Pseudomonas fluorescens increases mycorrhization and modulates expression of antifungal defense response genes in roots of aspen seedlings

Abstract

Background: Plants, fungi, and bacteria form complex, mutually-beneficial communities within the soil environment. In return for photosynthetically derived sugars in the form of exudates from plant roots, the microbial symbionts in these rhizosphere communities provide their host plants access to otherwise inaccessible nutrients in soils and help defend the plant against biotic and abiotic stresses. One role that bacteria may play in these communities is that of Mycorrhizal Helper Bacteria (MHB). MHB are bacteria that facilitate the interactions between plant roots and symbiotic mycorrhizal fungi and, while the effects of MHB on the formation of plant-fungal symbiosis and on plant health have been well documented, the specific molecular mechanisms by which MHB drive gene regulation in plant roots leading to these benefits remain largely uncharacterized.

Results: Here, we investigate the effects of the bacterium Pseudomonas fluorescens SBW25 (SBW25) on aspen root transcriptome using a tripartite laboratory community comprised of Populus tremuloides (aspen) seedlings and the ectomycorrhizal fungus Laccaria bicolor (Laccaria). We show that SBW25 has MHB activity and promotes mycorrhization of aspen roots by Laccaria. Using transcriptomic analysis of aspen roots under multiple community compositions, we identify clusters of co-regulated genes associated with mycorrhization, the presence of SBW25, and MHB-associated functions, and we generate a combinatorial logic network that links causal relationships in observed patterns of gene expression in aspen seedling roots in a single Boolean circuit diagram. The predicted regulatory circuit is used to infer regulatory mechanisms associated with MHB activity.

Conclusions: In our laboratory conditions, SBW25 increases the ability of Laccaria to form ectomycorrhizal interactions with aspen seedling roots through the suppression of aspen root antifungal defense responses. Analysis of transcriptomic data identifies that potential molecular mechanisms in aspen roots that respond to MHB activity are proteins with homology to pollen recognition sensors. Pollen recognition sensors integrate multiple environmental signals to down-regulate pollenization-associated gene clusters, making proteins with homology to this system an excellent fit for a predicted mechanism that integrates information from the rhizosphere to down-regulate antifungal defense response genes in the root. These results provide a deeper understanding of aspen gene regulation in response to MHB and suggest additional, hypothesis-driven biological experiments to validate putative molecular mechanisms of MHB activity in the aspen-Laccaria ectomycorrhizal symbiosis.

Keywords: Ectomycorrhiza; Laccaria bicolor; Mycorrhiza helper bacteria; Populus tremuloides; Receptors; Transcriptomics.

Fig. 1

Tripartite community phenotypes. (a) Aspen seedlings were grown in sand pots in four experimental conditions: aspen seedlings alone (Aspen), aspen with the mycorrhizal fungi Laccaria (Aspen+Lb), aspen with the MHB P. fluorescens SBW25 (Aspen+Pf), and aspen with both Laccaria and SBW25 (Aspen+Lb + Pf). A typical example of aspen under each experimental condition after 63 days of growth is shown. (b) Shoot biomass, (c) Root biomass, and (d) Percent mycorrhization of aspen seedling roots were measured in tripartite community systems (‘Aspen’ = aspen, ‘Lb’ = L. bicolor, ‘Pf’ = P. fluorescens). ‘*’ indicates a statistically significant difference between Percent mycorrhization with Laccaria and with Laccaria + SBW25

Fig. 2

Venn diagram of differentially expressed genes by experimental factor. Differential expression in aspen root transcriptome was determined by 2-factor ANOVA. Factors in ANOVA were Presence/Absence of Laccaria, Presence/Absence of SBW25, and Interaction between Laccaria and SBW25

Fig. 3

Average expression, relative to aspen-monoculture condition, for genes in co-regulated clusters. X-axis is labeled with co-regulated gene Cluster number. Number in parenthesis is the total number of genes in that cluster. Y-axis indicates the average log₂ fold difference between all genes within a cluster, relative to the aspen-only condition. ‘Aspen’ = aspen, ‘Lb’ = L. bicolor, ‘Pf’ = P. fluorescens

Fig. 4

Enriched GO-BP annotations in clusters of co-expressed aspen root genes. Tag clouds of significantly enriched GO-BP annotation terms in each cluster of co-regulated genes are presented with the size of annotation tag proportionate to the number of genes with that annotation in the cluster. Figures generated using web-based WordArt tool (https://wordart.com/). Enriched annotations can be found in tabular format in Additional file 4

Fig. 5

Predicted aspen root gene network for MHB activity. The proposed regulatory interactions between co-expressed gene clusters is represented as a logic circuit diagram. In the network, circles to left indicate presence or absence of Laccaria (red mushrooms) or SBW25 (blue microscopy image). Rectangles are co-regulated gene clusters. Cluster numbers reference cluster IDs as presented in Fig. 3 and cluster functions are draws from enriched gene annotations as presented in Fig. 4. Edges indicate predicted causal relationships between gene clusters inferred from observed patterns of gene regulation by Bayesian Inference analysis