Fast and reliable screening system to preselect candidate Dickeya solani Tn5 mutants in plant tissue-induced genes

Abstract

Here, we present a simple and fast screening method to preselect candidate Dickeya solani Tn5 mutants which carry transposon insertions in plant tissue-induced genes for the follow-up studies. The described method is faster and cost-effective in comparison to standard IVET, expression microarrays and RNAseq, and it does not require any specialized laboratory equipment and/or technical assistance. The Tn5 mutants are generated using mini-Tn5 transposon carrying inducible reporter gene (promoterless gusA coding for β-glucuronidase) and are screened for the β-glucuronidase positive phenotypes under non-inductive conditions with X-gluc as a glucuronidase substrate. The bacterial mutants negative in the first screen are then screened for β-glucuronidase phenotypes in the presence of plant tissues, viz. roots, stems and leaves, suspended in liquid bacterial growth medium supplemented with X-gluc in a 48-well microtiter plate assay. The mutants that are positive in the screen with plant tissues are selected for sequencing of the Tn5 insertion sites directly from bacterial genome. This method allows generation of a number of ready-to-use Tn5 mutants showing up-regulation by plant tissue which can be later selected for further studies. We used this method to evaluate interaction of D. solani IPO2222 (type strain) with bittersweet nightshade (Solanum dulcamara L.) and potato (Solanum tuberosum L.) tissues.