Background

Over the years, topics of ecology and evolution have been increasingly important when studying host-microbe interactions. In addition to simply assessing the virulence of bacterial pathogens, matters of fitness and adaptation to the environment are now being addressed. Recently, large-scale high-throughput screening tools, such as RB-TnSeq (Randomly-Barcoded Transposon insertion site Sequencing), have been developed to efficiently identify the genetic determinants of bacterial fitness. RB-TnSeq relies on the co-inoculation of a library of mutagenized bacteria, all carrying a unique barcode, to simultaneously evaluate the contribution of each gene to bacterial survival and growth (Wetmore et al., 2015). Such studies result in the identification of genes putatively implicated in bacterial adaptation to the host that then need to be confirmed by reverse genetics using simpler fitness assays.

Early on, it was proposed that competition assays could be used as an answer to this need. Typically, such competition assays are performed by co-inoculating two strains in a 1:1 ratio into a given environment and following the evolution of this ratio over time. If one of the strains grows more than the other, it is considered as more fit (in this specific environment) than its counterpart. The main limiting factor is then to be able to count separately the two mixed populations. A quite straightforward solution here is to use discriminating markers, such as antibiotic resistance, to count the abundance of each strain after plating on selective medium. Differential antibiotic resistances have been widely used in co-inoculation assays (Macho et al., 2007 and 2010; Feng et al., 2012), but it is known that such resistances often carry a strong cost of fitness that could interfere in studies that involve fine fitness mechanisms. Also, they require plating the samples twice, once on each selective medium, which can introduce a bias in the evaluation of the strains relative frequencies. Fluorescent markers provide an alternative to antibiotics but require costly imaging equipment (Perrier et al., 2019). Here, we describe a visual blue/white screening using the enzymatic properties of the beta-glucuronidase (GUS) enzyme. The assay is then performed by co-inoculating a GUS-expressing strain in competition with a non-tagged strain: after plating on medium containing X-gluc (the substrate of the GUS enzyme), colonies of tagged bacteria will turn blue while the others will remain white. A similar Bio-protocol had been proposed before using LacZ as a marker in the Vibrio cholerae animal pathogen (Fu and Mekalanos, 2014). However, even though the concept is similar, we feel that a detailed protocol for plant inoculations is very much needed.

Here, we chose the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) as a model to illustrate this protocol. Xcc is particularly interesting because it has the specificity to infect multiple organs and tissues of its cauliflower host (Brassica oleracea pv. botrytis cv. Clovis). After entry through the hydathodes, Xcc spreads through the xylem sap-conducting vessels and colonizes the leaf mesophyll of its host, where it causes the typical symptoms of the Black rot disease (Vicente and Holub, 2013; An et al., 2019). We recently performed an RB-TnSeq screening on Xcc during cauliflower infection (Luneau et al., 2022), which ultimately led us to set up a competition assay. In this protocol, we describe how this competition assay can be performed in the plant leaf mesophyll. We take the example of the well-described hrpX (transcriptional activator of the Type III Secretion System), hrcV (Type III Secretion System component), and xopAC (Type III Effector) mutants that display contrasted fitness phenotypes when confronted with the Xcc 8004 wild-type strain in planta. This protocol provides a basis that can be adapted for other plant compartments, such as hydathodes or xylem, as well as in vitro studies. Availability of such protocol is particularly relevant given the number of plant pathogens that enter through and thrive in their host leaves.