Expanding the Biological Role of Lipo-Chitooligosaccharides and Chitooligosaccharides in Laccaria bicolor Growth and Development

Abstract

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.

Keywords: Laccaria bicolor; chitooligosaccharides; lipo-chitooligosaccharides; polarized growth; proteomics.

Figure 1

LCOs and COs effect on growth and hyphal branching. (A) Growth of Laccaria bicolor over 21 days when exposed to LCOs or COs. Diameter measurements were taken every other day, starting on the day 3. Fungal growth is the area without the 1 cm² plug. Welch's ANOVA test was significant for 3 days post-inoculation (dpi) (p = 0.0058), 11 dpi (p = 0.0032), 13 dpi (p = 0.0079), 15 dpi (p = 0.01), 17 dpi (p = 0.0199), 19 dpi (p = 0.0005), and 21 dpi (p = 0.0004). Treatments were compared to the solvent controls with Welch's unpaired t-test. N is five biological replications. (B) Hyphal branching was determined by the average ratio of secondary branches (indicated by blue arrows and numbers) to 400 μm of the apical branch (red scale bar) counted after 3 dpi. (C) The ratios of secondary branches per apical branch in treatments were compared to the solvent controls with Welch's unpaired t-test (Welch's ANOVA test p = 0.0105). N is five biological replications with five technical replications each where five apical branches were counted. For all measurements containing significant treatments compared to the solvent control are as follows: (*) is a p < 0.05; (**) is a p < 0.01; (***) is a p < 0.001.

Figure 2

Proteomics analysis of L. bicolor under exposure of COs and LCOs. (A) Total number of proteins identified. (B) Venn diagram showing shared and unique proteins after data normalization and selection of proteins that were identified in at least 2 out of 3 biological replicates. (C) Principal component 2 with explained variance given as a percentage value after data normalization, filtration, and imputation. (D) Heatmap from the normalized abundance values of all identified proteins found in at least 2 out of 3 biological replicates per condition.

Figure 3

Proteome interrogation of L. bicolor growing with COs and LCOs compared to solvent control. (A) DiVenn diagram showing the numbers of up- (red color) and downregulated (blue color) proteins that are shared and/or unique in the comparisons between treatments and control samples. (B) GO enrichment analysis of the 254 common downregulated proteins shared between all treatments compared to control samples. (C) GO enrichment analysis of 144 common downregulated proteins in sLCO, CO4, and CO8-treated samples compared to control samples. Enrichment analyses performed with the Cytoscape plug-in ClueGO (Shannon et al., ; Bindea et al., 2009), terms reported were significant by Benjamini-Hochberg term p ≤ 0.05.

Figure 4

STRING network of all common downregulated proteins in treatments compared to control samples belonging to “cellular component assembly” and “establishment of cell polarity” GO categories. The network summarizes the predicted associations between proteins found by the STRING database. Each protein is represented by a node and the edges represent predicted functional associations. Edge colors indicate the confidence interaction score based on different types of evidence considered by STRING. Only protein-protein associations with high confidence scores (≥ 0.7) were considered in the predicted network. The node representing the CMGC/CDK/CDC2 protein kinase is highlighted in yellow. Disconnected proteins in the network are not shown.

Figure 5

Summary of the main physiological, proteomic, and metabolic changes observed in L. bicolor when exposed to COs and LCOs compared to control samples. (A) Reduced hyphal growth was observed in samples grown with nsLCOs, C18:1 sLCOs and CO4. (B) Most upregulated proteins quantified in this study were unique to each treatment and included cases of molecules involved in fungal growth control like Ras proteins, as well as proteins involved in mechanisms of cross-communication between fungi and other organisms; instead, the vast majority of downregulated proteins were found to be shared between treatments such as cyclin-dependent kinases, GTPases, and dyneins. Other examples discussed through this manuscript are shown. (C) Ten metabolites were significantly regulated in treated L. bicolor samples compared to controls and included cases of organic acid, fatty acids, sugars, and metabolites with unknown functions.