Tansley review. Nitrogen and phosphate metabolism in ectomycorrhizas. Review. Uwe Nehls 1 and Claude Plassard 2. Contents. Summary. I.

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1 Review Nitrogen and phosphate metabolism in ectomycorrhizas Author for correspondence: Uwe Nehls Tel: Received: 1 February 2018 Accepted: 1 May 2018 Uwe Nehls 1 and Claude Plassard 2 1 Botany, University of Bremen, Bremen 28359, Germany; 2 Eco & Sols, Universite de Montpellier, INRA, CIRAD, IRD, Montpellier SupAgro, Montpellier 34060, France Contents V. N/P storage and remobilization 3 Summary 1 I. Introduction 1 II. Mobilization of soil N/P by ECM fungi 2 III. N/P uptake 2 VI. Hyphal N/P efflux at the plant fungus interface 6 VII. Conclusion and research needs 8 Acknowledgements 9 References 9 IV. N/P assimilation 3 doi: /nph Key words: Baker s yeast, ectomycorrhizal fungal models, export, long-distance transport, N/P nutrition, nutrient homeostasis, vacuole. Summary Nutrient homeostasis is essential for fungal cells and thus tightly adapted to the local demand in a mycelium with hyphal specialization. Based on selected ectomycorrhizal (ECM) fungal models, we outlined current concepts of nitrogen and phosphate nutrition and their limitations, and included knowledge from Baker s yeast when major gaps had to be filled. We covered the entire pathway from nutrient mobilization, import and local storage, distribution within the mycelium and export at the plant fungus interface. Even when nutrient import and assimilation were broad issues for ECM fungi, we focused mainly on nitrate and organic phosphorus uptake, as other nitrogen/phosphorus (N/P) sources have been covered by recent reviews. Vacuolar N/P storage and mobilization represented another focus point of this review. Vacuoles are integrated into cellular homeostasis and central for an ECM mycelium at two locations: soil-growing hyphae and hyphae of the plant fungus interface. Vacuoles are also involved in long-distance transport. We further discussed potential mechanisms of bidirectional long-distance nutrient transport (distances from millimetres to metres). A final focus of the review was N/P export at the plant fungus interface, where we compared potential efflux mechanisms and pathways, and discussed their prerequisites. I. Introduction Climate, soil age and composition, as well as microbial activity, are central in determining the extent to which nitrogen (N) and phosphorus (P) constrain plant net primary production (Cleveland & Liptzin, 2007; LeBauer & Treseder, 2008; Rosling et al., 2016). In boreal and many temperate forests, N/P stocks are compartmented in organic forms by living cells or bound to the surface of soil particles (Callesen et al., 2007). Many forest trees have limited capacity to utilize complex organic substances for their nutrition (Jonasson & Shaver, 19; Perez-Moreno & Read, 2001). Therefore, saprotrophic decomposers, together with ectomycorrhizal (ECM) fungi, are central for nutrient cycling within these ecosystems. Released N/P sources are taken up in a 1

2 2 Review New Phytologist competitive manner by plant roots and soil organisms. Diffusion and mass flow (powered by the transpiration of host plants) are the main processes governing the movement of nutrients through the soil, and mass flow-driven movement of capillary water clearly modulates the composition of available sources (Oyewole et al., 2017). However, roots of forest trees are tightly associated with ECM fungi, and plants can thus access nutrients via two pathways: direct uptake of water-soluble nutrients from the soil solution and/ or fungus-based nutrient support. Nutrient mobilization from soil resources by ECM fungi (Courty et al., 2010; Rineau et al., 2012; Uroz et al., 2013; Lindahl & Tunlid, 2015) and increased host plant mineral nutrition on formation of ECM symbiosis have been demonstrated by numerous tracer studies (e.g. Melin & Nilsson, 1950, 1952; Finlay et al., 1988; Ek et al., 16). The fungal reward is host-derived carbohydrates (Melin & Nilsson, 1957; Finlay & Read, 1986a; for a recent review, see Nehls et al., 2016). However, as this type of symbiosis is not of monophyletic origin, but established independently in different fungal clades (Hibbett et al., 2000), fungal diversity must be taken into consideration when any aspect of ECM fungal lifestyle is discussed. Distinct saprotrophic capacity (Buee et al., 2007; Courty et al., 2010), mycelial organization and exploration strategies (Agerer, 2001) point towards major differences between ECM fungal species, and explain the variability in observed N/P to carbon exchange ratios (Chalot & Plassard, 2011). II. Mobilization of soil N/P by ECM fungi Even though boreal and temperate forest soils often contain large N stocks (Callesen et al., 2007), biotic processes are frequently limited by N availability (Hyvonen et al., 2008). In non-polluted forest ecosystems, most biodegradable N is found in living and dead organic residues. From this pool, nitrate (as a result of denitrification), ammonium, free amino acids, oligonucleotides, proteins and nucleic acids are released mainly by saprotrophic decomposers, which dominate in freshly produced organic matter, also rich in available carbohydrates and organic P (Talbot et al., 2013). However, apart from nitrate, such water-soluble N sources are usually only locally available because of their immobilization by humic substances (Yu et al., 2002). In particular, in deeper soil layers, where ECM fungi dominate (Talbot et al., 2013), N sources can be released by Fenton reaction-based redox processes (Rineau et al., 2012; Lindahl & Tunlid, 2015; Shah et al., 2016), for which Fe 3+ reduction next to soil organic matter is central. With involutin (Paxillus involutus), a secreted secondary metabolite has been identified that allows a fungus-controlled Fenton reaction (Shah et al., 2015). N release from complex organic sources may also occur by oxidation processes driven by the local generation of Mn 3+ by fungal peroxidases (Keiluweit et al., 2015; Lindahl & Tunlid, 2015; Kyaschenko et al., 2017). The potential of ECM fungi to explore and liberate N from a number of different sources has been extensively described in the literature (Courty et al., 2010; Rineau et al., 2012; Uroz et al., 2013; Lindahl & Tunlid, 2015; Shah et al., 2016) and is not targeted by this review. Next to N, the supply of phosphate from the environment remains a major constraint for many soil-living organisms, especially plants. It is assumed that roots of forest trees can only take up free orthophosphate ions () from the soil solution. However, even in P-rich agricultural soils, the concentration of free is rarely > 10 lm (Bieleski, 1973; Hinsinger, 2001), because easily form complexes or secondary minerals with cations under basic (with calcium) and acidic (with aluminium and iron oxides) conditions (Hinsinger, 2001). One way for organisms to increase availability in soil is therefore to dissolve P-containing minerals (Jones, 18; Plassard & Fransson, 2009; Courty et al., 2010; Cairney, 2011; Plassard et al., 2011). In addition to inorganic P, organic P (Po) compounds, such as phosphate esters (e.g. sugarphosphates, inositol-phosphates) or phosphate diesters (e.g. nucleic acids, phospholipids), can represent a great proportion of the total soil P (reviewed in George et al., 2018). Unlike organic N, the capability of ECM fungi to take up intact mono- or diesters has not been demonstrated so far experimentally. In contrast with ECM fungi, yeast cells are able to take up such compounds (glycerophosphoinositol and glycerophosphocholine), when supplied as sole P source (Patton-Vogt, 2007). Under these conditions, Baker s yeast expresses the glycerophosphoinositol transporter Git1p (Fisher et al., 2005). We have identified Git1p homologues (Supporting Information Table S1) of ECM, orchid, ericoid and arbuscular (Rhizophagus irregularis) fungal species in publically available genome databases (Mycocosm database at indicating the potential of mycorrhizal fungi to use Po directly. Whether such Git1p homologues are capable of importing phosphate diesters, however, remains unproven. III. N/P uptake A number of proton-coupled symporters for a wide range of potential N sources, present in forest soils, have been identified and functionally characterized in ECM fungi. As recent reviews are available (e.g. Lucic et al., 2008; Casieri et al., 2013; Garcia et al., 2016), we particularly focus on the transport of nitrate in this contribution. Nitrate is, in contrast with many other N sources, highly mobile in forest soils. Apart from disturbed areas (e.g. after fire), diffusion-based studies have indicated nitrate to be a minor N source for forest plants in native boreal forests (1 10% of total N). However, when mass flow, driven by plant transpiration, was taken into account experimentally, nitrate was observed to make up a much higher proportion of plant-available N (between 20 and 65%; Oyewole et al., 2017), indicating that nitrate has been underestimated as an N source for tree nutrition. On the other hand, a more or less dense fungal mantle covers tree fine roots in many ECM associations (Agerer, 15). Thus, whether or not nitrate can pass through the mantle to become available directly to the plant host is still a matter of debate. Although growth rates differ remarkably amongst taxa, ECM fungi have a widespread capability to grow on nitrate as sole N source (Nygren et al., 2008), and nitrate importer genes are ubiquitous in their genomes (Jargeat et al., 2003; Lucic et al., 2008; Kohler et al., 2015). However, nitrate is a non-favoured N source because of the high assimilation costs. Consequently, nitrate importer gene expression is repressed in the presence of ammonium (Morton & Macmillan, 1954; Jargeat et al., 2003). Nevertheless, as ammonium is found in soil water in

3 New Phytologist Review 3 only small amounts under mass flow conditions (Oyewole et al., 2017), ammonium-based repression of fungal nitrate importers might be of no significance. Furthermore, strong plant nitrate importer gene expression was observed in poplar Amanita muscaria mycorrhizas after nitrate application (Willmann et al., 2014), suggesting that nitrate reaches root cells. By contrast, the kinetics of nitrate uptake into Rhizopogon roseolus nus pinaster ECM roots indicated nitrate uptake mainly by fungal rather than by plant cells (Gobert & Plassard, 2007), suggesting that both funguscentred and plant-centred nitrate uptake can occur. Further research is needed to elucidate the impact of mass flow-based, direct nitrate utilization by plant hosts in ECM symbioses under natural conditions. Inorganic phosphate is taken up by fungal cells by transporters (Plassard & Dell, 2010; Casieri et al., 2013; Becquer et al., 2014), five of which are found within the genome of Saccharomyces cerevisiae. Two high-affinity transporters, Scpho84 (H + : symporter, Pht1 gene family) and Scpho89 ( : Na + symporter, Pht2 family), and two low-affinity transporters, Scpho87 and Scpho90, are localized in the plasma membrane (Bun-ya et al., 11; Martinez & Persson, 18; H urlimann et al., 2007). Another low-affinity transporter, ScPho91, is located at the tonoplast where it exports from the vacuole into the cytosol (H urlimann et al., 2007; Secco et al., 2012). All low-affinity transporters are members of the divalent anion symporter gene family and harbour an N-terminal SPX domain (Secco et al., 2012). We have identified protein homologues of high- and low-affinity transporters in the genomes ( of all investigated ECM fungal species (Fig. 1; Table S1). Although no low-affinity transporter has been functionally investigated so far, high-affinity H + : transporters have been characterized in a number of mycorrhizal fungal models. Two genes, HcPT1.1 (Garcia et al., 2013) and HcPT2, were identified in the basidiomycete Hebeloma cylindrosporum and were functionally characterized as proton-dependent importers (K m values of 50 lm for HcPT1.1 and 4 lm for HcPT2) by heterologous expression in yeast (Tatry et al., 2009). When mycelia were grown in axenic culture, the two transporters were differentially regulated. Similar to homologues in yeast (Scpho84; Secco et al., 2012) and Glomus versiforme (GvPT; Harrison & van Buuren, 15), HcPT1.1 transcript levels reflected the external availability. HcPT1.1 revealed high expression levels under deficiency, but low levels in -sufficient conditions. By contrast, HcPT2 was only weakly regulated by external availability. Both Hebeloma proteins belong to different phylogenetic subgroups (Fig. 1) and no HcPT2 homologues are present in the genomes of Rhizophagus irregularis, Cenococcum geophilum and A. muscaria (Fig. 1; Table S1). Remarkably, except for Tuber melanosporum (Hacquard et al., 2013; see Table S1), no homologues of the high-affinity : Na + transporter ScPho89 could be identified in the genomes of ECM fungal species (Mycocosm database). By contrast, the genomes of R. irregularis, Meliniomyces bicolor, M. variabilis, Oidodendron maius, Rhizoscyphus ericae, Sebacina vermifera and riformospora indica contain ScPho89 homologues (Fig. 1; Table S1). We therefore conclude that, for most ECM fungi, plasma membrane import depends on proton, but not sodium, symport. This corresponds well with the ecology of ECM species (except truffles), which are usually found in soils with ph values lower than 7. Another explanation might be that sodium/ cotransport results in hyphal sodium accumulation, which can become toxic at elevated concentrations. IV. N/P assimilation In plants and fungi, intracellular ammonium, obtained as a result of either nitrate reduction or the degradation of organic N sources, is assimilated into two key amino acids: glutamate (catalysed by NADP-dependent glutamate dehydrogenase, GDH) and glutamine (catalysed by glutamine synthetase, GS). In the ECM fungal model H. cylindrosporum, both GDH and GS genes are transcribed at low ammonium concentrations. However, high external ammonium concentrations resulted in repressed GDH expression in H. cylindrosporum and GDH was dispensable in many other ECM fungi (Morel et al., 2006), indicating that glutamine synthetase is the most important enzyme for ammonium assimilation. The primary amino acid glutamine further functions as a central N donor (Chalot et al., 14) for cellular N metabolism and storage. Intracellular phosphate is channelled into metabolism after initial assimilation in the form of nucleoside triphosphates (NTPs). However, can also accumulate within the cytosol and the vacuole (see below) to a given extent. As an example, free ranges from 0.4 up to 2.1 mg P g 1 DW in H. cylindrosporum, representing 16 40% of the total P of mycelia grown in pure culture (Rolin et al., 1984). In addition to, phosphate can also accumulate in the form of polyphosphates (polyp) in the cytosol and the vacuole. V. N/P storage and remobilization Balancing of N/P supply and demand locally (Kitamoto et al., 1988), as well as within the entire mycelium, is central for the mycelial lifestyle. Furthermore, strategies differ between extraradical hyphae (nutrient uptake) and hyphae of the functional interface of ectomycorrhizas (nutrient export). Apart from metabolism, the storage of assimilated nutrients is thus a central aspect of cellular homeostasis, and the vacuole is a major storage organ for N/P in fungal mycelia (Shi et al., 2002; Richards et al., 2012). Vacuolar N/ P storage is important for ECM fungi with respect to three different functions: local storage at the hyphal soil margin (for efficient N/P import), long-distance transport throughout the mycelium and export at the plant fungus interface. 1. N/P storage (at the hyphal front of soil mycelia) In yeast, a variety of amino acids are stored in the vacuole. However, glutamate, which is most abundant in the cytosol, is almost absent in the vacuole. Basic amino acids, such as arginine and histidine, are best suited for vacuolar storage, as they contain multiple N atoms per amino acid (4 and 3, respectively) and can function as charge antagonists of polyp. Indeed, N has been found to co-localize with

4 Review New Phytologist Subgroup III High-affinity :Na + transporters ScPho89 homologues Subgroup IV Low-affinity transporters ScPho87, ScPho90, ScPho91 homologues ScPho89 SvPT3 79 SvPT2 MbPT12 RePT12 OmPT13 MvPT15 80 MvPT1 TmPT2 AbPT1 ript4 AfPT2 TcPT9 NcPho4 RiPT4 RiPT3 OmPT10 SvPT ScPho90 ScPho87 TmPT3 CgPT CcPT LaPT6 LbPT8 ScPho91 RiPT5 MbPT13 60 MvPT16 78 RePT HcPT3 SvPT PnPT2 55 ript3 PmPT4 79 LbPT7 71 PtPT3 94 PrPT3 TcPT5 TcPT6 PrPT5 78 ScPT4 63 TcPT7 TcPT4 94 RePT10 PmPT MbPT7 98 MvPT11 56 CgPT6 OmPT12 Subgroup II P-diester transporters ScGit1p homologues ScPT5 PtPT MbPT9 57 RePT11 SvPT5 PT6 88 PT5 SlPT5 PcPT6 33 MvPT14 40 OmPT9 MvPT9 AmPT7 ript TcPT8 MbPT MvPT13 97 LaPT7 HcPT5 7 RePT9 LbPT MbPT MvPT12 OmPT11 ScGIt1p 94 HcPT4 97 LaPT5 PcPT5 72 SlPT4 95 PrPT4 97 LbPT PT RePT8 MbPT10 MvPT HcPT1.1 MvPT8 HcPT1.1(h 7) PnPT1 70 CgPT5 88 AbPT2 73 LaPT1 72 HcPT2(h7) LbPht LaPT2 LbPht15 13 LaPT3 HcPT2 50 LbPht13 LaPT4 PcPT4 16 PcPT2 PcPT3 86 TcPT PcPT1 LbPht11 AmPT2 94 HcPT1.2(h7) AmPT3 96 PmPT1 LbPht12 AmPT PtPT1 PmPT2 AmPT6 95 PT1 80 ScPT3 PrPT1 96 SlPT SlPT PrPT2 SlPT3 92 ScPT2 ScPT1 88 PmPT PtPT ript2 SvPT1 PT2 78 PT3 96 ript1 TcPT1 TcPT AmPT5 AmPT4 GigmPT 98 RiPT2 FmPT DePT RiPT RiPT1 OmPT3 RePT ScPho84 MbPT4 MvPT2 RePT3 OmPT2 OmPT4 MvPT4 82 RePT4 CgPT3 MvPT3 39 MbPT3 RePT1 TmPT1 CgPT2 AfPT1 AtPT-pho84 51 FoPho5 GzPho5 GfPho5 OmPT1 MbPT2 Ncpho5 CgPT1 MbPT1 RePT2 55 MbPT5 MvPT5 SpPT1 SpPT2 MvPT6 OmPT6 OmPT7 OmPT8 OmPT5 MbPT6 RePT6 RePT7 CgPT4 MvPT7 Subgroup I High-affinity H + : transporters Ia: HcPT1 homologues Ib: ScPho84 homologues Ic: HcPT2 homologues Fig. 1 Phylogenetic tree of selected phosphorus (P) transporter classes. The predicted proteins (names and accession numbers are given in Supporting Information Table S1) cluster into four subgroups: high-affinity H + : transporters (subgroup I), P-diester transporters (subgroup II), high-affinity : Na + transporters (subgroup III) and low-affinity transporters (subgroup IV). Protein sequences were retrieved from the Mycocosm database ( jgi.doe.gov/programs/fungi/index.jsf) and aligned using multiple sequence comparison by log-expectation alignment (MUSCLE). Phylogenetic trees (an optimal tree is shown in the figure) were calculated in MEGA7 (Kumar et al., 2016) using the minimum evolution (ME) method (Rzhetsky & Nei, 12). The percentage of replicate trees, in which the associated taxa clustered together in a bootstrap test (500 replicates), is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were calculated using the JTT matrix (Jones et al., 12) and the number of amino acid substitutions per site is shown. The ME tree was identified using the Close- Neighbour-Interchange algorithm (Nei & Kumar, 2000) at a search level of 1. Neighbour joining (Saitou & Nei, 1987) was used to generate the initial tree. The analysis involved 181 amino acid sequences. All ambiguous positions were removed for each sequence pair, leading to a total of 2140 positions in the final dataset. polyp in ECM fungi in symbiosis (Kottke et al., 15; B ucking et al., 18), and N accumulation could correspond to basic amino acids, but this has yet to be demonstrated. Similar to the plasma membrane, the tonoplast is characterized by an electrochemical proton gradient in plants and fungi. However, protons and positive charge are enriched inside the vacuole. As a consequence, proton-coupled amino acid antiporters of the Vacuolar Basic Amino Acid Transporter (VBA) gene family mediate the uptake of basic amino acids into the vacuole in yeast (Russnak et al., 2001; Shimazu et al., 2005; Sekito et al., 2008, 2014). Homologues of these genes are present in the genomes of ECM model fungi (U. Nehls, unpublished), but have not yet been functionally characterized. As a result of their pivotal role in cellular homeostasis, much attention has been devoted to polyp in mycorrhizal fungi. Under controlled laboratory conditions, as well as in the field, the presence of polyp was observed in vacuoles of free-living ECM fungal mycelium and mycorrhizas by a number of different techniques: in vivo nuclear magnetic resonance (NMR) (reviewed by Pfeffer et al., 2001), toluidine blue staining (Lapeyrie et al., 1984; Ashford et al., 14, 19; Torres-Aquino et al., 2017), energy-dispersive X-ray spectroscopy (EDX) (Frey et al., 17; B ucking & Heyser, 2000; Seven & Polle, 2014), particle-induced X-ray emission (PIXE) (Pallon et al., 2007) and electron energy loss spectroscopy (EELS) (Kottke & Martin, 14; Kottke et al., 15; Turnau et al., 16). Cations such as K + or Mg 2+, as well as basic amino acids, colocalized with polyp to compensate its strong negative charge. Furthermore, in metal-polluted environments, otherwise toxic metal cations (e.g. aluminium or cadmium) were found together with polyp in ECM root sections, counteracting polyp charge (Kottke & Martin, 14; Kottke et al., 15; Turnau et al., 16).

5 New Phytologist Review 5 In yeast, vacuolar polyp accumulation is linked to plasma membrane import, as deletion of Scpho84 showed a 50% decrease in polyp levels. This could explain why high external concentrations, which lead to Scpho84 suppression, result in lower polyp levels (H urlimann et al., 2007). A similar behaviour was observed for P. involutus (Lapeyrie et al., 1984) and recently also for H. cylindrosporum. Here, long-chain polyp synthesis occurred only when the mycelia had been grown previously under P-deficient conditions (Torres-Aquino et al., 2017). In Baker s yeast, polyp is synthesized through the Vacuolar Transporter Chaperone Complex (Vtc) comprising four subunits: Vtc1, Vtc2, Vtc3 and Vtc4. In vivo, Vtc4 is the catalytic subunit of a heterotrimeric Vtc complex consisting of Vtc1, Vtc4 and either Vtc2 or Vtc3 (Secco et al., 2012). Depending on the status of yeast cells, the Vtc2- containing complex is localized either in the tonoplast or in the plasma membrane, where it may lead to polyp efflux into the yeast apoplast. Interestingly, homologues of Vtc1, Vtc3 and Vtc4, but not Vtc2, have been identified in the ECM fungi Laccaria bicolor and A. muscaria (U. Nehls, unpublished). 2. N/P redistribution within the fungal mycelium Next to (local) assimilation, storage or biosynthesis at foraging margins, nutrient distribution within the mycelium (e.g. towards the ECM plant fungus interface) is a central part of the lifestyle of filamentous fungi. Advanced real-time imaging techniques (for a recent review, see Heaton et al., 2012) have shown a rapid redistribution of soluble nutrients in different directions within mycelial networks of wood-decaying (Jennings, 1987; Fricker et al., 2008; Heaton et al., 2012) and ECM (Ashford & Allaway, 2002; Wu et al., 2012; Teramoto et al., 2016) fungi. Within these hyphal networks, net transfer of N could be strongly correlated with that of P (Brandes et al., 18; Jentschke et al., 2001), suggesting that N and P could be transported together from the external hyphae towards the Hartig net. Distinct mechanisms (diffusion, vesicle-based transport, cytoplasmic streaming, mass flow), with different transport velocities, have been proposed for transport across varying distances in hyphal networks (centimetres, decimetres, metres; Fricker et al., 2008; Heaton et al., 2012). At short distances (millimetres to centimetres), transport is supposed to be organized at the level of individual hyphae by a combination of diffusion (in cytoplasm and tubular vacuoles), cytoplasmic (0.2 lms 1 ; Lew, 2005) and vacuolar streaming, and vesicular transport (Finlay & Read, 1986b; Cairney, 2005). In hyphae of ECM (Allaway & Ashford, 2001) and saprotrophic fungi, tubular vacuoles, spanning different individual cells, are commonly observed throughout the mycelium. Within such tubular structures, a concentration gradient-based, directed transport (diffusion) of individual substances (e.g. of carbohydrates and assimilated nutrients) occurs, which is uncoupled from cytoplasmic streaming (Darrah et al., 2006). In a wide range of fungi, individual vacuoles become interconnected by vesicle streaming (Rees et al., 14), allowing directed active transport within hyphae. For transport over longer distances (decimetres), individual hyphae self-organize into cord-like structures. At the highest level of organization, empty central hyphae of a cord allow a mass flow similar to that of xylem vessels of higher plants (55 69 lms 1 ; Cairney, 12; Heaton et al., 2012). As ECM fungi can be grouped into certain exploration types according to the size and growth behaviour of their mycelia (Agerer, 2001), these transport principles are supposed to be of different significance for individual fungal species. However, one aspect that is common to all ECM fungi is the intimate association of fungal mycelia with infected fine roots, which may lead to plant transpiration-driven flow within fungal hyphae. Data from Finlay & Read (1986b) for P transport/translocation by Suillus bovinus (Fr.) 0. Kuntze, however, suggest that transport is primarily driven by symplastic flow and is not influenced by plant transpiration. Instead, plant size and the number of mycorrhizal root tips were decisive. Measuring label (P) accumulation within plant tissue could, however, be misleading, as nutrient export at the plant fungus interface may follow different kinetics from hyphal transport. Depending on the experimental conditions and the combination of plant and fungal partners, the role of ECM fungi in plant N/P acquisition is highly variable. The application of labelled N sources to the fungal mycelium frequently results in strong N enrichment in ectomycorrhizas, but N delivery to the plant partner varies from 40% (He et al., 2004, 2005) to < 1% (Ek et al., 16; Teramoto et al., 2016) of labelled N taken up by the fungal partner. With regard to P, quantitative transfer rates are scarce because stable isotopes are missing. Hence, plants have been grown in two-compartment systems in which the external fungal mycelium is separated from plant roots. With Paxillus involutus cea abies (seedlings) as a model, the associated fungal mycelium contributed between 52% (Jentschke et al., 2001) and 76% (Brandes et al., 18) of total plant P, depending on the experimental conditions. Furthermore, strong variations in the N/P ratio, translocated by fungal hyphae towards the plant partner in ectomycorrhizas, were observed (Nara, 2005; Chalot & Plassard, 2011). With Laccaria amethystina, net transfer of P, but not of N, was found, whereas other fungal species (e.g. Hebeloma leucosarx or Scleroderma bovista) transferred N and P simultaneously or revealed neither N nor P transfer (Hebeloma mesophaeum). 3. Vacuolar N/P export Assuming that fungal vacuoles play a central role in local N/P storage at the plant fungus interface, several steps are necessary for fungal nutrient export towards the plant host: polyp metabolization in the vacuole, vacuolar export of N and P into the cytoplasm, N/P metabolization for cellular export and hyphal export towards the plant partner. In ECM fungi, many of these steps are poorly understood at the molecular level. Therefore, we outline potential mechanisms for fungal N/P export based on yeast as a nonmycorrhizal fungal model. For membrane-located transport processes discussed in this context, the following aspects must be considered: (1) the charge of the molecule to be transported; (2) its concentration gradient over the membrane; and (3) the orientation of the respective membrane potential with respect to the cytoplasm (see Box 1).

6 6 Review New Phytologist Box 1 Membrane transport and its energy demand Nutrient transport over a membrane can be passive (following a concentration gradient) or energy demanding (active transport against a concentration gradient). Active transport can be directly (coupling of ATP hydrolysis and transport) or indirectly energized. In plants and fungi, indirectly energized nutrient transport is linked to the transport of protons along their concentration gradient. If protons and nutrients are transported in the same direction, it is called symport; if protons are transported in the opposite direction, it is called antiport. For the transport of charged molecules, the membrane potential (orientation of the proton gradient) must be considered. The apoplastic face of the plasma membrane, but the inner side of the tonoplast and membranes of secretory vesicles, is positively charged. Charge transfer along with the membrane potential (e.g. import of positively charged molecules into the cytoplasm) reduces the energy demand for the transport of a nutrient against its concentration gradient. Not surprisingly, feeding experiments of ECM fungi with different 15 N sources have resulted primarily in glutamine labelling (see above; e.g. Martin et al., 14). However, arginine enrichment in mycorrhizal root tips has also been observed after 15 NH 4 + feeding (Finlay et al., 1988, 1989). As a result of the restricted spatial resolution of these measurements, it remains unclear whether labelled arginine is located within fungal vacuoles or present in root cells. However, in ECM fungi, large amounts of N were co-localized with vacuolar polyp (B ucking et al., 18). Furthermore, arginine was frequently found in vacuoles of yeast and other fungi (see above). Therefore, it can be supposed that arginine is, together with other basic amino acids, temporarily stored in vacuoles of ECM fungal mycelia at the plant fungus interface. The efflux of basic amino acids from yeast vacuoles is enabled by members of the AVT (A Family of Fungal Genes Related to Vesicular GABA Transporters-A) gene family (Russnak et al., 2001; Sekito et al., 2008, 2014). As a result of the orientation of the electrochemical gradient at the tonoplast, AVT members function as proton-coupled symporters to enable vacuolar amino acid efflux. Homologues of this family are found in the genomes of ECM model fungi (U. Nehls, unpublished), but their function remains to be proven experimentally. Similar to yeast (Harold, 1966; Trilisenko et al., 2002), a massive accumulation of vacuole-located, long-chain polyp was observed when a rich P source was applied to P-starved H. cylindrosporum (Torres-Aquino et al., 2017) or P. involutus (Lapeyrie et al., 1984) mycelia grown in pure culture. Long-chain polyp can be visualized only by staining with toluidine blue (Torres-Aquino et al., 2017), but not by in vivo or in vitro 31 P-NMR, as can short-chain polyp (Martin et al., 1985; Torres-Aquino et al., 2017). When H. cylindrosporum mycelia, preloaded with to generate longchain polyp, were brought into contact with roots of a mycorrhizal host plant (nus pinaster), a strong short-chain (average length of residues) polyp NMR signal became visible after 6 h. By contrast, no changes were observed in the presence of roots of a nonhost plant (Zea mays) or in free-living mycelia (Torres-Aquino et al., 2017). In addition to this polyp conversion, fungal mycelia in contact with P. pinaster roots also released much larger amounts of 32 P compared with controls. These data indicate a direct link between vacuolar polyp conversion and hyphal efflux in the presence of a host plant. In yeast, two potential polyp-degrading enzymes have been identified: the exo-polyphosphatase PPX1p and the endo-polyphosphatase Ppn1p (Persson et al., 2003). As PPX1p has no vacuolar targeting signal and Ppx1 transcript levels did not vary with P availability (Wurst et al., 15; Persson et al., 2003), Ppn1p is the best-known candidate for vacuolar polyp degradation. Ppn1p is a functional homodimer of a 35-kDa protein with proven activity in the yeast vacuole (Kumble & Kornberg, 16; Persson et al., 2003). The enzyme converts long polyp chains ( P residues) into shorter chains of 60 (P 60 ) and three (P 3 ) units. However, as no orthophosphate was generated, Ppn1p cannot fully explain the observed vacuolar efflux (see later). Furthermore, a ph optimum of 7.5 of the enzyme (Kumble & Kornberg, 16) leaves its vacuolar function unclear. By using yeast Ppn1p as a query, homologues were identified in the genomes of ECM model fungi (data not shown). However, no PPX or Ppn homologues have been functionally characterized in ECM fungi so far. In yeast, efflux through the tonoplast membrane into the cytoplasm is enabled by pho91 (H urlimann et al., 2007). As shown in Fig. 1 and Table S1, we identified protein homologues clustering with ScPho91 (but also with ScPho87 and ScPho90) in the Mycocosm database, suggesting that vacuolar orthophosphate efflux also occurs in ECM fungi. However, it remains unclear how the requested short-chain polyp conversion into is implemented in vacuoles of ECM fungi. VI. Hyphal N/P efflux at the plant fungus interface In the literature, two potential pathways for hyphal N and P export at the plant fungus interface have been discussed: (1) direct export at the plasma membrane through transporters; and (2) vesiclebased indirect export. Furthermore, for each of N and P, two potential efflux molecules have been considered in the literature for plant nutrition in symbiosis: amino acids and/or ammonia as N source, and and/or short-chain polyp as P source. There is a long history of proposals of amino acids as a potential N source delivered by fungal hyphae in ECM symbiosis (Smith & Read, 2008). This idea is mainly based on the activity of highaffinity amino acid importers in roots of forest trees (Smith & Read, 2008). However, a major criticism is that N support by amino acids would result in a severe carbon loss by fungal hyphae. Furthermore, no candidates for fungal plasma membrane-located amino acid efflux carriers have been described in the literature. A second potential N source delivered by fungal hyphae at the plant fungus interface is ammonia (Chalot et al., 2006; Dietz et al., 2011; Peter et al., 2016). This assumption is supported by: (1) Decreased amino acid pools (Blaudez et al., 18) in combination with a strong repression of glutamine synthetase expression (Wright et al., 2005) in hyphae next to root cells. (2) Enforced expression of genes encoding ammonia-permeable fungal major intrinsic proteins (MIPs) in ectomycorrhizas

7 New Phytologist Review 7 Fungal cell Common apoplastic space Plant cell Fig. 2 Scheme of possible pathways of orthophosphate ions () efflux from the fungal cells into the common apoplast at the Hartig net of ectomycorrhizas. The first step is the hydrolysis of vacuolar polyphosphates (polyp), followed by the efflux of or shortchain polyp into the fungal cytoplasm by as yet unknown enzymes and phosphorus (P) transporters (question marks). Cytosolic or short-chain polyp can be exported into the fungal apoplast by transporters ((1) lowaffinity : Na + or (2) high-affinity H + : transporters), (3) short-chain polyp efflux carrier (Vtc complex) or (4) vesicles, loaded by PHO1-like P transporters. It has been shown that ATPases are active at both fungal and plant membranes, and that released by the fungus is then taken up by H + : plant transporters. Cytosol Vacuole PolyPs N? P P P? 4 PHO1 type? Vtc ATP ADP + 2 Na + 2 Na + 1 P P P 3 P P P H + H + ATP ADP + 2 H + 2 H + 2 H + 2 H + 2 P Vtc? (Basidiomycota: Dietz et al., 2011; Ascomycota: Peter et al., 2016). In fungi, MIP-based ammonia permeability cannot be predicted from the primary protein sequence, but must be proven experimentally (Nehls & Dietz, 2014). (3) Upregulation of plant-located H + : ammonium importers in an ECM-dependent manner (Selle et al., 2005; Couturier et al., 2007). As in yeast (Whitney & Magasanik, 1973), a combination of arginase and urease (as part of the urea cycle) activities, followed by ornithine breakdown, has been proposed for arbuscular and ECM fungi at the plant fungus interface (Bago et al., 2001; Chalot & Plassard, 2011), resulting in the release of glutamate and three ammonium ions from a single arginine molecule. Catabolism of amino acids, such as glutamine, would result in two ammonium ions at the most. Depending on the N source, intracellular NH 4 + concentrations between 0.2 and 3 mm were determined in mycelia of H. cylindrosporum grown in pure culture (Javelle et al., 2003). The concentrations of ammonium and its corresponding ammonia are interlinked in a ph-dependent manner (pka of 9.4 for ammonium dissociation at 20 C; Martinelle & H aggstr om, 17). At a cytoplasmic ph of 6.8, typical for mycorrhizal hyphae, and an intracellular ammonium concentration of 200 lm, the resulting cytosolic ammonia concentration would be 0.5 lm. Ectomycorrhiza-expressed, ammonia-permeable fungal MIPs allow ammonia efflux into the apoplast in a gradient-dependent manner (Dietz et al., 2011; Peter et al., 2016). The acidic ph (5.5) within the apoplast of the Hartig net (Nenninger & Heyser, 18) leads to fast ammonia protonation, resulting in at least a 20-fold higher symplastic NH 3 concentration. This steep ammonia gradient would be maintained by the continuous ammonium uptake by plant importers (Selle et al., 2005; Couturier et al., 2007). There is, however, a gap in this outlined concept: in ectomycorrhizas of L. bicolor, distinct members of the ammonium : H + symporter (AMT) gene family are strongly induced (Lucic et al., 2008). The consequence of a MIP-based ammonia efflux and an AMT-based ammonium re-import would be a futile cycle of inorganic N, interfering with efficient N support of the host plant. However, both mycorrhiza-induced L. bicolor AMTs cluster in a phylogenetic branch that harbours only low-affinity ammonium importers (Lucic et al., 2008). Furthermore, localization studies of AMTs within ectomycorrhizas are missing. In addition, the corresponding proteins have not yet been functionally characterized, but we can speculate that their operation may be restricted to elevated external ammonium concentrations, perhaps to avoid extended N loss under certain conditions. In agreement with the model of ammonia efflux by hyphae at the plant fungus interface, transcript levels of a proven high-affinity AMT were suppressed on ectomycorrhiza formation in the ECM fungus A. muscaria (Willmann et al., 2007). Together, these data suggest a role of fungal AMTs in the avoidance of ammonium loss from the surface of ectomycorrhizas to the soil solution, but perhaps not in the retrieval of ammonium at the plant fungus interface. In S. cerevisiae, members of a second gene family (Ato) function as plasma membrane-located ammonium : H + antiporters (Ricicova et al., 2007) and allow energy-demanding ammonium efflux. Two homologues were found in the genome of the ECM fungus L. bicolor (Lucic et al., 2008). Expression analyses indicated high transcript levels for LbAto1 in free-living mycelium, which were further five-fold elevated in mycorrhizas. No LbAto2 transcripts at all were detected in mycorrhizas. The strong basal expression of LbAto1 is, however, not in agreement with the involvement of this exporter in mycorrhiza-specific ammonium efflux at the symbiotic interface. Several pathways can be proposed to mediate direct export at the plasma membrane (Fig. 2). The first relies on efflux mediated by low-affinity Na + : transporters, such as pho87 or pho90 of yeast. Homologues of these genes are found in the genomes of all investigated ECM fungal

8 8 Review New Phytologist species (Fig. 1). However, gene expression in ectomycorrhizas was observed only for the pho87 homologue of T. melanosporum, where transcript levels were slightly higher (9 2.7) in the Hartig net compared with the fungal sheath (Hacquard et al., 2013). This protein is thus a potential candidate for the mediation of a proton gradient-independent fungal efflux in symbiosis for T. melanosporum, but the transport properties of the protein remain to be determined. A second potential pathway is based on H + : symporters, homologues of pho84 present in all ECM fungal species. As a result of the orientation of the plasma membrane potential, import, but not efflux, is expected for such proteins. However, investigations of pho84-containing vesicles indicated that the protein could mediate bidirectional phosphate transport under certain conditions (Fristedt et al., 16). Nevertheless, it is doubtful whether close pho84 homologues play a role in efflux by ECM fungi, because the expression of the corresponding genes was repressed when hyphae faced good support (as expected at the plant fungus interface). However, HcPT2, located in a sister branch of pho84 homologues (Fig. 1), behaved differently. HcPT2 expression was both weakly affected by availability and upregulated in ectomycorrhizas. Furthermore, HcPT2 was located in hyphae of the Hartig net (Becquer et al., 2018). Therefore, the protein would fulfil many requirements of a efflux carrier. Recently, Schott et al. (2016) conducted a simplified in silico simulation of H + : and H + : sugar transport to define the constraints of nutrient and sugar exchange at the symbiotic interface. As a result of the small distance between plant and fungal plasma membranes at the Hartig net, plant and fungal H + : /H + : sugar symporters may work together in a concerted action. H + : import by plant and H + : sugar import by fungal symporters allow, in return, a reversion of the transport direction of fungal H + : and plant H + : sugar transporters. At ph 6 and a concentration of 10 lm in the apoplast, Schott et al. (2016) demonstrated the establishment of an equilibrium state in which 2H + :H 2 PO 4 influx into root cells would result in a corresponding efflux from fungal hyphae. The speed of equilibrium establishment, but not the equilibrium itself, was dependent on the apoplastic ph (a ph of c. 5.5 was indicated in the Hartig net; Nenninger & Heyser, 18). By contrast, ATPase activity and the concentration gradient of and sugar over the plasma membrane were decisive for the adjustment of export rates. A third potential pathway of efflux in symbiosis is based on polyp biosynthesis. Two assemblies of the Vtc complex are found in yeast. A complex of Vtc1, Vtc4 and Vtc3 localizes exclusively in the vacuolar membrane. However, a complex comprising Vtc2 is also found in the plasma membrane under -replete conditions. As a result of the orientation of the plasma membrane potential, polyp efflux from fungal hyphae into the common apoplast would be the consequence. Indeed, apoplastic location of polyp has been shown for a number of fungi (Werner et al., 2007). However, although homologues of Vtc1, Vtc3 and Vtc4 are present in the genomes of the ECM fungi L. bicolor and A. muscaria (U. Nehls, unpublished), homologues of Vtc2 could not be identified. Thus, the relevance of fungal polyp efflux for plant host provision remains uncertain in ECM symbiosis. Exocytotic vesicles enriched in (Fig. 2) could mediate a fourth potential pathway for hyphal P efflux. Arabidopsis thaliana plants defective in PHO1 are not able to transfer from root epidermal and cortical cells to the xylem (Hamburger et al., 2002). GFP- PHO1 fusions were localized in the Golgi and trans-golgi networks and, exclusively at very high P supply levels, at the plasma membrane (Arpat et al., 2012). Furthermore, plants overexpressing PHO1 in the vascular cylinder of roots and shoots accumulated ions in leaves, indicating an impact of this protein on vascular P export. However, the transport activity of PHO1 could not be proven experimentally by heterologous expression in non-plant systems (yeasts, Xenopus oocytes or liposomes; Arpat et al., 2012). PHO1 has no homology with H + : symporters of the plant PHT family, but does contain single SPX and EPX domains. These domains are known from low-affinity transporters in yeast, where they are involved in the regulation of accumulation (H urlimann et al., 2007, 2009). As the genomes of certain ECM fungi contain at least one PHO1 homologue with SPX and EPX domains (Table S2), the respective proteins could act as exporters in hyphae of the Hartig net. VII. Conclusion and research needs The available fungal genomes (Martin et al., 2011; oe.gov/jgi) cover a wide range of functional groups and are the basis for extended comparative analytics, which help us to describe and understand particular fungal functions. Although homologybased protein analyses are helpful in the classification of phylogenetic relationships and provide estimates of protein functions, they are often misleading with regard to details, such as the subcellular localization, substrate spectrum or conditions, direction and velocity of reactions. Saccharomyces cerevisiae and a few other fungal model species constitute powerful blueprints of fungal physiology and allow a detailed analysis of protein function (e.g. by heterologous expression) in a very well-defined functional context. Although yeast comparisons can help us to understand the basic principles of fungal physiology at the cellular level, they do not allow conclusions about the ECM lifestyle. Only selected ECM fungal models can fill this gap. However, > 1.5 billion fungal species exist in nature, as conservatively estimated (Hawksworth, 2001). Several thousand of these, covering a wide range of fungal taxa, have an ECM lifestyle. Apart from the basic concept of a more or less extended mycelium with extensive nutrient and metabolite exchange between often highly specialized sections, these fungi are often adapted to distinct functional niches. This requires a number of selected ECM fungal models for detailed investigations. Furthermore, soil hyphae face a completely different environment compared with hyphae at the plant fungus interface. The use of simplified systems, such as agar plate-grown ECM mycelia, to mimic certain mycorrhizal features is helpful to elucidate selected aspects, but only allows for limited scientific statements. Therefore, to provide a realistic picture, laboratory-based investigations must be supported by ecological studies. In the literature, the potential decomposer capacity of ECM fungi, as well as the import and primary assimilation of nutrients, has been relatively well investigated, even at the molecular level.

9 New Phytologist Review 9 However, mechanisms of nutrient export and the provision of metabolites required for this process are still vague, even though some concepts, as outlined in this review, have been developed. Furthermore, although research has covered some aspects of longdistance transport within fungal mycelia, the molecular details remain uncertain. ECM fungal transformation in combination with in vivo imaging techniques (using labelled metabolites as well as labelled proteins) is a suitable tool under development for such investigations of structure function relationships. The demand for localization studies was demonstrated in this review by addressing the impact of fungus-derived ammonia for plant N nutrition. Potential ammonia efflux carriers seem to be expressed together with ammonium importers in mycorrhizal hyphae. However, the impact of the respective gene products on fungal N efflux would strongly differ as a function of the protein localization in hyphal networks (plant fungus interface or the fungal mantle) and their subcellular localization (plasma membrane, tonoplast, vesicles). A similar situation is observed with regard to P efflux by fungal hyphae at the plant fungus interface. The in silico simulation of transport properties by Schott et al. (2016) clearly indicated that active and passive transport schemes within the same membrane segment would result in futile cycles. Therefore, distinct export strategies must be either separated in different membrane segments, or tightly controlled locally by protein modification (e.g. phosphorylation) or allosteric interaction with further proteins (Ye et al., 14). The in silico simulation of transport properties, furthermore, stresses the urgent demand for data on the cellular nutrient content, as well as investigations of cellular nutrient homeostasis and its regulation. Overall, we conclude that, although some mechanisms of fungus-based N/P nutrition of ECM host plants are relatively well understood (in particular, import mechanisms), many others (e.g. nutrient export and cellular nutrient homeostasis) remain unsolved. Acknowledgements We would like to thank the anonymous reviewers as well as the editor for their helpful suggestions, which substantially improved the manuscript. We would also like to thank the German Science Foundation (no. 332/14-1) for financial support of U.N. References Agerer R. 15. Anatomical characteristics of identified ectomycorrhizas: an attempt towards a natural classification. In: Varma AK, Hock B, eds. Mycorrhiza structure, function, molecular biology and biotechnology. 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