Summary
In ectomycorrhiza, root penetration and colonization of the intercellular space by symbiotic hyphae is thought to rely on the mechanical force that results from hyphal tip growth, enhanced by ...the activity of secreted cell‐wall‐degrading enzymes.
Here, we characterize the biochemical properties of the symbiosis‐induced polygalacturonase LbGH28A from the ectomycorrhizal fungus Laccaria bicolor. The transcriptional regulation of LbGH28A was measured by quantitative PCR (qPCR). The biological relevance of LbGH28A was confirmed by generating RNA interference (RNAi)‐silenced LbGH28A mutants. We localized the LbGH28A protein by immunofluorescence confocal and immunogold cytochemical microscopy in poplar ectomycorrhizal roots.
Quantitative PCR confirmed the induced expression of LbGH28A during ectomycorrhiza formation. Laccaria bicolor RNAi mutants have a lower ability to establish ectomycorrhiza, confirming the key role of this enzyme in symbiosis. The purified recombinant LbGH28A has its highest activity towards pectin and polygalacturonic acid. In situ localization of LbGH28A indicates that this endopolygalacturonase is located in both fungal and plant cell walls at the symbiotic hyphal front.
These findings suggest that the symbiosis‐induced pectinase LbGH28A is involved in the Hartig net formation and is an important determinant for successful symbiotic colonization.
Mycorrhizal symbioses-the union of roots and soil fungi-are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all ...depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains ∼20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.
Summary
The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, ...thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships.
Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed.
MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2‐regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis.
Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.
Rhizosphere-associated
are known plant growth promoting (PGP) and mycorrhizal helper bacteria (MHB) of many plants and ectomycorrhizal fungi. We investigated the spatial and temporal dynamics of ...colonization of mycorrhizal and non-mycorrhizal Aspen seedlings roots by the
strains SBW25, WH6, Pf0-1, and the
strain Pf-5. Seedlings were grown in laboratory vertical plates systems, inoculated with a fluorescently labeled
strain, and root colonization was monitored over a period of 5 weeks. We observed unexpected diversity of bacterial assemblies on seedling roots that changed over time and were strongly affected by root mycorrhization.
SBW25 and WH6 stains developed highly structured biofilms with internal void spaces forming channels. On mycorrhizal roots bacteria appeared encased in a mucilaginous substance in which they aligned side by side in parallel arrangements. The different phenotypic classes of bacterial assemblies observed for the four
strains were summarized in a single model describing transitions between phenotypic classes. Our findings also reveal that bacterial assembly phenotypes are driven by interactions with mucilaginous materials present at roots.
Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of β-propeller lectins conserved from bacteria to mammals that have been shown to bind ...bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans , suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1 , coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus , showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.
Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have ...therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin‐encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin‐encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.
The phytohormones jasmonate, gibberellin, salicylate, and ethylene regulate an interconnected reprogramming network integrating root development with plant responses against microbes. The ...establishment of mutualistic ectomycorrhizal symbiosis requires the suppression of plant defense responses against fungi as well as the modification of root architecture and cortical cell wall properties. Here, we investigated the contribution of phytohormones and their crosstalk to the ontogenesis of ectomycorrhizae (ECM) between grey poplar (Populus tremula x alba) roots and the fungus Laccaria bicolor. To obtain the hormonal blueprint of developing ECM, we quantified the concentrations of jasmonates, gibberellins, and salicylate via liquid chromatography–tandem mass spectrometry. Subsequently, we assessed root architecture, mycorrhizal morphology, and gene expression levels (RNA sequencing) in phytohormone‐treated poplar lateral roots in the presence or absence of L. bicolor. Salicylic acid accumulated in mid‐stage ECM. Exogenous phytohormone treatment affected the fungal colonization rate and/or frequency of Hartig net formation. Colonized lateral roots displayed diminished responsiveness to jasmonate but regulated some genes, implicated in defense and cell wall remodelling, that were specifically differentially expressed after jasmonate treatment. Responses to salicylate, gibberellin, and ethylene were enhanced in ECM. The dynamics of phytohormone accumulation and response suggest that jasmonate, gibberellin, salicylate, and ethylene signalling play multifaceted roles in poplar L. bicolor ectomycorrhizal development.
The ectomycorrhizal fungus Laccaria bicolor affects sensitivity to jasmonate in roots of Populus tremula x alba, while promoting responses to salicylate, gibberellin, and ethylene. Adjusted accumulation and perception of these phytohormones is necessary for the development of ectomycorrhizae.
Despite the pivotal role of jasmonic acid in the outcome of plant-microorganism interactions, JA-signaling components in roots of perennial trees like western balsam poplar (Populus trichocarpa) are ...poorly characterized. Here we decipher the poplar-root JA-perception complex centered on PtJAZ6, a co-repressor of JA-signaling targeted by the effector protein MiSSP7 from the ectomycorrhizal basidiomycete Laccaria bicolor during symbiotic development. Through protein-protein interaction studies in yeast we determined the poplar root proteins interacting with PtJAZ6. Moreover, we assessed via yeast triple-hybrid how the mutualistic effector MiSSP7 reshapes the association between PtJAZ6 and its partner proteins. In the absence of the symbiotic effector, PtJAZ6 interacts with the transcription factors PtMYC2s and PtJAM1.1. In addition, PtJAZ6 interacts with it-self and with other Populus JAZ proteins. Finally, MiSSP7 strengthens the binding of PtJAZ6 to PtMYC2.1 and antagonizes PtJAZ6 homo-/heterodimerization. We conclude that a symbiotic effector secreted by a mutualistic fungus may promote the symbiotic interaction through altered dynamics of a JA-signaling-associated protein-protein interaction network, maintaining the repression of PtMYC2.1-regulated genes.
Ectomycorrhizal fungi, living in soil forests, are required microorganisms to sustain tree growth and productivity. The establishment of mutualistic interaction with roots to form ectomycorrhiza ...(ECM) is not well known at the molecular level. In particular, how fungal and plant cell walls are rearranged to establish a fully functional ectomycorrhiza is poorly understood. Nevertheless, it is likely that Carbohydrate Active enZymes (CAZyme) produced by the fungus participate in this process.
Genome-wide transcriptome profiling during ECM development was used to examine how the CAZome of Laccaria bicolor is regulated during symbiosis establishment.
CAZymes active on fungal cell wall were upregulated during ECM development in particular after 4weeks of contact when the hyphae are surrounding the root cells and start to colonize the apoplast. We demonstrated that one expansin-like protein, whose expression is specific to symbiotic tissues, localizes within fungal cell wall.
Whereas L. bicolor genome contained a constricted repertoire of CAZymes active on cellulose and hemicellulose, these CAZymes were expressed during the first steps of root cells colonization. L. bicolor retained the ability to use homogalacturonan, a pectin-derived substrate, as carbon source. CAZymes likely involved in pectin hydrolysis were mainly expressed at the stage of a fully mature ECM.
All together, our data suggest an active remodelling of fungal cell wall with a possible involvement of expansin during ECM development. By contrast, a soft remodelling of the plant cell wall likely occurs through the loosening of the cellulose microfibrils by AA9 or GH12 CAZymes and middle lamella smooth remodelling through pectin (homogalacturonan) hydrolysis likely by GH28, GH12 CAZymes.
Relatively few comparative studies exist of nitrogen (N) uptake by ectomycorrhizal (EM) and nonmycorrhizal roots under controlled conditions. Instantaneous and long-term N uptake by EM and ...nonmycorrhizal roots of lodgepole pine seedlings (Pinus contorta (Douglas) var. latifolia (Engelm.)) were compared after 9-12 months of preconditioning under high or low N supply, with NH.sub.4.sup.+ and NO.sub.3.sup.- in proportions of 90:10 and 10:90. EM roots were inoculated with Amphinema byssoides or Laccaria bicolor. Instantaneous net NH.sub.4.sup.+, NO.sub.3.sup.-, and H.sup.+ fluxes were measured simultaneously on seedling roots with a microelectrode ion flux measurement system, and seedling organ biomass and N content were measured to infer long-term N uptake. Net NH.sub.4.sup.+ uptake was observed in EM roots of lodgepole pine but NH.sub.4.sup.+ efflux was observed in nonmycorrhizal roots. Greater instantaneous N uptake in EM roots was supported by higher root and shoot N contents in seedlings associated with L. bicolor than in noninoculated seedlings. Absence of NH.sub.4.sup.+ efflux in EM roots suggests that root colonization by EM fungi has the potential to reduce futile cycling of NH.sub.4.sup.+. EM lodgepole pine appeared to prefer NH.sub.4.sup.+ over NO.sub.3.sup.- as an N source and instantaneous NH.sub.4.sup.+ uptake rates were higher in NH.sub.4-starved roots. H.sup.+ efflux was observed in most roots. Future work should focus on a greater variety of EM species to explore differences among species in N uptake.
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