes are unknown.

Symbiosis-inducible ammonium transporters of the AMT2 family have been identified in several plant species (Gomez et al., 2009; Guether et al., 2009; Kobae et al., 2010; Hong et al., 2012; Ruzicka et al., 2012; Koegel et al., 2013; Pérez-Tienda et al., 2014). The Lotus japonicus and sorghum (Sorghum bicolor) transporters Lj-AMT2;2, Sb-AMT3;1, and Sb-AMT4 each complement growth of a yeast ammonium transporter mutant, and Lj-AMT2;2 showed a K_m for ammonium of 0.83 mM when expressed in Xenopus laevis oocytes (Guether et al., 2009; Koegel et al., 2013). Two ammonium transporters, soybean (Glycine max) AMT4;1, and S. bicolor AMT3;1, were localized in the periarbuscular membrane (Kobae et al., 2010; Koegel et al., 2013). Given these data, it seems very likely that these transporters function in ammonium transport across the periarbuscular membrane; however, the existence of multiple paralogs may complicate functional analyses.

Several phosphate transporter genes are induced transcriptionally during symbiosis (reviewed in Karandashov and Bucher, 2005; Harrison et al., 2010; Yang and Paszkowski, 2011), and members of the PHOSPHATE TRANSPORTER1 (PHT1) subfamily 1 have prominent roles in symbiosis (Javot et al., 2007; Yang et al., 2012; Xie et al., 2013). PHT1 subfamily 1 transporters PT4 of Medicago truncatula, PT11 of rice (Oryza sativa), and PT4 of Astragalus sinicus are localized in the periarbuscular membrane (Harrison et al., 2002; Paszkowski et al., 2002; Kobae and Hata, 2010; Xie et al., 2013), and the respective mutants fail to show increases in shoot Pi content or shoot growth during symbiosis (Javot et al., 2007; Yang et al., 2012; Xie et al., 2013). Furthermore, in os-pt11, tracer studies confirmed that Pi was not delivered via the fungus during symbiosis (Yang et al., 2012). Loss of transporter function also affected survival of the fungus, and mt-pt4, os-pt11, and As-PT4 RNA interference (RNAi) roots each contained a high proportion of degenerate arbuscules (Javot et al., 2007; Yang et al., 2012; Xie et al., 2013). In mt-pt4, arbuscule development occurred normally but degeneration was initiated prematurely within 48 h of fungal entry to the root. Premature arbuscule degeneration (PAD) resulted in the failure of the fungus to become established in the root and loss of symbiosis (Javot et al., 2007). These data indicate a causal relationship between loss of transporter function and arbuscule degeneration and support the hypothesis that symbiotic Pi transport is essential for maintenance of the symbiosis.

The mechanism underlying PAD is unknown, and initially it was proposed that PAD might result from a reduction in carbon supply arising from a failure in symbiotic Pi transfer. However, PAD still occurs when M. truncatula pt4 mutants are colonized by hyphae arising from a productive association with a nurse plant (Javot et al., 2011), which argues against a carbon starvation phenomenon. Although PAD cannot be suppressed with an external carbon supply, it is strongly influenced by the N status of the plant; PAD can be fully suppressed if the pt4 mutants are starved for N (Javot et al., 2011). Suppression occurs only in response to N starvation and not to S or K starvation (Javot et al., 2011). Here, we continue to investigate the connection between symbiotic nutrient transport, arbuscule life span, and maintenance of AM symbiosis. Analyses of double and triple Pi and ammonium transporter mutants reveal a role for an AMT2 family protein in arbuscule maintenance in pt4.

RESULTS

Identification of PT8, a Second AM Symbiosis-Inducible Pi Transporter

The observation that PAD in pt4 occurs only if the plants are grown with sufficient N was unexpected (Javot et al., 2011). If the hypothesis that symbiotic Pi transport is essential for maintenance of the symbiosis is true, then in pt4 mutants, another Pi transporter(s) should compensate for PT4 during growth in low N conditions. To identify additional Pi transporters, we performed in silico searches of the M. truncatula genome. These revealed a total of 13 Pi transporter genes (Supplemental Figure 1), seven of which (PT1-PT6 and PT9) have been reported previously (Liu et al., 1998, 2008; Chiou et al., 2001; Harrison et al., 2002; Xiao et al., 2006; Grunwald et al., 2009; Floss et al., 2013). The genes that were not reported previously included PT7, PT8, and PT10-13 (official nomenclature MEDtr;Pht1;7, MEDtr;Pht1;8, and MEDtr;Pht1;10, MEDtr;Pht1;11, MEDtr;Pht1;12, and MEDtr;Pht1;13). Of these 13 transporter genes, only PT4 and PT8 are induced in mycorrhizal roots (Figure 1A). Phylogenetic analysis showed that PT8 is a member of subfamily I and is an ortholog of A. sinicus PT1 (Xie et al., 2013) and of soybean Glyma14g36650, which has two names, Gm-PT7 (Tamura et al., 2012) and Gm-PT10 (Qin et al., 2012).

Figure 1.

Expression and Localization of PT8.

(A) Quantitative RT-PCR analysis showing the transcript levels of PT8 and PT4 relative to EF1-α in roots of M. truncatula A17 at 5 wpi with G. versiforme. Data are the means of three biological replicates ± se. Different letters indicate significant differences at (α = 0.01).

(B) Growth curves of the yeast phosphate transporter mutant strain PAM2 transformed with an empty vector (pWV3) or PT4 and PT8. The OD of three biological replicates was measured at 0 h, 10 h, 13 h 45 min, 17 h, 21 h 45 min, 32 h 45 min, 38 h, and 48 h.

(C) M. truncatula roots expressing MtPT8_pro:MtPT8-GFP colonized with G. versiforme. Upper panels: GFP signal from MtPT8_pro:MtPT8-GFP is observed in cells with arbuscules (arrow); the signal is weak or absent in cells with collapsed arbuscules (CA). Lower panels: A single cortical cell harboring an arbuscule displays PT8-GFP signal in the branch domain of the periarbuscular membrane. Left panel, GFP fluorescence obtained by confocal microscopy; middle panel, bright-field differential interference contrast; right panel, overlay. Upper panels, seven 1.22-μm optical sections; lower panels, seven 0.35-μm optical sections. Bars = 20 μm.

Based on its AM symbiosis-induced expression pattern, PT8 was the most promising candidate for a role in symbiotic Pi transport and was selected for further analysis. Expression of PT8 in the yeast Pi transport-defective strain PAM2 complemented the growth defect in PAM2, and PAM2/PT8 showed growth rates comparable to PAM2/PT4, suggesting that PT8 functions as a Pi transporter (Figure 1B). When expressed under the control of its native promoter, a PT8-GFP (green fluorescent protein) fusion was detected exclusively in cortical cells harboring arbuscules (Figure 1C; Supplemental Figures 2A and 2B) and localized in the periarbuscular membrane as observed previously for PT4. To evaluate the role of PT8 during AM symbiosis, a M. truncatula line harboring a transposon in the PT8 coding sequence was identified from the M. truncatula Tnt1 insertion mutant population. The 5.3-kb retrotransposon was inserted 657 nucleotides downstream of the first ATG (Supplemental Figure 3) and is predicted to result in a truncated protein with only 5 of the 12 membrane spanning domains. Following inoculation with Glomus intraradices, colonization levels and arbuscule morphology in pt8-1 did not differ from that of the wild type (Supplemental Figures 2C and 2D). Furthermore, arbuscule populations, as assessed by monitoring arbuscule length at 4, 6, and 8 d post-contact with primed spores (Lopez-Meyer and Harrison, 2006), did not differ significantly in pt8-1 and the wild type, illustrating that arbuscule development and arbuscule life span do not differ in these two lines (Supplemental Figure 2E). Thus, in contrast to PT4, loss of PT8 function does not result in PAD. Consistent with the appearance of a wild-type symbiosis, pt8-1 plants showed a similar benefit from the AM symbiosis as wild-type plants with an increase in shoot mass and shoot Pi content (Supplemental Figures 2F and 2G).

PT8 Is Not Required for the Suppression of PAD in pt4 in Low-N Conditions

To determine if PT8 was required for suppression of PAD in pt4 mutants, we created two independent pt4 pt8 double mutants. The first double mutant, pt4-2 pt8-1, results from a cross between the null allele, pt4-2 (A17 background), and pt8-1 (R108 background). The second double mutant, pt4-5 pt8-1, results from a cross between a null insertion allele of PT4, named pt4-5 (R108 background) (Supplemental Figure 3) and pt8-1 (R108 background). The phenotypes of the two double mutants were compared with the respective single mutants and with the two corresponding wild-type parents. When inoculated with Glomus versiforme, both of the double Pi transporter mutants displayed mycorrhizal phenotypes not significantly different from that of pt4. During growth in conditions with high N, the PAD phenotype, typified by a low percentage of large mature arbuscules and a high percentage of small arbuscules with septa, was apparent in both single pt4 alleles and in both pt4 pt8 double mutants (Figures 2A and 2B; Supplemental Figure 4A). During growth in low-N conditions, PAD was suppressed in both pt4 single mutants and also in the double Pi transporter mutants. The percentages of arbuscules in the 45- to 75-μm size class in the single pt4 mutants and double Pi transporter mutants were not significantly different from those in pt8-1 or in the wild-type parents (Figures 2C to 2E). Minor differences in the proportion of arbuscules in the small and mid-size arbuscule classes were observed in pt4-5 and pt4-5 pt8-1 double mutant but not in pt4-2 or the pt4-2 pt8-1 double mutant (Figures 2C and 2D). This may result from differences in timing for full suppression of PAD in the two ecotypes, likely resulting from differences in their responses to nutrient deprivation. The R108 ecotype shows symptoms of P and N starvation more readily than A17 and accumulates less Pi than the A17 ecotype. Because of the differential mineral nutrient sensitivity, the conditions to achieve N deprivation in the two ecotypes were slightly different (see Methods). In both ecotypes, low-N conditions led to higher shoot P levels (Supplemental Tables 1 and 2), which is consistent with the suppressive effect of N on shoot Pi accumulation reported for Arabidopsis thaliana (Kant et al., 2011). In both high-N and low-N conditions, the N content of the double mutants tended to be higher than the single mutants or wild-type plants. Otherwise, with respect to shoot weight and P content, the double mutants did not differ from the respective single mutants or their wild-type parents.

Figure 2.

Arbuscule Development in Pi Transporter Single and Double Mutants during Growth in High-N and Low-N Conditions.

(A) Arbuscule size distribution in arbuscule populations in A17 (wild type), pt4-2, and pt4-2 pt8-1 at 4 wpi with G. versiforme grown with high-N (15 mM) fertilization. Data represent the mean ± se of arbuscules from random infection units (IUs) from A17 (67 IUs), pt4-2 (54 IUs), and pt4-2 pt8-1 (38 IUs).

(B) Arbuscule size distribution in arbuscule populations in R108 (wild type), pt8-1 pt4-5, and pt4-5 pt8-1 at 4 wpi with G. versiforme grown with high-N (15 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from R108 (49 IUs), pt8-1 (49 IUs), pt4-5 (76 IUs), and pt4-5 pt8-1 (63 IUs).

(C) Arbuscule size distribution in arbuscule populations in A17 (wild type), pt4-2, and pt4-2 pt8-1 at 4 wpi with G. versiforme grown with low-N (1.5 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from A17 (61 IUs), pt4-2 (44 IUs), and pt4-2 pt8-1 (51 IUs).

(D) Arbuscule size distribution in arbuscule populations in R108 (wild type), pt8-1, pt4-5, and pt4-5 pt8-1 at 4 wpi with G. versiforme grown with high-N (15 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from R108 (83 IUs), pt8-1 (82 IUs), pt4-5 (94 IUs), and pt4-5 pt8-1 (65 IUs).

In (A) to (D), different letters indicate significant differences (α = 0.01).

(E) Fluorescence microscopy images of G. versiforme IUs in R108 (wild type), pt8-1, pt4-5, and pt4-5 pt8-1 at 4 wpi grown with low-N (1.5 mM) fertilization. Arrows indicate mature arbuscules. App indicates appressorium. Bars = 100 μm.

PAD and low-N suppression of PAD were also observed following colonization of the mutant with G. intraradices (Supplemental Figure 4B). Overall, the data indicate that the PAD phenotype in pt4 grown in high-N conditions is independent of PT8; furthermore, PT8 function is not required for suppression of PAD in low-N conditions. Current analyses suggest that PT4 and PT8 are the only Pi transporters present in the periarbuscular membrane in M. truncatula (Liu et al., 2008). Consequently, it appears that symbiotic Pi transport across the periarbuscular membrane is not required for arbuscule survival when the plants have a low-N status.

Suppression of PAD in pt4 Mutants Requires AMT2 Family Protein, AMT2;3 but Not AMT2;4

Given our findings that suppression of PAD occurs only in N-deprived pt4 and does not require PT8, we considered the possibility that symbiotic N transfer was required for suppression of PAD in pt4. The current data suggest that ammonium is the form of N transferred from the fungus to the plant (Govindarajulu et al., 2005; Cruz et al., 2007), and in several plants species, ammonium transporters of the AMT2 family are induced during AM symbiosis and are located in the periarbuscular membrane (Gomez et al., 2009; Guether et al., 2009; Kobae et al., 2010; Koegel et al., 2013). In M. truncatula, we identified six genes in the AMT2 family, four of which were reported recently (Straub et al., 2014); therefore, we follow their nomenclature. Our phylogenetic analysis revealed two distinct clades in the AMT2 family, the first clade contains AMT2;3, AMT2;4, and AMT2;5, while the second contains AMT2;1, AMT2;2, and AMT2;6 (Figure 3A). The single Arabidopsis AMT2 gene clusters with the second clade. AMT2;3, AMT2;4, and AMT2;5 transcripts are low or absent in noncolonized roots and increase significantly during AM symbiosis. In all three cases, transcript levels correlate with colonization and arbuscule levels as assessed by G. versiforme α-tubulin and PT4 transcript levels, respectively (Figure 3B). However, the relative abundance of the individual AMT2 transcripts differs substantially. For example, AMT2;3 transcripts are 30-fold less abundant than AMT2;5 transcripts (Figure 3B). In contrast, members of the second clade, AMT2;1, AMT2;2, and AMT2;6, are constitutively expressed and their transcripts are relatively abundant and do not change substantially during symbiosis. Previously, we showed that AMT2;3 transcripts were present in cells with arbuscules (Gomez et al., 2009); consequently, we focused initially on this member of the AMT2 family. To extend this initial analysis, we examined the expression patterns and location of the AMT2;3 protein. Surprisingly, noncolonized M. truncatula roots expressing an AMT2;3 promoter-GFP fusion construct showed GFP fluorescence in the cortex and endodermis (Figure 4A), although transcripts were barely detected in roots prior to symbiosis (Figure 3B). Following colonization, GFP fluorescence was restricted to cells containing arbuscules, which suggests changes in promoter activity during symbiosis (Figure 4B).

Figure 3.

Phylogeny of the M. truncatula AMT2 Family and Expression during AM Symbiosis.

(A) The approximately maximum likelihood phylogenetic tree was generated using the predicted full-length amino acid sequences of AMT2 transporters from Arabidopsis, tomato (Solanum lycopersicum), rice, sorghum, L. japonicus, soybean, and M. truncatula. Each branch division shows local support values with the Shimodaira-Hasegawa test (Price et al., 2010).

(B) Transcript levels of AMT2;1 to AMT2;6 genes in A17 roots either mock-inoculated (white bars) or colonized by G. versiforme (gray bars) at 2, 3, 4, and 5 wpp assayed by quantitative RT-PCR. Transcript levels of G. versiforme α-tubulin and PT4 provide an indication of the colonization level and arbuscule abundance respectively. Data are averages ± se (n ≥ 3 where n denotes the number of independent root samples). Significant differences (Student’s t test) between G. versiforme colonized roots and corresponding mock-inoculated control samples are indicated (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001). For AMT2;1, the 5 wpp control sample, n = 2 and, therefore, statistical analyses were not conducted for this sample.

Figure 4.

AMT2;3 Expression, Localization of AMT2;3-GFP, and Arbuscule Populations in amt2;3.

(A) and (B) M. truncatula roots expressing AMT2;3_pro:GFP. Control roots (A) and roots colonized with G. versiforme (B). Images are single optical sections and show GFP fluorescence overlayed on the corresponding differential interference contrast image. Cortex (c), endodermis (e), and cells containing arbuscules (a). Bars = 100 μm.

(C) and (D) Single cells from M. truncatula roots expressing AMT2;3_pro:AMT2;3-GFP and AMT2;3 _pro:GFP-AMT2;3 colonized with G. versiforme. Images are single optical sections and show GFP fluorescence overlayed on the corresponding differential interference contrast image. Arrow points to fluorescence associated with an arbuscule branch. Arrowhead points to fluorescence associated with the vacuole. The fluorescence patterns obtained with the two constructs did not differ significantly. Bars = 10 μm.

(E) Arbuscule size distribution in arbuscule populations in R108 (wild type) and amt2;3 at 4 wpi with G. versiforme grown with 15 mM (FS1), 7.5 mM (FS2), and 1.5 mM (FS3) N fertilization. Data represent the mean ± se of arbuscules from random infection units from R108 (28 IUs) and amt2;3 (26 IUs) at 15 mM N, R108 (12 IUs) and amt2;3 (25 IUs) at 7.5 mM N, and R108 (28 IUs) and amt2;3 (33 IUs) at 1.5 mM N. Different letters indicate significant differences (α = 0.01).

To examine the location of the AMT2;3 protein, N-terminal and C-terminal AMT2;3-GFP fusions expressed from the AMT2;3 promoter were analyzed. In both cases, we did not detect GFP fluorescence in control roots; however, Straub et al. (2014) reported weak, diffuse fluorescence from an AMT2;3-GFP fusion in control roots. In colonized roots, our analyses revealed that the GFP signal was extremely weak and was either associated with the arbuscule branches (Figure 4C) or visible as a diffuse haze (Figure 4D). Based on these data and the fact that other symbiosis-induced AMT2 family proteins have been localized to the periarbuscular membrane (Kobae et al., 2010; Koegel et al., 2013), we make a tentative conclusion that AMT2;3 is located in the periarbuscular membrane. The relatively high frequency of cells showing a diffuse, hazy signal suggests that the protein fusions are relatively unstable. Whether this is an inherent property of AMT2;3 or an effect of the GFP fusion is unclear. Stability issues were not reported for the AMT2-GFP fusions studied previously in soybean (Kobae et al., 2010; Koegel et al., 2013).

A M. truncatula line with a Tnt1 insertion positioned 465 bp downstream of the first ATG of the AMT2;3 gene was obtained from the M. truncatula Tnt1 insertion mutant population (Supplemental Figure 3). This insertion truncates the protein at amino acid 155, resulting in a protein with less than four transmembrane domains. The mutant was backcrossed to R108, and the mycorrhizal phenotype was evaluated in the BC1F3 generation. There were no apparent differences in the morphology of the symbiosis in amt2;3 roots relative to the R108 wild-type parent or to a wild-type segregant plant selected from the backcrossed amt2;3 F2 population. The number of infection units and numbers of arbuscules in amt2;3 and R108 roots were assessed during growth at three levels of N fertilization and no significant differences were observed (Table 1). Additionally, the structure of the arbuscule populations in amt2;3 did not differ from those in R108, and there was no evidence of a PAD phenotype in amt2;3 (Figure 4E; Supplemental Figures 5A to 5C) even in low N, high Pi conditions (Supplemental Figure 5D).

Table 1. Colonization of amt2;3 during Growth with Differing Levels of N Fertilization.

N Treatment	Genotype	No. of IUs (3 wpi)	No. of IUs (4 wpi)	No. of Arbuscules per Unit Area (3 wpi)	No. of Arbuscules per Unit Area (4 wpi)
15 mM	R108	20.33 ± 2.33a	68.00 ± 9.54a	5.22 ± 0.46a	4.39 ± 0.48a
	amt2;3	22.33 ± 3.93a	27.00 ± 3.51a	4.16 ± 0.46a	3.85 ± 0.46a
7.5 mM	R108	14.00 ± 2.65a’	37.67 ± 9.24a’	5.10 ± 0.54a’	4.67 ± 0.48a’
	amt2;3	20.00 ± 2.65a’	31.00 ± 2.00a’	4.92 ± 0.62a’	4.84 ± 0.58a’
1.5 mM	R108	25.00 ± 3.06a’’	30.67 ± 5.84a’’	6.87 ± 0.64a’’	3.93 ± 0.49a’’
	amt2;3	28.33 ± 2.33a’’	31.67 ± 3.28a’’	3.73 ± 0.60b’’	3.52 ± 0.37a’’

Infection unit number in R108 (wild type) and amt2;3 at 3 and 4 wpi with G. versiforme during fertilization with 15 mM N (FS1), 7.5 mM N (FS2), or 1.5 mM N (FS3) fertilizers. Data represent the mean ± se of three independent replicates. Different letters indicate significant differences between genotypes for a given treatment or parameter (α = 0.01).

From crosses of amt2;3 with pt4-2 and with pt4-5 pt8-1, we obtained two independent Pi transporters, ammonium transporter double mutants, pt4-2 amt2;3 and pt4-5 amt2;3, and a triple mutant, pt4-5 pt8-1 amt2;3. When grown in high-N conditions, the pt4-2 amt2;3 and pt4-5 amt2;3 double mutants and the Pi transporter single mutants pt4-2 and pt4-5 colonized with G. versiforme all showed PAD (Figures 5A and 5B; Supplemental Figure 6 and Supplemental Table 3), while arbuscule populations in the amt2;3 single mutant did not differ from those in the wild type. Consequently, we conclude that in high-N conditions, the pt4 PAD phenotype is independent of amt2;3. Surprisingly, during growth in low-N conditions, both double mutants also showed a typical PAD phenotype and the arbuscule populations differed significantly from those in the pt4 mutants, the amt2;3 mutant, and both wild-type lines (Figures 5C, 5D, and 5F; Supplemental Figure 7 and Supplemental Table 4). Thus, in low-N conditions, suppression of PAD in pt4 mutants is dependent on AMT2;3. Furthermore, analysis of the pt4-5 pt8-1 amt2;3 triple mutant further supports this conclusion; in low-N growth conditions, PAD was not suppressed in the triple mutant (Figure 5E; Supplemental Figure 8 and Supplemental Tables 5 to 7). Under the conditions of these experiments, there were no consistent differences in the shoot mass, shoot phosphorus content, and shoot N content of the wild type, single, and double mutant plants within the low- and high-N conditions (Supplemental Table 4). However in low-N conditions, infection unit length in the triple mutants was lower than that of the respective wild-type control, which is consistent with the PAD phenotype (Supplemental Table 7). Together, these data demonstrate that AMT2;3 function is required for arbuscule survival in pt4 mutants during growth in low-N conditions. These results were somewhat surprising given that AMT2;3 is one of three closely related AMT2 genes induced during symbiosis and that AMT2;3 transcript levels are significantly less abundant than those of AMT2;4 or AMT2;5. Insertions mutants in AMT2;5 were unavailable, but we were able to obtain an insertion mutant in AMT2;4. The transposon is inserted in the first exon after nucleotide 388 and is predicted to result in loss of function (Supplemental Figure 3). As observed for amt2;3, the amt2;4 mutant did not show any apparent defects in AM symbiosis, and its arbuscule population structure was similar to the wild type (Figure 6A). To establish whether AMT2;4 function influenced PAD or suppression of PAD, we generated a pt4-5 amt2;4 double mutant. In high N conditions, pt4-5 amt2;4 shows a typical PAD phenotype (Figure 6A), and in low-N conditions, PAD is suppressed as in the pt4-5 mutant (Figure 6B). These results were confirmed in an additional experiment in which both the pt4-5 amt2;3 and pt4-5 amt2;4 double mutants were grown in parallel (Supplemental Figure 9). Consequently, AMT2;3 but not AMT2;4 is required for arbuscule survival in pt4 in low-N conditions. This unexpected result prompted us to test the ability of AMT2;3 and AMT2;4 to complement growth of a yeast high-affinity ammonium transporter mutant strain, 31019b, referred to as the triple mep strain (Marini et al., 1997). This strain has been used to evaluate the ammonium transport activities of several plant AMT proteins (Straub et al., 2014), including symbiosis-induced ammonium transporters (Guether et al., 2009; Koegel et al., 2013) as well as fungal transporters such as G. intraradices (Gint)-AMT1 (López-Pedrosa et al., 2006). AMT2;3 and AMT2;4 were cloned into pYEUra3 under control of the GAL1 promoter, and as expected, triple mep transformants carrying the pYEUra3 empty vector, pYEUra3-MtAMT2;3, and pYEUra3-MtAMT2;4 all showed growth on medium containing 5 mM ammonium sulfate with galactose as a carbon source. However, only AMT2;4 complemented growth of the triple mep strain in low-N conditions with either 2 or 0.5 mM ammonium sulfate and galactose as a carbon source (Figure 7). To evaluate protein accumulation in yeast, we introduced GFP-MtAMT2;3 and GFP-AMT2;4 fusions into the triple mep stain; however, in both cases, the transformants showed only background autofluorescence and the fusion proteins were not detectable (Supplemental Figure 10). Analysis of AMT2;3 and AMT2;4 transcripts in the respective mep mutant transformants confirmed that both genes were expressed (Supplemental Figure 10).

Figure 5.

Arbuscule Development in pt4-5 and amt2;3 Single, Double, and Triple Mutants during Growth in High-N and Low-N Conditions.

(A) Arbuscule size distribution in arbuscule populations in A17 (wild type), pt4-2, and pt4-2 amt2;3 at 4 wpi with G. versiforme grown with high-N (15 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from A17 (76 IUs), pt4-2 (78 IUs), and pt4-2 amt2;3 (45 IUs).

(B) Arbuscule size distribution in arbuscule populations in R108 (wild type), amt2;3, pt4-5, and pt4-5 amt2;3 at 4 wpi with G. versiforme grown with high-N (15 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from R108 (65 IUs), amt2;3 (63 IUs), pt4-5 (34 IUs), and pt4-5 amt2;3 (26 IUs).

(C) Arbuscule size distribution in arbuscule populations in A17 (wild type), pt4-2, and pt4-2 amt2;3 at 4 wpi with G. versiforme grown with low-N (1.5 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from A17 (60 IUs), pt4-2 (86 IUs), and pt4-2 amt2;3 (80 IUs).

(D) Arbuscule size distribution in arbuscule populations in R108 (wild type), amt2;3, pt4-5, and pt4-5 amt2;3 at 4 wpi with G. versiforme grown with low-N (1.5 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from R108 (82 IUs), amt2;3 (22 IUs), pt4-5 (60 IUs), and pt4-5 amt2;3 (28 IUs). Different letters indicate significant differences (α = 0.02).

(E) Arbuscule size distribution in arbuscule populations in a wild-type segregant from the triple mutant population (WTseg) and pt4-5 pt8-1 amt2;3 at 4 wpi with G. versiforme grown with low-N (1.5 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs WTseg (29 IUs) and pt4-5 pt8-1 amt2;3 (32 IUs). In (A) to (C) and (E), different letters indicate significant differences (α = 0.01).

(F) Fluorescence microscopy images of G. versiforme infection units in A17 (wild type), pt4-2, amt2;3, pt4-2 amt2;3, and pt4-5 amt2;3 at 4 wpi with low-N (1.5 mM) fertilization. Arrows indicate mature arbuscules. Arrowheads indicate degenerate arbuscules. App indicates appressorium. Bars = 100 μm.

Figure 6.

Arbuscule Development in pt4-5 and amt2;4 Single and Double Mutants during Growth in High-N and Low-N Conditions.

(A) Arbuscule size distribution in arbuscule populations in R108 (wild type), amt2;4 pt4-5, and pt4-5 amt2;4 at 5 wpi with G. versiforme grown with high-N (15 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from R108 (14 IUs), amt2;4 (14 IUs), pt4-5 (9 IUs), and pt4-5 amt2;4 (12 IUs).

(B) Arbuscule size distribution in arbuscule populations in R108 (wild type), amt2;4, pt4-5, and pt4-5 amt2;4 at 5 wpi with G. versiforme grown with low-N (1.5 mM) fertilization. Data represent the mean ± se of arbuscules from random IUs from R108 (21 IUs), amt2;4 (19 IUs), pt4-5 (17 IUs), and pt4-5 amt2;4 (29 IUs). Different letters indicate significant differences (α = 0.01).

Figure 7.

Complementation of a Yeast Ammonium Transporter Mutant by AMT2;4 but Not AMT2;3.

Growth of the yeast 31019b (Δmep1; Δmep2; Δmep3) ammonium transporter triple mutant transformed with pYEUra3 (empty vector), pFL61-GintAMT1 (López-Pedrosa et al., 2006), pYEUra3-MtAMT2;3, and pYEUra3-MtAMT2;4 on YNB medium containing 5 mM (NH₄)₂SO₄, 2 mM (NH₄)₂SO₄, or 0.5 mM (NH₄)₂SO₄ at pH 4.5 with either glucose or galactose as a carbon source. pFL61-GintAMT1 serves as a positive control, and expression of Gint-AMT1 is constitutive (López-Pedrosa et al., 2006). In pYEUra3, the AMT2;3 and AMT2;4 genes are expressed from the GAL1 promoter.

DISCUSSION

Pi influences the AM symbiosis at multiple levels from regulation of the initial signaling to establishment, maintenance, and functioning of the symbiosis. Furthermore, there are both local and systemic effects; the nutritional status of the plant and the surrounding environment, as well as Pi delivered through the symbiosis, all have an impact on symbiotic development but the underlying mechanisms are mostly unknown (Koide and Schreiner, 1992; Breuillin et al., 2010; Hammer et al., 2011; Kiers et al., 2011; Kretzschmar et al., 2012; Yoneyama et al., 2012). Here, we focused on transporters active in the periarbuscular membrane and the influence of their function on arbuscule survival and maintenance of symbiosis.

With the availability of genome sequences and transcriptomes, it has become clear that plants possess at least two PHT1 family Pi transporters whose transcription is increased, sometimes substantially, during AM symbiosis. Furthermore, evolution of symbiosis-regulated Pi transporters has occurred differently among the plant families and variation in the subfamily and in the numbers of symbiosis-regulated transporters is apparent (reviewed in Harrison et al., 2010; Yang and Paszkowski, 2011). Here, we identify PT8, a second Pi transporter induced during AM symbiosis. Like PT4, PT8 shows Pi transport activity when assayed in yeast and the protein is located in the periarbuscular membrane. PT8 is an ortholog of A. sinicus PT1 and of soybean Glyma14g36650, which has two names, Gm-PT7 (Tamura et al., 2012) and Gm-PT10 (Qin et al., 2012). Both A. sinicus PT1 and soybean Glyma14g36650 are induced during AM symbiosis (Qin et al., 2012; Tamura et al., 2012; Xie et al., 2013). RNAi-mediated downregulation of PT1 in A. sinicus resulted in an alteration in AM symbiosis with a high proportion of small degenerate arbuscules (Xie et al., 2013). In contrast, we found that pt8 developed symbioses that did not differ from the wild type in fungal colonization levels, arbuscule morphology, symbiotic Pi accumulation, or growth responses. The difference in phenotype of mt-pt8 and As-PT1 RNAi roots is surprising and suggests that differences in function have arisen even between closely related transporters.

Although pt8 did not show an altered AM symbiosis phenotype, we hypothesized that PT8 function might be required for low-N suppression of PAD in pt4 mutants; however, analysis of pt4 pt8 double mutants indicated that PAD and low-N suppression of PAD are independent of PT8. Currently, the role of PT8 is unclear. Possibly the most striking finding from Pi transporter double mutants is that despite the importance of Pi in the symbiosis, it appears that symbiotic Pi transporters are not necessary for AM symbiosis in M. truncatula if the plants are grown in low-N conditions. In this growth condition, development and maintenance of symbiosis in the double Pi transporter mutant are phenotypically indistinguishable from the wild type grown under the same conditions. As noted previously, under these low-N conditions, wild-type plants do not show an obvious symbiotic growth response likely because N is limiting (Javot et al., 2011). To try to understand the mechanistic basis of these observations, we proposed a model in which Pi delivered to the root cortical cell acts, either directly or indirectly, as a signal that leads to a permissive cellular condition that enables arbuscule survival. The nature of the permissive cellular condition is currently unclear, but two possibilities are carbon release to the fungus or suppression of defenses. There are many examples of rapid cellular changes triggered by mineral nutrients or their primary assimilation products, so the model is not unprecedented (Crawford, 1995; Stitt, 1999; Krouk et al., 2010). Given the suppression of PAD by low-N conditions and the lack of requirement for PT8, we hypothesize that under low-N conditions, N transferred to the cell through the symbiosis also acts as a signal to induce appropriate cellular conditions for arbuscule survival. There is growing evidence for crosstalk between P and N signaling, although currently the underlying mechanisms are unknown (Scheible et al., 2004; Morcuende et al., 2007; Kant et al., 2011). Furthermore, nutritional analysis of AM symbioses suggests that exchange of the most limiting nutrient is maximized (Blanke et al., 2005; Johnson et al., 2010) and reciprocal effects of Pi or N availability on access to carbon have been observed (Kiers et al., 2011; Fellbaum et al., 2012), both of which support aspects of the model. To test aspects of the model, we started to disrupt the putative symbiotic ammonium transporters in the background of the pt4 mutants beginning with AMT2;3 (Gomez et al., 2009).

Mt-AMT2;3 is predicted to be an ortholog of G. max AMT4;4, and both genes are induced during symbiosis (Gomez et al., 2009; Kobae et al., 2010). Surprisingly, the Mt-AMT2;3 promoter was highly active in cortical cells of noncolonized roots. This was unexpected as AMT2;3 transcripts were barely detected prior to symbiosis. However, Straub et al. (2014) showed recently that an AMT2;3-GFP fusion protein could be detected at very low levels in control roots, suggesting that the protein is expressed. During symbiosis, AMT2;3-GFP fusion proteins were detected only in cells with arbuscules, and we provide initial evidence that AMT2;3 is located in the periarbuscular membrane as reported for two other symbiosis-inducible AMT2 family proteins, Gm-AMT4;1 and Sb-AMT3;1 (Kobae et al., 2010; Koegel et al., 2013). A similar finding was reported for S. bicolor; Sb-AMT3;1 transcripts were detected in cortical cells from noncolonized roots and in noncolonized cortical cells from mycorrhizal roots, but the protein was detected only in cells with arbuscules (Koegel et al., 2013).

We were unable to detect differences in arbuscule development or degeneration, or shoot N and P content in amt2;3 relative to its wild-type segregant control. However, AMT2;3 function is required for the low-N suppression of the PAD phenotype in pt4 and pt4 pt8 mutants. The shoot N content of the pt4 amt2;3 double and pt4 pt8 amt2;3 triple mutants was similar to that of the single mutants, which suggests that a failure to suppress PAD is probably not the result of a major change in symbiotic N transport, although a minor change cannot be ruled out. Measuring symbiotic N flow in these double and triple mutants is not feasible because of the severity of the PAD phenotype, which prevents robust development of an extraradical mycelium. Considering our original model, it could be envisaged that low-N suppression of PAD requires a particular level of symbiotic N transfer to the plant and removal of any single AMT2 transporter could potentially reduce symbiotic N flow sufficiently to prevent signaling. However, the finding that a second AM-inducible ammonium transporter, AMT2;4, is not required for PAD argues against this. Alternatively, AMT2;3 may have a specific sensing/signaling function not present in the other AMT transporters. When we tested the ability of AMT2;3 and AMT2;4 to complement a yeast high-affinity transport mutant, only AMT2;4 was able to complement growth in low N media. This suggests that AMT2;3 may have different activities from AMT2;4, although it is also possible that AMT2;3 is unable to function in yeast.

In yeast, the MEP/AMT transporter family contains several carriers that are referred to as transceptors because they sense as well as transport their substrates (Van Nuland et al., 2006; Rubio-Texeira et al., 2010). Similar observations have been made recently in plants. For example, in Arabidopsis, ammonium-induced lateral root branching is stimulated by a local ammonium supply and the effect is not correlated with overall ammonium uptake, suggesting that it is triggered by ammonium sensing rather than nutritional status. The effect is dependent on only one of the six ammonium transporters, AMT1;3 (Lima et al., 2010). Furthermore, in yeast, MEP2 activates signaling in N-starved cells through the protein kinase A pathway, which is also activated by PHO84 transceptor when Pi is resupplied to Pi-starved cells (Giots et al., 2003; Van Nuland et al., 2006). Consequently, we speculate that transport of Pi or ammonium through the respective symbiotic transporters acts not only to deliver nutrients to the root cell but also initiates signaling that enables conditions for maintenance of arbuscules. Furthermore, signaling involves only a subset of the transporter proteins. In this model, the nutritional status of the plant would determine whether symbiotic delivery of P or N would trigger signaling; this would provide flexibility for regulation of symbiosis during growth in conditions of varying fertility such as those seen in natural ecosystems. A direct role for the transporters in the signaling process is currently speculative but provides a framework for future experiments. Analysis of other pt4 ammonium transporter double mutants will help to determine whether suppression of PAD is the result of N nutrition or signaling.

METHODS

Medicago truncatula Mutants

In this study, two different subspecies of M. truncatula were used, M. truncatula ssp truncatula ecotype Jemalong (A17) and M. truncatula ssp tricycla (R108). The pt4-2 mutant allele described previously (Javot et al., 2007, 2011) is in the A17 background. The pt4-5, pt8, amt2;3, and amt2;4 alleles are all in the R108 background and are Tnt1 insertion alleles (Supplemental Figure 3) generated at the Noble Foundation or by Pascal Ratet (CNRS, France). The numbers of the M. truncatula Tnt1 lines are as follows: NF5213 (pt8-1), NF3229 (amt2;3), NF4575 (amt2;4), and TNK222 (pt4-5). The positions of the Tnt1 insertions in the M. truncatula lines are shown in Supplemental Figure 3. The primers used to genotype these lines are shown in Supplemental Table 8. All mutant lines were backcrossed to their respective parents. The following double mutants were generated by crossing the respective single mutants: pt8-1 pt-4-5, pt4-2 pt8-1, pt4-2 amt2;3, and pt4-5 amt2;4. A pt4-5 pt8-1 amt2;3 triple mutant was generated by crossing mtp4-5 mtpt8-1 with mtamt2;3. A wild-type segregant from this population was included as a control in the triple mutant analyses.

Plant Growth Conditions

Plants were grown in sterile Turface (calcined clay). At 15 d old, plants were inoculated with surface-sterilized spores of Glomus versiforme or Glomus intraradices, or mock-inoculated as described previously (Javot et al., 2007, 2011). Briefly, plants were transplanted to new pots or cones that were filled approximately two-thirds full with Turface. The Turface was covered with a 1-cm layer of fine sand. Plants were placed into the pots (or cones) with their roots on the sand, and surface-sterilized spores were pipetted directly onto the roots. The roots were covered with additional Turface, and the plants were maintained in a growth chamber with a 16-h-light (25°C)/8-h-dark (22°C) regime and were fertilized with a modified half-strength Hoagland solution containing 20 μM potassium phosphate unless otherwise described. In the double and triple mutant experiments, plants were inoculated with 300 spores of G. intraradices or 600 spores of G. versiforme. In pot experiments, each genotype had two pots each containing four plants. In cone experiments, each genotype had three cones each containing two plants. In “double cone” experiments (Lopez-Meyer and Harrison, 2006; Javot et al., 2007), each genotype possessed at least three biological replicates.

For the M. truncatula/G. versiforme time course (Figure 3), A17 seedlings were planted into cones containing a 1:1 mixture of gravel:sand with a layer of fine sand and 150 surface-sterilized G. versiforme spores positioned ∼6 cm below the top of the cone. Cones were fertilized twice weekly with half-strength Hoagland solution containing 100 μM potassium phosphate beginning 1 week after planting. Plants were harvested at 2, 3, 4, and 5 weeks post-planting (wpp), and the region of the root system 1 cm above and 1 cm below the sand/spore layer was harvested and frozen for RNA. A small sample was stained to view colonization. Cones harvested at 2 and 3 wpp contained five plants per cone. Cones harvested at 4 and 5 wpp contained four plants per cone. Mock-inoculated control cones received the last washing water from the G. versiforme surface sterilization procedure.

Nutrient Treatments

The high-N and low-N fertilizers were FS1, FS2, and FS3 as described previously (Javot et al., 2011). Prior to inoculation, all plants received FS2 fertilizer. After transplanting and inoculation, differential fertilizer treatments were applied beginning 1 week postinoculation (wpi). Fertilizers were applied twice weekly. Genotypes with an A17 background received FS1 (high-N treatment) or FS3 (low-N treatment) twice weekly for 3 weeks. Genotypes with an R108 background received FS2 for 1 week followed by either FS1 for 2 to 3 weeks (high-N treatment) or FS3 for 2 to 3 weeks (low-N treatment). Plants were harvested 4 to 5 wpi.

The double cone experiments were performed as described (Lopez-Meyer and Harrison, 2006; Javot et al., 2007) with the following modifications. Prior to contact between roots and spores, plants received FS2, twice weekly. Following contact between the roots and primed spores, plants received either FS3 solution (low-N condition) or FS1 (high-N condition). Plants were harvested 9 to 12 d post-contact with the spores.

Analysis of Colonization Parameters

Root colonization was assessed using the gridline intersect method and expressed as percentage of root length colonized (McGonigle et al., 1990). For analysis of amt2;3, the total number of infection units in the root systems was counted (data shown in Table 1).

Arbuscule size distribution in the arbuscule populations was estimated as described previously with minor modifications (Javot et al., 2011). Briefly, roots were stained with WGA-Alexafluor 488, and infection units were selected at random. One end of each infection unit was imaged at different planes (to capture images of all arbuscule) through the root using a 40× dry objective on a Leica DM5500 epifluorescence microscope, and images were captured using a Retiga 2000R color CCD camera. Images were delimited by the last arbuscule present at the end of the infection unit. The length of each arbuscule was measured using QCapture Pro 6.0 software. For measurements of infection unit length, random infection units were visualized with an Olympus SZX-12 stereomicroscope and images captured with a CCD camera. Infection unit length was then measured using ImageJ software.

Statistical Analysis

For the colonization levels, infection unit size, and arbuscule number and shoot weight, data shown are the means ± se. The data were tested using JMP software to verify the assumption that they follow a normal distribution. Then, multiple t tests were performed between the genotypes within a given treatment (Table 1; Supplemental Tables 1 to 7). The total P and total N data are means ± sd because the individual plants were pooled into two replicates of four plants for these analyses.

For analyses of arbuscule populations, arbuscules were placed into size classes based on their lengths and the percentage of arbuscules in each size class was calculated. Size classes were guided by previous analyses (Javot et al., 2007, 2011). For each class, the average and the se are graphed. Statistical analyses were conducted on the raw data, and differences between the genotypes were analyzed using a Generalized Linear Model with a Poisson distribution and a log link. The analysis was followed by contrasts to compare the genotypes within each arbuscule size class. A Bonferroni correction was used to adjust the P values for multiple comparisons (Figures 2A to 2D, 4E, 5A to 5E, and 6; Supplemental Figures 2E, 4B, 5D, 7, 8A, 8B. and 9).

Phosphorous and N Shoot Content

Shoot samples from a single pot (four plants) or single cone (two plants) were pooled, oven-dried for 2 d in a 70°C oven, and ground to a fine powder. The N content of the dried shoot powder was measured at the Cornell University Stable Isotope Laboratory (http://www.cobsil.com/index.php) where the samples were analyzed on an elemental analyzer (Carlo Erba NC2500). Total phosphorous content was determined by a phosphomolybdate colorimetric assay (Ames, 1966).

RNA Extraction, cDNA Synthesis, and Quantitative RT-PCR

Total RNA was extracted from root tissues using TRI reagent (Invitrogen) and treated with RNase-free TurboDNaseI (Ambion) followed by a chloroform purification step. First-strand cDNA was synthesized from 1 μg DNase-treated RNA using SuperScript III reverse transcriptase (Invitrogen) in a 20-μL reaction according to the protocol outlined by Gomez et al. (2009). For quantitative RT-PCR, transcripts were amplified from 2 μL of 10× diluted cDNA in a 10-μL reaction using 0.3 μM of each gene-specific primer (Supplemental Table 8) and SYBR Green PCR master mix in an ABI PRISM 7900 HT sequence detection system (Applied Biosystems). Quantitative RT-PCR was performed in 384-well plates with the following PCR cycling conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and a subsequent standard dissociation protocol to exclude primers generating nonspecific PCR products. The threshold cycle (CT) values were calculated using the automatic CT function available with the SDS 2.4 software (Applied Biosystems) and were normalized to the CT values of the housekeeping gene Mt-EF1α (ΔCT = CT_Mt-EF1α − CT_gene). Relative expression values were calculated (2^ΔCT) and were averaged over at least three biological replicates.

Vector Construction

UidA reporter fusions were created in a modified version of the pCAMBIA2301 vector. The UidA coding sequence was amplified by PCR with a forward primer adding either a 5′ PstI or 5′ HindIII restriction site and a reverse primer that included an endogenous BestEII site. The pCAMBIA2301 vector was digested with BstEII and either PstI or HindIII, releasing 35S:UidA, and the amplified fragment containing UidA but no promoter was ligated in its place. A genomic fragment encompassing 1139 nucleotides upstream of the PT8 ATG was amplified with primers adding 5′ BamHI and 3′ PstI restriction sites and cloned into the modified 5′ PstI:UidA vector creating MtPT8_pro:UidA. The MtPT8 _pro:MtPT8-GFP translational fusion vector was constructed in the background of a modified pCAMBIA2301 vector containing the S65T GFP variant (Chiu et al., 1996) in the multiple cloning site under control of the cauliflower mosaic virus 35S promoter and NOS terminator. A 2810-bp genomic fragment was amplified from M. truncatula A17 DNA corresponding to 1139 bp of sequence upstream of the PT8 ATG and the predicted open reading frame, excluding the stop codon, with primers that add flanking BamHI restriction enzyme sites. The modified pCAMBIA2301 GFP vector was digested with BamHI, which released the 35S promoter. BamHI digestion of the amplified PT8 genomic fragment and ligation into the prepared vector resulted in an in-frame fusion of the 3′ of MtPT8 to the 5′ ATG of GFP. All vectors were sequenced to confirm correct insertions and the absence of introduced mutations.

To create the AMT2;3 promoter GFP fusion, a 2-kb fragment 5′ of the AMT2;3 coding sequence was amplified and inserted between the AgeI and NcoI sites in pSAT6-AFP-C1 (1398). The resulting promoter-GFP construct with terminator was then excised with AgeI and NotI, recessed ends were filled in, and the fragment was inserted into SmaI-digested pCambia 2301. The C-terminal AMT2;3-GFP fusion was created by inserting a 4-kb fragment comprising the AMT2;3 5′ flanking sequence and AMT2;3 gene without the TAA termination codon (2262 bp) between the AgeI and SalI sites of pSAT6-AFP-N1 (1564). The resulting C-terminal fusion was excised with AgeI and NotI, recessed ends were filled in, and the fragment was inserted into SmaI-digested pCambia 2301. The N-terminal GFP-AMT2;3 fusion was created by adding the AMT2;3 cDNA (1446 bp) into the AMT2;3 promoter GFP fusion construct. The AMT2;3 open reading frame was amplified and inserted between the SacI and SalI sites of pSAT6-AFP-C1 (1398) already containing the AMT2;3 promoter GFP fusion. The resulting N-terminal fusion was excised with AgeI and NotI, recessed ends were filled in, and the fragment was inserted into SmaI-digested pCambia 2301.

Vectors were introduced into M. truncatula roots by Agrobacterium rhizogenes transformation as described previously (Boisson-Dernier et al., 2001; Pumplin et al., 2010).

Cloning and Complementation of PAM2, a Yeast Phosphate Transport-Defective Strain, with PT8

For analysis of phosphate transport activity in yeast, the PT8 coding sequence was inserted into the yeast expression vector, pWV3 (Versaw and Harrison, 2002) between the EcoRI and BamHI sites and transformed into the yeast double phosphate transporter mutant, PAM2 (Martinez and Persson, 1998). Growth of PAM2 expressing PT8 was compared with strains expressing PT4 (Harrison et al., 2002) and an empty vector control.

Cloning of AMT2;3 and AMT2;4 into pYEUra3 and Complementation of the Yeast Triple Ammonium Transporter Mutant Strain 31019b

To generate pYEUra3-MtAMT2;3 and pYEUra3-MtAMT2;4, the coding regions of AMT2;3 (1446 bp) and AMT2;4 (1443 bp) were amplified with the corresponding primers (Supplemental Table 8) and cloned into the yeast shuttle vector pWV3 between the BamHI/XhoI and XhoI/EcoRI sites, respectively. Subsequently, the AMT2;3 and AMT2;4 fragments were released by BamHI/XhoI and XhoI/EcoRI digestion respectively, blunt-ended by Klenow fill in, and subsequently ligated to the BamHI linearized and blunt-ended yeast plasmid pYEUra3. The sequences of the coding regions were verified.

The vectors were then transformed into the yeast high-affinity ammonium transporter triple mutant (strain 31019b; ΔΔΔmep1;2;3) (Marini et al., 1997; kindly provide by Nuria Ferrol, CSIC, Spain). Yeast transformants were selected, and single colonies were grown in liquid YNB medium containing 3% galactose for 2 d. A positive control strain, ΔΔΔmep1;2;3::pFL61-GintAMT1 (López-Pedrosa et al., 2006) (kindly provided by Nuria Ferrol, CSIC, Spain), was grown in YNB containing 2% glucose. The cells were harvested, washed, and diluted to an OD₆₀₀ of 0.2. Cell suspensions were spotted in 1- to 4-fold dilutions on YNB medium lacking amino acids at pH 4.5, supplemented with 5, 2, or 0.5 mM (NH₄)₂SO₄ as the sole nitrogen source and containing either glucose or galactose as a carbon source. The cells were then grown at 28°C for up to 6 d.

Generation and Analysis of GFP-AMT2;3 and GFP-AMT2;4 in Yeast

To create N-terminal GFP fusions for expression in yeast, the coding regions of AMT2;3 and AMT2;4 were amplified with the corresponding primers (Supplemental Table 8) and cloned into the CAMV35S-SGFP(S65T)-Nos plasmid between the BsrGI/NotI sites (Chiu et al., 1996). To release the GFP fusions, plasmids carrying GFP-AMT2;3 and GFP-AMT2;4 were digested with SalI/XhoI and BamHI/SalI, respectively, and subsequently ligated either to SalI/XhoI- or BamHI/SalI-digested pYEUra3 plasmid to generate GAL1_pro-GFP-MtAMT2;3 and GAL1_pro-GFP-MtAMT2;4, respectively. The constructs were introduced into the yeast high-affinity ammonium transporter triple mutant (strain 31019b; ΔΔΔmep1;2;3). Yeast transformants were selected and single colonies were grown to induce expression of the fusion proteins as described (Drew et al., 2008). Yeast cells were examined by confocal microscopy as described previously (Liu et al., 2008), and the emission spectra of the cells were evaluated by lambda scanning.

Phylogenetic Analysis

The approximately maximum likelihood phylogenetic trees were generated using the predicted full-length amino acid sequences of phosphate transporters or ammonium transporters (Supplemental Data Sets 1 and 2). Sequences were obtained from Phytozome10 (Arabidopsis thaliana, Oryza sativa, Sorghum bicolor, and Glycine max), Solgenomics (Solanum lycopersicum), Kazusa DNA Research Institute (Lotus japonicus), and JCVI (M. truncatula). Alignments were generated using Muscle v3.8.31 with default values (Supplemental Data Sets 1 and 2). FastTree version 2.1.15 was used with default values to generate the phylogenies using a neighbor joining method to create a starting tree and later refining the topology using maximum likelihood nearest-neighbor interchanges and minimum-evolution subtree-pruning-regrafting. Each branch division shows local support values with the Shimodaira-Hasegawa test (Price et al., 2010).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL libraries under the following accession numbers: PT6 (Medtr3g082700), PT7 (Medtr1g074940), PT8 (Medtr5g068140), PT9 (Medtr4g083960), PT10 (Medtr1g069930), PT11 (Medtr1g069935), PT12 (Medtr7g096870), PT13 (Medtr7g096880), AMT2;1 (Medtr7g069640), AMT2;2 (Medtr8g095040), AMT2;3 (Medtr8g074750), AMT2;4 (Medtr7g115050), AMT2;5 (Medtr1g036410), and AMT2;6 (Medtr4g017820).

Supplemental Data

• Supplemental Figure 1. M. truncatula Pi transporters of the PHT1 family.

• Supplemental Figure 2. Localization of PT8 and symbiosis phenotype of pt8-1.

• Supplemental Figure 3. M. truncatula Tnt1 lines with insertions in PT4, PT8 and AMT2;3 and AMT2;4.

• Supplemental Figure 4. Arbuscule images and arbuscule population structure in a Pi transporter double mutant.

• Supplemental Figure 5. Morphology of AM symbiosis in amt2;3 during growth in varying nitrogen and phosphate regimes.

• Supplemental Figure 6. Morphology of AM symbiosis in pt4-2 amt2;3 and pt4-5 amt2;3.

• Supplemental Figure 7. Arbuscule populations in pt4-2 amt2;3 and pt4-5 amt2;3 at 8 days post-contact with G. intraradices.

• Supplemental Figure 8. Arbuscule populations in a pt4-5 pt8-1 amt2;3 triple mutant.

• Supplemental Figure 9. Arbuscule populations in a pt4-5 amt2;4 double mutant.

• Supplemental Figure 10. Analysis of yeast 31019b (Δmep1; Δmep2; Δmep3) mep cells expressing AMT2;3 and AMT2;4 from the GAL1 promoter.

• Supplemental Table 1. Shoot fresh weight and P and N content of the pt4-2 pt8-1 and pt4-5 pt8-1 Pi transporter double mutants in high-N growth conditions.

• Supplemental Table 2. Shoot fresh weight and P and N content and infection unit length of the pt4-2 pt8-1 and pt4-5 pt8-1 Pi transporter double mutants in low-N growth conditions.

• Supplemental Table 3. Shoot fresh weight and P and N content of the pt4-2 amt2;3 and pt4-5 amt2;3 double mutants in high-N growth conditions.

• Supplemental Table 4. Shoot fresh weight and P and N content of the pt4-2 amt2;3 and pt4-5 amt2;3 double mutants in low-N growth conditions.

• Supplemental Table 5. Shoot fresh weight and P and N content of the pt4-5 pt8-1 amt2;3 triple mutant in low-N growth conditions.

• Supplemental Table 6. Shoot fresh weight and P and N content of the pt4-5 pt8-1 amt2;3 triple mutant, a wild-type segregant, and R108 in high-N growth conditions.

• Supplemental Table 7. Shoot fresh weight and N content of the pt4-5 pt8-1 amt2;3 triple mutant, a wild-type segregant, and R108 in low-N growth conditions.

• Supplemental Table 8. Primer sequences.

• Supplemental Data Set 1. PHT1 family alignment file.

• Supplemental Data Set 2. AMT2 family alignment file.

Glossary

AM

arbuscular mycorrhizal

polyP

polyphosphate

RNAi

RNA interference

PAD

premature arbuscule degeneration

wpp

weeks post-planting

wpi

week postinoculation

CT

threshold cycle

IU

infection unit