The role of lipo-chitooligosaccharides (LCOs) as signaling molecules that mediate the establishment of symbiotic relationships between fungi and plants is being redefined. New evidence suggests that the production of these molecular signals may be more of a common trait in fungi than what was previously thought. LCOs affect different aspects of growth and development in fungi. For the ectomycorrhizal forming fungi, Laccaria bicolor, the production and effects of LCOs have always been studied with a symbiotic plant partner; however, there is still no scientific evidence describing the effects that these molecules have on this organism. Here, we explored the physiological, molecular, and metabolomic changes in L. bicolor when grown in the presence of exogenous sulfated and non-sulfated LCOs, as well as the chitooligomers, chitotetraose (CO4), and chitooctaose (CO8). Physiological data from 21 days post-induction showed reduced fungal growth in response to CO and LCO treatments compared to solvent controls. The underlying molecular changes were interrogated by proteomics, which revealed substantial alterations to biological processes related to growth and development. Moreover, metabolite data showed that LCOs and COs caused a downregulation of organic acids, sugars, and fatty acids. At the same time, exposure to LCOs resulted in the overproduction of lactic acid in L. bicolor. Altogether, these results suggest that these signals might be fungistatic compounds and contribute to current research efforts investigating the emerging impacts of these molecules on fungal growth and development.

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.

Square Petri dishes with gridlines (Thomas Scientific) were filled with 50 ml of Pachlewski agar medium (P20). LCO treatments were synthesized resulting in LCO types: C16:0 sulfated (C16:0 sLCO), C16:0 non-sulfated (C16:0 nsLCOs), C18:1 sulfated (C18:1 sLCOs), and C18:1 non-sulfated (C18:1 nsLCOs) in 0.005% ethanol/water (v/v) as used before (Rush et al., 2020). COs were chitotetraose, tetra-N-acetyl (CO4) (IsoSep, Tullinge, Sweden—Product Number: 45/12-0050) and chitooctaose, octa-N-acetyl (CO8) (IsoSep, Tullinge, Sweden—Product Number: 57/12-0001) in 0.005% ethanol/water (v/v) (Rush et al., 2020). We used a concentration of 10−8 M because of its biological influence on fungi shown in previously studies (Maillet et al., 2011; Cope et al., 2019; Rush et al., 2020). Individually applied LCOs or COs treatments had a concentration of 10−8 M, were spread evenly across the agar medium with a sterile cell spreader and set to dry. The solvent control was 0.005% ethanol/water (v/v). A single fungal plug of L. bicolor was then cut with a sterile 1-cm2 area core borer and placed in the middle of the agar medium with the treatment. There were five biological replications per treatment. Inoculated plates were placed in a dark incubator at 25°C.

Laccaria bicolor strain S238N has radial hyphal growth patterns in culture (Labbe et al., 2014), therefore for the fungal growth data, diameter measurements were taken based on the cardinal points every odd day for 21 days post-inoculation (dpi). Growth area was measured as the area of fungal growth minus the area of the core borer. For the clamp connections data, there were five technical replications per biological replicate. Measurements were taken after 7, 15, and 21 dpi. The total number of clamp connections observed within a fixed area was counted per technical replication. A fixed area is all the clamp connections counted within the image taken. The average was used for the total number of clamp connections observed per biological replication. Hyphal branching measurements were taken 3 dpi, with five technical replicates per biological replicate. In addition, five random apical branches were counted for secondary branches within a technical replicate. Secondary branches were counted from 400 μm of the apical branch hyphae starting from the tip of the branch. The ratio is determined by the number of secondary branches counted within 400 μm of apical branch starting from the tip of the apical branch. The average of that ratio was used for the biological replication value. Statistical analyses were performed using GraphPad Prism software version 9.0.0 (GraphPad, San Diego, CA). Welch's one-way ANOVA was performed with an unpaired Welch's t-test per species, testing treatment group responses against solvent control responses.

Laccaria bicolor strain S238N was grown for 21 days at 25°C from a single fungal plug cut with a sterile 1 cm2 area core borer. Fungal plugs were shaken at 200 rpm in 250 ml Erlenmeyer flasks filled with 50 ml of Pachlewski medium and inoculated with a concentration of 10−8 M of individual LCOs or COs. LCO treatments were mixtures of sLCOs or nsLCOs purified from Sinorhizobium meliloti and Rhizobium sp. IRBG74, respectively, and resuspended in 0.005% ethanol/water (v/v), as previously published (Maillet et al., 2011; Mukherjee and Ane, 2011; Sun et al., 2015; Cope et al., 2019). COs were chitotetraose, tetra-N-acetyl (CO4), and chitooctaose, octa-N-acetyl (CO8) in 0.005% ethanol/water (v/v) (Rush et al., 2020). Each treatment had individually applied applications of LCOs or COs with three biological replications for proteomics analysis, and four replications used for metabolomics analysis. In addition, fungi grown in media inoculated with 0.005% ethanol/water were used as solvent controls.

Laccaria bicolor samples were suspended in 1 mL of SDS lysis buffer (2% sodium dodecyl sulfate in 100 mM NH4HCO3 solution). Samples were physically disrupted by ultrasonication using a pulse amplitude of 20% for 10 secs on and 10 secs off for a total of 2 min. Crude lysates were then boiled for 10 min at 95°C. Later, samples were centrifuged at 21,000 × g for 10 min to pre-clear the sample of DNA and other cellular debris and supernatants transferred to fresh Eppendorf tubes. Disulfide bond disruption was achieved by adjusting the pre-cleared protein extracts to 10 mM dithiothreitol and incubating the samples at 90°C for 10 mins. Cysteines were blocked by adjusting each sample to 30 mM iodoacetamide, followed by incubation in the dark for 15 min at room temperature. Proteins were precipitated using chloroform-methanol-water extraction. Dried protein pellets were resuspended in 250 μL of a 2% sodium deoxycholate (SDC) solution in 100 mM NH4HCO3, and protein amounts were estimated using a NanoDrop Onec spectrophotometer (Thermo Scientific). For each sample, aliquots of ~100 μg of protein, or all protein if less was obtained, were digested using sequencing-grade trypsin (Promega, 1:75 [wt/wt]) overnight under constant shaking at 37°C (600 rpm, Eppendorf Thermomixer). The second round of trypsin digestion was performed under the same conditions as before but for 3 h. Peptide mixtures were then adjusted to 0.5% formic acid to precipitate SDC. Hydrated ethyl acetate was added to each sample at a 1:1 (vol/vol) ratio three times to remove the SDC effectively. Samples were then placed in a SpeedVac concentrator (Thermo Fisher Scientific) to remove the ethyl acetate and further concentrate the sample. The peptide-enriched flow-through was quantified with the same NanoDrop OneC spectrophotometer as before.

Peptide samples were analyzed by automated one-dimensional LC-MS/MS analysis using a Vanquish ultra-HPLC (UHPLC) system plumbed directly in-line with a Q Exactive Plus mass spectrometer (Thermo Scientific) outfitted with a trapping column coupled to an in-house-pulled nanospray emitter. The trapping column (inner diameter, 100 μm) and the nanospray emitter (inner diameter, 75 μm) were packed with 5-μm Kinetex C18 reverse-phase resin (Phenomenex) to 10 and 30 cm, respectively. For each sample, peptides (2 μg) were loaded, desalted, separated, and analyzed across a 210-min organic gradient with the following parameters: sample injection followed by a 100% solvent A chase from 0 to 30 min (load and desalt), a linear gradient of 0–25% solvent B (70% acetonitrile, 30% water, and 0.1% formic acid) from 30 to 240 min (separation), a ramp to 75% solvent B from 240 to 250 min (wash), re-equilibration to 100% solvent A from 250 to 260 min, followed by maintaining 100% solvent A from 260 to 280 min. Eluting peptides were measured and sequenced by data-dependent acquisition with the Thermo Xcalibur v 4.2.47 software using the same parameters reported before (Johnson et al., 2017).

MS raw data files were searched against the Laccaria bicolor Uniprot protein database (ID. UP000001194, downloaded on November 23, 2020), to which commonly contaminated proteins had been added using Proteome Discover v2.3 (Thermo Fischer Scientific, USA). Each MS/MS raw data file was processed with the SEQUEST HT database search algorithm, and confidence in peptide-to-spectrum (PSM) matching was evaluated by Percolator (Kall et al., 2007). SEQUEST HT was configured to derive fully tryptic peptides with the following parameters: max 2 missed cleavages, minimum peptide length of 6 amino acids, the maximum number of charge states of 4, a precursor mass tolerance of 10 ppm (ppm), a fragment mass tolerance of 0.02 Da, a static modification on cysteines (iodoacetamide; +57.0214 Da), and dynamic changes on methionine (oxidation; 15.9949). Peptides and PSMs were considered identified at q < 0.01, and proteins were required to have at least one unique peptide sequence. Functional annotations of proteins in the L. bicolor database were generated with the OmicsBox v1.2.4 software (BLASTp against non-redundant NCBI database of fungal sequences, E-value cutoff