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Home , Arbuscular mycorrhiza, Root nodule

The Cell, Vol. 8, 1871-1883, October 1996 O 1996 American Society of Plant Physiologists

Plant Cell Responses to Arbuscular Mycorrhizal Fungi: Getting to the of the

Vivienne Gianinazzi-Pearson Laboratoire de Phytoparasitologie INRAICNRS, Station de Génétique et dAm6lioration des Plantes, lnstitut National de Ia Recherche Agronomique, BV 1540, 21034 Dijon cedex, France

INTRODUCTION

Since their colonization of terrestrial ecosystems, have essential genetic determinants for AM establishment are com- developed numerous strategies to cope with the diverse bi- mon to an extensive part of the plant kingdom. otic and abiotic challenges that are a consequence of their In contrast to their extremely wide host range and despite sedentary life cycle. One of the most successful strategies is their ancient origins, only six genera of fungi belonging to the the ability of systems to establish mutualistic and recipro- order Glomales of the Zygomycetes have evolved the ability cally beneficial symbiotic relationships with . to form AM (Morton and Benny, 1990). lnteractions between , the intricate associations roots form with specific an AM and a plant begin when a from a ger- fungal groups, are by far the most frequent of these and rep- minating soilborne spore comes into contact with a host root. resent the underground absorbing organs of most plants in This step is followed by induction of an appressorium, from (Gianinazzi-Pearson, 1984). Through their function in which an infection hypha penetrates deep into the parenchyma the efficient exploitation of mineral resources and their cortex (Figure lA), where inter- and intracellular proliferation bioprotective role against a number of common soilborne of is intense. Here, fungal development culminates , mycorrhizas are instrumental in the survival and in the differentiation of intracellular haustoria, known as ar- fitness of many plant taxa in diverse ecosystems, including buscules (Figure 16). These fungal structures, which establish many species (reviewed in Allen, 1991; Bethlenfalvay and a large surface of contact with the plant protoplast, are attrib- Linderman, 1992). uted a key role in reciproca1 exchange between the Severa1 kinds of mycorrhizal associations can be distin- plant cells and the AM fungal symbionts (Smith and Smith, guished according to their morphology and the plant and fungal 1990). However, arbuscules are ephemeral structures, and an taxa concerned. They fall almost exclusively into two broad individual arbuscule reaches full development within severa1 groups: (1) the of woody Angiosperms and days, after which it begins to senesce (Alexander et al., 1988). Gymnosperms, in which Basidiomycetes, Ascomycetes, or AM developinent continues within a root system as the fungus Zygomycetes develop intercellular hyphae from a mycelial spreads to newly emerging roots. In this way, fungal coloniza- sheath covering the surface of short lateral roots; and (2) the tion occurs concomitantly in different roots in an unsynchronized endomycorrhizas, characterized by intraradical mycelium manner. growth and intracellular fungal proliferation, which are formed There have been extensive studies of AM over the past two by Basidiomycetes in the Orchidaceae (orchidoid ), decades, but information concerning the cellular and molec- Ascomycetes in the Ericales (), and Zygomy- ular aspects has remained largely descriptive. This is partly cetes in most other terrestrial plant taxa (; dueto the complexity of the biological system and to the sta- reviewed in Harley and Smith, 1983). tus of AM fungi as obligate symbionts; thus, there is a lack Plant compatibility with mycorrhizal fungi is a generalized of knowledge about their and genetic constitution. and ancient phenomenon. Species in >80% of extant plant However, recently, significant steps have been made toward families are capable of establishing arbuscular mycorrhiza unraveling plant mechanisms governing AM symbioses (AM), and fossil evidence suggests that symbioses of this kind through approaches using mycorrhiza-defective plant mutants, existed >400 million years ago in the tissues of the first land molecular cytology and molecular biology techniques, and plants (Pirozynski and Dalpé, 1989; Remy et al., 1994). As such, comparisons of plant interactionswith microorganismsin other the ability of plants to form AM must be under the control of systems. This review highlights current knowledge about the mechanisms that have been conserved in new plant taxa as colonization process in AM, the properties of the ensuing sym- they appeared during evolution. This compatibility also implies biotic interface, the plant genes involved, and the molecular that selective recognition processes in plants discriminate be- nature of host plant responses to AM fungi. The impact of prog- tween beneficial and harmful microorganisms and that the ress in these different areas is discussed in relation to Plane Th 187t Cel2 l

Figure 1. Phenotypes of Interactions between Roots and an Arbuscular Mycorrhizal Fungus ( mosseae). (A) and (B) Wild-type genotype (cv Frisson). (A) shows an infection hypha penetrating outer root tissues from an appressorium (ap), and intercellu- lar proliferation (ih) in the cortical parenchyma where intracellular, highly branched arbuscules (ar) form (B). ) Myc~(C ' mutant (P2Frissonv c f )o . Appressoria (ap) differentiat rooe t develoth tt no ea surfac o d p t infectioebu n hyphae. ) Myc~(D 2 mutant (RisNod24 Finalev c f o ) . Intercellular hyphae (ih) coloniz corticae eth l parenchym f roota o t onlsbu y form stumpy branches (st) inside root cells. Funga t 90°a l 0.1n Cn i tissu mi staine %s 5 1 etrypawa r dfo n blue after clearin KOH% t 90°10 a r g.n Ci h root1 r sfo urn0 Bar2 .s=

perceiving not only the basis of widespread plant compatibil- (reviewed in Phillips and Tsai, 1992), they are not absolutely ity wit fungiM ht A als relevance bu ,o th f thieo s symbiosiso t essential to host-fungus interactions (Becard et al., 1995). the understanding of other plant-microbe interactions. The earliest defined recognition phenomeno fungue th n i s host-induces i d hyphal branching followe formatioe th y db n of appressoria at the root surface. These responses can occur ROOT COLONIZATION PROCESSES aafter sh earl6 r 3 inoculatios ya hose th f tn o plan t (Giovannetti et al., 1994). Cells of the and hypodermis in contact with these first infection structures sho significano wn t cyto- Recognitio Celd nan l Response logical modifications or responses typical of plant defense (Gianinazzi-Pearson et al., 1996a), and the fungus invades the The cellular adaptation necessary for morphological integra- root intercellularl intracellularlyr yo . Plant reactio hypha no t a reciprocatiod nan l functional compatibility betwee plane nth t crossin epiderman ga r hypodermao l l cel apparentls i l - yre and fungal cells in AM must depend on finely tuned recogni- stricted to the synthesis of a host-derived membrane and the tion processes and on highly evolved molecular coordination deposition of cell wall material continually around the fungus. betwee twoe nth . Signal exchang recognitiod ean n between Fungal developmen extremels i t y limite outedn i r root tissues host plants and AM fungi initiate before they come into physi- (Figure 1A), and hyphae quickly progress toward the inner l contactca . Metabolites exude y hosdb t roots specifically cortical parenchyma, where the mycelium proliferates inter- enhance spore germination and fungal growth, as compared cellularly along the root. Here, intercellular hyphae branch into with root from nonhost plants, which do not exert this root cells, where they differentiate inthighle oth y ramifie- dar effect active Th . e plant molecule t sbee havye t n eidentino - buscules characteristic of this symbiosis (Figure 1B). The fied. Although have been propose candidates da s restricted pattern of arbuscule development, which is a fea- Plant Responses to AM Fungi 1873

ture symbiosescommoM A l al no t , implies some sor f planto t interfacee iteth n d i impairmen e Th . f walo t l edificatioy nma control over fungal morphogenesis within root tissues, per- relatee b t leasd a acidificatio e parn th i t o t f thino s compart- haps specific to cortical parenchyma cells. Similar plant control ment (see discussioth e e presencn th belowo t f r o eo ) ove developmene rth f microbiao t l symbiont sees i othen i r polygalacturonas arbuscule th n ei e interface (Perett t al.oe , systems (reviewed in Long, 1996; Pawlowski and Bisseling, 1995). Whe arbuscule nth e begin senesceo t s fibrillae th , r 1996 thin i , s issue). material encapsulates the collapsed fungal structures. Sub- Extensive cytological modification inducee sar arbusculedn i - sequently, the host nucleus, cytoplasm, and recover containing cells, and these are reminiscent of those seen in their aspect before cell invasion (Jacquelinet-Jeanmougit ne undifferentiated meristematic cells or cells entering the cell al., 1987), demonstratin remarkable gth e developmental plastic- cycle vacuole Th . e decrease relative sizth n s i s ea e volume f itcorticayo l parenchyma cell rootsn si . of cytosol increases and cell (mitochondria, plastids, significance Th moleculae th f eo r compositio syme th f -no dictyosomes, and endoplasmic reticulum) proliferate. Concom- biotic interface is at present a matter of debate. The plant cell itant with arbuscule development and reactivation of the wall components may simply create a barrier that prevents di- cytoplasmic compartment, the plant nucleus moves to the cen- rect physical contact between the hyphal cell wall and plant cele th l te undergoef (Figurd ro an ) e2A s hypertrophy (Balestrini protoplast surfaces. However, the detection in this cell com- et al., 1992). Ther evidences i e that such reorganizatio hosf no t partment of the pathogenesis-related (PR) group-1 protein, cell contents requires synthesis of cytoskeletal components whicdepositet no hs i d around hyphae crossing epidermar o l becaus accompanies i et i transcriptionae th y db l activatiof no hypodermal cells reflecy ,ma nonspecifi a t c defense response a plant a-tubulin gene in the colonized cells (Bonfante et al., 1996). The plant cell nucleus is characterized by enhanced fluorochrome accessibility, increased nuclease sensitivity, and dispersion. All of these modifications reflect an in- A crease in chromatin decondensation, which is a sign of greater transcriptional activity of the plant genome in arbuscule- containing cells (see below; Berta et al., 1990; Sgorbati et al., 1993). The branching progressio fungue th f no s inthose oth t cell provokes de novo synthesis of the periarbuscular membrane (RAM). The RAM is derived from the peripheral plasma mem- brane and completely surrounds the arbuscule (Figure 2B). The resulting surface of contact between the plant protoplast fungae th d l celan l create symbiotie sth c interface special,a - ized apoplastic zon r nutrienfo e t exchange betweee th n mycorrhizal symbionts.

Symbiotie Th c Interface: From Structur Functioo et n

Extensive biosynthesis of the PAM to accommodate the de- veloping fungus is associated with wall-building activities, and as in epidermal or hypodermal cells, wall material is depos- ited agains invadine th t g fungal hypha adjacene th y eb t plant protoplast (Dexheime al.t re , 1979; Bonfante-Fasol al.t oe , 1990; Bonfante, 1994; Balestrin al.t e i , 1996; Gianinazzi-Pearsot ne al., 1996a). However, in cortical parenchyma cells, this wall material progressively thin t arounsou growine dth g fungus to form a matrix of disorganized fibrils that separates the PAM fro arbuscule mth e wall, creatin interfacian ga l0 zon10 f eo nm or less. The PAM has a neutral phosphatase activity, which Figur . einfrastructur2 Plant-Fungaf eo l Relationship Arbusculen a sn i - is a marker of glycosylation, and some of the interfacial matrix Containing Root Cell of Piaum sativum. molecules have been identifie typicas da l plant cell wall com- Cross-sectio) (A arbusculf no e branche surrounde) s(a organelley db - ponents (Tabl ; Jeanmaire1 t al.e , 1985) variatioe Th . n i planh nc t cytoplas centralla d man y positioned lobed nucleus (n), ih . interface components between different plants reflects varia- intercellular hypha; s, senescent arbuscule branches. tion thein si r cell wall makeup t thereforI . e appears thae th t Detai) (B finf o l e arbuscule branches surrounde periarbuscue th y db - symbiotic plant membrane continue synthesizo st e cell wall r membranla interfacian ea (pmd an ) l matrix (im). precursors, even though structured wall material is not depos-. nm 5 Bar1. = s 1874 The Plant Cell

active membrane comes from the observation of H+-ATPase Table 1. Plant Cell Wall Components ldentified in the Symbiotic lnterface and nonspecific phosphatase activities at the symbiotic inter- face of severa1 plants (Gianinazzi-Pearson and Gianinazzi, ComDonent Planta References 1988; Smith and Smith, 1990; Gianinazzi-Fearsonet al., 1991a). Xyloglucans +: onion, leek, Dexheimer et al H+-ATPase activity is not observed on the peripheral plasma cloverb (1979); membrane of cortical root cells, whether or not these are - : maize, tobaccoC Balestrini et al. colonized by an AM fungus, or around hyphae crossing epider- (1996) mal or hypodermal cells. The modified ATPase distribution in Galacturonic acid + : onionb Dexheimer et al. mycorrhizal roots of leek is mirrored by an overall and stable (1979) hyperpolarization of plant membranes, as compared with non- Nonesterified +: leek, maizeC Bonfante-Fasolo mycorrhizal roots (Fieschi et al., 1992). In nonmycorrhizalroots, pectins -: peaC et al. (1990); Balestrini et al. immunologically detectable plasma membrane H+-ATPase is (1994); located in the root cap, epidermis, and stele, but it is not pres- Gollotte et al. ent in cells of the cortical parenchyma (Parets-Soler et al., 1990; (1995b) Stenz et ai., 1993; Bouché-Pillon et al., 1994). This distribu- Cellulosel +: leek, maizeC Bonfante-Fasoloet al. tion has been corroborated by studies of the expression of two hemicelluloses (1990); H+-ATPase genes in promoter-P-glucuronidase (GUS) fusion Balestrini et al. assays: plasma membrane H+-ATPase promoters are appar- (1994) ently not active in differentiated root cortical parenchyma Hydroxyproline-rich + : leek, pea, Bonfante-Fasolo et al (DeWitt et al., 1991; Michelet et al., 1994). There is evidence glycoprotein maizeb (1991); that root plasma membrane H+-ATPase genes are upregu- Balestrini et al. lated during AM interactions. In barley, AM development is (1994) Glycocalyx +: peac Gianinazzi-Pearson accompanied by increased levels of transcripts hybridizing to oligosaccharide et al. (1990) an H+-ATPase homologous cDNA clone (Murphy et al., 1996), Arabinogalactan/ + : peaC Gollotte et al. and promoter-GUS fusion assays in tobacco indicate that at arabinogalactan + : maize, , (1995b); least two H+-ATPase genes are activated in arbuscule- protein tobaccoC Balestrini et al. containing cells (V. Gianinazzi-Pearson, L. Moriau, and M. -: leekC (1996) Boutry, unpublished data). a Presence (+) or absence (-) of each component. The major role of the H+-ATPases at the symbiotic interface Components identified cytochemically. should be in driving the two-way nutrient flow occurring across Components identified immunologically. the interfacial apoplast in AM. Loading of from the interface apoplast into the plant cell will be against a concen- tration gradient, and the proton motive force created by the that could contribute to the control of fungal spread within the H+-ATPase activity could provide the energy required for the host (reviewed in Gianinazzi-Pearson et al., 1996b). Alterna- transport of this anion in a symport system (Smith and Smith, tively, the molecules accumulating in the symbiotic interface 1990). However, increased proton pumping with the activation may act as signals for recognition between plant and fungus. of H+-ATPases in the PAM could acidify the interfacial Furthermore, until the heterotrophic capacities of AM fungi are apoplast (Smith and Smith, 1990). The ensuing drop in pH pos- known, the possibility cannot be excluded that wall precursors sibly contributes to the wall loosening observed during also provide a source of reduced carbon to the fungal symbi- arbuscule development, either by breaking acid-labile bonds ont (Smith and Smith, 1990). or by activating lytic enzymes in the wall (Navi et al., 1986; The chemical form of molecules transferred across the sym- Rayle and Cleland, 1992). Finally, modulationof a plasma mem- biotic interface is not defined. In the context of the whole plant, brane H+-ATPase by protein kinases has been invoked in the mycorrhizal tissue functions predominantly as a fungal sink signal transduction that regulates plant defense responses in for carbon supplied as photosynthateand as a source of phos- interactions with fungal pathogens (Xing et al., 1996). The pres- phate transported from soil into the roots via the fungal pathway ente of H+-ATPase activity in the symbiotic interface of AM (reviewed in Harley and Smith, 1983; Jakobsen, 1995; Shachar- might then be related to a signal process involved in the weak Hill et al., 1995). Mechanisms for controlled transport between or partia1 induction of plant defense responses by AM fungi the mycorrhizal symbionts are largely hypothetical and have (see the discussion below). been discussed in detail by Smith and Smith (1990) and Smith et al. (1994). Until experimental evidence arises to the con- trary, nutrient exchange can still be assumed to involve passive MOLECULAR OF AM movement of solutes into the interface followed by active up- take by symbiont cells across membranes (Woolhouse, 1974). The PAM is not structurally or cytochemically different from The lack of specificity between the plant and fungal taxa that the peripheral plasma membrane, and evidence that it is an form AM raises the question of the nature of the genetic deter- Plant Responses to AM Fungi 1875 minants involved. Gene-for-gene mechanisms like those 1993), which reverts to mycorrhiza formation under high light postulated to explain specificity in plant- interactions conditions. In this case, the Myc-I phenotype may result from are unlikely to be active in such extreme examples of recipro- alterations in genes involved in normal plant physiological cally compatible interactions, where the lack of adverse processes, such as sugar synthesis or transport, that are also selection pressures have most likely generated more gener- important for mycorrhiza development. alized, multigenic systems. The effects of genotypic variation lnitial recognition events occur between Myc-l plant mu- on AM colonization have been described in a wide number tants and fungi as appressoria differentiate on the root surface; of plant species. A particularly strong genetic basis for coloni- however, AM development is arrested at this stage (Figure 1C). zation ability has been demonstrated in wheat and its ancestral Because this resistance results from single gene mutations relatives (reviewed in Hetrick et al., 1995; Peterson and in Myc-l pea mutants, the corresponding wild-type genes Bradbury, 1995). These examples, however, mostly involve poly- could play a role in restricting plant defense responses so that genic variation. Plant mutants showing alterations in symbiosis can be established (Gollotte et al., 1993; Gollotte, mycorrhiza-forming abilities have been obtained (see below), 1994). Myc-l pea mutants challenged with AM fungi deposit but technical difficulties in observing large numbers of root abnormally thick cell wall appositions in epidermal and systems for defective phenotypes have limited the number of hypodermal cells. These structures contain wall-reinforcing and mutants that have been isolated to date. Nonetheless, muta- defense-related molecules, such as phenolics, callose, and tions affecting AM formation have been found in PR-1 protein (Gollotte et al., 1993, 1995a; Gollotte, 1994; species, although only in lines that are also altered in their Gianinazzi-Pearson et al., 1996b), and are reminiscent of the nodulating abilities. cell wall appositions induced at sites of resistance to patho- gen attack (Heath, 1980; Benhamou et al., 1989; TahiriAlaoui et al., 1993a; Collingeet al., 1994). It has been postulated that lsolation and Characterization of Mycorrhiza-Defective PR-related gene expression in epidermal tissues may contrib- Plant Mutants ute to defense in roots (Mylona et al., 1994), but whether resistance to AM fungi in Myc-l mutants is reinforced by ac- Almost half of nearly 50 nonnodulating (Nod-) ethyl tivation of other inducible plant defense processes has yet to methanesulfonate(EMS)-induced isogenic mutants of pea (P be investigated. sativum cvs Frisson, Finale, and Sparkle) and y-radiation mu- Two possible functions can be envisaged for the wild-type tants of truncatula have been found to be completely alleles of Myc-' mutated loci. First, they may be involved in resistant to AM fungi (Duc et al., 1989; Gianinazzi-Pearson et the production of a plant susceptibility factor that is elicited al., 1991b; Balaji et al., 1994; Sagan et al., 1995). The by the fungal symbiont and that negatively regulates the de- mycorrhiza-resistant phenotype of these mutants, presently fense response to establish interorganism compatibility, in a designated Myc-l, is the most frequently found among nodu- manner similar to that postulated for interactions between Mlo lation mutants. Myc-l mutations specifically affect plant alleles in barley and the biotrophic fungal pathogen ffysiphe interactions with AM fungi and because they are not graminis (Freialdenhoven et al., 1996). Alternatively, the acti- correlated with changes in the infection patterns of fungal, bac- vated symbiosis-relatedgenes may induce a fungal suppressor terial, or nematode root pathogens (Gollotte et al., 1993; of the plant defense system, as has been reported for certain Gianinazzi-Pearson et al., 1994). lnvestigations of the genetic plant pathogens (reviewed in Shiraishi et al., 1991). system determining the Myc-l character in pea have shown Another mycorrhiza mutant phenotype (MYC-~),affected at that mutations are each at a single locus and that they belong the stage of arbuscule development, has been identified in to three complementation groups (p, c, and a), which cor- two of -20 EMS-induced low- and late-nodulating mutants respond to the sym8, syml9 (described by LaRue and Weeden, (Nod+/-) of P sativum cv Finale. Only one recessive and ge- 1992), and sym30 nodulation genes (M. Sagan and G. Duc, netically stable mutation is known for Myc2 mutants (the s personal communication). For each complementation group, complementation group; G. Duc and M. Sagan, unpublished the mutation is recessive and genetically stable, and the data). In these mutants, the stages of appressorium formation, Myc-l trait does not segregate from the Nod- character in root penetration, and cortex colonization by intercellular hyphae large F2 progenies. This suggests that one gene controls both are completed. Although invasion of cortical parenchyma cells characters (Ducet al., 1989; Gianinazzi-Pearson et al., 1991b; was not described in initial investigations (Gianinazzi-Pearson G. Duc and A. Trouvelot, unpublished data). et al., 1991b), more detailed studies have since shown that in- The products of the genes defined by the Myc-"od- mu- tracellular fungal growth does occur. However, arbuscule tations must have a fundamental role in establishing formation is prevented, and hyphal growth is reduced to a few mycorrhizal and nodule symbioses because no field popula- stumpy branches (Figure 1D; Lherminier, 1993; Gollotte, 1994). tions or laboratory isolates of the microsymbionts have yet been The cellular processes affected by the Myc2mutation have found to infect Myc-"od- pea mutants, whatever the plant not yet been investigated fully, but there is cytological evidence growth conditions (Sagan et al., 1993; V. Gianinazzi-Pearson, that mycorrhiza function is impaired in Myc2mutants. This unpublished data). One exception is the unstable Myc-l is because ATPase activity is not elicited on the plant phenotype reported in a Nod- M. sativa line (Bradbury et al., membrane at the intracellular host-fungus interface of the Plane Th 187t Cel6 l

aborted or incomplete arbuscules (Gianinazzi-Pearson et al., et al., 1995), and the expression of genes encoding the early 1995). It is therefore possible that the wild-type gene corre- phenylpropanoid biosynthetic enzymes phenylalanine sponding to the Myc~2 mutated locus encodes a component -lyase (PAL), chalcone synthase (CHS) chalcond ,an e of signaling events leading to reciprocal morphofunctional com- isomerase (CHI) is enhanced concomitant with isoflavonoid patibility between the mycorrhizal symbionts. accumulation (Harrison and Dixon, 1993; Volpin et al., 1994, The precise roles of the mutated genes in the MycH and 1995). However suco ,n h changes have been observe beandn i , Myc~2 plant elucidatede b s o havt t presene ye Th . t indica- parsley, or potato roots (Figure 3; Lambais and Mehdy, 1993; tion from the pea system is that different genetic processes Franke Gnadingerd nan , 1994). Furthermore mycorrhizan i , l are active in the outermost tissues and in the cortical paren- Medicago species, therincreaso n es i transcriptn ei e th f so chym f hoso a t roots s definee fungaa ,th y b dl growth late enzyme reductase (IFR), which is specific to phenotypes resulting fro Myc"e m Myc~th d an 1 2 mutations, isoflavonoid biosynthesis, signifying an uncoordinated induc- respectively. Wild-type genes correspondin Myc~o gt 1 loci tion of genes in the phenylpropanoid pathway and contrasting may encode proteins that modify defense responses, thu- sal with the coordinated regulation that is a feature of pathogen lowing root colonizatio proceedno t , whereas Myc~2 y locma i interactions (Harrison and Dixon, 1993; Volpin et al., 1995). be involve subsequendn i t developmental processes tha- tes Thus, activatio phenylpropanoie th f no d pathway cannoe b t tablish the metabolic specialization of arbuscule-containing considered a general phenomenon linked to AM development. cells. Furthermore, the strong link between the genes deter- mining nodulatio mycorrhizd nan a formatio Myc~n i a pe 1 mutants point commoso t n mechanisms regulatin least ga t part plant-microbe oth f e interaction symbioseo tw e th n si s (see weeks the discussion below). There are undoubtedly many more plant 356 genes involved in the establishment of AM. The isolation and genetic analyse f additionaso l plant mutants wit hranga f eo mycorrhiza-defective phenotypes will leafullea do t r appreci- genetiatioe th f no c complexit systemf yo s regulating successful AM symbioses.

Molecular Analysi f AM-lnduceso d Modifications

Plant Defense Responses

Clearly plane ,th t symbiont exert sdegrea controf eo l oveM rA fungal development, restricting propagation and arbuscule for- matio corticae th no t l parenchyma. However formatioe ,th f no AM constitutes a massive fungal invasion of plant tissues. It is perhaps not surprising then that the first molecular modifi- cations investigate relatio n di developmene th o nt f thio t s NM symbiosis were those associated with plant defense responses. Lox The major inducible defense mechanisms that are triggered in plants as a general response to attack by a pathogen are M PAL cell wall modifications, enhancemen f secondaro t y metabo- lism accumulatiod an , proteinR P f no s (reviewe Bowlesdn i , NM PAL 1990; Dixon and Harrison, 1990; Collinge et al., 1994; Ryals et al., 1996, in this issue). The spatial and temporal expres- Figure 3. Modulation of Defense Gene Expression in Potato Roots Inoculated with an AM Fungus (Glomus sp). sion of these responses during the formation of AM have recently been described in a detailed review (Gianinazzi- RNA from mycorrhiza nonmycorrhizad an ) l(M l (NM) root sequens swa - Pearso t al.ne , 1996b). Consequently, onl mose yth t salient tially probed with ^P-labeled cDNA fragments encoding hydroxyprdine- feature discussee sar d here. rich glycoprotein (HRGP), acidic (A-Chi basid )an c chitinase (B-Chi), Isoflavonoid phytoalexins are antimicrobial phenylpropanoid acidic (A-Glu) and basic p-1,3-glucanase (B-Glu), lipoxygenase (Lox), phenylalanind an e ammonia-lyase (PAL) isolates wa . Tota- A daf RN l derivatives that often accumulate rapidly in plant tissues ex- week7 d an s , growt6 , 5 , f plantsh3 o te, 2 r , when root colonization hibiting resistance to pathogen attack (reviewed in Hahlbrock in G/omus-inoculated plants was 15,20,32,60, and 82%, respectively. and Scheel, 1989; Smith, 1996). Weak, slow, or transient ac- An loade s equawa totaf A dlo amounint) RN l ong eac0 t(2 h lane. Sig- cumulation of phytoalexins, or phytoalexin precursors, has been nals for RNA from NM roots with HRGP, A-Chi, B-Chi, and A-Glu probes reporte roodn i t system soybeaM A f sMedicagod o nan spe- wer same eth thoss ea B-Glr efo Tahiri-Alaoui. u(A Strittmatter. ,G d ,an cies (Morand t al.e i , 1984; Harriso Dixond nan , 1993; Volpin V. Gianinazzi-Pearson, unpublished data). Plant Responses to AM Fungi 1877

PR proteins involved in the defense response of plants in- velopment of the fungal symbionts is a matter for speculation clude the acidic or basic chitinases and p-1,3-glucanases, at this stage (for further discussion, see Gianinazzi-Pearson which are antimicrobial hydrolases(Collinge et al., 1994). En- et al., 1996b). In any event, the AM fungi fail to elicit the full hanced chitinase and S-1,3-glucanase activities coincide with cascade of nonspecific defense responses in host roots. This early plant-funga1 interactions in AM and strongly diminish is not due to an inability to elicit pathogen-like responses be- as root colonization proceeds (Spanu et al., 1989; Lambais cause AM fungi induce resistance reactions in roots of the and Mehdy, 1993; Vierheilig et al., 1994; Volpin et al., 1994). Myc-l pea mutants (see the discussion above). Thus, mech- This pattern of induction is also apparent at the level of tran- anisms should exist for the local suppression of defense script accumulation, suggesting that the enzymes are responses to levels compatible with symbiotic interactions. Re- transcriptionallyregulated during symbiosis development (Fig- juvenation of colonized plant cells, as occurs with arbuscule ure 3; see also Lambais and Mehdy, 1993). Differential development, has been postulated as a strategy to suppress activation of genes may reflect partia1 elicitation of a general or slow down resistance reactions in the case of biotrophic plant defense response to early stages of fungal invasion that pathogens (Johal et al., 1995). Such localized effects could is subsequently repressed as the symbiosis becomes estab- explain why AM fungi can develop in plants constitutively over- lished. Chitinase isoforms different from those related to plant expressing defense genes (reviewed in Gianinazzi-Pearson defense response to root pathogens are activated in the AM et al., 1996b). of severa1 plants in addition to constitutive root enzymes (Dumas-Gaudot et al., 1992; Dassi et al., 1996). The site of Nodulation Events activity and the role of the mycorrhiza-relatedisozymes in the symbiosis have yet to be determined. However, the mycorrhiza- There are a large number of structural and functional similari- related chitinase isoform can be weakly active in incompati- ties between AM and nodule symbioses. For example, cortical ble interactions in myc-’ mutants (Dumas-Gaudot et al., parenchyma cells are the same target for AM development 1994a), and it is different from isoforms detected in nodules and nodule organogenesis, and a plant-derived membrane (Slezack et al., 1996). borders the physiologically active interface between symbi- The expression of genes encoding hydroxyproline-richgly- onts in both systems (Mellor, 1989). Moreover, flavonoids, which coproteins can also be modulated during AM interactions elicit rhizobial nod genes, also enhance hyphal growth of AM (Figure 3; see also Franken and Gnadinger, 1994), and in fungi (reviewed in Phillipsand Tsai, 1992) and probably mediate tobacco the level of PR-1 gene transcripts accumulating dur- a -induced stimulation of mycorrhizal colonization ing AM development is considerably lower than in roots (Xie et al., 1995). Such similarities, together with the strong challenged with the fungal pathogen Chalara elegans link between some of the genes determining nodulation and (Gianinazzi-Pearsonet al., 1992). Moreover, the localization AM formation in , point to common mechanisms of defense-related gene expression is very restricted in fully regulating at least part of the interactions in the two symbioses. developed mycorrhizal tissues. As previously mentioned, PR-1 Some plant molecules associated with interactions between protein is detected only in the symbiotic interface between the rhizobia and legumes have been located in AM symbioses. PAM and the fungal wall (Gianinazzi-Pearson et al., 1992). Tran- In , for example, plant proteins and glycoproteins in the scripts of PAL, CHS, p-1,3-glucanase, chitinase, and PR-1 matrix surrounding in nodule infection threads are protein accumulate discretely in arbuscule-containing cells present in the host wall material around arbuscule hyphae, (Harrison and Dixon, 1994; Lambais and Mehdy, 1995; Blee and oligosaccharides or glycoconjugatesof the plant-derived and Anderson, 1996; Gianinazzi-Pearsonet al., 1996b), and membrane or interfacial matrix around bacteroids in nodule- expression of the gstl gene encoding glutathione-S-transferase infected cells are common to the PAM and arbuscule inter- (as detected by a gst7 promoter-GUS fusion) is restricted to face (Gianinazzi-Pearsonet al., 1991b, 1996a; Perotto et al., colonized cortical cells in AM transgenic potato roots (Strittmatter 1994; Gollotte et al., 1995b). Transcripts from mycorrhizal roots et al., 1996). This spatial pattern of defense gene expression of M. truncatula hybridize with a probe for the early nodulin is very different from that reported in plant-pathogen interac- gene €NOD72(M.J. Harrison, unpublished data), which codes tions, in which PR proteins accumulate throughout for a proline-rich protein involved in infection fungal-infected root tissues (Benhamou et al., 1989,1990; Tahiri- (Scheres et al., 1990). Using transgenic M. varia expressing Alaoui et al., 1993b), and defense gene expression is activated apENOD72-GUS fusion (Pichon et al., 1994), we have identi- in cells other than those containing the pathogen (Schmelzer fied the arbuscule-containing cells as the site of gene activation et al., 1989; Strittmatter et al., 1996). in mycorrhizal roots (V. Gianinazzi-Pearson,E.P. Journet, and The uncoordinated, transient, weak, and/or very localized D. Barker, unpublisheddata), suggesting that the encoded pro- expression of plant defense responses to AM fungi contrasts tein may be another common component of interactionsin the in many respects to those reported for either compatible or two symbioses. incompatible plant-pathogen interactions, which often differ A small nurnber of late nodulins have been identified among from each other only in timing and extent (Bowles, 1990; Dixon in vitro translation products from AM of . One of these and Harrison, 1990; Collinge et al., 1994). Whether plant proteins was postulated to be nodulin 26, a protein found in defense responses play any role in plant control over the de- the host membrane around bacteroids in nodules (Wyss et al., 1878 The Plant Cell

1990). cDNA sequences with high similarities to genes encod- AM development have been isolated by differential RNA dis- ing membrane intrinsic channel proteins, including nodulin 26, play from pea (Martin-Laurent et al., 1996) and screening of have recently been cloned from mycorrhizal roots of parsley, cDNA libraries from barley (Murphy et al., 1996). Interestingly, tobacco, and fava (Roussel, 1995; I? Franken, H. Roussel, apart from one upregulated sequence from barley that is highly and V. Gianinazzi-Pearson, unpublished data). Activation of similar to a plant H+-ATPase gene, sequence similarities have the corresponding genes in mycorrhizal roots may reflect the not been found, and the functions of the corresponding genes existence of a common transporter system in the symbiotic are as yet unknown. Predictions for secondary protein struc- interface of AM and nodules. Transcripts of another late nodu- ture indicate that one of the upregulated genes in pea may lin gene, VFLb29, have been found to be induced de novo in encode a putative transmembrane protein (F. Martin-Laurent, the nodules and AM of fava bean (Frühling, 1995; M. Frühling, D. van Tuinen, E. Dumas-Gaudot, V. Gianinazzi-Pearson, S. A.M. Perlick, H. Roussel, V. Gianinazzi-Pearson,and A. Puhler, Gianinazzi, and P. Franken, manuscript submitted). manuscript submitted). This gene codes for a Finally, a clone encoding a hexose transporter has been ob- that shares unusually low sequence identity with tained from a cDNA library of AM roots of M. truncatula other legume . Studies of its expression pat- (Harrison, 1996). Expression of the corresponding plant gene, tern within mycorrhizal roots will determine whether, and the which is normally active in phloem and root tip cells, increases extent to which, this protein modulates oxygen supply to the in arbuscule-containing cells or cells adjacent to intercellular fungal symbiont. hyphae. This does not occur in uncolonized regions of a mycorrhizal root system or during interactions with a mycor- Mycorrhiza-Specific Molecular Responses rhiza-resistant Myc- genotype of M. sativa, suggesting that the inducing signal operates over a short range and results The search for specific plant genes mediating symbiotic events from the presence of the fungus in the cortex. Such a change in AM formation and functioning is still in its infancy. Recent in cell-type expression may function to supply sugars to root advances have been made using two strategies: first, analy- cells containing the fungus in a functioning symbiosis ses of polypeptides for changes in protein biosynthesis; and (Harrison, 1996). These results set the pace for future advances second, cDNA cloning of genes that are differentially regu- toward identifying plant genes with a role in AM formation and lated during the symbiosis. f unction. Differential biosynthesis of soluble polypeptides accompa- nies AM development, and numerous quantitative and qualitative modifications have been observed in early and late CONCWSIONS stages of plant-funga1 interactions in AM (Dumas et al., 1990; Arines et al., 1993; Simoneau et al., 1994; Samra et al., 1996). Proteins that increase and decrease in amount or disappear Significant progress in the analysis of plant systems modulat- have been described. lnduced polypeptides are most frequent ing symbiotic interactions in AM has been made through the and can represent -4% of resolved polypeptides (Dumas- application of genetic, ultracytological, biochemical, and mo- Gaudot et al., 1994b; Samra et al., 1996). Although such lecular biology techniques. Mycorrhiza-defective legume mycorrhiza-induced polypeptides have been called mycor- mutants provide direct evidence for specific symbiosis-related rhizins, in analogy to nodulins, actinorhizins, and haustorins gene control and afford the potential for analyzing some host in other plant-microbe interactions (verma et al., 1992; Roberts genes and gene functions involved in early and late events. et al., 1993; Seguin and Lalonde, 1993), those of plant origin However, the range of mutant phenotypes available is still ex- have not been conclusively identified. Mycorrhiza-resistantpea tremely limited. Expansionof the genetic approach to additional mutants respond differently when challenged with an AM mutations is essential to understand more fully the molecular fungus, the major modification being the disappearance of poly- mechanisms leading to morphological integration and recipro: peptides normally present in uninoculated plants (Samra et cal functional compatibility between AM symbionts. Moreover, al., 1996). Hence, the lack of repression of genes that are con- mutagenesis programs, including mycorrhiza-forming nonle- stitutively expressed in plants may be an essential component gumes such as maize or tomato, should indicate to what extent of compatible interactions leading to AM establishment AM development is determined by processes other than those (Gianinazzi et al., 1995). common to nodulation. Quantitative and qualitative alterations in gene expression Despite obvious similarities between the infection processes have been confirmed in onion and pea AM by in vitro RNA of AM fungi and rhizobia in legumes, important differences translation (Garcia-Garrido et al., 1993) and differential RNA also exist. A number of nonnodulating legume genotypes are display (Martin-Laurent et al., 1995), respectively. The fre- able to form mycorrhiza (Duc et al., 1989; Wyss et al., 1990; quency of qualitative modifications in differential display Gianinazzi-Pearson et al., 1991b), and some nodule infection products between mycorrhizal and nonmycorrhizal pea roots thread components are not synthesized around the fungal sym- is similar to that of polypeptide changes (Martin-Laurent et al., bionts in mycorrhizal roots (Gianinazzi-Pearson et al., 1990; 1996; Samra et al., 1996). cDNA clones corresponding to plant Perotto et al., 1994). Evidence for AM symbioses appears in genes that are differentially expressed during early stages of the fossil record before the evolution of legume species. This Plant Responses to AM Fungi 1879

raises the interesting possibility that during evolution, rhizo- was supported through collaborative projects (PROCOPE No. 93134, bia may have exploited plant processes involved in symbiotic COST Action 8.21, and AIP-INRA No. 30) and by the Burgundy Re- interactions with arbuscular mycorrhizal fungi and modified gional Council (contrat de plan No. 3060A). them to a completely different purpose, that of nodule devel- opment (Gollotte et al., 1995b). REFERENCES Use of heterologous probes for genes that mediate other plant-microbe interactions has provided much information on cell-cell interactions in AM. This approach has been particu-. Alexander, T., Meier, R., Toth, R., and Weber, H.C. (1988). Dynamics larly effective in evaluating the restricted expression of defense of arbuscule development and degeneration in mycorrhizas of Tri- responsesthat is essential for compatibility between plant and cum aestivum L. and Avena sativa L. with reference to Zea mays fungal symbionts. 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