Genome wide identification of bacterial genes required for plant infection by Tn-seq

Soft rot enterobacteria ( Dickeya and Pectobacterium ) are major pathogens that cause diseases on plants of agricultural importance such as potato and ornamentals. Long term studies to identify virulence factors of these bacteria focused mostly on plant cell wall degrading enzymes secreted by the type II secretion system and the regulation of their expression. To identify new virulence factors we performed a Tn-seq genome-wide screen of a transposon mutant library during chicory infection followed by high-throughput sequencing. This allowed the detection of mutants with reduced but also increased fitness in the plant. Virulence factors identified differed from those previously known since diffusible ones (secreted enzymes, siderophores or metabolites) were not detected by this screen. In addition to genes encoding proteins of unknown function that could be new virulence factors, others could be assigned to known biological functions. The central role of the FlhDC regulatory cascade in the control of virulence was highlighted with the identification of new members of this pathway. Scarcity of the plant in certain amino acids and nucleic acids required presence of the corresponding biosynthetic genes in the bacteria. Their products could be targets for the development of antibacterial compounds. Among the genes required for full development in chicory we also identified six genes involved in the glycosylation of the flagellin FliC, glycosylation, which in other plant pathogenic bacteria contributes to virulence. Author summary Identification of virulence factors of plant pathogenic bacteria has relied on the test of individual mutants on plants, a time-consuming method. New methods like transcriptomic or proteomic can now be used but they only allow the identification of genes induced during the infection process and non-induced genes may be missed. Tn-seq is a very powerful method to identify genes required for bacterial growth in their host. We used for the first time this method in a plant pathogenic bacteria to identify genes required for the multiplication of Dickeya dadantii in chicory. We identified about 100 genes with decreased or increased fitness in the plant. Most of them had no previously described role in bacterial virulence. We unveiled important metabolic genes and regulators of motility and virulence. We showed that D. dadantii flagellin is glycosylated and that this modification confers fitness to the bacteria during plant infection. Our work opens the way to the use of Tn-seq with bacterial phytopathogens. Assay by this method of large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.