Mycorrhizal associations are important mutualisms between plant roots and fungi that allow plants to acquire water and nutrients from the environment in exchange for photosynthates (Jeffries et al., 2003; Bonfante and Anca, 2009; Plett and Martin, 2018; Tedersoo et al., 2020). In the past years, there has been a continuous effort to advance the understanding of the molecular signaling mechanisms used by fungi to colonize plants in both natural and agricultural environments (Bonfante and Genre, 2010; Pan et al., 2013; Kamel et al., 2017; Maclean et al., 2017; Choi et al., 2018). An example is the production of lipo-chitooligosaccharides (LCOs—also known as Nod factors) that arbuscular mycorrhizal (AM) and ectomycorrhizal fungi (ECM) produce to stimulate colonization of host plant roots (Maillet et al., 2011; Cope et al., 2019; Khokhani et al., 2021). The production of LCOs was also shown to cause diverse effects on a variety of plants with or without mycorrhizal fungi (Tanaka et al., 2015).

LCOs were first discovered and characterized in rhizobia bacteria (Lerouge et al., 1990; Dénarié et al., 1996; Poinsot et al., 2016) and later found to be produced by most fungi (Maillet et al., 2011; Cope et al., 2019; Rush et al., 2020). These amphiphilic molecules are polymers made of three to five N-acetyl glucosamine (GlcNAc) residues with β-(1,4) linkages modified with a long-chain fatty acyl group and various other functional groups (Lerouge et al., 1990; Dénarié et al., 1996; Malkov et al., 2016). Most fungi produce sulfated LCOs (sLCOs) or non-sulfated LCOs (nsLCOs) with a palmitic (C16:0) or oleic (C18:1) acid (fatty acid chain) attached to the first chitin monomer and have either a chitotetraose, tetra-N-acetyl (CO4), or chitopentaose, penta-N-acetyl (CO5) backbone (Rush et al., 2020). CO4 and CO5 are N-acetyl chitooligosaccharides and should not be confused with chitosan oligomers (Yin et al., 2016). Other chemical substitutions have been identified in fungal LCOs, but their role has not yet been determined (Rush et al., 2020). LCOs and COs are perceived on the surface of the root cells of host plants by a group of lysine-motif receptor-like kinases (LysM) (Buendia et al., 2018) which in return activate a central plant signaling cascade known as the Common Symbiosis Pathway (CSP) (Feng et al., 2019; Cope et al., 2021; Wu et al., 2021). The immediate response of plants to the perception of LCOs or COs, produced by symbiotic or beneficial endophytic fungi, are oscillations in nuclear calcium (Ca⁺²) concentration levels in the epidermal cells of host roots, which subsequently cause Ca⁺²/calmodulin-dependent protein kinases to regulate the activity of several transcription factors necessary for the establishment of symbiosis or mutualistic associations (Chabaud et al., 2011; Genre et al., 2013; Buendia et al., 2016; Luginbuehl and Oldroyd, 2017; Choi et al., 2018; Cope et al., 2019, 2021; Feng et al., 2019; Skiada et al., 2020). It remains unknown if LCOs and COs from saprotrophic and pathogenic fungi trigger the CSP and if they could use this signaling pathway for colonization of a host plant.

In addition to their impact on their host plants, LCOs and COs can alter fungal physiology and transcriptomics. The observed fungal behavior may be attributed to sensing diffusible chemical signals (Aleklett and Boddy, 2021). For example, in the saprotrophic and opportunistic human pathogen, Aspergillus fumigatus, growth with C16:0 sulfated LCOs (C16:0 sLCOs) resulted in differential expression of genes encoding proteins associated with cell membrane activities and cell wall processes that led to significantly reduced hyphal branching (Rush et al., 2020). Also, C16:0 sLCOs were shown to influence fungal behavior in other opportunistic human pathogens like increased pseudohyphae formation in Candida glabrata and increased cell proliferation in Rhodotorula mucilaginosa (Rush et al., 2020). When a fungus is co-inoculated with a host plant, LCOs were shown to increase disease incidence of Sclerotinia stem rot on susceptible lines of soybeans (Marburger et al., 2018), increased colonization from arbuscular mycorrhizal fungi in legumes (Maillet et al., 2011), and increased mantle width and Hartig net formations in L. bicolor colonizing poplar roots (Cope et al., 2019). Although few investigations reported the role of LCOs on fungal development or their influence on co-colonization within a host, the recent onset of evidence is pointing toward alternative roles of LCOs and COs outside of symbiosis. These molecules were reported to be produced by fungi on substrate media in the absence of a host to potentially influence the development of the producing organism (Rush et al., 2020). However, the mechanisms by which these molecules are triggering these physiological changes remained largely unknown. Therefore, the goal of this study is to provide a baseline cognizance of the structure, function, and regulation of fungal behavior caused by LCOs and COs.

Ectomycorrhizal fungi, such as Laccaria bicolor, play a crucial role in various forest ecosystems. They provide a nutritional benefit to their host plants and help mitigate the impact of a wide range of biotic and abiotic stresses (Martin et al., 2008). Given the recognition of LCOs and COs as critical signaling molecules in regulating plant and fungal behaviors, we set to study these signals' influence on L. bicolor physiology, protein abundance changes, and metabolite production. We hypothesize that when the fungus is living within a soil microbiome, it uses specific types of LCOs or COs to regulate its development, regardless of its symbiosis with the host plant. Previously, different LCO structures were shown to increase lateral root development in poplar or improve colonization of L. bicolor with its poplar host (Cope et al., 2019). However, understanding how LCOs or COs influence L. bicolor development by itself has not been investigated. Therefore, we expose the fungus to the same sLCOs and nsLCOs purified from rhizobia, and to the same chitooligosaccharides chitotetraose (CO4) and chitooctaose (CO8) used by Cope et al. (2019). A key consideration for these experiments is that various LCO types are used for specific host symbiosis and phenotypic responses (D'Haeze and Holsters, 2002). This is noteworthy because as previously shown, sLCOs and nsLCOs extracted from rhizobia or numerous fungi were shown to cause root hair branching in Medicago truncatula and Vicia sativa, respectively (Cope et al., 2019; Rush et al., 2020), indicating that the structure of LCOs is potentially similar enough between rhizobia and fungi to elicit this phenotype in legumes. Therefore, for this manuscript, we assume that the sLCOs and nsLCOs used are not recognized as foreign signals, but commonly produced molecules by rhizobia and most fungi, including L. bicolor. Later, we tested the effect of synthetically made LCOs on L. bicolor to mimic the structure of LCOs abundantly produced by this fungus. We also examined the effects from CO4, one of the possible backbone molecules of an LCO (Lerouge et al., 1990), and CO8, an elicitor for plant defense responses, but not a backbone molecule of LCOs (Kuchitsu et al., 1997; Buendia et al., 2018; Feng et al., 2019). Lastly, since rhizobia produce LCOs to initiate symbiosis with legumes under field conditions (Kidaj et al., 2012; Siczek et al., 2014), presumably, L. bicolor will perform a similar function with poplar. Therefore, the findings from our in vitro studies would help understanding the influence of LCOs and COs on L. bicolor in field studies. Finally, a proteomic approach was subsequently used to provide molecular insights into the changes in L. bicolor protein abundance when growing with or without the various forms of LCOs and COs. Differential protein abundance underpinning reduced growth for specific LCOs and COs represent a substantial alteration in proteome expression in biological processes related to polarized growth.

The integrated characterization of L. bicolor physiology with molecular and metabolic measurements provides new insights into how fungal behavior is altered in the presence of LCOs and COs. Moreover, the in-depth proteome analysis serves as a crucial data resource and a first step toward understanding how these secondary metabolites influence fungal behavior in the absence of a host. Overall, each treatment decreased the total number of proteins, with nsLCOs being the treatment that had the least impact 21 days post-induction (dpi). The underlying protein regulation and associated functional annotations might explain the reduced fungal growth compared to solvent control samples. Exposure of L. bicolor to LCOs or COs treatments resulted in the downregulation of proteins involved in polarized growth compared to solvent control samples. These proteins were part of “cellular component assembly” and “establishment of cell polarity” GO categories that included kinases, transcription factors, GTPases, nuclear transport-related proteins, dynein chains, and others (Supplementary Table S2). Network-based integration of these proteins using protein-protein interactions in the STRING v11.5 database suggested biologically meaningful clusters of proteins, as observed in Figure 4. Amongst these, one cluster showed the highest number of protein-protein connections and contained several protein kinases. We noticed the presence of a protein annotated as a CMGC/CDK/CDC2 protein kinase (Accession ID. B0DHF2), which was the most connected node in the resulting network (Supplementary Table S4). In general, cyclin-dependent kinases (Cdks) are essential for hyphal development under specific hypha-inducing conditions and transcription of hypha-specific genes (Loeb et al., 1999; Chen et al., 2000). In C. albicans, disruption of a gene coding for a CDC2-related kinase (Crk1) have led to defective hyphal formation under various hypha-inducing conditions suggesting an active role in the transcriptional regulation of hypha-specific genes (Chen et al., 2000). In S. cerevisiae, the activity of another Cdk, CDC28, has been shown to regulate various aspects of filamentous growth depending on its associated cyclins (Edgington et al., 1999; Loeb et al., 1999; Rua et al., 2001). A closer look in sequence space of the L. bicolor CMGC/CDK/CDC2 protein kinase identified here revealed a higher level of similarity to CDC28 from S. cerevisiae (67% similarity) than to the reported Crk1 in C. albicans (23% similarity) and the visualization provided by the STRING network seems to suggest that this protein is critical to initiate a cascade of phosphorylation events that might lead to the regulation of polarized growth. Interestingly, although not connected to the kinases mentioned previously, another cluster of proteins contained several translation-initiation factors, strengthening the idea of this regulatory network (Figure 4).

Additionally, all treated samples caused several small GTPases to be downregulated compared to solvent control samples (Supplementary Table S2). These proteins play essential roles as molecular switches that can turn various cell polarity pathways on or off (Ranocchi and Amicucci, 2021). In S. cerevisiae, evidence has suggested that CDK complexes act as crucial regulators of small GTPases. For example, for the Rho family GTPase Cdc42, which is considered a master player in the process of yeast budding and its associated polarized growth (Johnson, 1999; Park and Bi, 2007), it is hypothesized that CDKs exert control on it by either direct phosphorylation or by regulation of its effectors and adaptor proteins (Pedraza et al., 2019). Although our STRING analysis did not show any evidence of interaction between protein kinases and small GTPases, the identifications provided in this study should be considered for investigations into potential associations between these groups of enzymes in L. bicolor. Additionally, other proteins related to filamentous growth development were downregulated in all-treated samples. Amongst these, we identified dynein heavy chains. Dynein is a type of cytoskeleton-dependent motor protein that has various roles in transporting proteins, vesicles, and organelles (Yamamoto and Hiraoka, 2003; Xiang and Fischer, 2004; Fischer et al., 2008). In filamentous fungi, the disruption of dynein expression causes defects in typical fungal developmental structures (Seiler et al., 1999; Riquelme et al., 2002).

The determination of the specific regulatory network underlying the effects on L. bicolor polarized growth when exposed to LCOs or COs is out of the scope of this manuscript; however, these observations suggest that the growth effects observed here may be orchestrated by the coordinated actions of protein kinases, small GTPases and other effectors. To our knowledge, nutrient starvation is not playing a significant role in these molecular responses, as all samples kept growing past 21 days. The induction of hyphal branching in response to plant or fungal signals has been documented. However, the only other study showing that exogenous LCOs signals can actively suppress filamentous branching in fungi was the study mentioned previously, investigating the effects of LCOs or COs in non-symbiotic fungi (Rush et al., 2020). It is plausible that the production of exogenous LCOs or COs by other microbes in the vicinity of L. bicolor acts to counteract the spatial growth of the fungus in some sort of competition for resources. This hypothesis is also supported by the upregulation of L. bicolor proteins involved in cross-communication or defense response mechanisms observed in specific treatments, like a terpenoid synthase in sLCO-treated samples, tectonin-2 in CO8-treated samples, or an isochorismatase family protein in nsLCO-treated samples. The latter is part of a group of enzymes speculated to reduce external responses such as the accumulation of salicylic acid secreted by plants in response to pathogen attack (Soanes et al., 2008).

The treatments that resulted in the most significant growth effects (CO4, CO8, and sLCOs), shared protein regulation in processes related to the repression of polarized growth in L. bicolor, such as proteins associated with the GO categories “autophagy” and “cleistothecium development” (Supplementary Table S2). In general, autophagy is an essential intracellular turnover mechanism involving the lysosomal/vacuolar pathway (Levine and Klionsky, 2004; Pollack et al., 2009). In filamentous fungi, autophagy appears to be involved in nutrient recycling during starvation and normal developmental processes (Pollack et al., 2009). In several fungal species, autophagy has been implicated in determining cell architecture during differentiation and development (Levine and Klionsky, 2004) and morphogenesis and morphology (Pollack et al., 2009). Few examples of downregulated proteins associated with the autophagy category include a WD40 repeat like-protein (Uniprot ID. B0DVN6) with 35% sequence identity to the S. cerevisiae autophagy-related gene protein ATG18, alongside vacuolar protein sorting-associated proteins (VPS) VPS13 (B0CPT6), VPS1 (B0D3D9), and a GTP-binding protein ypt (B0CQK4) with sequence identities above 30% to VPS13, VPS1, and VPS21 in yeast, respectively. ATGs have been intensively investigated in S. cerevisiae, and up to 42 proteins are known. Among these, 18 ATG have been previously implicated in different steps of autophagy (Lin et al., 2019). For example, yeast ATG18 is involved in vesicle nucleation, whereby proteins and lipids concentrate on forming the pre-autophagosomal structure. VPS proteins act as controllers directing the specific delivery of cellular material targeted for vacuole degradation (Kim et al., 2015). In filamentous fungi, such as Fusarium graminearum and Magnaporthe oryzae, mutants lacking VPS74 were strikingly reduced in hyphal growth and fungal virulence on host plants. Both mutants showed abnormal hyphal morphology with swollen hyphal tips (Kim et al., 2015). As such, the observed downregulation of VPS proteins implies that the exogenous application of specific COs and LCOs could alter cellular trafficking and sorting processes that are important for the delivery of matrix components necessary for hyphal elongation (Wessels, 1990).

Among regulated proteins associated with the “cleistothecium development” GO term there were several 26S proteasome subunits. “Cleistothecium development” category may be an artifact of the gene annotation, since L. bicolor is a basidiomycete and will not develop a cleistothecium. A previous study in yeast filamentous fungi suggested that the 26S proteasome controls filamentous-form cell properties through the regulation of the Rpn4 subunit protein. When the RPN4 gene was deleted in this organism, cells grow slower compared to the wild type (Prinz et al., 2004). Although we did not detect in our study the Rpn4 protein specifically, we observed downregulation of other 26S proteasome subunits, including P43, Rpn1, and Rpn7. The latter was also reported to be significantly induced in filamentous forms of yeast cells (Prinz et al., 2004). Thus, it is plausible to support the idea that in L. bicolor, similarly to yeast, the induction of specific proteasomal subunits could exert different types of regulatory control on filamentous growth.

In addition to the reported observations at the proteome level, metabolite analysis provided another layer of evidence supporting the suppression of L. bicolor filamentous growth. In this study, the abundance of three fatty acids (palmitic, stearic, and tetracosanoic acids) was significantly lower in some LCOs and COs treatments compared to the solvent control samples (Supplementary Figure S3). Fatty acids are components of membranes and storage lipids. In filamentous fungi, many lipids accumulate in hyphae as they tend to be used as carbon and energy sources during starvation (Laczko et al., 2004; Reich et al., 2009). Thus, it is plausible to hypothesize that the downregulation of fatty acids observed here may be contributing to an alteration in the production of material necessary for the formation of new hyphae (i.e., new membrane). Interestingly, these metabolite observations can be linked to the downregulation of the fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two enzymes required for the stepwise synthesis of fatty acids in L. bicolor (Reich et al., 2009).

Low molecular weight organic acids (LMWOAs) are commonly found in rhizospheric exudates and play a role in soil fertility, plant growth, and organization of microbial communities (Macias-Benitez et al., 2020). For mycorrhizal forming fungi, the expression of LMWOAs are dependent on different environmental conditions (Cumming et al., 2001; Eldhuset et al., 2007; Machuca et al., 2007; Johansson et al., 2008). For example, it has been proposed that the production of organic acids like citric, malate, citrate, and oxalate by mycorrhizal forming fungi can play essential roles in metal complexation, which helps in improved metal resistance (Plassard and Fransson, 2009). In our study, differential production of some organic acids was observed. The production of citric, malic, and oxalic acid was downregulated in almost all treatments compared to solvent control samples. In contrast, lactic acid was upregulated in both LCO treatments (Supplementary Figure S3). Alternative to the metal complexation hypothesis, the regulation of organic acids might be recruiting beneficial microbes as previously shown (Rodriguez-Morgado et al., 2017; Macias-Benitez et al., 2020), yet this potential association garners further investigation.

As more investigations are conducted on COs and LCOs, the roles of these molecules in fungal biology have expanded. They were first characterized as the primary signals allowing root colonization by rhizobia or mycorrhizal fungi. Now LCOs were shown to have a great influence on fungal development even in organisms with no known ability to form symbiotic associations with plants (Rush et al., 2020; Khokhani et al., 2021). The observations reported here in L. bicolor suggest that a cascade of phosphorylation events are potentially responsible for the reduced growth observed in samples grown 21 dpi in presence of LCOs and COs.

In the L. bicolor—Populus sp. ectomycorrhizal association, the application of sLCOs but not nsLCOs was reported to increase the ratio of mantle width to root diameter as well the ratio of Hartig net boundary to root circumference (Cope et al., 2019). This observation was puzzling as Populus sp. is known to be more sensitive to nsLCOs than sLCOs (Cope et al., 2019, 2021). Given the data presented in this manuscript, it is likely that this result was potentially due to the effect of sLCOs on L. bicolor rather than on its host plant.

Here we suggest that specific LCOs and COs are possibly fungistatic agents that inhibit the growth of the fungus without killing it (Figure 5). Therefore, it can be the reason why most fungi produce LCOs (Rush et al., 2020) to inhibit the growth of nearby microbes and explain why the growth and development of some fungi are influenced by certain LCOs and not others (Marburger et al., 2018; Rush et al., 2020). Surprisingly, we observed that the LCOs predominantly produced by the same organism have the most significant influence on it (Rush et al., 2020). That was the C16:0 LCOs in A. fumigatus (Rush et al., 2020) and the C18:1 LCOs as reported here in L. bicolor. These observations remain inexplicable as why a fungus would produce a fungistatic compound to inhibit its growth. Additional data are needed to support or disprove this suggestion. Lastly, further experiments are required to determine if these changes in fungal development are not xenobiotic responses since sLCOs and nsLCOs were purified from rhizobia (Maillet et al., 2011; Mukherjee and Ane, 2011; Sun et al., 2015; Cope et al., 2019).

The results presented here provide baseline evidence that an LCO-producing organism might be controlled by its own chemical signal. To understand these mechanisms better, it would be ideal to generate mutant strains in which the genes responsible for LCO production are overexpressed or knocked out. This would allow to examine the behavior of the fungus in the absence or overproduction of its own LCOs. However, the genes responsible for LCO production in fungi have not been characterized yet. To conclude, this study document potential role for LCOs outside of symbiosis and open new avenues for research on the biological function of LCOs and their implication in fungal growth and development.

This manuscript has been authored by Q21 UT-Battelle, LLC under Contract No. DE-AC05- 00OR22725 with the U.S. Department of Energy. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

All proteomics MS/MS raw and searched data files used in this study were deposited at the 574 MassIVE repository (https://massive.ucsd.edu/) as public dataset MSV000088201.

TR, PA, RH, and JL planned and designed the research. SC, SF, JM, and JMA provided LCO molecules. TR and MS did the inoculations and provided samples for proteomics and metabolomics analysis. TR performed all L. bicolor growth experiments and observations. MV performed all proteomics sample preparation and analysis and wrote the manuscript. NE and TT performed metabolite extraction and analysis. All authors contributed to the revision of the manuscript.

This research was sponsored by the Genomic Science Program, US Department of Energy (DOE), Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Areas at the Oak Ridge National Laboratory (ORNL). ORNL is managed by UT-Battelle LLC for DOE under contract DE-AC05-00OR22725. This work was also supported by the NSF award # 1546742 as well as USDA Hatch #WIS03041 to JMA.

This study received funding from the Genomic Science Program, US Department of Energy (DOE), Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Areas at the Oak Ridge National Laboratory (ORNL). ORNL is managed by UT-Battelle LLC for DOE under contract DE-AC05-00OR22725. This work was also supported by the NSF award # 1546742 as well as USDA Hatch #WIS03041 to JMA. Lastly, partial financial support from the LABEX ARCANE and CBH-EUR-GS (ANR-17-561 EURE-0003), Glyco@Alps (ANR-15-IDEX-02), and PolyNat Carnot Institut (ANR-16-CARN562 0025-01) for SF and SC. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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