# Pseudomonas syringae Lipopolysaccharide Synthesis Gene wbpL Displays Heterogeneous Expression Within In Vitro and In Planta Populations

## TL;DR

This study shows that the wbpL gene in Pseudomonas syringae is expressed differently among genetically identical bacteria, both in lab and plant environments, which may affect infection and immunity.

## Contribution

The study reveals phenotypic heterogeneity in wbpL expression, a key gene for lipopolysaccharide synthesis in Pseudomonas syringae, in both in vitro and in planta conditions.

## Key findings

- wbpL expression in Pseudomonas syringae is heterogeneous, with ON and OFF subpopulations in minimal medium.
- In planta, wbpL expression is also heterogeneous, with intermingled ON/OFF cells showing similar viability.
- Smaller microcolonies in planta show higher wbpL expression, suggesting a possible link between expression and growth or defense responses.

## Abstract

Phenotypic heterogeneity usually refers to phenotypic variation not associated with genetic variation, nor induced by environmental stimuli. The phenotypic heterogeneity processes described for some complex bacterial traits are causing a shift in how bacterial phenotypes are studied, from traditional assessments by averaging populations to single‐cell analysis focused on bacterial individual phenotypes and how these distribute within the population. The structure of the lipopolysaccharide (LPS) layer on the outer membrane in Gram‐negative bacteria is often subjected to phenotypic variation, a process that is critical for virulence in animal pathogens. Here, we apply single‐cell expression analyses to wbpL, a conserved Pseudomonas syringae glycosyltransferase‐encoding gene essential for the synthesis of the O‐antigen component of LPS. We show that expression of wbpL displays phenotypic heterogeneity in P. syringae pv. phaseolicola growing in rich medium and reaches bistable expression in minimal medium, where the population splits into WbpLON and WpbLOFF subpopulations. In planta, wbpL expression is also heterogeneous, displaying intermingled ON/OFF with comparable viability. Finally, we followed the expression of wbpL within the spatial context of apoplastic microcolonies, and not only detected heterogeneity within each microcolony, but also found that microcolonies displayed overall differences in fluorescence intensity that correlated with size, with smaller microcolonies displaying higher levels of wbpL expression.

Genetically identical bacteria in the same environment sometimes behave differently. This happens for the expression of wbpL, a gene of plant pathogenic Pseudomonas syringae that shapes the bacterial surface and is important for infection and immunity. whose expression varies among individual bacteria, with ON/OFF bacteria coexisting with similar viability. In planta, the smaller size of microcolonies with higher wbpL expression suggests wbpL expression might slow growth or trigger stronger defenses. Phenotypic heterogeneity of lipopolysaccharide genes is a common strategy for plant and animal pathogens.