Complete Genome Sequence Analysis of Enterobacter sp. SA187, a Plant Multi-Stress Tolerance Promoting Endophytic Bacterium

Enterobacter sp. SA187 is an endophytic bacterium that has been isolated from root nodules of the indigenous desert plant Indigofera argentea. SA187 could survive in the rhizosphere as well as in association with different plant species, and was able to provide abiotic stress tolerance to Arabidopsis thaliana. The genome sequence of SA187 was obtained by using Pacific BioScience (PacBio) single-molecule sequencing technology, with average coverage of 275X. The genome of SA187 consists of one single 4,429,597 bp chromosome, with an average 56% GC content and 4,347 predicted protein coding DNA sequences (CDS), 153 ncRNA, 7 rRNA, and 84 tRNA. Functional analysis of the SA187 genome revealed a large number of genes involved in uptake and exchange of nutrients, chemotaxis, mobilization and plant colonization. A high number of genes were also found to be involved in survival, defense against oxidative stress and production of antimicrobial compounds and toxins. Moreover, different metabolic pathways were identified that potentially contribute to plant growth promotion. The information encoded in the genome of SA187 reveals the characteristics of a dualistic lifestyle of a bacterium that can adapt to different environments and promote the growth of plants. This information provides a better understanding of the mechanisms involved in plant-microbe interaction and could be further exploited to develop SA187 as a biological agent to improve agricultural practices in marginal and arid lands

The Lys‐motif receptor LYK4 mediates Enterobacter sp. SA187 triggered salt tolerance in Arabidopsis thaliana

International audienceSummary Root endophytes establish beneficial interactions with plants, improving holobiont resilience and fitness, but how plant immunity accommodates beneficial microbes is poorly understood. The multi‐stress tolerance‐inducing endophyte Enterobacter sp. SA187 triggers a canonical immune response in Arabidopsis only at high bacterial dosage (>10 8 CFUs ml −1 ), suggesting that SA187 is able to evade or suppress the plant defence system at lower titres. Although SA187 flagellin epitopes are recognized by the FLS2 receptor, SA187‐triggered salt tolerance functions independently of the FLS2 system. In contrast, overexpression of the chitin receptor components LYK4 and LYK5 compromised the beneficial effect of SA187 on Arabidopsis, while it was enhanced in lyk4 mutant plants. Transcriptome analysis revealed that the role of LYK4 is intertwined with a function in remodelling defence responses with growth and root developmental processes. LYK4 interferes with modification of plant ethylene homeostasis by Enterobacter SA187 to boost salt stress resistance. Collectively, these results contribute to unlock the crosstalk between components of the plant immune system and beneficial microbes and point to a new role for the Lys‐motif receptor LYK4 in beneficial plant–microbe interaction

Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2-keto-4-methylthiobutyric acid production.

Several plant species require microbial associations for survival under different biotic and abiotic stresses. In this study, we show that Enterobacter sp. SA187, a desert plant endophytic bacterium, enhances yield of the crop plant alfalfa under field conditions as well as growth of the model plant Arabidopsis thaliana in vitro, revealing a high potential of SA187 as a biological solution for improving crop production. Studying the SA187 interaction with Arabidopsis, we uncovered a number of mechanisms related to the beneficial association of SA187 with plants. SA187 colonizes both the surface and inner tissues of Arabidopsis roots and shoots. SA187 induces salt stress tolerance by production of bacterial 2-keto-4-methylthiobutyric acid (KMBA), known to be converted into ethylene. By transcriptomic, genetic and pharmacological analyses, we show that the ethylene signaling pathway, but not plant ethylene production, is required for KMBA-induced plant salt stress tolerance. These results reveal a novel molecular communication process during the beneficial microbe-induced plant stress tolerance

Ethylene induced plant stress tolerance by <i>Enterobacter</i> sp. SA187 is mediated by 2‐keto‐4‐methylthiobutyric acid production

<div><p>Several plant species require microbial associations for survival under different biotic and abiotic stresses. In this study, we show that <i>Enterobacter</i> sp. SA187, a desert plant endophytic bacterium, enhances yield of the crop plant alfalfa under field conditions as well as growth of the model plant <i>Arabidopsis thaliana in vitro</i>, revealing a high potential of SA187 as a biological solution for improving crop production. Studying the SA187 interaction with Arabidopsis, we uncovered a number of mechanisms related to the beneficial association of SA187 with plants. SA187 colonizes both the surface and inner tissues of Arabidopsis roots and shoots. SA187 induces salt stress tolerance by production of bacterial 2-keto-4-methylthiobutyric acid (KMBA), known to be converted into ethylene. By transcriptomic, genetic and pharmacological analyses, we show that the ethylene signaling pathway, but not plant ethylene production, is required for KMBA-induced plant salt stress tolerance. These results reveal a novel molecular communication process during the beneficial microbe-induced plant stress tolerance.</p></div

Ion content in Arabidopsis seedlings.

<p>Shoot Na⁺ content (A), shoot K⁺ content (B) and shoot Na⁺/K⁺ ratio (C) of 17-day-old mock- or SA187-inoculated Arabidopsis seedlings exposed for 12 days to ½ MS with or without 100 mM NaCl (48 > n > 36). Root Na+ content (D), root K⁺ content (E) and root Na⁺/K⁺ ratio (F) of 17-day-old mock- or SA187-inoculated Arabidopsis seedlings exposed for 12 days to ½ MS with or without 100mM NaCl (48 > n > 12). All plots represent the mean of three biological replicates, and error bars represent SE. Asterisks indicate a statistical difference based on the Mann-Whitney test (* P < 0.05; ** P < 0.01; *** P < 0.001).</p

Ethylene signaling is important for the beneficial effect of SA187 under salt stress.

<p>(A) Fresh weight and beneficial index (a ratio between fresh weight of SA187- and mock-inoculated seedlings) of mutants in hormonal pathways transferred from ½ MS to ½ MS + 100 mM NaCl (5+12 days). <i>acs</i> = heptuple mutant <i>acs1-1 acs2-1 acs4-1 acs5-2 acs6-1 acs7-1 acs9-1</i>, and <i>pyr1/pyl</i> = quadruple mutant <i>pyr1 pyl1 pyl2 pyl4</i>. All plots represent the mean of three biological replicates (n > 36). Error bars represent SE. (B) qPCR expression analysis of four ethylene-associated genes in 17-day-old mock- and SA187-inoculated Arabidopsis seedlings exposed for 12 days to ½ MS with or without 100 mM NaCl. Normalized expression indicates the linear fold change compared to mock-treated plants on ½ MS. Values represent means of three biological experiments, each in three technical replicates. Error bars indicate SE. (C) 100 nM ACC partially mimics the effect of SA187 on salt stress tolerance improvement in Arabidopsis seedlings. Five-day-old-seedlings were transferred to ½ MS + 100 mM NaCl with or without ACC and evaluated after 12 days. SA187-inoculated plants were used for comparison. (D) Total fresh weight of mock- and SA187-inoculated 18-day-old Arabidopsis seedlings on ½ MS with 100 mM NaCl supplemented with the ethylene synthesis inhibitor AVG or ethylene signaling inhibitor AgNO₃. Error bars representing SE and beneficial index (%) are displayed. Asterisks indicate a statistical difference based on the Student’s t-test (* P < 0.05; ** P < 0.01; *** P < 0.001).</p

Colonization of Arabidopsis seedlings with GFP-expressing SA187 visualized by confocal microscopy.

<p>(A) Root colonization of agar-grown seedlings starts in the elongation zone. Large colonies then occur in the differentiation zone. MIP; bar = 100 μm. (B) Colonies first established themselves in grooves between root epidermal cells. MIP; bar = 10 μm. (C) Large colonies in the differentiation zone grow out from the grooves. MIP; bar = 10 μm. (D) Root colonization of soil-grown seedlings exhibit a more random pattern in comparison to agar-grown seedlings. MIP; bar = 50 μm. (E) Lateral root emergence allows SA187 to enter the root and colonize the lateral root base (marked by arrowheads). A selected confocal section from a Z-stack with top and side orthogonal views. Bar = 20 μm. (F) Scattered SA187 colonies occur inside the root tissues in two-week-old seedlings (marked by arrowheads). A single confocal section. Bar = 20 μm. (G) In cotyledons, SA187 colonizes grooves between epidermal cells (left side) as well as the extracellular space between mesophyll cells (right side; marked by arrowheads). A single oblique confocal section is shown. Bar = 20 μm. (H) SA187 colonization of the hypocotyl epidermis. MIP; bar = 20 μm. (I) SA187 cells enter hypocotyl via stomata, move freely among hypocotyl cells and occasionally establish colonies inside. A selected confocal section from a Z-stack with top and side orthogonal views. Bar = 50 μm. Green–SA187-GFP; Magenta–cells walls (propidium iodide labeling); Blue–chloroplasts (autofluorescence); MIP–maximum intensity projection of a confocal Z-stack.</p

KMBA as a potential ethylene precursor in the plant-SA187 interaction.

<p>(A) qPCR analysis of the methionine salvage pathway gene expression of SA187 colonizing plants in control or salt stress conditions compared to SA187 cultivated alone in ½ MS with or without 100 mM NaCl. Values represent means of three biological experiments, each in three technical replicates. Error bars indicate SE. (B) KMBA partially mimics the effect of SA187 on salt stress tolerance improvement in Arabidopsis seedlings. Plants were transferred 5 days after germination to ½ MS + 100 mM NaCl with or without KMBA and evaluated after 12 days. SA187-inoculated plants transferred to ½ MS + 100 mM NaCl were used as a positive control. (C, D) Total fresh weight of mock- and SA187-inoculated 17-day-old Arabidopsis seedlings grown on ½ MS medium (C) or ½ MS with 100 mM NaCl (D) supplemented with 3 μM DNPH. All plots represent the mean of four biological replicates (n > 75). Error bars representing SE, beneficial index (%) is displayed. Asterisks indicate a statistical difference based on the Student’s t-test (*** P < 0.001).</p

Transcriptome analysis of Arabidopsis response to SA187.

<p>Hierarchical clustering of up- and down-regulated genes in Arabidopsis seedlings in response to SA187, salt (100 mM NaCl) or both treatments based on the RNA-Seq analysis. For every gene, FPKM values were normalized. Heat map colors indicate expression levels. For the most relevant clusters, gene families significantly enriched are indicated based on gene ontology.</p