Bacterial endosymbionts protect beneficial soil fungus from nematode attack

Abstract

Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.

Keywords: microbial interactions; natural products; symbiosis.

Fig. 1.

Bacterial origin of cytotoxic benzolactones from M. verticillata cultures. (A) Cytotoxic lactone compounds assigned to endofungal symbionts from the fungus R. microsporus (1–4), M. verticillata (3–4), Pseudomonas sp. (5), and a tunicate and the bacterium Gynuella sunshinyii (6). (B) Metabolic profiles of extracts from Burkholderia sp. strain B8 and M. verticillata NRRL 6337 as symbiont or cured strain as total ion chromatograms in the negative mode. (C) Fluorescence micrograph depicting endosymbionts living in the fungal hyphae; staining with Calcofluor White and Syto9 Green. (D) Phylogenetic relationships of Mortierella symbionts, Burkholderia sp. strain B8, and other bacteria based on 16S rDNA. BRE, Burkholderia-related endosymbiont of Mortierella spp. (E) Metabolic profiles of extracts from M. verticillata NRRL 6337 and other necroxime-negative M. verticillata strains analyzed for endosymbionts in this study as total ion chromatograms in the negative mode. M, medium component. (F) Growth of symbiotic M. verticillata NRRL 6337 in comparison to the cured strain.

Fig. 2.

Comparative genomic analyses of Mycoavidus spp. (A) Number of orthologous proteins among the four Mycoavidus strains at 70% identity. (B) Circos plot of shared protein orthologs, and secondary metabolite loci (detected by antiSMASH v5) in Mycoavidus genomes. Outer blocks (orange, brown, yellow, green) represent genome sizes, while the inner blocks represent genomic positions of secondary metabolite loci. Lines linking the three genomes show position of genes whose proteins are orthologous at 70% identity. Depicted are the genome sequences of M. cysteinexigens strains AG77, B1-EB, B2-EB, and Ca. M. necroximicus (Ca. M. nec.). (C) Number of gene clusters putatively coding for natural products in Mycoavidus spp. detected by antiSMASH and by manual assignment. (D) BGCs and their encoded assembly lines identified from the endofungal Ca. M. necroximicus are displayed. A, adenylation; AT, acyltransferase; C, condensation; DH, dehydratase; E, epimerization; Gnat, GCN5-related N-acetyltransferase; KR, ketoreductase; KS, ketosynthase; MT, methyltransferase; OX, oxygenase; TE, thioesterase domains. Acyl carrier (light blue) and peptidyl carrier proteins (dark blue) are shown as circles without designators. (E) Homologous benzolactone BGCs in the genome of Burkholderia strain B8 and Ca. M. necroximicus.

Fig. 3.

Nematocidal activity of symbiont-derived toxins. (A) Viability assay of C. elegans in presence of extract fractions of symbiotic and cured M. verticillata NRRL 6337. HPLC profiles of extracts are shown with corresponding effect on nematodes, measured as effect on the E. coli optical density (OD). When nematode growth is impaired by the fraction, E. coli cells are not consumed, and thus the OD₆₀₀ is not altered (error bars represent mean of three biological replicates). The red asterisk represents 4. (B) Toxicity screening of 4 against C. elegans. The red line marks IC₅₀ at 11.3 µg ⋅ mL⁻¹ (24.66 µM; 95% CI, 21.45 to 28.37 µM; error bars as mean of five biological replicates). (C) Nematode counts from propagation assay of M. verticillata and A. avenae cocultures. Bars represent relative nematode numbers compared to the mean of the nematode count from cured M. verticillata NRRL 6337 cultures. cur., cured; sym., symbiotic. *P < 0.02; ***P < 0.001; ****P < 0.0001. Data represent three biological replicates with three technical replicates each. (D) Workflow of image analysis and mathematical evaluation of A. avenae mobility in fungal–nematodal coincubations. Processing of time series is demonstrated by one time frame. Exemplary images of nematodes from two time frames (frame 1 and 26) are shown to illustrate differences in motility. Results of calculated mobility ratios (MR) were used for live or paralyzed/dead categorization. (E) Results of image analysis and mathematical quantification of nematode movement. Bars show ratio between moving/living nematodes and paralyzed/dead nematodes, which were harvested from cocultures of A. avenae with symbiotic M. verticillata NRRL 6337 cultures, cured NRRL 6337 cultures, or CSB 225.35 cultures. Numbers and error bars were calculated from minimal 176 worms from three biological replicates. (F) Stereomicroscopic images and schematic picture of chemical complementation assay with a magnitude of 25×. Sample of nematodes harvested from plates containing symbiotic, cured, or cured and with 4 chemically complemented M. verticillata NRRL 6337 cultures. (G) Schematic summary of tripartite interaction between fungal host, bacterial endosymbiont, and mycophagous nematodes.