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The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. We generated a set of 25 906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and454sequencing. NRVTswere used to construct an oligoarray for investigating gene expression. We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

• An in vitro targeted inoculation technique has been developed for studying the earliest stages of arbuscular endomycorrhizal (AM) infection of Medicago truncatula roots, and in particular the spatio-temporal expression of the early nodulin gene MtENOD11. • Agrobacterium rhizogenes transformed root explants were derived either from Myc+ M. truncatula or from the infection-defective Myc- mutant TR26 (dmi2-2), both expressing the pMtENOD11-gusA fusion. The normal positive geotropism of these roots, coupled with the negative geotropism of Gigaspora germ tubes allowed oriented growth of the two symbiotic partners, facilitating the identification of initial fungal/root contacts. • Early infection events at the stage of appressoria and/or internal hyphae could be observed for over 50% of the inoculated explants, revealing that MtENOD11 is expressed transiently in both epidermal and cortical cells at sites of hyphal penetration in Myc+ roots, but not in epidermal cells in contact with appressoria in Myc- roots. • We propose that a direct link exists between MtENOD11 gene expression and cellular events required for fungal penetration, thereby extending analogies between rhizobial and AM host root infection processes.

Both the plant and the fungus benefit nutritionally in the arbuscular mycorrhizal symbiosis: The host plant enjoys enhanced mineral uptake and the fungus receives fixed carbon. In this exchange the uptake, metabolism, and translocation of carbon by the fungal partner are poorly understood. We therefore analyzed the fate of isotopically labeled substrates in an arbuscular mycorrhiza (in vitro cultures of Ri T-DNA-transformed carrot [Daucus carota] roots colonized by Glomus intraradices) using nuclear magnetic resonance spectroscopy. Labeling patterns observed in lipids and carbohydrates after substrates were supplied to the mycorrhizal roots or the extraradical mycelium indicated that: (a) 13C-labeled glucose and fructose (but not mannitol or succinate) are effectively taken up by the fungus within the root and are metabolized to yield labeled carbohydrates and lipids; (b) the extraradical mycelium does not use exogenous sugars for catabolism, storage, or transfer to the host; (c) the fungus converts sugars taken up in the root compartment into lipids that are then translocated to the extraradical mycelium (there being little or no lipid synthesis in the external mycelium); and (d) hexose in fungal tissue undergoes substantially higher fluxes through an oxidative pentose phosphate pathway than does hexose in the host plant.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the roots of over 80% of plants in all terrestrial environments. Understanding why AM fungi do not complete their life cycle under free-living conditions has significant implications for the management of one of the world's most important symbioses. We used (13)C-labeled substrates and nuclear magnetic resonance spectroscopy to study carbon fluxes during spore germination and the metabolic pathways by which these fluxes occur in the AM fungus Glomus intraradices. Our results indicate that during asymbiotic growth: (a) sugars are made from stored lipids; (b) trehalose (but not lipid) is synthesized as well as degraded; (c) glucose and fructose, but not mannitol, can be taken up and utilized; (d) dark fixation of CO2 is substantial; and (e) arginine and other amino acids are synthesized. The labeling patterns are consistent with significant carbon fluxes through gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid cycle, glycolysis, non-photosynthetic one-carbon metabolism, the pentose phosphate pathway, and most or all of the urea cycle. We also report the presence of an unidentified betaine-like compound. Carbon metabolism during asymbiotic growth has features in between those presented by intraradical and extraradical hyphae in the symbiotic state.

Sporisorium reilianum f.sp. zeae, the causal agent of head smut, infects the roots of the maize plantlets. Little information is available concerning the development of the fungus in soil, although this saprophytic phase is an important part of the life cycle. This paper reports that water potential also affects hyphal induction, and this effect on the fungus may influence disease transmission. In response to a decrease in water potential from 0 to –1.52 MPa in presence of variable molecular weight polyethylene glycols, haploid hyphae develop from the haploid yeast. Hyphal extension is fastest at low water potentials (–1.2 MPa) controlled with high molecular weight polyethylene glycols, PEG-3350 and PEG-8000. Formation of parasitic dikaryotic hyphae following fusion between haploid hyphae was possible at low water potential (–1.2 MPa) and was not inhibited by water stress. These results are consistent with the hypothesis that the effects of low soil water potential on yeast–hyphal transition and hyphal growth facilitate the convergence of compatible haploid strains, and that this may increase disease severity.

An in vitro system using Ri T-DNA transformed roots and the vesicular-arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall has been developed to study the initial events of mycorrhiza formation. Sucrose, sodium and phosphorus were found to be critical components of the medium used to establish the dual culture. Using a single spore as inoculum it was consistently possible to obtain colonization of a preselected point on the root and to time the colonization process (within 5 days). Abundant viable and aseptic spores can be obtained. The system is especially appropriate for studying the triggering of the fungal biotrophy towards the root.

Strigolactones are rhizosphere signalling compounds that mediate host location in arbuscular mycorrhizal (AM) fungi and parasitic plants. Here, the regulation of the biosynthesis of strigolactones is studied in tomato (Solanum lycopersicum). Strigolactone production under phosphate starvation, in the presence of the carotenoid biosynthesis inhibitor fluridone and in the abscisic acid (ABA) mutant notabilis were assessed using a germination bioassay with seeds of Orobanche ramosa; a hyphal branching assay with Gigaspora spp; and by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. The root exudates of tomato cv. MoneyMaker induced O. ramosa seed germination and hyphal branching in AM fungi. Phosphate starvation markedly increased, and fluridone strongly decreased, this activity. Exudates of notabilis induced approx. 40% less germination than the wild-type. The LC-MS/MS analysis confirmed that the biological activity and changes therein were due to the presence of several strigolactones; orobanchol, solanacol and two or three didehydro-orobanchol isomers. These results show that the AM branching factors and parasitic plant germination stimulants in tomato root exudate are strigolactones and that they are biosynthetically derived from carotenoids. The dual activity of these signalling compounds in attracting beneficial AM fungi and detrimental parasitic plants is further strengthened by environmental conditions such as phosphate availability.

Using dual cultures of arbuscular mycorrhizal (AM) fungi and Medicago truncatula separated by a physical barrier, we demonstrate that hyphae from germinating spores produce a diffusible factor that is perceived by roots in the absence of direct physical contact. This AM factor elicits expression of the Nod factor-inducible gene MtENOD11, visualized using a pMtENOD11-gusA reporter. Transgene induction occurs primarily in the root cortex, with expression stretching from the zone of root hair emergence to the region of mature root hairs. All AM fungi tested (Gigaspora rosea, Gigaspora gigantea, Gigaspora margarita, and Glomus intraradices) elicit a similar response, whereas pathogenic fungi such as Phythophthora medicaginis, Phoma medicaginis var pinodella and Fusarium solani f.sp. phaseoli do not, suggesting that the observed root response is specific to AM fungi. Finally, pMtENOD11-gusA induction in response to the diffusible AM fungal factor is also observed with all three M. truncatula $\\text{Nod}^{-}/\\text{Myc}^{-}$ mutants (dmi1, dmi2, and dmi3), whereas the same mutants are blocked in their response to Nod factor. This positive response of the $\\text{Nod}^{-}/\\text{Myc}^{-}$ mutants to the diffusible AM fungal factor and the different cellular localization of pMtENOD11-gusA expression in response to Nod factor versus AM factor suggest that signal transduction occurs via different pathways and that expression of MtENOD11 is differently regulated by the two diffusible factors.

During spore germination, arbuscular mycorrhizal (AM) fungi show limited hyphal development in the absence of a host plant (asymbiotic). In the presence of root exudates, they switch to a new developmental stage (presymbiotic) characterized by extensive hyphal branching. Presymbiotic branching of the AM fungus Gigaspora rosea was induced in liquid medium by a semipurified exudate fraction from carrot (Daucus carota) root organ cultures. Changes in RNA accumulation patterns were monitored by differential display analysis. Differentially appearing cDNA fragments were cloned and further analyzed. Five cDNA fragments could be identified that show induced RNA accumulation 1 h after the addition of root exudate. Sequence similarities of two fragments to mammalian Nco4 and mitochondrial rRNA genes suggested that root exudates could influence fungal respiratory activity. To support this hypothesis, additional putative mitochondrial related-genes were shown to be induced by root exudates. These genes were identified after subtractive hybridization and putatively encode a pyruvate carboxylase and a mitochondrial ADP/ATP translocase. The gene GrosPyc1 for the pyruvate carboxylase was studied in more detail by cloning a cDNA and by quantifying its RNA accumulation. The hypothesis that respiratory activity of AM fungi is stimulated by root exudates was confirmed by physiological and cytological analyses in G. rosea and Glomus intraradices. Oxygen consumption and reducing activity of both fungi was induced after 3 and 2 h of exposition with the root factor, respectively, and the first respiration activation was detected in G. intraradices after approximately 90 min. In addition, changes in mitochondrial morphology, orientation, and overall biomass were detected in G. rosea after 4 h. In summary, the root-exuded factor rapidly induces the expression of certain fungal genes and, in turn, fungal respiratory activity before intense branching. This defines the developmental switch from asymbiosis to presymbiosis, first by gene activation (0.5-1 h), subsequently on the physiological level (1.5-3 h), and finally as a morphological response (after 5 h).

Two cvs of alfalfa (Medicago sativa L.), Gilboa and Moapa 69, were inoculated in glasshouse pots with three arbuscular mycorrhizal (AM) fungi to investigate the efficacy of mycorrhizas with respect to the extent of colonization and sporulation. Paspalum notatum Flugge also was inoculated to describe fungal parameters on a routine pot culture host. Percentage root length of P. notatum colonized by Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe, Glomus intraradices Schenck & Smith, and Gigaspora margarita Becker & Hall increased from 10 to 21 wk, and all fungi sporulated during that period. In alfalfa, only colonization by G. intraradices increased over that time period, and it was the only fungus to sporulate in association with alfalfa at 10 wk. Glomus mosseae did not sporulate after 16–21 wk despite having colonized 30–35% of the root length of both alfalfa cvs. In vitro experiments in which Ri T-DNA-transformed roots of alfalfa were inoculated with AM fungi showed normal mycorrhizal formation by G. intraradices and a hypersensitivity-like response to Gi. margarita. Colonized cells became necrotic, and HPLC analysis indicated increased concentrations of phenolics and isoflavonoids in these root segments. These data strongly support the existence of a degree of specificity between AM fungi and host that might rely on specific biochemical regulatory processes initiated in the host as a result of the attempts at colonization by the fungus.