Preparing for the KIL: Receptor Analysis of Pseudomonas syringae pv. porri Phages and Their Impact on Bacterial Virulence

Abstract

The prevalence of Pseudomonas syringae pv. porri (Pspo) in Belgium continues to increase and sustainable treatments for this pathogen remain unavailable. A potentially attractive biocontrol strategy would be the application of bacteriophages. The ideal application strategy of phages in an agricultural setting remains unclear, especially in a field-based production such as for leek plants in Flanders. Therefore, more insight in bacteria–phage interaction is required, along with the evaluation of different application strategies. In this study, we further characterized the infection strategy of two Pspo phages, KIL3b and KIL5. We found that both phages recognize lipopolysaccharide (LPS) moieties on the surface of the bacterium. LPS is an important pathogenicity factor of Pspo. Our data also suggest that KIL5 requires an additional protein in the bacterial cytoplasmatic membrane to efficiently infect its host. Virulence tests showed that this protein also contributes to Pspo virulence. Furthermore, a cocktail of both phages was applied in a seed bioassay. A combination of KIL3b and KIL5 reduced the bacterial concentration 100-fold. However, in vitro Pspo resistance against phage infection developed quite rapidly. However, the impact of this phage resistance might be mitigated as is suggested by the fact that those resistance mutations preferably occur in genes involved in LPS metabolism, and that the virulence of those mutants is possibly reduced. Our data suggest that the phage cocktail has promising potential to lower the prevalence of Pspo and to be integrated in a pest management strategy. Targeted research is needed to further explore the applicability of the phages in combination with other disease control strategies.

The potential of bacteriophages to control Xanthomonas campestris pv. campestris at different stages of disease development

“…Our data further suggest that knocking out the genes encoding WxcE and an epimerase/dehydratase protein slightly reduces the virulence of Xcc. This reduced virulence in phage‐resistant mutants has previously been established for different pathosystems (Schoonejans et al ., 1987; Filippov et al ., 2011; Olszak et al ., 2019; Sumrall et al ., 2019; Holtappels et al ., 2020). We further analysed the diversity of this wxc gene cluster within our strains and found that the current collection is split into three groups.…”

“…Phage receptor analysis using a Tn5 knock-out mutant library and fluorescence microscopy Phage receptors were determined as previously described (Holtappels et al, 2020). Briefly, a Tn5 knockout library was made using the EZ-Tn5<KAN-2>Tnp Transposome TM kit (Epicentre, Madison, WI, USA).…”

The future of phage biocontrol in integrated plant protection for sustainable crop production

“…porri against a cocktail of two phages emerged at the level of receptor recognition. Here, seed treatment based on phages was used and a subset of resistant mutants accumulated in the lipopolysaccharide metabolism of the bacteria, the known receptor for the phages within the cocktail [25]. It is thought that the use of multiple phages targeting different receptors could limit this resistance emergence, since resistance development often involves a fitness cost.…”

“…In this sense, artificially infected leek seeds with Pseudomonas syringae pv. porri were primed in a phage solution showing a log 2 reduction of the bacterial titer [25]. Another approach in this perspective is based on seed coatings.…”

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the Pseudomonas syringae species complex

“…It has been shown recently that there is a high degree of variation in the sensitivity to tailocins within P. syringae (Baltrus et al ., 2019). Understanding the molecular basis of this variation is important if we are to implement novel methods such as the use of phage therapy or tailocins for biological control of this pathogen (Frampton et al ., 2014; Frampton et al ., 2015; Baltrus et al ., 2019; Pinheiro et al ., 2019; Rooney et al ., 2020; Holtappels et al ., 2020).…”