Transcriptomic analysis of Pseudomonas ogarae F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption

Abstract

Rhizosphere colonization by bacteria involves molecular and cellular mechanisms, such as motility and chemotaxis, biofilm formation, metabolic versatility, or biosynthesis of secondary metabolites, among others. Nonetheless, there is limited knowledge concerning the main regulatory factors that drive the rhizosphere colonization process. Here we show the importance of the AmrZ and FleQ transcription factors for adaption in the plant growth-promoting rhizobacterium (PGPR) and rhizosphere colonization model Pseudomonas ogarae F113. RNA-Seq analyses of P. ogarae F113 grown in liquid cultures either in exponential and stationary growth phase, and rhizosphere conditions, revealed that rhizosphere is a key driver of global changes in gene expression in this bacterium. Regarding the genetic background, this work has revealed that a mutation in fleQ causes considerably more alterations in the gene expression profile of this bacterium than a mutation in amrZ under rhizosphere conditions. The functional analysis has revealed that in P. ogarae F113, the transcription factors AmrZ and FleQ regulate genes involved in diverse bacterial functions. Notably, in the rhizosphere, these transcription factors antagonistically regulate genes related to motility, biofilm formation, nitrogen, sulfur, and amino acid metabolism, transport, signalling, and secretion, especially the type VI secretion systems. These results define the regulon of two important bifunctional transcriptional regulators in pseudomonads during the process of rhizosphere colonization.

Keywords: AmrZ; FleQ; Pseudomonas ogarae; regulation; rhizosphere; transcriptomics.

Fig. 1.

Gene expression comparison of RNA-Seq samples. Principal Component Analysis (PCA) plot (a) and similarity-based clustering (b) for RNA-Seq samples. Hierarchical clustering is based on Euclidean sample-to-sample distance. WT: wild-type; Exp: Exponential; St: Stationary; Rhiz: Rhizosphere. Different replicates for each sample and condition are represented (R1: replicate 1, R2: replicate 2).

Fig. 2.

Differential gene expression in P. ogarae F113 and derivatives in different growth conditions. Upset plot showing the intersections of shared and specific up- and down-regulated DEGs among P. ogarae F113 grown under culture conditions (a) and the amrZ and fleQ mutants under rhizosphere conditions (b). DEGs were calculated using F113 wild-type under rhizosphere condition as a reference. DEGs were selected applying the following threshold: log₂ Fold-Change ≤−1/≥1 and a p-adjusted value ≤ 0.001. Set size indicates the total number of DEGs found in each condition. Exp: exponential; St: stationary.

Fig. 3.

Functional analysis of differentially expressed genes in amrZ and fleQ mutants during rhizosphere colonization. Dot plots showing the expression of genes belonging to distinct functional categories according to KEGG (a) and GO (b) databases during rhizosphere growth in the amrZ and fleQ mutants compared to P. ogarae F113 wild-type. Up- and down-regulated KEGG pathways with categories including more than two annotated genes were selected. The dot size is based on the numbers of annotated DEGs found in each case, and the dot colour depicts the ratio of DEGs versus the total of annotated genes in each category (a). Dot plot shows the up- and down-regulated GO terms of Biological processes with a P-value < 0.001 in the classic Fisher’s test. The dot size shows the number of genes in each term and the colour the ratio between the number of significant genes and the number of annotated genes for each term. The top ten GO terms for each experiment were selected (b).

Fig. 4.

Differential gene expression of relevant pathways during the rhizosphere colonization process in the amrZ and fleQ mutants.(a) Volcano plot representing the differential gene expression analysis in the amrZ and fleQ mutant data sets versus wild-type under rhizosphere conditions. The x-axis shows log₂ Fold-Change (FC) expression and the y-axis the odds of a gene being differentially expressed as the -log₁₀(P-adjusted value). Black lined shapes indicate whether genes meet the following threshold: log₂ FC ≤−1/≥1 and a P-adjusted value≤0.001 whereas grey points indicate genes that do not meet either of the two criteria . Distinct point shapes indicate differentially expressed genes in one of the mutants versus the wild-type strain belonging to a relevant functional pathway: biofilm formation, nitrogen metabolism, c-di-GMP, motility, chemotaxis and T6SS. (b) Heatmap representation of genes belonging to relevant functional pathways in the amrZ and fleQ mutants in the rhizosphere. Pseudomonas ogarae F113 annotated genes with a log₂FC (mutant/wild-type) ≤−1/≥1 and belonging to the KEGG database categories specified above are represented. The gene name or locus (‘PSF113_’) is shown. Values, locus, and annotations are listed in File S3.