Differential Expression of Genes Involved in Host Recognition, Attachment, and Degradation in the Mycoparasite Tolypocladium ophioglossoides

Abstract

The ability of a fungus to infect novel hosts is dependent on changes in gene content, expression, or regulation. Examining gene expression under simulated host conditions can explore which genes may contribute to host jumping. Insect pathogenesis is the inferred ancestral character state for species of Tolypocladium, however several species are parasites of truffles, including Tolypocladium ophioglossoides. To identify potentially crucial genes in this interkingdom host switch, T. ophioglossoides was grown on four media conditions: media containing the inner and outer portions of its natural host (truffles of Elaphomyces), cuticles from an ancestral host (beetle), and a rich medium (Yeast Malt). Through high-throughput RNASeq of mRNA from these conditions, many differentially expressed genes were identified in the experiment. These included PTH11-related G-protein-coupled receptors (GPCRs) hypothesized to be involved in host recognition, and also found to be upregulated in insect pathogens. A divergent chitinase with a signal peptide was also found to be highly upregulated on media containing truffle tissue, suggesting an exogenous degradative activity in the presence of the truffle host. The adhesin gene, Mad1, was highly expressed on truffle media as well. A BiNGO analysis of overrepresented GO terms from genes expressed during each growth condition found that genes involved in redox reactions and transmembrane transport were the most overrepresented during T. ophioglossoides growth on truffle media, suggesting their importance in growth on fungal tissue as compared to other hosts and environments. Genes involved in secondary metabolism were most highly expressed during growth on insect tissue, suggesting that their products may not be necessary during parasitism of Elaphomyces. This study provides clues into understanding genetic mechanisms underlying the transition from insect to truffle parasitism.

Keywords: Elaphomyces; PTH11 GPCR; RNA-Seq; adhesin; chitinase.

Figure 1

Phylogeny of PTH11-related GPCRs in T. ophioglossoides and closely related insect pathogens. PTH11-related GPCR phylogeny and numbers per taxon sampled. Genes that are upregulated in T. ophioglossoides on EMP are colored blue and marked with a truffle, and those that are upregulated on CUT are colored red and marked with a beetle larva. Genes that are upregulated in Metarhizium spp. on grasshopper or roach hosts are colored red and marked with the host(s) eliciting expression of those genes. The boxed area highlights the T. ophioglossoides homolog (TOPH_01767) of M. acridum (MAC_00494), which are both upregulated on insect cuticles. CUT, O. sulcata cuticle media; EMP, E. muricatus peridium media; GPCRs, G-protein-coupled receptors; Osin, O. sinensis; MAA, M. robertsii; MAC, M. acidrum; TINF, T. inflatum; TOPH, T. ophioglossoides.

Figure 2

Phylogenetic relationships of expressed chitinases. Phylogeny of chitinases showing two major fungal classes. Green branches highlight those chitinases of T. ophioglossoides. The T. ophioglossoides chitinase (TOPH_09828) upregulated on EMP (E. muricatus peridium media, represented by an asterisk), which is closely related to one of the four Tr. virens chitinases upregulated during mycoparasitism (represented by stars), as identified by Gruber et al. (2011). The most highly expressed T. ophioglossoides chitinase (TOPH_05538) in all conditions (represented by a ↑) is part of the Class III chitinases, a phylogenetically distinct group. Taxa sampled: Tr. virens, Tr. atroviride, Tr. reesei, Beauveria bassiana, Cordyceps militaris, Epichloë festucae, Claviceps purpurea, M. acridum, M. robertsii, O. sinensis, T. inflatum, and T. ophioglossoides.

Figure 3

Mad1 expression. (A) RPKM and corresponding 95% confidence intervals of Mad1 homolog in T. ophioglossoides during growth in four experimental conditions. (B) Upregulation of Mad1 and Mad2 homologs in three hypocrealean spp. from data collected in this study combined with data from M. robertsii and M. acridum in other studies (Wang and Leger 2005; Wang et al. 2005; Gao et al. 2011). CUT, O. sulcata cuticle medium; EMG, E. muricatus gleba media; EMP, E. muricatus peridium media; RPKM, reads per thousand base pairs per million reads; YM, Yeast Malt medium.

Figure 4

Upregulated Gene Ontology (GO) categories in EMP (E. muricatus peridium media). BiNGO network of statistically overrepresented GO categories in the 360 T. ophioglossoides genes upregulated in EMP compared to YM (Yeast Malt medium). Colors are based on p-values and follow the scale provided.

Figure 5

Secondary metabolite expression. Heatmap of RPKM of secondary metabolite core genes (NRPS, PKS, Terpene Synthases) in T. ophioglossoides under the experimental conditions. Row scaled Z-scores are based on normalized standard deviation units. CUT, O. sulcata cuticle media; EMG, E. muricatus gleba medium; EMP, E. muricatus peridium medium; NRPS, nonribosomal peptide synthetases; PKS, polyketide synthases; RPKM, reads per thousand base pairs per million reads; YM, Yeast Malt medium.