Analysis of arbuscular mycorrhizas using symbiosis-defective plant mutants


  • John F. Marsh,

    1. The Plant Laboratory, Department of Biology, University of York, PO Box 373, York, YO10 5YW, UK
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  • Michael Schultze

    Corresponding author
    1. The Plant Laboratory, Department of Biology, University of York, PO Box 373, York, YO10 5YW, UK
      Author for correspondence: Michael Schultze Tel: +44 1904434 302 Fax: +44 1904434 312
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Author for correspondence: Michael Schultze Tel: +44 1904434 302 Fax: +44 1904434 312


Arbuscular mycorrhizas (AM), mutualistic plant–fungus symbioses formed on the majority of land plants, appear to play an important role in plant mineral nutrition, plant health, and might influence the structure of plant communities. Progress in understanding the genetic and molecular basis of this important symbiotic association has been hampered by the obligate biotrophy of the fungal partner, and by the lack of mycorrhiza formation on the plant model species Arabidopsis thaliana. Recently, legume plants, such as Medicago truncatula and Lotus japonicus, have been chosen as experimental systems for research of plant–microbe symbioses. The application of both reverse- and traditional forward-genetic approaches, in conjunction with genomics initiatives in model legumes, will rapidly increase our understanding of the plant-encoded genetic determinants of the arbuscular mycorrhiza. An essential step in this process has been the generation, isolation and analysis of mycorrhizal mutants. This review provides an introduction to existing plant mutants affected in AM formation.


Around two-thirds of all plant species form arbuscular mycorrhizas, a symbiosis between plant roots and glomalean fungi that leads to the formation of intraradical organs of nutrient exchange and an extraradical network of fungal hyphae effectively extending the plant root system. The mycorrhiza can play a key role in plant health by increasing acquisition of immobile mineral nutrients, particularly phosphate (see Smith & Read, 1997), enhancing drought tolerance (Subramanian et al., 1995) and promoting resistance of plants to root pathogens (Newsham et al., 1995; Cordier et al., 1998). On the other side, the fungal partner profits from carbon supplied by the plant. Depending on mycorrhizal fungal diversity, mycorrhiza formation influences the function of ecosystems as well as the biodiversity of plant communities (Helgason et al., 1998; Van der Heijden et al., 1998).

Distinct morphological stages have been defined for the development of the AM (Smith & Read, 1997). These stages are represented schematically in Fig. 1 and described briefly here. The process begins with spore germination and limited hyphal growth which may occur in the absence of the plant root (Fig. 1a,b). However, extensive preinfection branching (Fig. 1c) and sustained hyphal growth require the presence of host plant roots. Upon contact with the root epidermis, hyphal tips swell and form appressoria (Fig. 1d). Hyphal outgrowth at the point of contact between the root and appressoria initiates proliferation of intraradical hyphae (Fig. 1e). Cortical cells are subsequently penetrated and highly branched haustoria, the arbuscules, develop (Fig. 1f). Within the arbuscules the fungus remains in the apoplastic compartment surrounded by an interfacial matrix (IM) and the periarbuscular membrane (PAM). The IM and the PAM are continuous with the plant cell wall and plasma membrane, respectively. It is across this interface, between the plant-derived PAM and the fungal cell wall and plasma membrane, that bi-directional nutrient transfer (particularly phosphate transfer from fungus to plant) is assumed to occur. Having reached the end of its life span within as few as seven days, the arbuscule degenerates allowing the host cell to recover and occasionally be recolonized. Ultimately, colonization of the root promotes extensive growth of external hyphae which gives the root system the macroscopically visible structure of a mycorrhiza (Fig. 1g). Depending on the plant species two major morphological types of AM are observed. In the ‘Paris’ type, hyphae grow relatively slowly and primarily intracellularly. Direct spread occurs from cell to cell and the formation of coiled hyphae with only limited development of arbuscules is typical. The ‘Arum’ type is characterized by the rapid growth of intercellular hyphae followed by extensive formation of the intracellular arbuscules. This type is formed in the plants from which mycorrhizal mutants have been isolated.

Figure 1.

Stages of arbuscular mycorrhiza development. Spore germination and limited hyphal growth may occur in the absence of the plant root (a,b), whereas extensive preinfection branching and sustained hyphal growth require the presence of host plant roots (c). Upon contact with the root epidermis, hyphal tips swell and form appressoria (d). This is followd by the penetration of the root and the proliferation of intraradical hyphae. Cortical cells are subsequently penetrated and arbuscules develop (f). Colonisation of the root promotes extensive growth of external hyphae (g). This scheme represents the ‘Arum’ type of AM. This type is formed on the model plant species M. truncatula. Note that under natural conditions infections do not need to originate from germinating spores, but occur predominantly from previously infected root segments covered with external hyphae.

In the mycorrhizal symbiosis, growth and differentiation of the plant root and the fungal hyphae must be tightly coordinated. This requires a reciprocal recognition, either via the exchange of diffusible signals or a direct cell-to-cell interaction. Toward the characterization of these processes at the molecular level, abnormal AM phenotypes have been identified in mutant plant lines, most frequently in legumes. These include both defective AM phenotypes (Myc) and enhanced mycorrhizal phenotypes (Morandi et al., 2000; Solaiman et al., 2000). Interestingly, the mycorrhizal symbiosis shares common steps with the plant-Rhizobium symbiosis, as demonstrated by plant mutants affected in both root nodule and mycorrhiza development. Mycorrhizal mutant screens have been performed almost exclusively on hosts deficient in root nodule formation (Nod) or function (Nod+/Fix) resulting in the isolation of mutants in which both nodulation and the AM are altered. In this review we summarize the current state of knowledge concerning the existing collection of Myc mutants (Table 1) and discuss the future prospect of isolating mycorrhizal specific mutants, as well as the techniques that may be used to generate and identify them.

Table 1.  Summary of plant Myc mutants
  1. Column headings are defined as follows. Host: plant species and cultivar in which mutant phenotype was identified, including references relevant to assessment of mycorrhizal colonization. Mutant: designation of AM mutant plant lines. Locus/Allele: designation of genetic loci and alleles mutated in AM mutant plant lines. Grey highlighting distinguishes independent loci, with the exception of Ljsym71–1, 71–2 and 72 which have not been demonstrated to be distinct from those of Wegel et al. (1998). Mutagen: method used to mutagenize wild-type (WT) host; Transfer DNA (T-DNA), ethylmethane sulphonate (EMS), fast neutron (FN), gamma irradiation (GAMMA), nitrosoethylurea (NEU), nitrosomethylurea (NMU), or spontaneous mutation (SM). Inoculum: fungi used to assess AM phenotype; Gigaspora margarita (Gi.m), Glomus clarum (G.c), Glomus intraradices (G.i), Glomus mosseae (G.m), Glomus R-10 (G.R-10) or Glomusversiforme (G.v). Myc: stage of mycorrhizal colonization affected by mutation; no penetration of root epidermis (Pen), penetration of epidermal cells, but absence of cortex invasion (Coi), exodermis colonized, but absence of inner cortex invasion (Ici), cortex colonized in the absence of arbuscules (Arb) or arbuscule development is abnormal (Ard). Nod/Fix: Nodulation/nitrogen fixation phenotype of mutant; does not form nodules (Nod) or forms nonnitrogen fixing nodules (Nod+/Fix). *Phenotype is inferred from alleles in which the affected stage has been established. **Phenotype is predicted from the biochemical epistasis between alleles revealed by their affect on intracellular calcium spiking (Wais et al., 2000).

Lotus japonicus      
B-129 Gifu      
Wegel et al. (1998)282–287Lj sym2–1T-DNAG.iCoiNod
Schauser et al. (1998)282–288Lj sym2–2T-DNAG.iCoiNod
Wegel et al. (1998)2557–1Ljsym3–2T-DNAG.iCoiNod
Schauser et al. (1998)282–227Ljsym4–1T-DNAG.i,Gi.mCoiNod
Bonfante et al. (2000)EMS 1749Ljsym4–2EMSG.i,Gi.mCoiNod
Senoo et al. (2000a)mcbexLjsym71–1EMSG. R-10IciNod
 mcbexLjsym71–2EMSG. R-10IciNod
Senoo et al. (2000a)mcbepLjsym72EMSG. R-10PenNod
Lycopersicon esculentum      
cv 76R      
Barker et al. (1998)rmcrmcFNG.i,Gi.m,G.mPenN/A
Medicago sativa      
Bradbury et al. (1991)MN NN-1008MN NN-1008SMG.i,G.vPenNod
 MN IN-3811MN IN-3811SMG.i,G.vArbNod+/Fix
Medicago trunculata      
cv Jemalong      
Catoira et al. (2000)C71domi/dmi1–1EMSN/APen**Nod
Penmetsa & Cook (1997)B129dmi1–2EMSN/APen**Nod
Catoira et al. (2000)P1dmi2–1EMSN/APen*Nod
Sagan et al. (1995)TR25dmi2–2GAMMAG.i,G.mPenNod
Catoira et al. (2000)TRV25dmi3–1GAMMAG.i,G.mPenNod
Sagan et al. (1998)      
Phaseolus vulgaris      
cv OAC Rico      
Shirtliffe & Vessey (1996)R69R69EMSG.m,G.cArbNod+/Fix
Pisum sativum      
cv. Finale      
Gianinazzi-Pearson (1996)RisNod24sEMSG.mArdNod+/Fix
cv. Frisson      
Duc et al. (1989)P1aEMSG.i,G.mPenNod
Sagan et al. (1994)F4-1 (P53)a/sym30EMSG.i,G.mPenNod
Schneider et al. (1999)F4-141 (P55)c/sym19EMSG.i,G.mPenNod
Duc et al. (1989)P4cEMSG.i,G.mPenNod
Duc et al. (1989)P6fEMSG.i,G.mPenNod
cv. Rondo      
Weeden et al. (1990)K24c/sym19EMSN/APen*Nod
cv. Sparkle      
Gianinazzi-Pearson (1996)N/Aa/sym30N/AN/APen*Nod
Balaji et al. (1994)R72b/sym9GAMMAGi.mPenNod
Weeden et al. (1990)NMU1c/sym19NMUN/APen*Nod
Albrecht et al. (1998)R19p/sym8GAMMAGi.mPenNod
Gianinazzi-Pearson (1996)E140p/sym8EMSGi.mPenNod
Balaji et al. (1994)R25p/sym8GAMMAGi.mPenNod
Vicia faba     Nod
Duc et al. (1989)Indian 778sym1SMG.i,G.mPenNod+/Fix

Current AM host mutants

To date the vast majority of mutants are defective in the earliest stage of AM formation and block fungal penetration (Pen) of the root epidermis. These include the first AM mutants to be isolated from Pisum sativum and Vicia faba populations (Duc et al., 1989), as well as subsequent mutants identified in Medicago truncatula (Sagan et al., 1995), Medicago sativa (Bradbury et al., 1991), Phaseolus vulgaris (Shirtliffe & Vessey, 1996), Lycopersicon esculentum (Barker et al., 1998) and most recently, Lotus japonicus (Senoo et al., 2000a). In pea (Pisum sativum), mutations in as many as six loci result in a Pen phenotype and at least three separate loci, recently renamed dmi1, dmi2 and dmi3, are linked to the same defect in the model legume M. truncatula (Catoira et al., 2000). Although single Pen mutants have been isolated from V. faba (Duc et al., 1989) and P. vulgaris (Shirtliffe & Vessey, 1996), it is expected that saturating mutagenesis in conjunction with exhaustive screening would reveal additional mutants in these legumes.

Common to Pen mutants is the formation of complex and somewhat abnormal appressoria relative to the wild-type (WT). This can be seen clearly in the mcbep (mycorrhizal colonization blocked at epidermis) mutants of L. japonicus (Senoo et al., 2000a), the rmc (reduced mycorrhizal colonization) mutant of tomato (Lycopersicon esculentum) (Barker et al., 1998), the alfalfa (Medicago sativa) Nod line MN-NN1008 (Bradbury et al., 1991), and the P2 (sym30) mutant of pea (Duc et al., 1989). Irrespective of inoculum (Table 1) each of these mutations prevent penetration of the root epidermis resulting in the formation of complex hyphal branching, multiple and unusually swollen appressoria. It has been suggested that hyphal branching and multiple appressoria, in particular, are a consequence of the continuing but unsuccessful attempts by the fungus to penetrate the root surface (Bradbury et al., 1991).

It has also been observed that the number of appressoria varies dramatically between Pen mutants. Compared with the number forming on wild-type roots, groups have reported finding more (Bradbury et al., 1991; Senoo et al., 2000a), fewer (Duc et al., 1989; Shirtliffe & Vessey, 1996) or identical numbers of appressoria (Bradbury et al., 1991; Bonfante et al., 2000; Senoo et al., 2000a). In alfalfa, the nonnodulating MN-NN1008 and inefficiently nodulating MN-IN3811 mutants both support formation of a greater number of appressoria following inoculation with G. versiforme, but no significant difference in numbers when grown in the presence of G. intraradices suggesting that both fungal and plant host genotypes play a role in this process (Bradbury et al., 1991). In general, the difficulty of unambiguously identifying and quantitating appressoria may also contribute to the tremendous variation that has been reported.

As the isolation of genes from AM mutants has not yet been reported, it is not possible to determine their precise mode of action. However, evidence suggests that plant defence responses and signalling events are altered in Pen mutants. In the P2 (sym30) mutant of pea (Gianinazzi-Pearson, 1996), cell wall thickening and a coincident increase in the deposition of callose and phenolics by epidermal and hypodermal cells was observed at the points of contact with appressoria (Gollotte et al., 1993). Furthermore, the level of endogenous salicylic acid, as well as, the steady state transcript levels of a number of defence related genes in P2 roots were significantly higher than in the WT (Blilou et al., 1999; Ruiz-Lazano et al., 1999). These observations are consistent with the possible exclusion of the fungus by the inappropriate expression of defence mechanisms in the P2 mutant and other sym30 alleles. In addition to this phenomenon which may be related directly to the Pen phenotype, a variety of other effects are seen in these mutants. Several mutants (dmi1 and dmi2 in M. truncatula;sym8 and sym19 in pea) are defective in calcium spiking in root hairs, one of the earliest responses to rhizobial nodulation factors (Wais et al., 2000; Walker et al., 2000). This raises the question as to whether calcium spiking is stimulated by mycorrhizal fungal signals. Intriguingly, chitin oligomers, which may arise as breakdown products from fungal cell walls, have been shown to elicit calcium spiking in pea although the response was qualitatively different from that induced by Nod factors (Walker et al., 2000). Additional analysis of the sym8 pea mutants E140, R19 and R25 demonstrate that SYM8 is essential for the induction of the early nodulation (ENOD) genes, PsENOD5 and PsENOD12A in roots inoculated with Gigaspora margarita (Albrecht et al., 1998). Similarly, transcript levels of a sugar transporter, normally induced in AM roots, do not increase in the alfalfa mutant MN-NN1008 (Harrison, 1996). Which if any of these processes are altered in the rmc mutant of the nonlegume host, tomato, is of particular interest. Although phenotypically similar to other Pen mutants selected from pre-existing Nod populations, the rmc mutant was identified by direct screening for abnormal AM. It may therefore harbour a mutated gene specific to the mycorrhizal symbiosis.

By contrast to mutants in which the infection process aborts following appressoria formation, a large collection of mutants were recently described in L. japonicus which are blocked prior to cortex invasion (Coi), but form normal appressoria and are capable of penetrating the root (Wegel et al., 1998; Bonfante et al., 2000; Parniske et al., 2000). Complementation analysis has revealed that mutations in at least six distinct loci (Ljsym2, Ljsym3, Ljsym4, Ljsym5, Ljsym23, and Ljsym30) result in the Coi phenotype characterized by arrested hyphal development following epidermal cell penetration. Based on a detailed cytological analysis of the progress of mycorrhiza formation of WT L. japonicus roots, it appears that fungal penetration into one or more epidermal cells prior to intercellular hyphal development within the exodermis is a prerequisite for normal colonization and arbuscule development in the underlying inner cortex (Bonfante et al., 2000). The authors suggest that such a detour is necessary to activate a general accommodation programme. Perhaps the most interesting aspect of this class of mutation is that they are both stage and cell type specific. This is demonstrated by the fact that weak alleles allow occasional colonization of the inner cortex, producing hyphae and arbuscules that are indistinguishable from those formed in the WT root. Another significant feature of these mutants is that epidermal cell penetration ultimately leads to death of both the host cell and fungal hyphae. Though reminiscent of the hypersensitive response (HR) in plants, no evidence of a defence response was observed in these mutants.

In addition to Pen and Coi phenotypes, a number of genetic lesions have been identified which affect intercellular proliferation of hyphae to varying degrees. These include phenotypes in which inner cortex invasion (Ici) does not occur as in the case of the mcbee (mycorrhizal colonization blocked between epidermis and exodermis) and the mcbex (mycorrhizal colonization blocked in exodermis) mutants of L. japonicus (Senoo et al., 2000b). While other mutants allow the colonization of the inner cortex but do not go on to form arbuscules (Arb), as exemplified by R69 of P. vulgaris (Shirtliffe & Vessey, 1996) and the Nod+/Fix, MN-IN3811 mutant of alfalfa (Bradbury et al., 1991). It should be noted, that in the case of the mcbex mutants, although they predominantly expresses an Ici phenotype, these mutants are also characterized by the overproduction of deformed appressoria and occasional formation of abnormal arbuscules. The molecular basis of these mutant phenotypes is unknown.

Compared with the significant number of mutants affecting fungal penetration and hyphal proliferation, only two mutants have been isolated in which the phenotype appears to be restricted to an effect on arbuscule development (Ard). The most extreme phenotype of the two is exhibited by the RisNod24 mutant of pea in which truncated arbuscules form (Gianinazzi-Pearson, 1996). RisNod24 is clearly defective only in the later stage of colonization as appressoria formation, root penetration and hyphal proliferation within the cortex are normal. In contrast, the mcbco (mycorrhizal colonization blocked in cortex) alleles of L. japonicus form arbuscules that are normal in appearance, but lead to premature senescence (Senoo et al., 2000b). Although the genes corresponding to these mutations have not been identified, differential RNA display analysis of RisNod24 revealed increased expression of the gene Psam4 which is predicted to encode a proline-rich protein (Lapopin et al., 1999). Increased expression of proline-rich proteins (normally down-regulated in AM) are associated with defence against plant pathogens. Despite the apparent divergence of the form and function of arbuscules and nodules, even at this late stage of AM development the two symbioses share common genetic determinants. This situation may reflect the large degree of conservation of the symbiosome between nodulation and mycorrhiza formation (Parniske, 2000).

Future AM host mutants

The current body of AM mutants share the common origin of having been isolated from pre-existing Nod pools (with the noted exception of rmc of tomato). Consequently, these mutants are affected in genes that play a role in both mycorrhiza formation and nodulation. It has become clear that a substantial portion of gene products required for endomycorrhizal and rhizobial colonization are shared between these two quite distinct symbioses. The mutant lines reviewed here represent nearly 40 mutations in at least 7–10 separate loci and constitute roughly half of the total Nod loci from which they were selected (Duc et al., 1989; Wegel et al., 1998; Catoira et al., 2000; Senoo et al., 2000a). The remainder of these Nod lines contain mutations that specifically affect the rhizobial symbiosis, but do not alter mycorrhiza formation. By analogy, we expect a number of AM mutants to be identified that will be specific to the mycorrhizal symbiosis.

Mutations that are unique to the AM symbiosis might be expected to affect precolonization events and arbuscule development and function especially. For example, mutations in plant genes involved in the production of plant signals triggering preinfection branching and appressorium formation are yet to be discovered. It has been demonstrated using isolated cell walls of carrot that appressoria formation is dependent on host epidermal cell wall factors (Nagahashi & Douds, 1997). Such preappressoria processes including precontact signalling, contact/recognition and adhesion of the fungus to the root are likely to be mycorrhiza formation-specific and be controlled in part by the plant host genotype. Because of the specialized role that the arbuscule is believed to play in phosphate transport, one would also expect a number of plant-encoded gene products to specifically contribute to arbuscule development and function. Other mutations may result in phenotypes similar to those described so far (i.e. lack of penetration or arbuscule formation), but may occur in genes acting specifically in the mycorrhizal pathways (Fig. 2). Among the potential targets are those involved in signalling (i.e. receptors, components of signal transduction including elements acting in feedback control), components of plant defence pathways, structural components of cell walls and the periarbuscular membrane, and proteins involved in arbuscule function, such as ion pumps and transporters (see Gianinazzi-Pearson, 1996; Harrison, 1999).

Figure 2.

Hypothetical model depicting common and specific pathways in plant-microbe symbioses. Nod, Myc+ mutant phenotypes represent mutations in nodulation-specific steps (black arrows). Nod, Myc phenotypes (blue arrows) are specified by at least seven genetic complementation groups and originate from mutations within genes involved in both nodulation and mycorrhiza development. Nod+, Myc mutant phenotypes have not been reported so far. However, it is possible that mycorrhiza-specific steps are as abundant as nodulation-specific steps. The model implies the existence of several parallel pathways that would not substitute for each other, but would all be required for the development of a fully functional symbiosis. Some of the mutations identified in Medicago truncatula (Mt), Lotus japonicus (Lj) and Pisum sativum (Ps) are shown at their putative site of action (also see Table 1).

To date, it is not possible to predict the number of mycorrhiza-specific plant genes that could be identified through mutant screens. However, given the fact that nodulation is essentially restricted to one plant family, whereas arbuscular mycorrhizas are widespread, a significant number of mycorrhiza-specific genes must exist. Moreover, despite apparent similarities in the accommodation of bacterial and fungal symbionts (Parniske, 2000), the infection process and arbuscule formation is sufficiently different from nodule formation so as to require specific gene functions. As multiple perception systems have been suggested to exist for rhizobial lipochitooligosaccharide nodulation factors (Schultze & Kondorosi, 1998), it is conceivable that there are also multiple (nonredundant) signalling pathways leading from the perception of putative fungal signals to the formation of arbuscular mycorrhizas. As outlined in the model in Fig. 2, parallel pathways may exist that control different processes required during the development of a complex symbiotic association.

Methods for the generation and isolation of AM mutants

Direct screening for mycorrhizal mutants requires microscopic analysis of mutant families, a labour intensive procedure. Nevertheless, the feasibility of this approach has been demonstrated (Barker et al., 1998). In the plant-Rhizobium symbiosis nodulation-defective mutants can be conveniently identified through macroscopic screens based on growth retardation in the absence of combined nitrogen, or by visual inspection of the root system for the appearance of nodules (e.g. Duc & Messager, 1989; Catoira et al., 2000). Under phosphate-limiting conditions, macroscopic screens based on growth responses might be feasible for mycorrhizas as well. However, plants are not absolutely dependent on mycorrhizas for the uptake of phosphate. Any growth advantage conferred by mycorrhizas may be too small to be used as a reliable criterion in mutant screens. Alternative, specialized, approaches could be developed. For instance, one may envisage performing mutagenesis on transgenic plant lines carrying mycorrhiza-inducible reporter gene fusions and screening for alterations in reporter gene expression. Similarly, insertion mutagenesis using gene trapping vectors may help identifying mutant alleles in the absence of visible mutant phenotypes (Webb et al., 2000). Recently, highly efficient transformation protocols have been established for the model legume M. truncatula (Kamatéet al., 2000; Trinh et al., 1998; Trieu et al., 2000). To date, M. truncatula is the only plant species, besides A. thaliana, for which a simple in planta infiltration protocol for Agrobacterium-mediated transformation has been described (Trieu et al., 2000). This will allow extensive insertional mutagenesis. To date several laboratories, including ours, are on the way to producing large numbers of T-DNA tagged M. truncatula lines using promoter-, enhancer- and activation-tagging vectors (Frugoli & Harris, 2001). Once large enough populations of insertion lines have been generated, functional genomics combined with reverse genetics may become a major tool for the identification of mutant alleles in model legumes. Unlike T-DNA mutagenesis, transposon mutagenesis has not yet been established in M. truncatula, whereas it has been successful in the other model legume, L. japonicus (Schauser et al., 1999, 1998). In addition to gene tagging strategies, positional cloning of genes from chemically induced mutants is becoming increasingly effective, based on the progress in genome mapping and sequencing (Bell et al., 2001; Frugoli & Harris, 2001). Whatever approach is taken for generating mutants, the concerted effort on model legumes is likely to lead to a rapid progress, in the near future, in identifying plant gene functions involved in AM formation.