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Keywords:

  • symbiosis;
  • Lotus japonicus;
  • rust;
  • biotroph;
  • arbuscular mycorrhiza;
  • intracellular;
  • SYMRK

Obligate biotrophic fungi are typically unable to grow in the absence of a host plant, and comprise a taxonomic diversity of basidio-, asco-, zygo- and oomycetes (Voegele & Mendgen, 2003; Schulze-Lefert, 2004). Although many biotrophic pathogens (such as rust fungi, powdery mildew fungi and oomycetes) cause devastating diseases of crop plants, other biotrophs (such as arbuscular mycorrhizal fungi) engage in beneficial mutualisms that actually improve plant health (Parniske, 2004). Considering their impact on a global ecology (Vance, 2001), this symbiosis is probably the most important plant–fungus interaction.

One common feature of many biotrophic interactions is the formation of elaborate intracellular accommodation structures that maintain an intimate coexistence of both fungal and plant cells over a prolonged period (Panstruga, 2003). These structures, called haustoria in pathogenic interactions, or arbuscules in the arbuscular mycorrhiza symbiosis, have superficially similar structural and functional properties, and are believed to be the main site of nutrient flow between interacting cells (Mendgen & Hahn, 2002).

The commonalities between these intracellular biotrophic interactions have kindled speculation about their evolutionary and genetic relationship (Parniske, 2000). Genetic analyses of biotrophy are most advanced for the arbuscular mycorrhiza symbiosis, in which seven plant genes have been identified that are required to support infection (Kistner et al., 2005). These plant genes, which are also required for nitrogen-fixing root nodule symbiosis with bacteria, are referred to as the common symbiosis (SYM) genes, and encode proteins that carry the hallmarks of signalling molecules (Oldroyd & Downie, 2004). This, together with the phenotype of the corresponding plant mutants, indicates that the common SYM genes are involved in a signalling process that is required for successful infection of plant cells by symbiotic fungi and bacteria.

The AM symbiosis is believed to be at least as old as the earliest land plants (Remy et al., 1994; Redecker et al., 2000), and the radiation of the basidiomycetes and ascomycetes that cause plant diseases occurred later (Berbee & Taylor, 1993; Taylor et al., 1999). One of our major unanswered questions about biotrophic interactions is whether the plant uses a similar or the same genetic program for the intracellular hosting of pathogenic and symbiotic fungi (Parniske, 2000). Although it might seem unlikely that plants would maintain genes to assist infection by pathogenic biotrophs, it is entirely possible that parasites may exploit some aspects of the mutualistic plant program that evolved much earlier, but that still provide a strong selective advantage for the plant. Research on biotrophic pathogenic interactions has mostly focused on resistance processes, therefore plant genetics of compatibility in these systems is still in its infancy (Schulze-Lefert & Panstruga, 2003; Schulze-Lefert, 2004). In the current study we used a genetic approach to unravel the overlap between mutualistic and parasitic biotrophy. In order to determine whether pathogenic rust fungal infection is dependent on plant genes required for mutualistic symbioses, we inoculated Lotus japonicus symbiosis-defective mutants representing six different common SYM genes with a compatible isolate of the Lotus rust fungus Uromyces loti.

The symbiosis receptor-like kinase SYMRK (DMI2 in Medicago truncatula) has an extracellular domain that is potentially involved in the perception of a fungal or bacterial signalling molecule (Endre et al., 2002; Stracke et al., 2002). Because of this predicted structure and mutant phenotype, SYMRK has been placed conceptually as one of the most upstream components in a symbiotic signalling pathway (Parniske, 2004). Although mutant plants are somewhat responsive to bacterial symbiotic signalling molecules (lipochitin oligosaccharides) called Nod factors, inoculated root hairs do not respond to mutualistic bacteria by producing microbe-accommodating infection threads or with the rapid transcriptional upregulation of symbiotic marker genes (Stracke et al., 2002; Kistner et al., 2005). Although SYMRK is also required for mycorrhizal infection, it was not important for infection by the phytopathogenic Lotus rust fungus. We observed hyphal proliferation and sporulation on symRK mutant plants. Both in timing and appearance, fungal growth on symRK mutants was indistinguishable from that on wild-type leaves (Table 1; Fig. 1).

Table 1.  Compatible growth of the rust fungus Uromyces loti on common symbiosis mutants of Lotus japonicus
Mutant alleleLine designationReferencesIdentity (if known)
symRK-2EMS61Stracke et al. (2002); Szczyglowski et al. (1998)Receptor-like kinase
castor-2EMS1749Bonfante et al. (2000); Imaizumi-Anraku et al. (2005)Plastid-localized cation transporter
pollux-2EMS167Imaizumi-Anraku et al. (2005); Szczyglowski et al. (1998)Plastid-localized cation transporter
ccamk-2cac57.3Schauser et al. (1998); Tirichine et al. (2006)Calcium and calmodulin-dependent protein kinase
nup133-3EMS247Kanamori et al. (2006); Szczyglowski et al. (1998)Nucleoporin
sym6-3EMS126Szczyglowski et al. (1998)?
image

Figure 1. Leaves of Lotus japonicus wild-type Gifu and several common sym mutants infected with the Lotus rust fungus Uromyces loti. Successful colonization of Lotus cotyledons results in the formation of rust pustules (arrows, left panels) 9 d after inoculation. Note the formation of healthy, non-encased, fungal haustoria (h) in the light micrographs of fixed, cleared rust-infected Lotus cotyledons (right panels, bar = 10 µm). Leaves were fixed and decolourized in 95% (v/v) ethanol, stained with trypan blue-lactophenol to visualize fungal structures, and cleared in saturated chloral hydrate before being mounted in modified Hoyer's medium, as described previously (Stumpf & Heath, 1985).

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Perception of bacterial Nod factors is quickly followed (within 10 min) by intracellular calcium oscillations known as ‘calcium spiking’ (Oldroyd & Downie, 2004). Two closely related putative cation transporters called CASTOR and POLLUX (Imaizumi-Anraku et al., 2005) are required for this calcium response and for the arbuscular mycorrhizal symbiosis. Importantly, these plastid-localized transporters are not required for infection by the Lotus rust fungus (Table 1). However, measurements of calcium levels in single susceptible rust-infected cowpea vein epidermal cells revealed no increased calcium levels at early stages of wall penetration and intracellular infection (Xu & Heath, 1998). This, together with pharmacological data, has led to the suggestion that changes in intracellular calcium concentration are not likely to have an influence on the outcome of compatible interactions with phytopathogenic rust fungi. It will be interesting to determine whether infection by powdery mildew fungi that appears to be dependent on calcium/calmodulin signalling through the susceptibility determinant MLO (Kim et al., 2002) has a comparable or different response on the castor and pollux mutants.

Physical infection by rhizobia and arbuscular mycorrhizal fungi involves the active participation of the plant, which probably includes the localized release of cell wall-degrading enzymes such as pectinases that allow the microbe successfully to colonize the inter- and intracellular spaces of the root. For arbuscular mycorrhizal interactions, one such response is the epidermal opening response that is dependent on the presence of the Lotus gene CCamK (Demchenko et al., 2004; Tirichine et al., 2006). We clearly demonstrate that the presence of CCamK is not required for infection by the rust U. loti (Table 1; Fig. 1), a fungus that is more likely to enter plant cells through a combination of physical force and secretion of cell wall-degrading enzymes (Tucker & Talbot, 2001).

In brief, Lotus mutants defective for SYMRK, CCamK, CASTOR, POLLUX, NUP133 and SYM6, which are dysfunctional in establishment of symbiotic infection by mutualistic mycorrhizal fungi and mutualistic bacteria, were all successfully colonized by the Lotus rust fungus (Table 1). More importantly, there was no evidence of fungal cell death or fungal encasement in mesophyll cells of mutant plants, and there were no obvious signs of any adverse plant responses, such as cell death or browning (Fig. 1). Together, our cytological investigation revealed nothing to distinguish rust fungal interactions with symbiosis-defective mutant plants from that seen in the wild-type Lotus accession Gifu.

Our data suggest there is little, if any, significant overlap in the initial signalling processes between root mutualistic vs leaf phytopathogenic biotrophic interactions. One caveat of our study is that the response of different tissues (root vs leaf) was compared. It is therefore still possible that genes parologous to those involved in root-associated mutualisms may affect infection by foliar leaf pathogens, which would not have been identified in the current study. Given that pathogen species may exhibit different infection mechanisms in roots vs leaves (Sesma & Osbourn, 2004), it is possible that different plant genes are involved in accommodating infection by biotrophs in different plant organs.

That rust fungi, powdery mildew fungi and oomycetes all successfully infect the model plant Arabidopsis thaliana in the absence of clear orthologues of, for example, SYMRK (Kevei et al., 2005) or CCaMK (Levy et al., 2004; Mitra et al., 2004) in the completely sequenced Arabidopsis genome appears to support this view. Recent data in rust fungal systems suggest that successful establishment of compatible infection is determined at a very early stage of infection, when the fungus is just beginning to penetrate the plant cell wall (Mould & Heath, 1999), but that success or failure of infection depends more on the presence or absence of plant defence responses (Mellersh & Heath, 2001, 2003) than on any genetically predetermined plant accommodation program. It will perhaps be of greater future interest to focus on those later stages of mutualistic and phytopathogenic biotrophic interactions (during or after formation of intracellular infection structures), where changes in plant cell metabolism affecting the exchange (either uni- or bidirectional) of nutrients and signals between host and microbe could shed more light on shared elements involved in the establishment of biotrophic compatibility across a variety of microbial taxa.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. References

The authors thank Matthias Hahn, University of Kaiserslautern for providing the isolate of Uromyces loti. Research at The Sainsbury Laboratory was funded by the Gatsby Charitable Foundation.

References

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