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PLANTS INTERACTING WITH OTHER ORGANISMS

The Mycorrhizal Fungus Gigaspora margarita Possesses a CuZn Superoxide Dismutase That Is Up-Regulated during Symbiosis with Legume Hosts¹

Dipartimento di Biologia Vegetale, Università di Torino, 10125 Turin, Italy (L.L., M.N., P.B.); and Istituto per la Protezione delle Piante, Sezione di Torino, CNR, 10125 Turin, Italy (P.B.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
A full-length cDNA showing high similarity to previously described CuZn superoxide dismutases (SODs) was identified in an expressed sequence tag collection from germinated spores of the arbuscular mycorrhizal fungus Gigaspora margarita (BEG 34). The corresponding gene sequence, named GmarCuZnSOD, is composed of four exons. As revealed by heterologous complementation assays in a yeast mutant, GmarCuZnSOD encodes a functional polypeptide able to confer increased tolerance to oxidative stress. The GmarCuZnSOD RNA was differentially expressed during the fungal life cycle; highest transcript levels were found in fungal structures inside the roots as observed on two host plants, Lotus japonicus and Medicago truncatula. These structures also reacted positively to 3,3'-diaminobenzidine, used to localize H₂O₂ accumulation. This H₂O₂ is likely to be produced by CuZnSOD activity since treatment with a chelator of copper ions, generally used to inhibit CuZnSODs, strongly reduced the 3,3'-diaminobenzidine deposits. A slight induction of GmarCuZnSOD gene expression was also observed in germinated spores exposed to L. japonicus root exudates, although the response showed variation in independent samples. These results provide evidence of the occurrence, in an arbuscular mycorrhizal fungus, of a functional SOD gene that is modulated during the life cycle and may offer protection as a reactive oxygen species-inactivating system against localized host defense responses raised in arbuscule-containing cells.

ROS and SODs are involved in disease resistance mechanisms other than HR. In the systems Cladosporium fulvum-tomato (Lycopersicon esculentum) and Erisyphe graminis-barley (Hordeum vulgare), evidence suggests that the oxidative burst can be decoupled from the HR and that H₂O₂-mediated cross-linking of cell wall proteins or phenolic substances is of crucial importance to establish resistance (Vallélian-Bindschedler et al., 1998; Kwon and Anderson, 2001). Even though a typical HR does not occur in the biotrophic system Claviceps purpurea-rye (Secale cereale), accumulation of superoxide and H₂O₂ is observed and lignification is pronounced at the host-pathogen interface (Moore et al., 2002).

To add a further level of complexity, many pathogens have themselves developed ROS-inactivating systems, where catalases and peroxidases, which break down H₂O₂ to H₂O and O₂, are the principal enzymatic antioxidants together with SODs. The role of ROS and ROS-inactivating systems in microbe pathogenicity and in overcoming host resistance is still an open question. Several examples in animal systems show that bacterial pathogen virulence is correlated with the secretion of ROS-scavenging enzymes (Mandell, 1975; De Groote et al., 1997). A similar result was recently found for the pathogenic yeast Cryptococcus neoformans, which causes meningoencephalitis: a CuZnSOD, dispensable in its saprobic life, is critical for pathogenesis of the fungus (Narasipura et al., 2003). Necrotrophic fungi are hypothesized to cope with a hostile environment thanks to an array of ROS-inactivating enzymes such as SOD, peroxidase, catalase, and perhaps laccase and polyphenol oxidases (Mayer et al., 2001). However, in Claviceps purpurea the lack of a CuZnSOD, evidenced by targeted gene disruption, does not significantly reduce fungal virulence on rye (Moore et al., 2002). That appears to be the only report showing that a SOD is not essential for a fungal pathogen, and its significance is uncertain.

ROS production and antioxidant systems from the plant and/or the microorganism also play a role during symbiotic interactions. In root nodules a delicate equilibrium is required to supply the energy demands of nitrogen reduction and to protect the nitrogenase complex from the ROS produced, suggesting that antioxidant activities from both plants and bacteria are essential to establish a functional symbiosis (Matamoros et al., 2003). A critical protective role of a bacterial SOD was described in the Sinorhizobium-legume symbiosis: SOD effects extend far beyond protection of the nitrogenase complex since defects in the mutant strain were observed in all steps of symbiosis, including infection, nodulation, and bacteroid differentiation. This suggests that oxidative stress, unless counteracted by SOD, interferes at several steps in symbiosis (Santos et al., 2000). The demonstration that Nod factors do not elicit an early oxidative burst in Medicago truncatula roots provides further evidence of the differences between pathogenic and symbiotic interactions (Shaw and Long, 2003).

Arbuscular mycorrhiza (AM) is the most widespread type of symbiosis and a unique example of a fully compatible interaction between plants and fungi (Harrison, 1999; Gadkar et al., 2001), but there is little information in this field about ROS production and ROS inactivation. The intimate association of fungal and plant tissues implies that the fungus must be recognized by the plant, which sets up a complex accommodation process whose genetic determinants have, at least in part, been identified (Parniske, 2004). The induction/suppression of mechanisms associated with plant defense also plays a key role in AM colonization compatibility with the host. Expression of genes related to plant defense, investigated using specific probes (for review, see Garcia-Garrido and Ocampo, 2002) or large-scale approaches (Journet et al., 2002; Liu et al., 2003), demonstrates a transient and weak defense response during the initial phases of colonization, usually followed by activation of defense-related genes in cells containing arbuscules.

Changes in profiles of antioxidative enzymes such as SOD, catalases and peroxidases, have been observed in mycorrhizal roots, and CuZnSOD activity was hypothesized for Glomus mosseae spores (Palma et al., 1993; Blilou et al., 2000). The molecular data refer only to host-plant genes. Catalase and peroxidase expression, detected in arbuscule-containing cells of bean (Phaseolus vulgaris) and wheat (Triticum aestivum) colonized by Glomus intraradices, was suggested to result from localized activation of defense mechanisms (Blee and Anderson, 2000). Gene fragments encoding two MnSOD and one FeSOD were identified in lettuce (Lactuca sativa; Ruiz-Lozano et al., 2001). Their expression was down-regulated in normal conditions, while under drought stress the MnSOD II gene was induced in mycorrhizal roots, suggesting a protective mechanism against drought. Genes expressed during development of AM symbiosis between M. truncatula and Glomus versiforme have recently been analyzed by cDNA macroarray (Liu et al., 2003). Two clones, AW587301 and AW584200, showing high similarity to plant CuZnSOD and FeSOD, respectively, did not show significant changes of their expression during mycorrhizal development (Liu et al., 2003). In contrast with ectomycorrhizal fungi where SOD genes have been identified and characterized (Jacob et al., 2001), molecular data on genes coding for ROS-scavenging enzymes in AM fungi are not available.

Here, we describe the cloning and the functional characterization of a CuZnSOD gene from the AM fungus Gigaspora margarita and present evidence that this gene is differentially expressed during the fungal life cycle.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Identification and Characterization of GmarCuZnSOD

A full-length cDNA showing high similarity to previously described CuZnSODs (SOD; EC 1.15.1.1) was identified in an expressed sequence tag collection from G. margarita germinated spores (Lanfranco et al., 2000). This cDNA was named GmarCuZnSOD according to the nomenclature proposed for AM fungal genes (Franken, 2002). To confirm the fungal origin of the cDNA the genomic sequence of GmarCuZnSOD was obtained from PCR experiments carried out on spore genomic DNA with oligonucleotide primers designed on the 5'- and 3'-untranslated regions of the GmarCuZnSOD cDNA. Comparison between genomic and cDNA sequences showed that three introns, limited by consensus splice junctions, are present in the GmarCuZnSOD gene at positions 162, 736, and 936 (Fig. 1). The introns, shown in lowercase letters in Figure 1, are 379, 93, and 82 bp long, respectively. The GC content of the genomic sequence is relatively low (33.76%) and characteristic for AM fungi (Hosny et al., 1997).

View larger version (69K): [in this window] [in a new window]   Figure 1. Nucleotide and deduced amino acid sequence of GmarCuZnSOD. The intron sequence is shown in lowercase letters. Amino acid residues important for the catalytic activity are underlined.

A phylogenetic analysis was carried out on CuZnSOD amino acid sequences ranging from bacteria to humans, available in databases (Fig. 2). Sequences from higher eukaryotes, plants, and animals form distinct clusters. Fungal sequences group together and, surprisingly, GmarCuZnSOD forms a group distinct from this cluster. These two clusters are supported by a tree branch also including some sequences from animals, although its bootstrap value is low (below 50%).

View larger version (51K): [in this window] [in a new window]   Figure 2. Neighbor-joining tree obtained from the alignment of GmarCuZnSOD gene product from G. margarita (in bold) and CuZnSODs sequences of other organisms retrieved from database. Salmonella typhimurium AAB62385was used as outgroup taxon to root the tree. Bootstrap values above 50% are indicated (1,000 replicates). Branch lengths are proportional to genetic distance which is indicated by a bar at the bottom left.

To test whether GmarCuZnSOD codes a functional protein, a complementation assay was performed in a yeast mutant defective in the CuZnSOD gene. The mutant was transformed with the complete open reading frame of GmarCuZnSOD cDNA placed under the control of a constitutive yeast promoter in the expression vector pFL61 (Minet et al., 1992). As a control, the yeast strain was also transformed with the vector alone. To induce oxidative stress, transformed yeasts were plated on synthetic dextrose (SD)-agar medium supplemented with CdSO₄ (Brennan and Schiestl, 1996). The two yeast transformants were streaked onto SD minus uracil plates containing 0 or 100 µM CdSO₄. As shown in Figure 3, cells carrying the pFL61 vector grew only in the medium without cadmium. By contrast, GmarCuZnSOD-transformed cells also developed colonies in the presence of heavy metal (Fig. 3). This result indicates that the gene, at least in a heterologous system, can confer tolerance to oxidative stress.

View larger version (115K): [in this window] [in a new window]   Figure 3. Increased oxidative stress tolerance conferred by GmarCuZnSOD in a yeast mutant defective of CuZnSOD. Yeast mutants harboring either the pFL61-GmarCuZnSOD plasmid (pFLSOD) or the empty pFL61 vector (pFL61) were grown on SD (–uracil) agar plates with 0 or 100 µM cadmium sulfate (100 µM Cd).

To understand whether GmarCuZnSOD was differentially expressed, colonization experiments were done using root segments of M. truncatula and Lotus japonicus, considered model plants when plant/microbe interactions are investigated, due to their symbiotic capacities. A mutant of L. japonicus was also used. Colonization by G. margarita was successful with both the wild-type plants. To evaluate the intensity of root colonization, four parameters were considered: frequency of mycorrhization (F%), intensity of mycorrhization (M%), percentage of arbuscules in the colonized portion of the root (a%), and percentage of arbuscules in the root overall (A%; Trouvelot et al., 1986). As shown in Table I, the confined-sandwich method led to an excellent percentage of colonization in L. japonicus wild type, in comparison with the pot culture of M. truncatula. As expected, the fungus did not develop in the L. japonicus mutant Ljsym4-2 (Novero et al., 2002). In this mutant, fungal penetration is limited to epidermal cells that appeared, after infection attempts, morphologically dead. Abortion of colonization also was associated with localized death of fungal hyphae (Bonfante et al., 2000).

GmarCuZnSOD mRNA expression levels were analyzed throughout the fungal life cycle by real-time reverse transcription (RT)-PCR. Specific primers were designed on GmarCuZnSOD and on the ribosomal gene 18S of G. margarita, considered as a housekeeping gene. To exclude cross-hybridizations with plant material, conventional PCR reactions were performed on L. japonicus and M. truncatula genomic DNAs; all primers gave negative results with these (data not shown).

cDNA samples were prepared from quiescent spores, germinated spores, extraradical mycelium collected from L. japonicus and M. truncatula mycorrhizal roots, and from mycorrhizal root pieces with external hyphae removed. In addition, samples of G. margarita mycelium grown on the mutant Ljsym4-2 were also tested. Samples were calibrated using the fungal 18S rRNA transcript, and to calculate relative expression, quiescent spores were taken as a reference sample. The results obtained from L. japonicus are summarized in Figure 4A. A slight induction was observed in samples corresponding to germinating spores and extraradical mycelium compared to quiescent spores. However, these values were not statistically significant (P = 0.261 and P = 0.280, respectively). The external mycelium growing on Ljsym4-2 did not show any difference when compared to extraradical mycelium growing on a wild-type plant (data not shown). The highest induction was observed in colonized roots from which external mycelium was eliminated. Statistical analyses of data indicated a significant difference in the expression level between this condition and quiescent spores (P = 0.000), germinating spores (P = 0.003), and external hyphae (P = 0.002). Similar expression profiles were obtained in M. truncatula samples (Fig. 4B); the GmarCuZnSOD transcript was significantly (P < 0.05) more abundant in intraradical fungal structures compared to the other three conditions.

View larger version (21K): [in this window] [in a new window]   Figure 4. Real-time RT-PCR analysis of the GmarCuZnSOD mRNA during asymbiotic phases (quiescent and germinating spores) and symbiotic phases (extraradical and intraradical mycelium from L. japonicus [A] or M. truncatula [B]). Relative expression levels were obtained with the Ct method (see "Materials and Methods" for details) and were normalized with respect to GmarCuZnSOD levels in quiescent spores.

We also investigated whether GmarCuZnSOD could be involved in the early stages of interaction and be part of signaling events occurring before a direct contact with host roots. In particular, we analyzed whether GmarCuZnSOD gene expression responds to plant molecules that are present in root exudates and have been shown to induce hyphal branching in AM fungi (Buee et al., 2000; Tamasloukht et al., 2003). The GmarCuZnSOD expression level was thus studied in germinated spores treated with root exudates prepared from L. japonicus seedlings as described by Buee et al. (2000).

An induction was observed in spores exposed to root exudates (Fig. 5), although statistical analyses indicated no significant differences (P = 0.187). We hypothesized that the high variability existing among batches of G. margarita (Jargeat et al., 2004; V. Bianciotto, personal communication) could explain the different response to root exudates.

View larger version (15K): [in this window] [in a new window]   Figure 5. Real-time RT-PCR analysis of the GmarCuZnSOD mRNA in germinating spores after exposure to water or L. japonicus root exudates. Relative expression levels were obtained with the Ct method (see "Materials and Methods" for details) and were normalized with respect to GmarCuZnSOD levels in spores treated with water.

To answer the question whether fungal SOD activity was responsible for localized H₂O₂ production, we did 3,3'-diaminobenzidine (DAB) assays. To evaluate whether H₂O₂ was produced by CuZnSODs, root samples were pretreated with sodium diethyldithiocarbamate (DDC), a copper ion chelator generally used to inhibit CuZnSODs (Delledonne et al., 2001). Production of H₂O₂ was investigated in mycorrhizal roots of M. truncatula grown in pot cultures and of L. japonicus (wild type and mutant Ljsym4-2) grown in the Millipore sandwich system (Giovannetti et al., 1993) as well as in nonmycorrhizal control roots.

Dark deposits that mark the DAB reaction and indicate H₂O₂ production were consistently observed in mycorrhizal tissues (Figs. 6A and 7A), whereas nonmycorrhizal roots never reacted, with the exception of vascular tissues and meristematic regions (Figs. 6, G and H, and 7G). This latter unexpected result is in agreement with a preliminary observation reported by Shaw and Long (2003).

View larger version (115K): [in this window] [in a new window]   Figure 6. L. japonicus roots colonized by G. margarita after DAB reaction. A, Dark deposits, which are the marker of the DAB reaction and are indicative of H2O2 production, are consistently observed associated with many of the intraradical (Ih) and extraradical hyphae (Eh). Bar corresponds to 200 µm. B, Higher magnification of the previous image where the dark deposits associated with the intraradical hyphae (Ih) and arbuscules (A) are particularly evident. The host cells do not show any reaction (cc). Bar corresponds to 100 µm. C, The reaction is evident in mature arbuscules (Ma), while other younger arbuscules (Ya, arrowheads), which are located in close cortical cells, are less reactive. The labeling seems to be more intense on the trunks (T). A diffuse yellow color is present in the host cells. Bar corresponds to 50 µm. D, Younger arbuscules (Ya) where the thin branches are still visible are not reactive as well as some of the intercellular hypha (Ih). In this section the dark deposit is particularly evident on the trunks (T). Bar corresponds to 60 µm. E, The treatment with DDC, a copper chelator usually used to block CuZnSOD, strongly inhibits the reaction associated to the intraradical fungal structures: both arbuscules (A) and intercellular hyphae. Bar corresponds to 70 µm. F, After DDC treatment the dark precipitate is only weakly quenched in the extraradical hyphae (Eh). Bar corresponds to 200 µm. G, Differentiated tissues from nonmycorrhizal roots do not show any reactivity to DAB staining. Bar corresponds to 200 µm. H, Root tips with the meristematic regions (Me) are the only root regions to be reactive to DAB staining. Bar corresponds to 500 µm.

View larger version (116K): [in this window] [in a new window]   Figure 7. M. truncatula roots colonized by G. margarita after DAB reaction. A, The dark precipitates, indicative of H2O2 production, are associated with the intercellular hyphae (Ih) as well as with the arbuscules (A). Bar corresponds to 220 µm. B, When the roots are treated with the copper chelator, DDC, the reaction is fully quenched in all the intraradical structures (Ih and A). Bar corresponds to 270 µm. C, The dark precipitates are associated with extraradical hyphae (Eh). Bar corresponds to 100 µm. D, When the roots are treated with DDC, the reaction is only partly quenched. Eh, Extraradical hyphae. Bar corresponds to 100 µm. E, Detail of two arbuscules (arrowheads) differently reactive to the DAB staining. The trunk (T) of the bottom one is clearly reactive as well as the intercellular hyphae (Ih). Bar corresponds to 60 µm. F, Detail of a collapsing arbuscule (Ca) were the DAB reaction has produced a homogenous black deposit filling up the whole cell lumen. Bar corresponds to 60 µm. G, Nonmycorrhizal root tissues do not show any reaction in the differentiated regions, with the exception of vascular tissues (Vv). Bar corresponds to 200 µm.

Similar results were obtained on M. truncatula mycorrhizal roots, growing in pots and probably in a more natural situation. The extraradical mycelium, intercellular hyphae, and arbuscules often reacted strongly to DAB staining (Fig. 7, A, C, E, and F), whereas the plant tissues were never stained with the exception of vascular tissues (Fig. 7G) and meristem cells (data not shown). Staining was particularly intense on collapsing arbuscules (Fig. 7F), where the DAB reaction was homogenous. By contrast, other arbuscules reacted weakly. When the roots were treated with DDC, the reaction was limited to extraradical mycelium (Fig. 7D), being absent in the intraradical mycelium (Fig. 7B).

Taken together, the morphological data show that the H₂O₂ is produced in both intraradical and extraradical compartments by the fungus. However, following the use of DDC, only the H₂O₂ associated with intraradical structures seemed to be produced by the fungal CuZnSOD. Other metabolic pathways may have been responsible for the DAB deposits associated with the extraradical mycelium (Neill et al., 2002; Mittler et al., 2004).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Due to their superoxide detoxifying capacities, SODs are universal protective tools well characterized in prokaryotes and eukaryotes. By contrast, little is known about SODs in filamentous fungi. While most eukaryotes possess multiple CuZnSODs, yeasts and fungi seem to have no more than one CuZnSOD gene (Moore et al., 2002). We characterized a CuZnSOD gene from an AM fungus. It is presumably the only CuZnSOD gene in G. margarita. Southern-blot analysis, however, was not performed due to the limited amount of material available for this obligate biotroph.

GmarCuZnSOD presents amino acid residues typical of CuZnSODs and important for enzyme structure and activity (Steinman and Ely, 1980; Chary et al., 1990). The sequence does not contain an N-terminal signal peptide for secretion, reinforcing the claim that fungal CuZnSODs have a cytoplasmic localization. There is, however, evidence that CuZnSODs lacking an N-terminal signal peptide in Aspergillus fumigatus, C. neoformans, and Claviceps purpurea are nevertheless secreted (Hamilton et al., 1996; Hamilton and Holdom, 1997, Moore et al., 2002). Phylogenetic analysis shows that GmarCuZnSOD does not cluster with the other fungal sequences. This could be due to the limited number of fungal sequences covering all different phyla present in data banks (most of them belong to Ascomycetes) or to the fact that AM fungi are a very ancient group clearly distinct from Ascomycetes and Basidiomycetes (Schußler et al., 2001).

Functional characterization of GmarCuZnSOD showed that the gene can confer increased tolerance to oxidative stress in a yeast mutant defective with respect to CuZnSOD. Functional analysis of AM fungal genes has to be performed in heterologous systems since no mutants are available at the moment for this group of organisms. Transformation technology has been applied to AM fungi with promising results (Harrier and Millam, 2001). In the near future new techniques such as RNA interference, already successfully applied in several other systems (Hannon, 2002), may make it possible to silence specific genes also in AM fungi and to check their functions directly.

GmarCuZnSOD Is Up-Regulated during Symbiosis

GmarCuZnSOD expression is regulated during the different phases of the fungal life cycle. A low expression level was observed in quiescent spores as well as in germinated ones. The last result was to be expected since the sequence was identified as an expressed sequence tag (EST) clone from germinated spores (Lanfranco et al., 2000). Other genes involved in stress responses were found within this EST collection, suggesting that in vitro germination is not the most favorable growth condition for G. margarita (Lanfranco et al., 2002).

Irrespective of the plant genotype, the transcript was detected in the external mycelium, which was also positive in the DAB reaction used to detect H₂O₂ accumulation. The dark precipitate, indicating H₂O₂ production, was however not fully quenched by DDC, a copper chelator, suggesting that H₂O₂ was produced by mechanisms that did not exclusively involve CuZnSOD. This seems a consistent result, as external hyphae growing on different host plants and in different experimental conditions behave in a similar way. It might be that H₂O₂ is produced following electron transport in mitochondria, during which it is though to be generated from superoxide presumably in an uncontrolled manner (Neill et al., 2002). However, at least in plants, there is likely to be more than one enzymatic source of H₂O₂ produced in response to specific abiotic and biotic stimuli. Potential candidates include NADPH oxidases, cell wall peroxidases, amine oxidases, oxalate oxidases, and flavin-containing oxidases (Neill et al., 2002; Mittler et al., 2004). Deeper investigation of ROS-generating and ROS-scavenging systems from AM fungi will help to clarify why external hyphae are so reactive to DAB.

The highest transcript levels were found in fungal structures developing inside the root. This was obtained from two host plants colonized under two experimental conditions. Up-regulation of a fungal SOD was not reported in the M. truncatula-G. versiforme system where a global analysis of genes expressed during the development of AM symbiosis was performed by cDNA macroarray (Liu et al., 2003). However, as the authors stated, only 5% of the genes investigated belonged to the fungal genome. On the plant side, expression of at least a CuZnSOD and an FeSOD did not significantly change during mycorrhizal development (Liu et al., 2003). Interestingly, up-regulation of a fungal SOD in mycorrhizal tissues as compared to the free-living mycelium was found by cDNA microarray analyses in the interaction between the ectomycorrhizal fungus Paxillus involutus and Betula pendula (Johansson et al., 2004).

In our study, the intraradical fungal structures (intercellular hyphae and arbuscules) of both mycorrhizal plants were also DAB-positive, whereas plant tissues were only faintly so. The H₂O₂ production is likely due to CuZnSOD activity since treatment with DDC strongly reduced the DAB deposits. A previous study using the same DAB assay showed H₂O₂ accumulation in cells of M. truncatula colonized by G. intraradices (Salzer et al., 1999). However, H₂O₂ production was hypothesized by these authors to be a consequence of activation of a plant plasma membrane NADPH oxidase, analogous to what occurs during the HR (Bolwell et al., 2002). Molecular evidence for elicitation of a HR and supporting Salzer's hypothesis is not so far available, and further investigations of H₂O₂-producing systems in plants challenged by AM fungi are needed to clarify this issue. Possible candidates might be germin-like proteins (GLPs), which have been found to be up-regulated in mycorrhizal roots (Doll et al., 2003). Similar results came from a large-scale analysis of gene expression based on EST collection from M. truncatula (http://medicago.toulouse.inra.fr/Mt/EST/). Some true germins from Gramineae display oxalate oxidase activity producing H₂O₂ and CO₂ from oxalic acid; other GLPs have been identified as SODs (Yamahara et al., 1999; Carter and Thornburg, 2000, Christensen et al., 2004), but until now most attempts to ascribe oxalate oxidase/SOD activity to GLPs from dicotyledonous plants have been negative (Ohmiya et al., 1998; Carter and Thornburg, 2000).

In our experiments, quantitative expression analysis and DAB staining showed a similar trend, suggesting that GmarCuZnSOD could be responsible for localized H₂O₂ accumulation in intracellular fungal structures. We can speculate about the possible roles of fungal SOD activity. Two possible explanations, not necessarily exclusive, can be proposed. Several in situ hybridization-based studies have shown that in mycorrhizal roots induction of a number of defense-related genes is confined to arbuscule-containing cells (Harrison and Dixon, 1994; Blee and Anderson, 1996, 2000; Balestrini et al., 1999; Franken et al., 2000; Bonanomi et al., 2001). Fungal ROS-scavenging systems, such as SOD, might be required to control and overcome the range of plant defense responses at a site crucial to AM symbiotic function. The host plants can contribute to maintain arbuscule function. In fact, a gene coding for a plant leghemoglobin, Vfl29, was recently shown to be specifically up-regulated in arbuscule-containing cells (Vieweg et al., 2004). Beside the role in oxygen supply, plant leghemoglobins are thought to bind NO, a crucial molecule involved in plant defense against pathogens (Delledonne et al., 2001; Neill et al., 2002). The authors speculated that this leghemoglobin gene, through the ability to encode NO-scavenging activity, could help to suppress defense responses in arbuscule-containing cells (Vieweg et al., 2004). Another interpretation relies on the evidence that arbuscules are terminal fungal structures; after arbuscule formation the fate of these fine hyphal branches is to collapse (Bonfante, 1984). Nothing is known about the molecular mechanisms underlying this process, but nuclei in the fine arbuscule hyphae are pycnotic and electrondense with a morphology typical of cells undergoing apoptosis (Balestrini et al., 1992). We can speculate that ROS, and in particular H₂O₂, may act in arbuscules as a molecule signaling programmed cell death, as it has been described in plant systems (Delledonne et al., 2001; Neill et al., 2002).

Does GmarCuZnSOD Respond to Root Exudates?

A key topic in AM research is the identification of molecular signals exchanged between the plant and the fungus during early stages of the interaction (Parniske, 2004). The existence of a fungal factor has recently been proposed on the basis of the transcriptional activation of a nodulin gene by a diffusible molecule originating from germinating hyphae of AM fungi (Kosuta et al., 2003). On the other side, some investigations have been performed on the plant factor. Although the chemical structure of the specific compound or compounds is still unknown, it has been shown that root exudates from mycotrophic plants can enhance growth and elicit hyphal branching (Buee et al., 2000).

The observation that the expression levels of GmarCuZnSOD are enhanced following exposure to plant root exudates is quite interesting, since it could represent a tool for the analysis of plant-fungus interaction before the colonization process. However, we observed a large variability in spore response. Each spore sample, consisting of about 100 spores coming from different batches, is not a very homogeneous biological material. Over the last decade a high genetic diversity within AM species and even within individual AM fungal spores, including this G. margarita isolate (Lanfranco et al., 1999a), has been reported (Sanders, 2004). It is still unknown whether this genetic variation may reveal important biological consequences on the phenotype and the fitness of the fungus (Sanders, 2004). As far as the specific G. margarita strain used in this study is concerned, different batches of spores have large phenotypic variability for example in germination capability (V. Bianciotto, personal communication) and in the abundance of endosymbiotic bacteria (Jargeat et al., 2004). This intrinsic genetic diversity may explain the variability in the response to root exudates of different batches of germinating spores.

An early event in root exudate perception is stimulation of fungal respiratory activity, mirrored by the concomitant induction of mitochondria-related genes (Tamasloukht et al., 2003). The higher transcript level of GmarCuZnSOD might reflect a need to remove the excess ROS generated during the respiration increase. It is tempting to speculate that, in addition to the protection against ROS, CuZnSOD might be involved in root exudate perception and might mediate fungal morphogenetic responses. Evidence for involvement of CuZnSOD in the control of morphogenetic processes comes from Neurospora crassa, in which specific inactivation of a CuZnSOD has been shown to affect morphogenetic responses to light, such as carotenoid synthesis and perithecium polarity (Yoshida and Hasunuma, 2004).

	CONCLUSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
We have identified a functional homolog of a CuZnSOD in an AM fungus. The gene is differentially expressed during the interaction with the host plant, suggesting potentially different roles. GmarCuZnSOD is expressed when the fungus is metabolically active, leading to a basal production of H₂O₂ similar to that reported in plants (Shaw and Long, 2003). During the symbiotic phase it seems to be responsible for a microlocalized oxidative burst mainly associated with the collapsing of arbuscule branches. In this specific site, H₂O₂ may also act as a factor required for signaling fungal cell death.

In conclusion, our results provide evidence that fungal ROS-scavenging systems, such as SOD, may be components of the plant/fungus dialogue, allowing functional and structural compatibility between the partners.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Biological Materials

Spores of Gigapora margarita (BEG 34) were collected from pot cultures of mycorrhizal Trifolium repens and sterilized with 3% (w/v) chloramine T/0.03% (w/v) streptomycin, plus four rounds of sonication. To induce germination, spores were incubated in water at 26°C for 2 weeks.

Medicago truncatula (J5) mycorrhizal roots were obtained in pot cultures. Seeds were surface sterilized with 5% (v/v) sodium hypochlorite for 3 min, rinsed thoroughly with distilled water, and placed in petri dishes with 0.6% (w/v) agar to germinate. Seedlings were then placed in a pot of sterilized quartz sand and 80 to 100 spores. Plants were grown in a growth chamber as described in Bianciotto et al. (2004). Phosphate was added as Na₂HPO₄ 12 H₂O at 0.0032 mM concentration. Roots were sampled after 3 months. Mycorrhizal roots were obtained from Lotus japonicus (Regel) Larsen wild-type and mutant Ljsym4-2 (Bonfante et al., 2000) in a sandwich system as described by Giovannetti et al. (1993); in this case phosphorus was added as 0.0016 mM Na₂HPO₄ 12 H₂O concentration. Plants were kept in a growth chamber (Novero et al., 2002), and the roots were harvested after 1 month. After inspection with the stereomicroscope, 100 1-cm-long root segments of M. truncatula and 50 similar root segments for each genotype of L. japonicus were sampled; they were stained with cotton blue and used to evaluate the intensity of root colonization according to Trouvelot et al. (1986).

Root exudates were obtained as described by Buee et al. (2000) from L. japonicus plants grown in water-agar for 15 d. Germinated spores were treated with root exudates for 2.5 h in the dark at 26°C.

The G. margarita (germinated spores) cDNA library, with an estimated complexity of 50,000 recombinant clones, was constructed into the TriplEx II vector using the SMART cDNA library construction kit (CLONTECH Laboratories, Palo Alto, CA). Individual clones were randomly selected and sequenced (Lanfranco et al., 2000).

H₂O₂ Localization

H₂O₂ production was examined by a DAB assay (Thordal-Christensen et al., 1997). Mycorrhizal roots were incubated in DAB solution (1 mg mL^–1) at room temperature in the dark for 12 h. Samples were then clarified by 1-h wash in lactic acid, mounted on slides, and observed under a light microscope (Eclipse E400; Nikon, Tokyo). To inhibit CuZnSODs, samples were treated before DAB staining with 2 mM DDC for 2 h at room temperature (Delledonne et al., 2001).

PCR Amplifications on Genomic DNA

Genomic DNA was extracted from spores, mycorrhizal roots, or leaves as described by Lanfranco et al. (1999b). PCR reactions were carried out in a final volume of 50 µL containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.1 mM MgCl₂, 0.01% (w/v) gelatin, 200 µM each dNTPs, 1 µM of each primer, 50 to 100 ng of genomic DNA, and 2 units of REDTaq DNA polymerase (Sigma, St. Louis). The PCR program was as follows: 95°C for 3 min (1 cycle), 92°C for45 s, 45 s annealing at temperatures indicated below, 72°C for 45 s (30 cycles), 72°C for 5 min (1 cycle). To amplify the genomic sequence, primers SG1 (5'-AGTTGTGATAATGTCTCAAAAGTC-3') and SG2 (5'-ATCGTCCTTTGATCGCAATCG-3') were employed at an annealing temperature of 50°C.

Oligonucleotides specifically recognizing the G. margarita 18S ribosomal gene, 18S/283 forward (5'-GAATTTCTACCTTCTGGGGAACT-3') and 18S/388 reverse (5'-TCAGACGTAAGCCTGCTTTG-3'), and oligonucleotides specific for GmarCuZnSOD, SOD/229 forward (5'-GCTGGACCTCATTTCAATCCAC-3') and SOD/341 reverse (5'-TGTTCTTTAGCAACGCCATTCAC-3'), were used at an annealing temperature of 60°C. PCR products were separated on 1.2% to 2% (w/v) agarose gels and visualized by ethidium bromide staining. Negative controls for all PCR experiments consisted of reaction mixtures from which template DNA was omitted.

Cloning and Sequence Analysis

The PCR product amplified from genomic DNA was extracted and purified from agarose gels using the QIAEX II gel extraction kit (Qiagen, Hilden, Germany) and directly cloned into the pGEM-T vector (Promega, Madison, WI). XL-2 Blue ultracompetent cells (Stratagene, La Jolla, CA) were transformed and plated onto selective medium following the manufacturer's instructions. Plasmid DNAs were prepared with the Qiagen plasmid mini kit. DNA sequences were determined by GeneLab (Rome) using T7 and Sp6 primers. The sequence of GmarCuZnSOD has been submitted to the GenBank database under accession number AJ640199. DNA sequence analyses were performed with Sequencer (Gene Codes, Ann Arbor, MI) and BLASTX software available through the National Center for Biotechnology Information.

Phylogenetic analysis was performed with CuZnSOD sequences obtained from GenBank databases. Multiple alignment was carried out using ClustalX (version 1.81; Thompson et al., 1994) with the following parameters: matrix, pam; gap open, 10; gap extension, 7.5. Neighbor-joining analysis was performed with the PAUP program (Phylogenetic Analysis Using Parsimony, version 4.0b10; Sinaur Associates, Sunderland, MA; Swofford, 2003), and the phylogenetic tree was constructed and edited with TreeView (Page, 1996). Accession numbers of sequences used in the alignment are given in Figure 2.

Yeast Complementation Assays

The full-length GmarCuZnSOD sequence was amplified under standard PCR conditions using the NotI site-containing primers FLS1 (5'-TGACATTGCGGCCGCATAATGTCTCAAAAGTCTC-3') and FLS2 (5'-ACTTCGAGCGGCCGCTTATTTAAGGTACCCAATA-3') at an annealing temperature of 50°C. The resulting product was digested with NotI and cloned into the dephosphorylated NotI site of the yeast expression vector pFL61 (Minet et al., 1992). The pFL61-GmarCuZnSOD construct or the empty pFL61 vector were then transformed (Rose et al., 1990) into chemically competent DTY116-SOD1 yeast mutant (MAT , trp1-1::SOD1deletion::TRP1 leu2-3,-112 gal1 ura3-50 his-CUP1s, kindly given by Professor T.J. Thiele, Duke University, Durham, NC). Transformants were grown at 30°C for 3 d on selective (minus uracil) SD-agar medium, before being transferred to SD-agar plates containing or not containing 100 µM CdSO₄. Plate assays were conducted in triplicate on three independent transformants.

Real-Time RT-PCR Analyses

For RNA extraction extraradical mycelium and root pieces with external hyphae removed using forceps were collected under a binocular microscope and immediately frozen in liquid nitrogen. RNA was extracted from about 100 quiescent or germinated spores, 50-mg mycorrhizal root pieces, and extraradical mycelium using the SV Total RNA Isolation System kit (Promega). The RNA was precipitated by adding an equal volume of 2 M LiCl, centrifuged at 10,000g for 30 min and resuspended in 25 µL of diethyl pyrocarbonate-treated sterile water. All RNA samples were routinely checked for DNA contamination by RT-PCR analyses conducted with a one-step RT-PCR kit (Qiagen). Reactions were carried out in a final volume of 25 µL containing 5 µL of 5x buffer, 5 µL of Q-solution, 400 µM dNTPs, 0.6 µM of each G. margarita 18S rRNA-specific primer (18S/283+ and 18S/388–), 0.5 µL of one-step RT-PCR Enzyme Mix (Qiagen), and 1 µL of total RNA. Samples were incubated for 30 min at 50°C followed by a 15-min incubation at 95°C. Samples corresponding to RT minus were kept on ice instead of 50°C. Amplification reactions (92°C for 45 s, 60°C for 45 s, 72°C for 45 s) were run for 35 cycles.

To obtain cDNAs from the different samples, RT reactions were performed in a final volume of 20 µL containing 2 µL of 10x buffer, 0.5 mM each dNTPs, 10 µM random primer (Invitrogen, Carlsbad, CA), 1 µL of Sensiscript reverse transcriptase (Qiagen), and 8 µL of RNA. Samples were incubated 60 min at 37°C. To minimize potential differential efficiency of the enzyme, at least two separate RT reactions were pooled for each RNA preparation. cDNAs, prior real-time PCR experiments, were tested in conventional PCR experiments with G. margarita ribosomal primers (18S/283+ and 18S/388–) as described above.

Real-time reactions were carried out in a final volume of 25 µL containing 12.5 µL of iQ SYBR Green Supermix 2X (Bio-Rad Laboratories, Hercules, CA; 100 mM KCl, 40 mM Tris-HCl, pH 8.4, 0.4 mM dNTPs, 50 units/mL iTaq DNA polymerase, 6 mM MgCl₂, 20 nM SYBR Green I, 20 nM fluorescein), 0.3 µM of each oligonucleotide (18S/283 forward and 18S/388 reverse for G. margarita 18S rRNA or SOD/229 forward and SOD/341 reverse for GmarCuZnSOD), and an appropriate amount of cDNAs. The following program was run: 95°C for 3 min (1 cycle) and 95°C for 15 s, 60°C for 30 s (50 cycles) in an iCycler iQTM real-time PCR detection system (Bio-Rad Laboratories). All reactions were performed at least in duplicate. Data were analyzed with the iCycler software. Single amplicons (106- and 113-bp long for the 18S rRNA and GmarCuZnSOD, respectively) were produced by both primer sets. A melting curve (55°C–95°C with a heating rate of 0.5°C per 10 s and continuous fluorescence measurement) was generated at the end of every run to ensure correct identity of the amplified product (Ririe et al., 1997). Standard curves were obtained using recombinant plasmids containing a portion of G. margarita 18S ribosomal gene or the GmarCuZnSOD sequence.

RNA extractions were performed on at least two independent biological samples. Real-time PCR reactions were carried out in triplicate and only comparative threshold cycle (Ct) values leading to a Ct mean with a SD below 0.2 were considered. The Ct method was used to calculate relative GmarCuZnSOD expression levels with the 18S rRNA as a reference (Rasmussen, 2001). Statistical analysis of data has been performed using the program two-way ANOVA with Tukey test as a post-hoc test.

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AJ640199.

	ACKNOWLEDGMENTS

 
We thank Stefano Ghignone for the phylogenetic analysis, Professor Massimo Delledonne for M. truncatula seeds and critical reading of the manuscript, and Dr. Robert Milne for the linguistic revision.

Received July 29, 2004; returned for revision December 1, 2004; accepted December 20, 2004.

	FOOTNOTES

 
¹ This work was supported by grants from the Italian Progetti Ricerca Interesse Nazionale–Ministero Istruzione Università Ricerca and Firb Project (Plant-Microbe Interactions), Cassa di Risparmio di Torino, and Centro Eccellenza Biosensoristica Vegetale Microbica (grant no. D.M. 193/2003).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.050435.

^* Corresponding author; e-mail p.bonfante{at}ipp.cnr.it; fax 39–011–6705962.

	LITERATURE CITED

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