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Keywords:

  • symbiotic signaling;
  • beneficial fungi;
  • ectomycorrhizal (ECM) fungi;
  • yeast two-hybrid screens;
  • symbiosis-regulated Ras-like protein;
  • Laccaria bicolor

Mycorrhizal associations enhance net photosynthesis, offer the potential for increased below ground carbon sequestration under elevated carbon dioxide concentrations, and hold promise for phytoremediation (Lussenhop et al., 1998; Fitter et al., 2000; Rufyikiri et al., 2002). Despite all this, very little is known about the mechanistic details of host-symbiont recognition, the pathways leading to symbiosis-specific development and subsequent nutrient exchange. The research presented in this issue by Sundaram et al. (pp. 525–534) – showing the results of yeast two-hybrid screens identifying protein partners interacting with a symbiosis-regulated Ras-like protein, Lbras, from the ECM fungus Laccaria bicolor– is therefore a significant step forward.

‘Ras proteins are involved in a dizzying array of regulatory activities in fungal systems including nucleotide and carbohydrate metabolism, cell division and DNA synthesis, pathogenicity, defense, membrane organization, cell death and aging’

Symbiotic signaling in a broad context

  1. Top of page
  2. Symbiotic signaling in a broad context
  3. Fungal regulatory genes
  4. Ras-interacting yeast two-hybrid mycorrhizal clones
  5. Perspectives
  6. References

The mycorrhizal fungi are thought to have played a significant role in the earliest evolution of land-based plants based on fossil records at least 460 million yr old with indications that their origins may be twice as old (Redecker et al., 2000; Schussler, 2002). Today a few hundred species of (AM) fungi colonize more than a hundred thousand different species of plants, creating a mutually beneficial biochemical economy (Smith & Read, 1997). The lack of specificity in AM partnerships contrasts with the exquisite specificity of the Rhizobium–legume symbioses (Soltis et al., 1995; Hirsch et al., 2001; Doyle & Luckow, 2003). Ironically, several plant mutations affecting early steps in symbiotic signaling with rhizobia are also defective for mycorrhizal colonization, suggesting the nitrogen-fixing symbiosis evolved utilizing pre-existing pathways required for AM symbiosis. One example is the receptor kinase-like proteins identified in pea and Lotus that are members of a large family of plant and animal receptors containing an extracellular leucine-rich motif (Stracke et al., 2002; Endre et al., 2002). Another plant gene required for symbiosis, the Sym4 protein from Lotus (Bonfante et al., 2000), appears to exert its effects through changes in the cytoskeleton mediating compatibility responses in the epidermal tissues of the plant host (Genre & Bonfante, 2002). These findings and others underscore the importance of studying ECM and AM symbiotic signaling in the broadest possible context in order to increase our understanding of the biochemistry and ecology of these organisms (Martin et al., 2001).

Fungal regulatory genes

  1. Top of page
  2. Symbiotic signaling in a broad context
  3. Fungal regulatory genes
  4. Ras-interacting yeast two-hybrid mycorrhizal clones
  5. Perspectives
  6. References

The search for ECM and AM fungal regulatory genes is beginning to bear fruit, as the work of Sundaram et al. demonstrates. Lbras was first identified in an EST screen and the full length Lbras was shown to possess several remarkable characteristics, including the ability to complement a yeast Ras2 mutant and transform mouse embryonic stem cells, altering their growth rate and morphology to an oncogenic phenotype (Sundaram et al., 2001). Expression of the single copy Lbras requires interaction with host root factors with transient expression observable within 6 h and stable transcription after 24 h. The protein is localized within membranous regions of the Hartig net and mantle particularly along the periphery of the cell. It is not yet known if Lbras is modified by acylation events as occurs with other Ras proteins. Interestingly, while the Arabidopsis genome encodes many genes belonging to the Ras superfamily (Rab, Rho, Arf, and Ran) there are no true ras genes to be found (Vernoud et al., 2003).

Ras proteins are involved in a dizzying array of regulatory activities in fungal systems including nucleotide and carbohydrate metabolism, cell division and DNA synthesis, pathogenicity, defense, membrane organization, cell death and aging. In yeast, the Ras2 gene encodes a homolog of the mammalian oncogene RAS and is highly related to the yeast RAS1 gene (Kataoka et al., 1984). Ras2p is a small GTP-binding protein localized to the yeast plasma membrane as a result of the modification of its C-terminus with palmitoyl and farnesyl groups (Bhattacharya et al., 1995). Ras2p regulates processes such as sporulation, filamentous growth and the nitrogen starvation response through its effects on yeast adenylate cyclase (encoded by the CYR1 gene). In the activated, GTP-bound form Ras2p directly stimulates the production of cAMP by adenylate cyclase (Broek et al., 1985). Cdc25p binds to and activates Ras2p by directly stimulating the exchange of GDP for GTP (Lai et al., 1993). Conversely, the redundant proteins Ira1p and Ira2p inactivate Ras2p by stimulating hydrolysis of GTP to GDP (Parrini et al., 1996). In the alfalfa fungal phytopathogen Colletotrichum trifolii, suppression of Ras activity significantly decreases fungal germination frequencies and hyphal growth rates (Ha et al., 2003). Ras is also known to regulate spore germination in Neurospora and Aspergillus (d’Enfert, 1997) and is essential for morphogenetic switching to hyphal growth and pathogenesis in Candida albicans (Rocha et al., 2001). Three facts become abundantly clear from these examples:

  • 1
    Ras is a multifunctional protein regulating developmental pathways in free-living, symbiotic and pathogenic fungi.
  • 2
    Control of Ras activity depends not only on its pattern of expression, but also on factors that regulate Ras interaction with GDP (inactive) and GTP (active).
  • 3
    The key to understanding symbiosis-specific Ras regulation will be the identification of its protein partners.

Ras-interacting yeast two-hybrid mycorrhizal clones

  1. Top of page
  2. Symbiotic signaling in a broad context
  3. Fungal regulatory genes
  4. Ras-interacting yeast two-hybrid mycorrhizal clones
  5. Perspectives
  6. References

Three distinct Lbras-interacting gene products were identified by Sundaram et al. and designated RythmA, RythmB and Rythm C (Ras interacting yeast two hybrid mycorrhizal clones). The RythmB sequence showed no significant similarities to known genes and the RythmC sequence had weak similarity to a receptor kinase. Interestingly RythmA resembles the eukaryotic AP180 protein family. This family displays an Asn-Pro-Phe motif required for protein–protein interactions and several sites suggestive of phosphorylation sites for canonical casein kinase II, protein kinase C, and tyrosine kinases. The C-terminus of the RythmA protein contains a fungal hydrophobin motif as well, found in other fungal symbiotic proteins, although the functional significance of this observation is not yet clear.

Immuno-localization of LbRAS protein revealed a clustering in the vicinity of dolipore-septum regions in the Hartig net region. The dolipore allows the cytoplasm of adjacent ECM cells to mix, yet another novel feature of hyphal biology. It is tempting to speculate that Lbras-RythmA proteins are involved in the regulation of vesicle traffic in this region. If so, this would indicate that Lbras has an activity resembling plant and mammalian Rab GTPases that control vesicle assembly and transport (Sohn et al., 2003). The probability that Lbras will manifest other regulatory activities is high, given the diversity of Ras functions in different organisms and the isolation of RythmB and RythmC from L. bicolor in the two-hybrid screen. The Lbras protein is most closely related to the Aras protein from Aspergillus nidulans, yet a BLAST search of the A. nidulans genome with the RythmA sequence failed to identify a gene product with any significant degree of homology. Thus, it seems likely that the genetic differences along the continuum of pathogenic and symbiotic fungi may be productively explored by focusing attention on novel proteins that interact with key common regulatory components.

Perspectives

  1. Top of page
  2. Symbiotic signaling in a broad context
  3. Fungal regulatory genes
  4. Ras-interacting yeast two-hybrid mycorrhizal clones
  5. Perspectives
  6. References

Future work will be needed to elucidate the mechanism of Lbras regulation and determine which other symbiotic pathways are directly or indirectly regulated by this key protein. Critical questions include the issue of Ras-dependent regulation of cAMP levels, and whether the ECM utilize the MAP kinase signaling pathways as well. Analysis of the Lbras and RythmA promoters for cis and trans elements that regulate its symbiosis-specific expression should reveal components of the pathway that links ECM gene expression to diffusible plant root signaling compounds. Finally, given the nutrient exchanges that lie at the heart of ECM and AM fungal symbioses, an understanding of the mechanisms of long-distance inter- and intracellular transport will require understanding not just biochemical pathways but also the regulated movement of organelles and nutrient ‘bodies’ like glycogen and lipid bodies (Sohn et al., 2003). The work of Sundaram et al. provides an important and exciting starting point for these investigations.

References

  1. Top of page
  2. Symbiotic signaling in a broad context
  3. Fungal regulatory genes
  4. Ras-interacting yeast two-hybrid mycorrhizal clones
  5. Perspectives
  6. References
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