Identification by Tn‐seq of Dickeya dadantii genes required for survival in chicory plants

Abstract

Summary The identification of the virulence factors of plant‐pathogenic bacteria has relied on the testing of individual mutants on plants, a time‐consuming process. Transposon sequencing (Tn‐seq) is a very powerful method for the identification of the genes required for bacterial growth in their host. We used this method in a soft‐rot pathogenic bacterium to identify the genes required for the multiplication of Dickeya dadantii in chicory. About 100 genes were identified showing decreased or increased fitness in the plant. Most had no previously attributed role in plant–bacterium interactions. Following our screening, in planta competition assays confirmed that the uridine monophosphate biosynthesis pathway and the purine biosynthesis pathway were essential to the survival of D. dadantii in the plant, as the mutants ∆ carA , ∆ purF , ∆ purL , ∆ guaB and ∆ pyrE were unable to survive in the plant in contrast with the wild‐type (WT) bacterium. This study also demonstrated that the biosynthetic pathways of leucine, cysteine and lysine were essential for bacterial survival in the plant and that RsmC and GcpA were important in the regulation of the infection process, as the mutants ∆ rsmC and ∆ gcpA were hypervirulent. Finally, our study showed that D. dadantii flagellin was glycosylated and that this modification conferred fitness to the bacterium during plant infection. Assay by this method of the large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.

Genome-wide identification of Pseudomonas syringae genes required for fitness during colonization of the leaf surface and apoplast

“…Similar to these observations, biosynthetic genes for tryptophan (and its precursor anthranilate) were identified as important for fitness in a TnSeq study examining Pantoea stewartii colonization of maize xylem (53). A TnSeq screen in Dickeya dadantii in rotting plant tissue also noted a significant decrease in competitive fitness in planta for leucine, cysteine, and lysine auxotrophs that could be negated through the external addition of amino acids (54). Since most amino acids are apparently present at relatively low concentrations in the bean apoplast (9), it could be expected that many auxotrophs are incapable of growth without the ability to synthesize these essential nonsubstitutable metabolites, a model supported by the observations of this study.…”

Genome-wide identification ofPseudomonas syringaegenes required for competitive fitness during colonization of the leaf surface and apoplast

“…Similar to our observations, biosynthetic genes for tryptophan (and its precursor anthranilate) were identified as important for fitness in a TnSeq study examining Pantoea stewartii colonization of maize xylem (49). A TnSeq screen in Dickeya dadantii in rotting plant tissue also noted a significant decrease in competitive fitness in planta for leucine, cysteine, and lysine auxotrophs that could be negated through the external addition of amino acids (50). Since most amino acids are apparently present at relatively low concentrations in the bean apoplast (9), it could be expected that many auxotrophs are incapable of growth without the ability to synthesize these essential non-substitutable metabolites, a model supported by the observations of this study.…”

Application of Transposon Insertion Sequencing to Agricultural Science

“…Another pathogenic lifestyle is the necrotrophic growth of rot-causing pathogens such as Dickeya dadantii. A Tn-Seq study of this pathogen showed that the biosynthetic pathways for uridine monophosphate, purines, and the amino acids leucine, cysteine and lysine, are essential for survival on chicory plants (Royet et al, 2019). The study also determined that the RsmC and GcpA regulators are important in the infection process and glycosylation of flagellin confers fitness during plant infection.…”

“…To further our understanding of plant-microbe and microbemicrobe interactions a faster method of determining microbial gene function is needed. The traditional methods of ascribing functions to genes (for example, making single gene knockouts or heterologous expression) are laborious and very slow as mutants are screened individually (Royet et al, 2019). In addition, these methods can be problematic, for example, gene expression in a heterologous bacterial host may exhibit an altered phenotype due to the different genomic context of the original organism (Levy et al, 2018).…”