Root-microbe systems: the effect and mode of interaction of Stress Protecting Agent (SPA) Stenotrophomonas rhizophila DSM14405(T.)

Abstract

Stenotrophomonas rhizophila has great potential for applications in biotechnology and biological control due to its ability to both promote plant growth and protect roots against biotic and a-biotic stresses, yet little is known about the mode of interactions in the root-environment system. We studied mechanisms associated with osmotic stress using transcriptomic and microscopic approaches. In response to salt or root extracts, the transcriptome of S. rhizophila DSM14405(T) changed drastically. We found a notably similar response for several functional gene groups responsible for general stress protection, energy production, and cell motility. However, unique changes in the transcriptome were also observed: the negative regulation of flagella-coding genes together with the up-regulation of the genes responsible for biofilm formation and alginate biosynthesis were identified as a single mechanism of S. rhizophila DSM14405(T) against salt shock. However, production and excretion of glucosylglycerol (GG) were found as a remarkable mechanism for the stress protection of this Stenotrophomonas strain. For S. rhizophila treated with root exudates, the shift from the planktonic lifestyle to a sessile one was measured as expressed in the down-regulation of flagellar-driven motility. These findings fit well with the observed positive regulation of host colonization genes and microscopic images that show different colonization patterns of oilseed rape roots. Spermidine, described as a plant growth regulator, was also newly identified as a protector against stress. Overall, we identified mechanisms of Stenotrophomonas to protect roots against osmotic stress in the environment. In addition to both the changes in life style and energy metabolism, phytohormons, and osmoprotectants were also found to play a key role in stress protection.

Keywords: FISH–CLSM; PGPR; SPA; oilseed rape; plant-microbe interaction; root exudates; transcriptomics.

Figure 1

The effect of salt shock on the gene expression of various functional gene groups in S. rhizophila DSM14405^T. A total of 912 and 1521 genes were significantly up and down-regulated, respectively. The impact of salt stress on most functional gene groups is clearly pronounced, as a given functional group shows either an increase or decrease in the transcription of genes belonging to that group. Genes involved in translation, synthesis of the cell wall, outer or cytoplasm membrane, nucleotide and amino acid transport and metabolism, energy production and conversion are up-regulated. In contrast, genes involved in cell motility, secretion, and intracellular trafficking, defense mechanisms, and transport and metabolism of carbohydrates and inorganic ions are down-regulated. Genes involved in lipid metabolism, and the hypothetical genes are rather ambiguously affected by salt stress, as some of these are up and others down-regulated. The values above each column correspond to the percentage abundance of the corresponding functional group relative to the total count of the up and down-regulated genes. The transcription fold change for each CDS corresponds to the ratio calculated for S. rhizophila under salt shock compared with the control. Data are presented as the mean value of two independent replicates. The error bar shown on each functional group corresponds to the mean value of errors for all genes belonging to that functional group.

Figure 2

The effect of oilseed rape seedling exudates on gene expression of various functional gene groups in S. rhizophila DSM14405^T. A total of 763 and 246 genes were significantly up and down-regulated, respectively. While some functional groups are both positively and negatively regulated by root exudates, others show a clear and pronounced alteration, as the majority of the corresponding genes are either up or down-regulated. For example, genes responsible for amino acid, nucleotide, and carbohydrate transport and metabolism, and those involved in cell wall, outer-membrane or cytoplasmic membrane biogenesis and transport as well as genes responsible for the transport of secondary metabolites and coenzymes are mainly up-regulated. In contrast, genes involved in cell motility and secretion, and those responsible for the transport and metabolism of inorganic ions are mainly down-regulated. The value above each column corresponds to percentage abundance of the corresponding functional group in the total count of the up or down-regulated genes.

Figure 3

Model showing the response of S. rhizophila DSM14405^T to osmotic stress: salt shock and root exudates. Functional gene groups shared in the response to oilseed rape root exudates and salt shock are presented. Several functional gene groups are up-regulated as a result of both oilseed rape root exudates and salt shock including those responsible for the synthesis and transport of cell wall, outer membrane, and cytoplasmic membrane, the metabolism and transport of amino acids, nucleotide, and secondary metabolites, and energy production. In contrast, genes responsible for cell motility, secretion and intracellular trafficking, and the transport and metabolism of inorganic ions are down-regulated.

Figure 4

The impact of salt stress on the capability of S. rhizophila DSM14405^T to colonize the oilseed rape rhizosphere visualized using FISH-CLSM. S. rhizophila DSM14405^T intensely colonizes the oilseed rape rhizosphere (left) while the treatment of seeds with 1.25% NaCl (right) severely decreases the colonization capability. An equimolar ratio of the FISH probes EUB338, EUB338 II, and EUB338 III labeled with the fluorescent dye Cy3 was used in the hybridization step. Microscopic images were captured using a Leica TCS SPE confocal microscope. The Leica ACS APO 63X OIL CS objective (NA: 1.30) was used to acquire confocal stacks by applying a z-step of 0.4–0.8 μm.