Rhizobiales as functional and endosymbiontic members in the lichen symbiosis of Lobaria pulmonaria L

Abstract

Rhizobiales (Alphaproteobacteria) are well-known beneficial partners in plant-microbe interactions. Less is known about the occurrence and function of Rhizobiales in the lichen symbiosis, although it has previously been shown that Alphaproteobacteria are the dominating group in growing lichen thalli. We have analyzed the taxonomic structure and assigned functions to Rhizobiales within a metagenomic dataset of the lung lichen Lobaria pulmonaria L. One third (32.2%) of the overall bacteria belong to the Rhizobiales, in particular to the families Methylobacteriaceae, Bradyrhizobiaceae, and Rhizobiaceae. About 20% of our metagenomic assignments could not be placed in any of the Rhizobiales lineages, which indicates a yet undescribed bacterial diversity. SEED-based functional analysis focused on Rhizobiales and revealed functions supporting the symbiosis, including auxin and vitamin production, nitrogen fixation and stress protection. We also have used a specifically developed probe to localize Rhizobiales by confocal laser scanning microscopy after fluorescence in situ hybridization (FISH-CLSM). Bacteria preferentially colonized fungal surfaces, but there is clear evidence that members of the Rhizobiales are able to intrude at varying depths into the interhyphal gelatinous matrix of the upper lichen cortical layer and that at least occasionally some bacteria also are capable to colonize the interior of the fungal hyphae. Interestingly, the gradual development of an endosymbiotic bacterial life was found for lichen- as well as for fungal- and plant-associated bacteria. The new tools to study Rhizobiales, FISH microscopy and comparative metagenomics, suggest a similar beneficial role for lichens than for plants and will help to better understand the Rhizobiales-host interaction and their biotechnological potential.

Keywords: Lobaria pulmonaria; Rhizobiales; Rhizobiales-specific FISH probe; endosymbiont; lichen symbiosis; metagenomics.

Figure 1

Lobaria pulmonaria from a mountain maple in an Abieti-Fagetum forest in Johnsbach (Austria) at an altitude of 1200 m above sea level. (Styria, Austria, 47°32′29.7″N 14°37′36.6″E).

Figure 2

Rhizobiales taxa within the Lobaria-associated metagenome are presented in a multi-level chart. Taxonomic information was retrieved through blastn assignments of assembled contigs, followed by processing with MEGAN. Circles represent taxonomic classifications in ascending order up to the species level (outermost circle). Less abundant taxa are listed outside the charts together with their relative abundance.

Figure 3

Selected functions within the Lobaria-associated metagenome were assigned to taxa at order (Rhizobiales) and family (Methylobacteriaceae, Bradyrhizobiaceae and Rhizobiaceae) level together with hits without assignment to a specific taxon. Taxonomic information was retrieved through blastx assignments of assembled contigs, followed by processing with MEGAN.

Figure 4

TestProbe 3.0 (Silva ribosomal RNA database) analysis showing the targeted coverage of bacterial taxa in a comparison of RHIZ3r- and RHIZ1244- FISH Probes. The maximum amount of mismatches was set to 0 (see also Table S3 for RDP Probmatch comparison).

Figure 5

Confocal laser scanning microscopy (CLSM) micrographs showing the bacterial colonization of the lichen L. pulmonaria stained by fluorescence in situ hybridization (FISH). Green, algae; blue/purple, fungi; yellow, Rhizobiales; red, other bacteria. (A,B,D), three-dimensional models made of isosurfaces and spheres; (C), volume rendering; (E), maximum projection; (F), single optical slice. In (B): white box indicates the region shown in (D). Arrow in (F) indicates an endophytic bacterial cell (see also Figure S1). Scale bars: (A-C) (30 μm); (D-F) (5 μm).