The Dark Side of Orchid Symbiosis: Can Tulasnella calospora Decompose Host Tissues?

Abstract

Photosynthetic orchids associate with mycorrhizal fungi that can be mostly ascribed to the "rhizoctonia" species complex. Rhizoctonias' phylogenetic diversity covers a variety of ecological/nutritional strategies that include, beside the symbiosis establishment with host plants, endophytic and pathogenic associations with non-orchid plants or saprotrophic soil colonization. In addition, orchid mycorrhizal fungi (OMF) that establish a symbiotic relationship with an orchid host can later proliferate in browning and rotting orchid tissues. Environmental triggers and molecular mechanisms governing the switch leading to either a saprotrophic or a mycorrhizal behavior in OMF remain unclear. As the sequenced OMF genomes feature a wide range of genes putatively involved in the degradation of plant cell wall (PCW) components, we tested if these transitions may be correlated with a change in the expression of some PCW degrading enzymes. Regulation of several genes encoding PCW degrading enzymes was evaluated during saprotrophic growth of the OMF Tulasnella calospora on different substrates and under successful and unsuccessful mycorrhizal symbioses. Fungal gene expression in planta was investigated in two orchid species, the terrestrial Mediterranean Serapias vomeracea and the epiphytic tropical Cattleya purpurata. Although we only tested a subset of the CAZyme genes identified in the T. calospora genome, and we cannot exclude therefore a role for different CAZyme families or members inside a family, the results showed that the degradative potential of T. calospora is finely regulated during saprotrophic growth and in symbiosis, often with a different regulation in the two orchid species. These data pose novel questions about the role of fungal PCW degrading enzymes in the development of unsuccessful and successful interactions.

Keywords: CAZymes; gene expression; orchid mycorrhiza; orchid symbiosis; saprotrophic growth.

Figure 1

Protocorms of Serapias vomeracea inoculated with Tulasnella calospora at different stages: from the typical features (a) to a brown-dark/rot aspect (b–d). Bars = 2mm.

Figure 2

Semi-thin sections of Serapias vomeracea protocorms colonized by Tulasnella calospora. (a) Stage where protocorms appeared with the typical features and color. At cellular level, typical colonization pattern with T. calospora is evident with the presence of coils at different developmental stages. c, coil; cc, collapsed coil. (b,c) Subsequent stages where protocorms are becoming brown. The fungal colonization pattern is still evident as well as host cell features. (d) Section of a dark/soft protocorm. Cell borders are not well-defined and the fungal hyphae are widespread in the tissues without a typical colonization pattern. Bars = 33, 13, 45, 25 μm for (a), (b), (c) and (d), respectively.

Figure 3

Number of CAZymes encoding-genes annotated in a number of Basidiomycota genomes (upper panel) and heatmap of expression levels across different tissues and conditions (bottom panel). Expression values are reported in TPM (transcripts per million) and columns are clustered by using Euclidean distances. Fungal species with different lifestyles were considered: orchid mycorrhizal (OM), ecto-mycorrhizal (ECM), white rot (WR), plant pathogens.

Figure 4

CAZymes expression under different experimental conditions. Letters indicate significant differences after Kruskal-Wallis test and Dunn’s post-hoc test (p < 0.05). NRQ, normalized relative quantities; mean ± standard error (SE) is plotted.

Figure 5

T. calospora symbiosis marker genes in C. purpurata and S. vomeracea orchid mycorrhiza interactions. Letters indicate significant differences after Kruskal-Wallis test and Dunn’s post-hoc test (p < 0.05). NRQ, normalized relative quantities; mean ± standard error (SE) is plotted.