Comparative Transcriptomics of Bacillus mycoides Strains in Response to Potato-Root Exudates Reveals Different Genetic Adaptation of Endophytic and Soil Isolates

Abstract

Plant root secreted compounds alter the gene expression of associated microorganisms by acting as signal molecules that either stimulate or repel the interaction with beneficial or harmful species, respectively. However, it is still unclear whether two distinct groups of beneficial bacteria, non-plant-associated (soil) strains and plant-associated (endophytic) strains, respond uniformly or variably to the exposure with root exudates. Therefore, Bacillus mycoides, a potential biocontrol agent and plant growth-promoting bacterium, was isolated from the endosphere of potatoes and from soil of the same geographical region. Confocal fluorescence microscopy of plants inoculated with GFP-tagged B. mycoides strains showed that the endosphere isolate EC18 had a stronger plant colonization ability and competed more successfully for the colonization sites than the soil isolate SB8. To dissect these phenotypic differences, the genomes of the two strains were sequenced and the transcriptome response to potato root exudates was compared. The global transcriptome profiles evidenced that the endophytic isolate responded more pronounced than the soil-derived isolate and a higher number of significant differentially expressed genes were detected. Both isolates responded with the alteration of expression of an overlapping set of genes, which had previously been reported to be involved in plant-microbe interactions; including organic substance metabolism, oxidative reduction, and transmembrane transport. Notably, several genes were specifically upregulated in the endosphere isolate EC18, while being oppositely downregulated in the soil isolate SB8. These genes mainly encoded membrane proteins, transcriptional regulators or were involved in amino acid metabolism and biosynthesis. By contrast, several genes upregulated in the soil isolate SB8 and downregulated in the endosphere isolate EC18 were related to sugar transport, which might coincide with the different nutrient availability in the two environments. Altogether, the presented transcriptome profiles provide highly improved insights into the life strategies of plant-associated endophytes and soil isolates of B. mycoides.

Keywords: Bacillus mycoides; endophyte; root colonization; root exudates; transcriptome.

FIGURE 1

Root colonized by Bacillus mycoides observed by CLSM. GFP-tagged B. mycoides EC18 (A), RFP-tagged B. mycoides SB8 (B), and a 1:1 mixture of the two strains (C) were inoculated to the seedlings. Root samples were inspected 1 and 3 days after inoculation (DAI). (D,G) Root inoculated with B. mycoides EC18 at 1 and 3 DAI. (E,H) Root inoculated with B. mycoides SB8 at 1 and 3 DAI. (F,I) Root inoculated with a 1:1 mixture of B. mycoides EC18 and B. mycoides SB8 at 1 and 3 DAI. (J–L) Three dimensional model of roots inoculated with EC18, SB8, and a 1:1 mixture of the two strains 3 DAI.

FIGURE 2

Venn diagram showing the core genome and the genes specific of the B. mycoides strain ATCC 6462, EC18, and SB8.

FIGURE 3

Analyses of differentially expressed genes (DEGs) of B. mycoides in response to potato root exudates. (A,B) Volcano diagrams of DEGs of EC18 and SB8. Spots in the right and left quadrant indicate significantly up and down regulated genes, respectively. Spots in the gray area were neither over- or under-expressed genes. (C,D) The number of up and downregulated DEGs of B. mycoides EC18 and SB8. (E) Venn diagram showing the numbers of overlapped and non-overlapped DEGs.

FIGURE 4

Functional categories of genes regulated of strain EC18 and SB8 according to COGs (Clusters of Orthologous Groups). Categorization of all altered genes was done on the Genome2D server (http://genome2d.molgenrug.nl).

FIGURE 5

Overlapping DEGs of B. mycoides strain EC18 and SB8 in response to root exudates. Red represents significant upregulation and blue indicates significant downregulation of gene expression.

FIGURE 6

Differentially expressed genes show opposite trends in B. mycoides strain EC18 and SB8 in response to root exudates. Red represents significant upregulation and blue indicates significant downregulation of gene expression.

FIGURE 7

Differentially expressed genes specifically present in endophytic strain B. mycoides EC18 in response to root exudates. Red represents significant upregulation and blue indicates significant downregulation of gene expression.

FIGURE 8

Differentially expressed genes specifically present in the soil strain B. mycoides SB8 in response to root exudates. Red represents significant upregulation and blue indicates significant downregulation of gene expression.