the mycorrhizal fungus gigaspora margarita possesses a cuzn … · the mycorrhizal fungus gigaspora...

The Mycorrhizal Fungus Gigaspora margarita Possessesa CuZn Superoxide Dismutase That Is Up-Regulatedduring Symbiosis with Legume Hosts1

Luisa Lanfranco, Mara Novero, and Paola Bonfante*

Dipartimento di Biologia Vegetale, Universita 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.)

A full-length cDNA showing high similarity to previously described CuZn superoxide dismutases (SODs) was identified in anexpressed 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 heterologouscomplementation assays in a yeast mutant, GmarCuZnSOD encodes a functional polypeptide able to confer increasedtolerance to oxidative stress. The GmarCuZnSOD RNA was differentially expressed during the fungal life cycle; highesttranscript levels were found in fungal structures inside the roots as observed on two host plants, Lotus japonicus and Medicagotruncatula. These structures also reacted positively to 3,3#-diaminobenzidine, used to localize H2O2 accumulation. This H2O2 islikely to be produced by CuZnSOD activity since treatment with a chelator of copper ions, generally used to inhibitCuZnSODs, strongly reduced the 3,3#-diaminobenzidine deposits. A slight induction of GmarCuZnSOD gene expression wasalso observed in germinated spores exposed to L. japonicus root exudates, although the response showed variation inindependent samples. These results provide evidence of the occurrence, in an arbuscular mycorrhizal fungus, of a functionalSOD gene that is modulated during the life cycle and may offer protection as a reactive oxygen species-inactivating systemagainst localized host defense responses raised in arbuscule-containing cells.

Superoxide dismutases (SODs; EC 1.15.1.1) are me-talloproteins found in all aerobic organisms, rapidlyconverting superoxide ðO2

2Þ to hydrogen peroxide(H2O2) and molecular oxygen (Fridovich, 1995). Theyprevent damage to cellular membranes caused byreactive oxygen species (ROS), acting as a primarydefense during oxidative stresses to which organismsare exposed (Natvig et al., 1996). Due to their super-oxide detoxifying capacities, SODs are considered ahallmark of plant defense responses to pathogens.Research has been largely focused on the hypersensi-tive response (HR), which occurs when avirulentpathogens incompatibly interact with resistant hostplants to produce a localized lesion (Lamb and Dixon,1997). In these interactions, the oxidative burst,markedby the production of ROS such as superoxide, hydroxylradicals, and hydrogen peroxide, is thought to inducethe HR either directly by oxidative killing or indirectlyby activating genes involved in programmed cell death(Levine et al., 1994, 1996). Convincing evidence showsthat H2O2 acts as a signal molecule in plants (Alvarezet al., 1998; Neill et al., 2002). Interestingly, Delledonne

et al. (2001) found that in plants, unlike the animalsystems studied, HR is triggered only by a balancedproduction of nitric oxide (NO) and ROS and thatH2O2 enzymatically generated from superoxide byCuZnSOD is crucial for triggering cell death.

ROS and SODs are involved in disease resistancemechanisms other than HR. In the systems Cladospo-rium fulvum-tomato (Lycopersicon esculentum) andErisyphe graminis-barley (Hordeum vulgare), evidencesuggests that the oxidative burst can be decoupledfrom the HR and that H2O2-mediated cross-linking ofcell wall proteins or phenolic substances is of crucialimportance to establish resistance (Vallelian-Bindsche-dler et al., 1998; Kwon and Anderson, 2001). Eventhough a typical HR does not occur in the biotrophicsystem Claviceps purpurea-rye (Secale cereale), accumu-lation of superoxide and H2O2 is observed andlignification is pronounced at the host-pathogen in-terface (Moore et al., 2002).

To add a further level of complexity,manypathogenshave themselves developed ROS-inactivating systems,where catalases and peroxidases, which break downH2O2 to H2O and O2, are the principal enzymaticantioxidants together with SODs. The role of ROS andROS-inactivating systems in microbe pathogenicityand in overcoming host resistance is still an openquestion. Several examples in animal systems showthat bacterial pathogen virulence is correlated with thesecretion of ROS-scavenging enzymes (Mandell, 1975;De Groote et al., 1997). A similar result was recentlyfound for the pathogenic yeastCryptococcus neoformans,which causes meningoencephalitis: a CuZnSOD,

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

* Corresponding author; e-mail [email protected]; fax 39–011–6705962.

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

Plant Physiology, April 2005, Vol. 137, pp. 1319–1330, www.plantphysiol.org � 2005 American Society of Plant Biologists 1319

dispensable in its saprobic life, is critical for pathogen-esis of the fungus (Narasipura et al., 2003). Necrotro-phic fungi are hypothesized to cope with a hostileenvironment thanks to an array of ROS-inactivatingenzymes such as SOD, peroxidase, catalase, and per-haps laccase and polyphenol oxidases (Mayer et al.,2001). However, in Claviceps purpurea the lack ofa CuZnSOD, evidenced by targeted gene disruption,does not significantly reduce fungal virulence on rye(Moore et al., 2002). That appears to be the only reportshowing that a SOD is not essential for a fungalpathogen, and its significance is uncertain.

ROS production and antioxidant systems from theplant and/or the microorganism also play a role dur-ing symbiotic interactions. In root nodules a delicateequilibrium is required to supply the energy demandsof nitrogen reduction and to protect the nitrogenasecomplex from the ROS produced, suggesting thatantioxidant activities from both plants and bacte-ria are essential to establish a functional symbiosis(Matamoros et al., 2003). A critical protective role ofa bacterial SOD was described in the Sinorhizobium-legume symbiosis: SOD effects extend far beyondprotection of the nitrogenase complex since defects inthe mutant strain were observed in all steps of sym-biosis, including infection, nodulation, and bacteroiddifferentiation. This suggests that oxidative stress, un-less counteracted by SOD, interferes at several steps insymbiosis (Santos et al., 2000). The demonstration thatNod factors do not elicit an early oxidative burst inMedicago truncatula roots provides further evidence ofthe differences between pathogenic and symbioticinteractions (Shaw and Long, 2003).

Arbuscular mycorrhiza (AM) is the most wide-spread type of symbiosis and a unique example ofa fully compatible interaction between plants andfungi (Harrison, 1999; Gadkar et al., 2001), but thereis little information in this field about ROS productionand ROS inactivation. The intimate association offungal and plant tissues implies that the fungusmust be recognized by the plant, which sets upa complex accommodation process whose geneticdeterminants have, at least in part, been identified(Parniske, 2004). The induction/suppression of mech-anisms associated with plant defense also plays a keyrole in AM colonization compatibility with the host.Expression of genes related to plant defense, investi-gated using specific probes (for review, see Garcia-Garrido and Ocampo, 2002) or large-scale approaches(Journet et al., 2002; Liu et al., 2003), demonstratesa transient and weak defense response during theinitial phases of colonization, usually followed byactivation of defense-related genes in cells containingarbuscules.

Changes in profiles of antioxidative enzymes suchas SOD, catalases and peroxidases, have been ob-served in mycorrhizal roots, and CuZnSOD activitywas hypothesized for Glomus mosseae spores (Palmaet al., 1993; Blilou et al., 2000). The molecular data referonly to host-plant genes. Catalase and peroxidase

expression, detected in arbuscule-containing cells ofbean (Phaseolus vulgaris) and wheat (Triticum aestivum)colonized by Glomus intraradices, was suggested toresult from localized activation of defense mecha-nisms (Blee and Anderson, 2000). Gene fragmentsencoding two MnSOD and one FeSOD were identifiedin lettuce (Lactuca sativa; Ruiz-Lozano et al., 2001).Their expression was down-regulated in normal con-ditions, while under drought stress theMnSOD II genewas induced in mycorrhizal roots, suggesting a pro-tective mechanism against drought. Genes expressedduring development of AM symbiosis betweenM. truncatula and Glomus versiforme have recentlybeen analyzed by cDNA macroarray (Liu et al.,2003). Two clones, AW587301 and AW584200, showinghigh similarity to plant CuZnSOD and FeSOD, re-spectively, did not show significant changes of theirexpressionduringmycorrhizal development (Liu et al.,2003). In contrast with ectomycorrhizal fungi whereSOD genes have been identified and characterized(Jacob et al., 2001), molecular data on genes coding forROS-scavenging enzymes in AM fungi are not avail-able.

Here, we describe the cloning and the functionalcharacterization of a CuZnSOD gene from the AMfungus Gigaspora margarita and present evidence thatthis gene is differentially expressed during the fungallife cycle.

RESULTS

Identification and Characterization of GmarCuZnSOD

A full-length cDNA showing high similarity topreviously described CuZnSODs (SOD; EC 1.15.1.1)was identified in an expressed sequence tag collectionfrom G. margarita germinated spores (Lanfranco et al.,2000). This cDNAwas named GmarCuZnSOD accord-ing to the nomenclature proposed for AM fungalgenes (Franken, 2002). To confirm the fungal originof the cDNA the genomic sequence of GmarCuZnSODwas obtained from PCR experiments carried out onspore genomic DNA with oligonucleotide primersdesigned on the 5#- and 3#-untranslated regions of theGmarCuZnSOD cDNA. Comparison between genomicand cDNA sequences showed that three introns,limited by consensus splice junctions, are present inthe GmarCuZnSOD gene at positions 162, 736, and 936(Fig. 1). The introns, shown in lowercase letters inFigure 1, are 379, 93, and 82 bp long, respectively. TheGC content of the genomic sequence is relatively low(33.76%) and characteristic for AM fungi (Hosny et al.,1997).

The protein sequence (158 amino acids) showsamino acid residues that are important for enzymestructure and activity: six His (H) and one Asp (D) areresponsible for binding copper (Cu) and zinc (Zn)ions, two Cys (S) are involved in the formation ofa disulfide bridge, and one Arg residue constitutes the

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catalytic site of the enzyme (Steinman and Ely, 1980;Chary et al., 1990). No evidence for signal peptides inthe N-terminal sequence was obtained using theSignalP program available at the Expasy MolecularBiology Server (http://www.expasy.org), suggestingthat it is an intracellular SOD.A phylogenetic analysis was carried out on CuZnSOD

amino acid sequences ranging from bacteria to hu-mans, available in databases (Fig. 2). Sequences fromhigher eukaryotes, plants, and animals form distinctclusters. Fungal sequences group together and, sur-prisingly, GmarCuZnSOD forms a group distinct fromthis cluster. These two clusters are supported by a treebranch also including some sequences from animals,although its bootstrap value is low (below 50%).

Complementation Assays

To test whether GmarCuZnSOD codes a functionalprotein, a complementation assay was performed ina yeast mutant defective in the CuZnSOD gene. Themutant was transformed with the complete openreading frame of GmarCuZnSOD cDNA placed underthe control of a constitutive yeast promoter in the ex-pression vector pFL61 (Minet et al., 1992). As a control,the yeast strain was also transformed with the vectoralone. To induce oxidative stress, transformed yeastswere plated on synthetic dextrose (SD)-agar mediumsupplemented with CdSO4 (Brennan and Schiestl,

1996). The two yeast transformants were streakedonto SD minus uracil plates containing 0 or 100 mM

CdSO4. As shown in Figure 3, cells carrying the pFL61vector grew only in the medium without cadmium. Bycontrast, GmarCuZnSOD-transformed cells also de-veloped colonies in the presence of heavy metal (Fig.3). This result indicates that the gene, at least ina heterologous system, can confer tolerance to oxida-tive stress.

Quantification of Mycorrhizal Intensity

To understand whether GmarCuZnSOD was differ-entially expressed, colonization experiments weredone using root segments of M. truncatula and Lotusjaponicus, considered model plants when plant/microbe interactions are investigated, due to theirsymbiotic capacities. A mutant of L. japonicus wasalso used. Colonization by G. margarita was successfulwith both the wild-type plants. To evaluate the in-tensity of root colonization, four parameters wereconsidered: frequency of mycorrhization (F%), inten-sity of mycorrhization (M%), percentage of arbusculesin the colonized portion of the root (a%), and percent-age of arbuscules in the root overall (A%; Trouvelotet al., 1986). As shown in Table I, the confined-sandwich method led to an excellent percentage ofcolonization in L. japonicus wild type, in comparisonwith the pot culture of M. truncatula. As expected, thefungus did not develop in the L. japonicus mutantLjsym4-2 (Novero et al., 2002). In this mutant, fungalpenetration is limited to epidermal cells that appeared,after infection attempts, morphologically dead. Abor-tion of colonization also was associated with localizeddeath of fungal hyphae (Bonfante et al., 2000).

GmarCuZnSOD mRNA Expression Analysis:

Presymbiotic and Symbiotic Phase

GmarCuZnSOD mRNA expression levels were ana-lyzed throughout the fungal life cycle by real-timereverse transcription (RT)-PCR. Specific primers weredesigned on GmarCuZnSOD and on the ribosomalgene 18S of G. margarita, considered as a housekeepinggene. To exclude cross-hybridizations with plant ma-terial, conventional PCR reactions were performedon L. japonicus and M. truncatula genomic DNAs; allprimers gave negative results with these (data notshown).

cDNA samples were prepared from quiescentspores, germinated spores, extraradical mycelium col-lected from L. japonicus and M. truncatula mycorrhizalroots, and from mycorrhizal root pieces with externalhyphae removed. In addition, samples of G. margaritamycelium grown on the mutant Ljsym4-2 were alsotested. Samples were calibrated using the fungal 18SrRNA transcript, and to calculate relative expression,quiescent spores were taken as a reference sample. Theresults obtained from L. japonicus are summarized inFigure 4A. A slight induction was observed in samples

Figure 1. Nucleotide and deduced amino acid sequence of Gmar-CuZnSOD. The intron sequence is shown in lowercase letters. Aminoacid residues important for the catalytic activity are underlined.

A CuZn Superoxide Dismutase from an Arbuscular Mycorrhizal Fungus

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corresponding to germinating spores and extraradi-cal mycelium compared to quiescent spores. How-ever, these values were not statistically significant(P 5 0.261 and P 5 0.280, respectively). The externalmycelium growing on Ljsym4-2 did not show anydifference when compared to extraradical mycelium

growing on a wild-type plant (data not shown). Thehighest induction was observed in colonized rootsfrom which external mycelium was eliminated. Statis-tical analyses of data indicated a significant differ-ence in the expression level between this conditionand quiescent spores (P 5 0.000), germinating spores

Figure 2. Neighbor-joining tree obtained from the alignment of GmarCuZnSOD gene product from G. margarita (in bold) andCuZnSODs sequences of other organisms retrieved from database. Salmonella typhimurium AAB62385 was used as outgrouptaxon to root the tree. Bootstrap values above 50% are indicated (1,000 replicates). Branch lengths are proportional to geneticdistance which is indicated by a bar at the bottom left.

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(P 5 0.003), and external hyphae (P 5 0.002). Similarexpression profiles were obtained in M. truncatulasamples (Fig. 4B); the GmarCuZnSOD transcript wassignificantly (P , 0.05) more abundant in intraradicalfungal structures compared to the other three condi-tions.

GmarCuZnSOD mRNA Expression Analysis:

The Effects of Root Exudates

We also investigated whether GmarCuZnSOD couldbe involved in the early stages of interaction andbepartof signaling events occurring before a direct contactwith host roots. In particular, we analyzed whetherGmarCuZnSOD gene expression responds to plantmolecules that are present in root exudates and havebeen shown to induce hyphal branching in AM fungi(Buee et al., 2000; Tamasloukht et al., 2003). TheGmarCuZnSOD expression level was thus studied ingerminated spores treatedwith root exudates preparedfrom 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 5 0.187). Wehypothesized that the high variability existingamong batches of G. margarita (Jargeat et al., 2004;V. Bianciotto, personal communication) could explainthe different response to root exudates.

H2O2 Localization

To answer the question whether fungal SOD activitywas responsible for localized H2O2 production, we did3,3#-diaminobenzidine (DAB) assays. To evaluatewhether H2O2 was produced by CuZnSODs, rootsamples were pretreated with sodium diethyldithio-carbamate (DDC), a copper ion chelator generally usedto inhibit CuZnSODs (Delledonne et al., 2001). Pro-duction of H2O2 was investigated in mycorrhizal rootsof M. truncatula grown in pot cultures and of L.japonicus (wild type and mutant Ljsym4-2) grown inthe Millipore sandwich system (Giovannetti et al.,1993) as well as in nonmycorrhizal control roots.

Dark deposits that mark the DAB reaction andindicate H2O2 production were consistently observedin mycorrhizal tissues (Figs. 6A and 7A), whereasnonmycorrhizal roots never reacted, with the excep-tion of vascular tissues and meristematic regions (Figs.6, G and H, and 7G). This latter unexpected result is inagreement with a preliminary observation reported byShaw and Long (2003).

The DAB reaction in mycorrhizal wild-type L. japo-nicus plants was associated with extraradical hyphae(Fig. 6A) aswell as intraradical fungal structures (Fig. 6,A–D) and was particularly evident in mature arbus-cules (Fig. 6C), where labeling appeared more intenseon the trunks and collapsing terminal branches. Otheryounger arbuscules located in adjacent cortical cellswere less reactive (Fig. 6, C and D) or not labeled at all.In all experiments, host cells were very weakly reactiveor not at all. DDC treatment strongly inhibited thereaction of intracellular arbuscules (Fig. 6E), whereas itwas still, at least in part, present in extraradical hyphae(Fig. 6F). The same pattern (extracellular hyphaestrongly labeled by DAB but not inhibited by DDCtreatment) was observed when the fungus profuselydeveloped at the surface of the mutant Ljsym4-2 (datanot shown). Plant tissues, including epidermal cells ofthe mutant line Ljsym4-2 (data not shown), reacted toDAB similarly to nonmycorrhizal roots.

Figure 3. Increased oxidative stress tolerance conferred by Gmar-CuZnSOD in a yeast mutant defective of CuZnSOD. Yeast mutantsharboring either the pFL61-GmarCuZnSOD plasmid (pFLSOD) or theempty pFL61 vector (pFL61) were grown on SD (2uracil) agar plateswith 0 or 100 mM cadmium sulfate (100 mM Cd).

Table I. Intensity of root colonization by G. margarita

Four parameters were considered according with Trouvelot et al. (1986).

F%a M%b a%c A%d

L. japonicus wild type 72.3 31.4 72.9 22.9L. japonicus Ljsym4-2 0 0 0 0M. truncatula 15 2.59 66 1.71

aIndicates the percentage of root segments showing internal colonization. bIndicates the averagepercent colonization of roots segments. cIndicates the average presence of arbuscules within theinfected areas. dIndicates the presence of arbuscules in the whole root apparatus.



Similar results were obtained on M. truncatulamycorrhizal roots, growing in pots and probably ina more natural situation. The extraradical mycelium,intercellular hyphae, and arbuscules often reactedstrongly to DAB staining (Fig. 7, A, C, E, and F),whereas the plant tissues were never stained with theexception of vascular tissues (Fig. 7G) and meristemcells (data not shown). Staining was particularly in-tense on collapsing arbuscules (Fig. 7F), where theDAB reaction was homogenous. By contrast, otherarbuscules reacted weakly. When the roots weretreated with DDC, the reaction was limited to extra-radical mycelium (Fig. 7D), being absent in the intra-radical mycelium (Fig. 7B).

Taken together, the morphological data show thatthe H2O2 is produced in both intraradical and extra-radical compartments by the fungus. However, fol-lowing the use of DDC, only the H2O2 associated withintraradical structures seemed to be produced by thefungal CuZnSOD. Other metabolic pathways mayhave been responsible for the DAB deposits associatedwith the extraradical mycelium (Neill et al., 2002;Mittler et al., 2004).

DISCUSSION

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

GmarCuZnSOD presents amino acid residues typ-ical of CuZnSODs and important for enzyme structureand activity (Steinman and Ely, 1980; Chary et al.,1990). The sequence does not contain an N-terminalsignal peptide for secretion, reinforcing the claim thatfungal CuZnSODs have a cytoplasmic localization.There is, however, evidence that CuZnSODs lackingan N-terminal signal peptide in Aspergillus fumigatus,C. neoformans, and Claviceps purpurea are neverthelesssecreted (Hamilton et al., 1996; Hamilton and Holdom,1997, Moore et al., 2002). Phylogenetic analysis showsthat GmarCuZnSOD does not cluster with the otherfungal sequences. This could be due to the limitednumber of fungal sequences covering all differentphyla present in data banks (most of them belong toAscomycetes) or to the fact that AM fungi are a veryancient group clearly distinct from Ascomycetes andBasidiomycetes (Schußler et al., 2001).

Functional characterization of GmarCuZnSODshowed that the gene can confer increased toleranceto oxidative stress in a yeast mutant defective withrespect to CuZnSOD. Functional analysis of AM fungalgenes has to be performed in heterologous systemssince no mutants are available at the moment for thisgroup of organisms. Transformation technology hasbeen applied to AM fungi with promising results(Harrier and Millam, 2001). In the near future new

Figure 4. Real-time RT-PCR analysis of the GmarCuZnSOD mRNAduring asymbiotic phases (quiescent and germinating spores) andsymbiotic phases (extraradical and intraradical mycelium from L.japonicus [A] or M. truncatula [B]). Relative expression levels wereobtained with the Ct method (see ‘‘Materials and Methods’’ for details)and were normalized with respect to GmarCuZnSOD levels inquiescent spores.

Figure 5. Real-time RT-PCR analysis of the GmarCuZnSOD mRNA ingerminating spores after exposure to water or L. japonicus rootexudates. Relative expression levels were obtained with the Ct method(see ‘‘Materials and Methods’’ for details) and were normalized withrespect to GmarCuZnSOD levels in spores treated with water.

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techniques such as RNA interference, already success-fully applied in several other systems (Hannon, 2002),may make it possible to silence specific genes also inAM fungi and to check their functions directly.

GmarCuZnSOD Is Up-Regulated during Symbiosis

GmarCuZnSOD expression is regulated duringthe different phases of the fungal life cycle. A lowexpression level was observed in quiescent spores aswell as in germinated ones. The last result was tobe expected since the sequence was identified as anexpressed sequence tag (EST) clone from germinatedspores (Lanfranco et al., 2000). Other genes involved instress responses were found within this ESTcollection,suggesting that in vitro germination is not the mostfavorable growth condition for G. margarita (Lanfrancoet 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 producedby mechanisms that did not exclusively involve CuZn-SOD. This seems a consistent result, as externalhyphae growing on different host plants and in dif-ferent experimental conditions behave in a similar way.It might be that H2O2 is produced following electrontransport in mitochondria, during which it is thoughto be generated from superoxide presumably in anuncontrolled manner (Neill et al., 2002). However, atleast in plants, there is likely to be more than oneenzymatic source of H2O2 produced in response tospecific abiotic and biotic stimuli. Potential candidatesinclude NADPH oxidases, cell wall peroxidases, amineoxidases, oxalate oxidases, and flavin-containing oxi-dases (Neill et al., 2002; Mittler et al., 2004). Deeperinvestigation of ROS-generating and ROS-scavenging

Figure 6. L. japonicus roots colonized byG. margarita after DAB reaction. A, Darkdeposits, which are the marker of the DABreaction and are indicative of H2O2 pro-duction, are consistently observed associ-ated with many of the intraradical (Ih) andextraradical hyphae (Eh). Bar correspondsto 200 mm. B, Higher magnification of theprevious image where the dark depositsassociated with the intraradical hyphae(Ih) and arbuscules (A) are particularlyevident. The host cells do not show anyreaction (cc). Bar corresponds to 100 mm.C, The reaction is evident in mature ar-buscules (Ma), while other younger arbus-cules (Ya, arrowheads), which are locatedin close cortical cells, are less reactive.The labeling seems to be more intense onthe trunks (T). A diffuse yellow color ispresent in the host cells. Bar correspondsto 50 mm. D, Younger arbuscules (Ya)where the thin branches are still visibleare not reactive as well as some of theintercellular hypha (Ih). In this section thedark deposit is particularly evident on thetrunks (T). Bar corresponds to 60 mm. E,The treatment with DDC, a copper chela-tor usually used to block CuZnSOD,strongly inhibits the reaction associatedto the intraradical fungal structures: botharbuscules (A) and intercellular hyphae.Bar corresponds to 70 mm. F, After DDCtreatment the dark precipitate is onlyweakly quenched in the extraradical hy-phae (Eh). Bar corresponds to 200 mm. G,Differentiated tissues from nonmycorrhi-zal roots do not show any reactivity toDAB staining. Bar corresponds to 200 mm.H, Root tips with the meristematic regions(Me) are the only root regions to be re-active to DAB staining. Bar corresponds to500 mm.



systems from AM fungi will help to clarify whyexternal hyphae are so reactive to DAB.

The highest transcript levels were found in fungalstructures developing inside the root. This was ob-tained from two host plants colonized under twoexperimental conditions. Up-regulation of a fungalSOD was not reported in the M. truncatula-G. versi-forme system where a global analysis of genes ex-pressed during the development of AM symbiosis wasperformed by cDNA macroarray (Liu et al., 2003).However, as the authors stated, only 5% of the genesinvestigated belonged to the fungal genome. On theplant side, expression of at least a CuZnSOD and anFeSOD did not significantly change during mycorrhi-zal development (Liu et al., 2003). Interestingly, up-regulation of a fungal SOD in mycorrhizal tissues ascompared to the free-living mycelium was found bycDNA microarray analyses in the interaction betweenthe ectomycorrhizal fungus Paxillus involutus andBetula pendula (Johansson et al., 2004).

In our study, the intraradical fungal structures (in-tercellular hyphae and arbuscules) of bothmycorrhizalplants were also DAB-positive, whereas plant tissueswere only faintly so. The H2O2 production is likely due

to CuZnSOD activity since treatment with DDCstrongly reduced the DAB deposits. A previous studyusing the same DAB assay showed H2O2 accumulationin cells of M. truncatula colonized by G. intraradices(Salzer et al., 1999). However, H2O2 production washypothesized by these authors to be a consequence ofactivation of a plant plasma membrane NADPH oxi-dase, analogous to what occurs during theHR (Bolwellet al., 2002). Molecular evidence for elicitation of a HRand supporting Salzer’s hypothesis is not so far avail-able, and further investigations of H2O2-producingsystems in plants challenged by AM fungi are neededto clarify this issue. Possible candidates might begermin-like proteins (GLPs), which have been foundto be up-regulated in mycorrhizal roots (Doll et al.,2003). Similar results came from a large-scale analysisof gene expression based on EST collection fromM. truncatula (http://medicago.toulouse.inra.fr/Mt/EST/). Some true germins from Gramineae displayoxalate oxidase activity producing H2O2 and CO2 fromoxalic acid; other GLPs have been identified as SODs(Yamahara et al., 1999; Carter and Thornburg, 2000,Christensen et al., 2004), but until nowmost attempts toascribe oxalate oxidase/SOD activity to GLPs from

Figure 7. M. truncatula roots colonized by G.margarita after DAB reaction. A, The dark pre-cipitates, indicative of H2O2 production, areassociated with the intercellular hyphae (Ih) aswell as with the arbuscules (A). Bar correspondsto 220 mm. B, When the roots are treated withthe copper chelator, DDC, the reaction is fullyquenched in all the intraradical structures (Ih andA). Bar corresponds to 270 mm. C, The darkprecipitates are associated with extraradical hy-phae (Eh). Bar corresponds to 100 mm. D, Whenthe roots are treated with DDC, the reaction isonly partly quenched. Eh, Extraradical hyphae.Bar corresponds to 100 mm. E, Detail of twoarbuscules (arrowheads) differently reactive tothe DAB staining. The trunk (T) of the bottom oneis clearly reactive as well as the intercellularhyphae (Ih). Bar corresponds to 60 mm. F, Detailof a collapsing arbuscule (Ca) were the DABreaction has produced a homogenous black de-posit filling up the whole cell lumen. Bar corre-sponds to 60 mm. G, Nonmycorrhizal root tissuesdo not show any reaction in the differentiatedregions, with the exception of vascular tissues(Vv). Bar corresponds to 200 mm.

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dicotyledonous plants have been negative (Ohmiyaet al., 1998; Carter and Thornburg, 2000).In our experiments, quantitative expression analy-

sis and DAB staining showed a similar trend, sug-gesting that GmarCuZnSOD could be responsible forlocalized H2O2 accumulation in intracellular fungalstructures. We can speculate about the possible rolesof fungal SOD activity. Two possible explanations, notnecessarily exclusive, can be proposed. Several in situhybridization-based studies have shown that in my-corrhizal 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 sys-tems, such as SOD, might be required to control andovercome the range of plant defense responses at asite crucial to AM symbiotic function. The host plantscan contribute to maintain arbuscule function. In fact,a gene coding for a plant leghemoglobin, Vfl29, wasrecently shown to be specifically up-regulated inarbuscule-containing cells (Vieweg et al., 2004). Besidethe role in oxygen supply, plant leghemoglobins arethought to bind NO, a crucial molecule involved inplant defense against pathogens (Delledonne et al.,2001; Neill et al., 2002). The authors speculated thatthis leghemoglobin gene, through the ability to en-code NO-scavenging activity, could help to suppressdefense responses in arbuscule-containing cells(Vieweg et al., 2004). Another interpretation relies onthe evidence that arbuscules are terminal fungal struc-tures; after arbuscule formation the fate of these finehyphal branches is to collapse (Bonfante, 1984). Noth-ing is known about the molecular mechanisms un-derlying this process, but nuclei in the fine arbusculehyphae are pycnotic and electrondense with amorphology typical of cells undergoing apoptosis(Balestrini et al., 1992). We can speculate that ROS,and in particular H2O2, may act in arbuscules as amolecule signaling programmed cell death, as it hasbeen 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 ofmolecular signals exchanged between the plant andthe fungus during early stages of the interaction(Parniske, 2004). The existence of a fungal factor hasrecently been proposed on the basis of the transcrip-tional activation of a nodulin gene by a diffusiblemolecule originating from germinating hyphae of AMfungi (Kosuta et al., 2003). On the other side, someinvestigations have been performed on the plantfactor. Although the chemical structure of the specificcompound or compounds is still unknown, it has beenshown that root exudates frommycotrophic plants canenhance growth and elicit hyphal branching (Bueeet al., 2000).

The observation that the expression levels of Gmar-CuZnSOD are enhanced following exposure to plantroot exudates is quite interesting, since it could repre-sent a tool for the analysis of plant-fungus interactionbefore the colonization process. However, we observeda large variability in spore response. Each spore sam-ple, consisting of about 100 spores coming from differ-ent batches, is not a very homogeneous biologicalmaterial. Over the last decade a high genetic diver-sity within AM species and even within individualAM fungal spores, including this G. margarita isolate(Lanfranco et al., 1999a), has been reported (Sanders,2004). It is still unknownwhether this genetic variationmay reveal important biological consequences on thephenotype and thefitness of the fungus (Sanders, 2004).As far as the specific G. margarita strain used in thisstudy is concerned, different batches of spores havelarge phenotypic variability for example in germina-tion capability (V. Bianciotto, personal communica-tion) and in the abundance of endosymbiotic bacteria(Jargeat et al., 2004). This intrinsic genetic diversitymayexplain the variability in the response to root exudatesof different batches of germinating spores.

An early event in root exudate perception is stim-ulation of fungal respiratory activity, mirrored by theconcomitant induction of mitochondria-related genes(Tamasloukht et al., 2003). The higher transcript levelof GmarCuZnSOD might reflect a need to remove theexcess ROS generated during the respiration increase.It is tempting to speculate that, in addition to theprotection against ROS, CuZnSOD might be involvedin root exudate perception and might mediate fungalmorphogenetic responses. Evidence for involvementof CuZnSOD in the control of morphogenetic pro-cesses comes from Neurospora crassa, in which specificinactivation of a CuZnSOD has been shown to affectmorphogenetic responses to light, such as carotenoidsynthesis and perithecium polarity (Yoshida andHasunuma, 2004).

CONCLUSION

We have identified a functional homolog of a CuZn-SOD in an AM fungus. The gene is differentiallyexpressed during the interaction with the host plant,suggesting potentially different roles. GmarCuZnSODis expressed when the fungus is metabolically active,leading to a basal production of H2O2 similar to thatreported in plants (Shaw and Long, 2003). During thesymbiotic phase it seems to be responsible for a micro-localized oxidative burst mainly associated with thecollapsing of arbuscule branches. In this specific site,H2O2 may also act as a factor required for signalingfungal cell death.

In conclusion, our results provide evidence thatfungal ROS-scavenging systems, such as SOD, may becomponents of the plant/fungus dialogue, allowingfunctional and structural compatibility between thepartners.



MATERIALS AND METHODS

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 in-

spection 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 lTriplEx II

vector using the SMART cDNA library construction kit (CLONTECH Labo-

ratories, Palo Alto, CA). Individual clones were randomly selected and se-

quenced (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 mgmL21) 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 DNAwas 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 mL containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.1 mM

MgCl2, 0.01% (w/v) gelatin, 200 mM each dNTPs, 1 mM 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 for

45 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#-ATCGTCCTTT-

GATCGCAATCG-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 spe-

cific for GmarCuZnSOD, SOD/229 forward (5#-GCTGGACCTCATTTCAATC-

CAC-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 stain-

ing. Negative controls for all PCR experiments consisted of reaction mixtures

from which template DNAwas 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 Informa-

tion.

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: ma-

trix, pam; gap open, 10; gap extension, 7.5. Neighbor-joining analysis was

performed with the PAUP program (Phylogenetic Analysis Using Parsi-

mony, 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#-TGACA-

TTGCGGCCGCATAATGTCTCAAAAGTCTC-3#) and FLS2 (5#-ACTTCGAG-

CGGCCGCTTATTTAAGGTACCCAATA-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-

DSOD1 yeast mutant (MAT a, trp1-1TSOD1deletionTTRP1 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 mM 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. RNAwas extracted from about 100

quiescent or germinated spores, 50-mg mycorrhizal root pieces, and extra-

radical mycelium using the SV Total RNA Isolation System kit (Promega). The

RNAwas precipitated by adding an equal volume of 2 M LiCl, centrifuged at

10,000g for 30 min and resuspended in 25 mL of diethyl pyrocarbonate-treated

sterile water. All RNA samples were routinely checked for DNA contamina-

tion by RT-PCR analyses conducted with a one-step RT-PCR kit (Qiagen).

Reactions were carried out in a final volume of 25 mL containing 5 mL of 53

buffer, 5 mL of Q-solution, 400 mM dNTPs, 0.6 mM of each G. margarita 18S

rRNA-specific primer (18S/2831 and 18S/3882), 0.5 mL of one-step RT-PCR

Enzyme Mix (Qiagen), and 1 mL 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�Cfor 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 mL containing 2 mL of 103 buffer, 0.5 mM each dNTPs,

10 mM random primer (Invitrogen, Carlsbad, CA), 1 mL of Sensiscript reverse

transcriptase (Qiagen), and 8 mL 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/2831 and 18S/3882) as described

above.

Real-time reactions were carried out in a final volume of 25 mL

containing 12.5 mL 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 mM 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�Cfor 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

Lanfranco et al.


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 Gmar-

CuZnSOD expression levels with the 18S rRNA as a reference (Rasmussen,

2001). Statistical analysis of data has been performed using the program two-

way ANOVAwith 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.

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