Specific modulation of the root immune system by a community of commensal bacteria

Abstract

Plants have an innate immune system to fight off potential invaders that is based on the perception of nonself or modified-self molecules. Microbe-associated molecular patterns (MAMPs) are evolutionarily conserved microbial molecules whose extracellular detection by specific cell surface receptors initiates an array of biochemical responses collectively known as MAMP-triggered immunity (MTI). Well-characterized MAMPs include chitin, peptidoglycan, and flg22, a 22-amino acid epitope found in the major building block of the bacterial flagellum, FliC. The importance of MAMP detection by the plant immune system is underscored by the large diversity of strategies used by pathogens to interfere with MTI and that failure to do so is often associated with loss of virulence. Yet, whether or how MTI functions beyond pathogenic interactions is not well understood. Here we demonstrate that a community of root commensal bacteria modulates a specific and evolutionarily conserved sector of the Arabidopsis immune system. We identify a set of robust, taxonomically diverse MTI suppressor strains that are efficient root colonizers and, notably, can enhance the colonization capacity of other tested commensal bacteria. We highlight the importance of extracellular strategies for MTI suppression by showing that the type 2, not the type 3, secretion system is required for the immunomodulatory activity of one robust MTI suppressor. Our findings reveal that root colonization by commensals is controlled by MTI, which, in turn, can be selectively modulated by specific members of a representative bacterial root microbiota.

Keywords: MAMP; SynCom; flg22; plant immunity; root microbiome.

Fig. 1.

A synthetic community comprised of 35 bacterial strains (SynCom35) modulates a sector of the Arabidopsis immune system. (A) Canonical analysis of principal coordinates (CAP) based on Bray–Curtis dissimilarities of 16S rDNA profiling reveals no significant effect of flg22 in the composition of root bacterial community. Significance of model terms was determined with a PERMANOVA analysis using 5,000 permutations (α = 0.05). (B) Number of differentially expressed genes (DEG) identified by RNA sequencing in roots treated with flg22 or not in the absence of bacteria, with heat-killed SynCom35 or with SynCom35 alive. (C) Overlap among the sets of flg22-responsive genes in the “No bacteria,” “Heat-killed SynCom35,” and “SynCom35” conditions. (D) Pearson correlation among flg22 responses in plants grown axenically (no bacteria) or in the presence of dead or living SynCom35. Note the low correlation between “SynCom35” and the other two conditions. (E) CAP coordinates shows that plants grown in the presence of SynCom35 display a unique transcriptional signature that is not differentiated by addition of flg22. (F) Hierarchical clustering of the 716 genes that responded to flg22 in at least one of the experimental conditions. SynCom35 triggers a transcriptional signature that is very similar to the flg22 response except for three sets of genes (Cluster SC1, Cluster SC7, and Cluster SC8), which display a remarkably different expression profile when compared to control plants. (G) Expression pattern of each of the clusters defined in the heatmap. The P values on the bottom refer to the comparison between groups (flgs22 vs. mock) for each condition using two-tailed t tests. (H) Gene ontology enrichment analyses showing enriched biological processes in each of the clusters. Cluster SC1, which is suppressed by SynCom35, contains defense genes that are activated by flg22. Cluster SC7, which is activated by SynCom35, is enriched in auxin metabolism. All results refer to plants exposed to the treatments for 12 d. HK, heat-killed SynCom35; FDR, false-discovery rate; NB, no bacteria; SC, SynCom35. The complete enrichment analysis and the associated statistics are shown in Dataset S3.

Fig. 2.

Suppression of the flg22 response is a common feature among members of SynCom35. (A) Arabidopsis root length in the presence of each strain of SynCom35. While some strains have a neutral effect on the plant root elongation, others are able to suppress the flg22-induced RGI or induce the same phenotype even in the absence of flg22. The taxonomic class of each strain and the presence or not of a T3SS are annotated as colored boxes on the left of the heatmap. The images (Right) show the plant phenotype in the presence of a representative neutral (MF2), suppressor (MF374), and inducer strain (MF362). The boxplots summarize the effect of each group from the heatmap on root length (multiple comparisons were performed with ANOVA followed by a Tukey test, α = 0.05). (B) PCA based on 428 genes that responded to flg22 in axenic plants demonstrates that some, but not all, strains of SynCom35 interfere with the transcriptional regulation of the flg22 regulon. Ellipses show the parametric smallest area around the mean that contains 70% of the probability mass for each group (blue: mock; red: 1 μM flg22). Only controls (“no bacteria” and “SynCom35”) and suppressors of this gene set are labeled for clarity. Underlined labels indicate that the strain also suppressed the RGI phenotype triggered by flg22 shown in A. (C) Hierarchical clustering based on the expression of the same 428 genes in seedlings grown in the presence of each strain from SynCom35 along with controls (no bacteria, heat-killed SynCom35, and SynCom35). Five clusters were defined and their profiles in seedlings grown under axenic conditions or with SynCom35 are shown on the bottom. The P values refer to the comparison between groups (flg22 vs. mock) using two-tailed t tests. The boxes on the top of the heatmap indicate the taxonomic classification of the inoculated strain and treatment (orange) or not (white) with flg22. Lanes on the right of the heatmap indicate whether each gene was suppressed by SynCom35 in roots (cluster SC1) (Fig. 1F) or whether they belong to the set of 84 core flg22 marker genes (SI Appendix, Fig. S6). Representative biological processes that are enriched in each cluster are shown on the right. The complete enrichment analysis and the associated statistics are shown in Dataset S4.

Fig. 3.

Microbiota members that suppress the plant immune system are better colonizers of Arabidopsis roots either in monoassociation or in the context of SynComs. (A) Summary of the assays performed to define suppression of the flg22 response in this study. Strains are clustered based on their response to a total of five assays that are illustrated as rows of boxes to the right. RGI: the ability of a strain to suppress flg22-induced RGI or induce this phenotype even in the absence of the MAMP (Fig. 2A). Regulon: the ability to interfere with the overall expression signature of 428 flg22-responsive genes defined in control (no bacteria) seedlings (Fig. 2B). Core: the ability of each strain to interfere specifically with a set of 84 defense-related genes that are consistently induced by flg22 under diverse experimental conditions (SI Appendix, Fig. S6C). Acute GUS: the ability of each strain to interfere with the acute response to flg22 (5 h treatment) specifically in roots based on the pCYP71A12::GUS reporter line (SI Appendix, Fig. S6D). Clusters 3, 4, and 5: the suppression ability of each strain on specific transcriptional response sectors of the flg22 regulon (Fig. 2C). Members of SynCom5-S1 and SynCom5-NS1 are marked with red and blue arrowheads, respectively. (B) Suppressors are better colonizers of Arabidopsis roots in monoassociation experiments. The growth of five suppressors (members of SynCom5-S1) and taxonomically matching nonsuppressors (members of SynCom5-NS1) were individually assayed in Arabidopsis roots after 12 d of colonization in three biological replicates. Each dot represents an individual strain/replicate. Strains are colored based on their behavior in the context of SynCom35 in a previous study (22). Note that all robust endophytic compartment colonizers are suppressor strains. A Kruskal–Wallis test was performed on the CFU counts from three independent experiments each with three technical reps. n = 9 for each strain and n = 45 for each group being compared. (C) A SynCom made of five suppressors (SynCom5-S1) colonizes Arabidopsis roots better than a SynCom made of five taxonomically matching nonsuppressors (SynCom5-NS1). Different symbols represent independent experimental repetitions. Letters indicate significantly different groups based on an ANOVA followed by a Tukey test (α = 0.05). (D) Seedlings grown with SynCom5-NS1 activate the expression of flg22 marker genes while seedlings inoculated with SynCom5-S1 do not. Statistical significance was determined using a permutation approach, where the actual group mean differences were compared to the group mean differences of 10,000 permutations of the gene counts. The group mean differences that were greater than 95% of the group mean differences calculated in the permutations were considered significant (asterisks). n.s. = not significant. (E) Suppressor (SynCom5-S1), but not nonsuppressor (SynCom5-NS1), strains are enriched in the endophytic compartment of Arabidopsis roots relative to agar in the context of a synthetic community. Suppressors n = 80, nonsuppressors n = 68.

Fig. 4.

Immunomodulatory bacteria enhance the root colonization ability of other commensal strains. (A) Cartoon representation of the experimental system employed. Arabidopsis seedlings were grown axenically, in the presence of a community of five suppressors (SynCom5-S2) or in the presence of five nonsuppressors (SynCom5-NS2) and then inoculated with one of the commensal strains. (B) Quantification of Ochrobactrum sp. MF370 cells (kanamycin resistant) from Arabidopsis roots (n = 9). (C) Quantification of P. viridiflava OTU5 p5.e6 cells (gentamycin resistant) from Arabidopsis roots (n = 12). Different symbols represent independent experimental repetitions. Multiple comparisons were performed with ANOVA followed by a Tukey test (α = 0.05).

Fig. 5.

D. japonica MF79 requires the T2SS to suppress the root response to flg22. (A) The T3SS is not required for MTI suppression by D. japonica MF79. Two independent mutants (ΔT3SS_RSTU and ΔT3SS_full) retained the ability to prevent activation of the pCYP71A12::GUS reporter in roots exposed to 100 nM flg22 for 5 h. (B) The screening of a transposon insertion library revealed two major components of the bacterial T2SS (GspD and GspE) as required for suppression of the root response to flg22. The diagram (Upper) summarizes the strategy employed in the screening. Additional mutants and replicates are shown in SI Appendix, Fig. S10B. (C) The cell-free supernatant of wild-type D. japonica MF79, but not of T2SS mutants, is sufficient for the suppression of the root response to flg22. (D) The molecule responsible for the suppression activity is larger than 10 kDa. The diagram (Left) illustrates the preparation of 10-kDa retentate and flow-through from bacterial cultures. (E) The 10-kDa retentate of wild-type D. japonica MF79 prevents the flg22-mediated reduction of Ochrobactrum sp. MF370 colonization. Four independent experiments were performed (n = 42 per condition). Diamonds represent means with two times SE. P values were determined with the Wilcoxon test. Root images were captured at 43× magnification on a Leica M205 FA fluorescence stereo microscope.