Bacteria respond to changing environments by altering gene expression. Some responses display probabilistic cell-to-cell variation within isogenic populations. A few paradigmatic examples in animal pathogens have demonstrated that this phenotypic heterogeneity has biological relevance for virulence. We investigate single-cell flagellar expression in relation to type III secretion expression in the model plant pathogen Pseudomonas syringae and describe that both systems undergo phenotypic heterogeneity throughout plant colonization. We establish that high expression of these system carries growth penalties. Stochastic, spatial and time factors shape dynamics of a phenotypically diverse population which displays division of labor during colonization: T3SSON bacteria effectors act as ‘common goods` to suppress immunity, allowing the increase of motile bacteria that actively leave the infected tissue before necrosis. This study provides a comprehensive view of how processes underlying bacterial specialization play out in the context of complex and changing environments of biological and applied relevance such as host colonization.

Epigenetic regulation as a means for bacterial adaptation is receiving increasing interest in the last decade. Significant efforts have been directed towards understanding the mechanisms giving raise to phenotypic heterogeneity within bacterial populations and its adaptive relevance. Phenotypic heterogeneity mostly refers to phenotypic variation not linked to genetic differences nor to environmental stimuli. Recent findings on the relevance of phenotypic heterogeneity on some bacterial complex traits are causing a shift from traditional assays where bacterial phenotypes are defined by averaging population-level data, to single-cell analysis that focus on bacterial individual behavior within the population. Fluorescent labeling is a key asset for single-cell gene expression analysis using flow cytometry, fluorescence microscopy, and/or microfluidics. We previously described the generation of chromosome-located transcriptional gene fusions to fluorescent reporter genes using the model bacterial plant pathogen Pseudomonas syringae. These fusions allow researchers to follow variation in expression of the gene(s) of interest, without affecting gene function. In this report, we improve the analytic power of the method by combining such transcriptional fusions with constitutively expressed compatible fluorescent reporter genes integrated in a second, neutral locus of the bacterial chromosome. Constitutively expressed fluorescent reporters allow for the detection of all bacteria comprising a heterogeneous population, regardless of the level of expression of the concurrently monitored gene of interest, thus avoiding the traditional use of stains often incompatible with samples from complex contexts such as the leaf.

The plant immune system is constituted by two functionally interdependent branches that provide the plant with an effective defense against microbial pathogens. They can be considered separate since one detects extracellular pathogenassociated molecular patterns by means of receptors on the plant surface, while the other detects pathogen-secreted virulence effectors via intracellular receptors. Plant defense depending on both branches can be effectively suppressed by host-adapted microbial pathogens. In this review we will focus on bacterially driven suppression of the latter, usually known as ETI for Effector-Triggered Immunity and depending on diverse NOD-like receptors, or NLRs. We will examine how some effectors secreted by pathogenic bacteria carrying Type III Secretion Systems can be subject to specific NLR-mediated detection, which can be evaded by the action of additional co-secreted effectors (suppressors), implying that virulence depends on the coordinated action of the whole repertoire of effectors of any given bacteria, and their complex epistatic interactions within the plant. We will consider how, to avoid ETI activation, suppressors can directly alter compromised cosecreted effectors, modify plant defense-associated proteins, or occasionally both. We will also comment on the potential assembly within the plant cell of multi-protein complexes comprising both bacterial effectors and defense protein targets.

Here we describe the generation of fluorescently labeled derivatives of the plant pathogen Pseudomonas syringae DC3000 and 1449b strains, with each derivative constitutively expressing either the enhanced green (eGFP), enhanced cyan (eCFP), or Discosoma sp. red (dsRED) fluorescent proteins. The fluorophore-expressing cassetes are stably located in a neutral locus in the chromosome, and its expression does not affect bacterial fitness, while allowing efficient detection by microscopy or flow cytometry. We have generated these strains as a complementary set of labeled strains to those previously generated in our laboratory, thus extending the range of applications.