Dual-Fluorescence Chromosome-Located Labeling System for Accurate In Vivo Single-Cell Gene Expression Analysis in Pseudomonas syringae.

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.

Suppression of NLR-mediated plant immune detection by bacterial pathogens.

The plant immune system is constituted of two functionally interdependent branches that provide the plant with an effective defense against microbial pathogens. They can be considered separate since one detects extracellular pathogen-associated 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 focus on bacterially driven suppression of the latter, known as effector-triggered immunity (ETI) and dependent on diverse NOD-like receptors (NLRs). We 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 bacterium and their complex epistatic interactions within the plant. We consider how ETI activation can be avoided by using suppressors to directly alter compromised co-secreted effectors, modify plant defense-associated proteins, or occasionally both. We also comment on the potential assembly within the plant cell of multi-protein complexes comprising both bacterial effectors and defense protein targets.

A bacterial type III effector targets plant vesicle-associated membrane proteins.

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector.

Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.

Fluorescently labeled Pseudomonas syringae DC3000 and 1449b wild-type strains constitutively expressing either eGFP, eCFP, or dsRED.

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.

ER Bodies Are Induced by Pseudomonas syringae and Negatively Regulate Immunity.

ER bodies are endoplasmic reticulum-derived organelles present in plants belonging to the Brassicales order. In Arabidopsis thaliana, ER bodies are ubiquitous in cotyledons and roots and are present only in certain cell types in rosette leaves. However, both wounding and jasmonic acid treatment induce the formation of ER bodies in leaves. Formation of this structure is dependent on the transcription factor NAI1. The main components of the ER bodies are ß-glucosidases (BGLUs), enzymes that hydrolyze specialized compounds. In Arabidopsis, PYK10 (BGLU23) and BGLU18 are the most abundant ER body proteins. In this work, we found that ER bodies are downregulated as a consequence of the immune responses induced by bacterial flagellin perception. Arabidopsis mutants defective in ER body formation show enhanced responses upon flagellin perception and enhanced resistance to bacterial infections. Furthermore, the bacterial toxin coronatine induces the formation of de novo ER bodies in leaves and its virulence function is partially dependent on this structure. Finally, we show that performance of the polyphagous beet armyworm herbivore Spodoptera exigua increases in plants lacking ER bodies. Altogether, we provide new evidence for the role of the ER bodies in plant immune responses.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The bacterial effector HopZ1a acetylates MKK7 to suppress plant immunity.

The Pseudomonas syringae type III secretion system translocates effector proteins into the host cell cytosol to suppress plant basal immunity. Effector HopZ1a suppresses local and systemic immunity triggered by pathogen-associated molecular patterns (PAMPs) and effectors, through target acetylation. HopZ1a has been shown to target several plant proteins, but none fully substantiates HopZ1a-associated immune suppression. Here, we investigate Arabidopsis thaliana mitogen-activated protein kinase kinases (MKKs) as potential targets, focusing on AtMKK7, a positive regulator of local and systemic immunity. We analyse HopZ1a interference with AtMKK7 by translocation of HopZ1a from bacteria inoculated into Arabidopsis expressing MKK7 from an inducible promoter. Reciprocal phenotypes are analysed on plants expressing a construct quenching MKK7 native expression. We analyse HopZ1a-MKK7 interaction by three independent methods, and the relevance of acetylation by in vitro kinase and in planta functional assays. We demonstrate the AtMKK7 contribution to immune signalling showing MKK7-dependent flg22-induced reactive oxygen species (ROS) burst, MAP kinas (MAPK) activation and callose deposition, plus AvrRpt2-triggered MKK7-dependent signalling. Furthermore, we demonstrate HopZ1a suppression of all MKK7-dependent responses, HopZ1a-MKK7 interaction in planta and HopZ1a acetylation of MKK7 with a lysine required for full kinase activity. We demonstrate that HopZ1a targets AtMKK7 to suppress local and systemic plant immunity.

A bacterial effector protein uncovers a plant metabolic pathway involved in tolerance to bacterial wilt disease.

Bacterial wilt caused by the soil-borne plant pathogen Ralstonia solanacearum is a devastating disease worldwide. Upon plant colonization, R. solanacearum replicates massively, causing plant wilting and death; collapsed infected tissues then serve as a source of inoculum. In this work, we show that the plant metabolic pathway mediated by pyruvate decarboxylases (PDCs) contributes to plant tolerance to bacterial wilt disease. Arabidopsis and tomato plants respond to R. solanacearum infection by increasing PDC activity, and plants with deficient PDC activity are more susceptible to bacterial wilt. Treatment with either pyruvic acid or acetic acid (substrate and product of the PDC pathway, respectively) enhances plant tolerance to bacterial wilt disease. An effector protein secreted by R. solanacearum, RipAK, interacts with PDCs and inhibits their oligomerization and enzymatic activity. Collectively, our work reveals a metabolic pathway involved in plant resistance to biotic and abiotic stresses, and a bacterial virulence strategy to promote disease and the completion of the pathogenic life cycle.

A bacterial effector protein prevents MAPK-mediated phosphorylation of SGT1 to suppress plant immunity.

Nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as sensors that perceive pathogen molecules and activate immunity. In plants, the accumulation and activation of NLRs is regulated by SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1). In this work, we found that an effector protein named RipAC, secreted by the plant pathogen Ralstonia solanacearum, associates with SGT1 to suppress NLR-mediated SGT1-dependent immune responses, including those triggered by another R. solanacearum effector, RipE1. RipAC does not affect the accumulation of SGT1 or NLRs, or their interaction. However, RipAC inhibits the interaction between SGT1 and MAP kinases, and the phosphorylation of a MAPK target motif in the C-terminal domain of SGT1. Such phosphorylation is enhanced upon activation of immune signaling and contributes to the activation of immune responses mediated by the NLR RPS2. Additionally, SGT1 phosphorylation contributes to resistance against R. solanacearum. Our results shed light onto the mechanism of activation of NLR-mediated immunity, and suggest a positive feedback loop between MAPK activation and SGT1-dependent NLR activation.

A Bacterial Effector Protein Hijacks Plant Metabolism to Support Pathogen Nutrition.

Many bacterial plant pathogens employ a type III secretion system to inject effector proteins within plant cells to suppress plant immunity. Whether and how effector proteins also co-opt plant metabolism to support extensive bacterial replication remains an open question. Here, we show that Ralstonia solanacearum, the causal agent of bacterial wilt disease, secretes the effector protein RipI, which interacts with plant glutamate decarboxylases (GADs) to alter plant metabolism and support bacterial growth. GADs are activated by calmodulin and catalyze the biosynthesis of gamma-aminobutyric acid (GABA), an important signaling molecule in plants and animals. RipI promotes the interaction of GADs with calmodulin, enhancing the production of GABA. R. solanacearum is able to replicate efficiently using GABA as a nutrient, and both RipI and plant GABA contribute to a successful infection. This work reveals a pathogenic strategy to hijack plant metabolism for the biosynthesis of nutrients that support microbial growth during plant colonization.

An immune receptor complex evolved in soybean to perceive a polymorphic bacterial flagellin.

In both animals and plants, the perception of bacterial flagella by immune receptors elicits the activation of defence responses. Most plants are able to perceive the highly conserved epitope flg22 from flagellin, the main flagellar protein, from most bacterial species. However, flagellin from Ralstonia solanacearum, the causal agent of the bacterial wilt disease, presents a polymorphic flg22 sequence (flg22Rso) that avoids perception by all plants studied to date. In this work, we show that soybean has developed polymorphic versions of the flg22 receptors that are able to perceive flg22Rso. Furthermore, we identify key residues responsible for both the evasion of perception by flg22Rso in Arabidopsis and the gain of perception by the soybean receptors. Heterologous expression of the soybean flg22 receptors in susceptible plant species, such as tomato, enhances resistance to bacterial wilt disease, demonstrating the potential of these receptors to enhance disease resistance in crop plants.

Protocol: an improved method to quantify activation of systemic acquired resistance (SAR).

BACKGROUND: Plant responses triggered upon detection of an invading pathogen include the generation of a number of mobile signals that travel to distant tissues and determine an increased resistance in distal, uninfected tissues, a defense response known as systemic acquired resistance (SAR). The more direct means of measuring activation of SAR by a primary local infection is the quantification of pathogen multiplication in distal, systemic sites of secondary infection. However, while such assay provides a biologically relevant quantification of SAR, it is hampered by experimental variation, requiring many repetitions for reliable results. RESULTS: We propose a modification of the SAR assay based on the Arabidopsis-Pseudomonas syringae pathosystem exploiting the knowledge of source-sink relationships (orthostichies), known to centralize SAR-competency to upper leaves in the orthostichy of a lower primary infected leaf. Although many sources of variation such as genotypes of plant and pathogen, inoculation procedure, or environmental conditions are already taken into account to improve the performance of SAR assays, a strict leaf selection based on source-sink relationships is not usually implemented. We show how enacting this latter factor considerably improves data reliability, reducing the number of experimental repetitions for results. CONCLUSIONS: Direct selection of leaves for both primary and secondary inoculation exclusively within the orthostichy of the primary infected leaf is a key element on reducing the number of experimental repetitions required for statistically relevant SAR activation results.

Suppression of HopZ Effector-Triggered Plant Immunity in a Natural Pathosystem.

Many type III-secreted effectors suppress plant defenses, but can also activate effector-triggered immunity (ETI) in resistant backgrounds. ETI suppression has been shown for a number of type III effectors (T3Es) and ETI-suppressing effectors are considered part of the arms race model for the co-evolution of bacterial virulence and plant defense. However, ETI suppression activities have been shown mostly between effectors not being naturally expressed within the same strain. Furthermore, evolution of effector families is rarely explained taking into account that selective pressure against ETI-triggering effectors may be compensated by ETI-suppressing effector(s) translocated by the same strain. The HopZ effector family is one of the most diverse, displaying a high rate of loss and gain of alleles, which reflects opposing selective pressures. HopZ effectors trigger defense responses in a variety of crops and some have been shown to suppress different plant defenses. Mutational changes in the sequence of ETI-triggering effectors have been proposed to result in the avoidance of detection by their respective hosts, in a process called pathoadaptation. We analyze how deleting or overexpressing HopZ1a and HopZ3 affects virulence of HopZ-encoding and non-encoding strains. We find that both effectors trigger immunity in their plant hosts only when delivered from heterologous strains, while immunity is suppressed when delivered from their native strains. We carried out screens aimed at identifying the determinant(s) suppressing HopZ1a-triggered and HopZ3-triggered immunity within their native strains, and identified several effectors displaying suppression of HopZ3-triggered immunity. We propose effector-mediated cross-suppression of ETI as an additional force driving evolution of the HopZ family.

Confocal microscopy reveals in planta dynamic interactions between pathogenic, avirulent and non-pathogenic Pseudomonas syringae strains.

Recent advances in genomics and single-cell analysis have demonstrated the extraordinary complexity reached by microbial populations within their hosts. Communities range from complex multispecies groups to homogeneous populations differentiating into lineages through genetic or non-genetic mechanisms. Diversity within bacterial populations is recognized as a key driver of the evolution of animal pathogens. In plants, however, little is known about how interactions between different pathogenic and non-pathogenic variants within the host impact on defence responses, or how the presence within a mixture may affect the development or the fate of each variant. Using confocal fluorescence microscopy, we analysed the colonization of the plant apoplast by individual virulence variants of Pseudomonas syringae within mixed populations. We found that non-pathogenic variants can proliferate and even spread beyond the inoculated area to neighbouring tissues when in close proximity to pathogenic bacteria. The high bacterial concentrations reached at natural entry points promote such interactions during the infection process. We also found that a diversity of interactions take place at a cellular level between virulent and avirulent variants, ranging from dominant negative effects on proliferation of virulent bacteria to in trans suppression of defences triggered by avirulent bacteria. Our results illustrate the spatial dynamics and complexity of the interactions found within mixed infections, and their potential impact on pathogen evolution.

Generating Chromosome-Located Transcriptional Fusions to Fluorescent Proteins for Single-Cell Gene Expression Analysis in Pseudomonas syringae.

The last decade has seen significant effort directed toward the role of phenotypic heterogeneity in bacterial adaptation. Phenotypic heterogeneity usually refers to phenotypic diversity that takes place through nongenetic means, independently of environmental induced variation. Recent findings are changing how microbiologists analyze bacterial behavior, with a shift from traditional assays averaging large populations to single-cell analysis focusing on bacterial individual behavior. Fluorescence-based methods are often used to analyze single-cell gene expression by flow cytometry, fluorescence microscopy and/or microfluidics. Moreover, fluorescence reporters can also be used to establish where and when are the genes of interest expressed. In this chapter, we use the model bacterial plant pathogen Pseudomonas syringae to illustrate a method to generate chromosome-located transcriptional gene fusions to fluorescent reporter genes, without affecting the function of the gene of interest.

Pseudomonas syringae Differentiates into Phenotypically Distinct Subpopulations During Colonization of a Plant Host.

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non-mammalian host.

Competitive Index: Mixed Infection-Based Virulence Assays for Genetic Analysis in Pseudomonas syringae-Plant Interactions.

When studying bacterial plant pathogens, the genetic analysis of the contribution of virulence factors to the infection process has traditionally been hindered by their high degree of functional redundancy. In recent years, it has become clear that the use of competitive index in mixed infections provides an accurate and sensitive manner of establishing virulence phenotypes for mutants for which other assays have failed. Such increases in sensitivity and accuracy are due to the direct comparison between the respective growths of the co-inoculated strains within the same infection, each strain replicating as they would in individual infections. Interferences between the co-inoculated strains must be therefore avoided using the appropriate experimental settings. In this chapter, we will present the optimal experimental conditions to achieve maximum sensitivity on virulence assays using the phytopathogenic bacterium Pseudomonas syringae, as well as some additional considerations to ensure the correct interpretations of the results.

Auto-acetylation on K289 is not essential for HopZ1a-mediated plant defense suppression.

The Pseudomonas syringae type III-secreted effector HopZ1a is a member of the HopZ/YopJ superfamily of effectors that triggers immunity in Arabidopsis. We have previously shown that HopZ1a suppresses both local [effector-triggered immunity (ETI)] and systemic immunity [systemic acquired resistance (SAR)] triggered by the heterologous effector AvrRpt2. HopZ1a has been shown to possess acetyltransferase activity, and this activity is essential to trigger immunity in Arabidopsis. HopZ1a acetyltransferase activity has been reported to require the auto-acetylation of the effector on a specific lysine (K289) residue. In this paper we analyze the relevance of autoacetylation of lysine residue 289 in HopZ1a ability to suppress plant defenses, and on the light of the results obtained, we also revise its relevance for HopZ1a avirulence activity. Our results indicate that, while the HopZ1a(K289R) mutant is impaired to some degree in its virulence and avirulence activities, is by no means phenotypically equivalent to the catalytically inactive HopZ1a(C216A), since it is still able to trigger a defense response that induces detectable macroscopic HR and effectively protects Arabidopsis from infection, reducing growth of P. syringae within the plant. We also present evidence that the HopZ1a(K289R) mutant still displays virulence activities, partially suppressing both ETI and SAR.