ABSTRACT
BACKGROUND: Bacteria of the genus Xanthomonas cause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverse Xanthomonas species, particularly beyond type III secretion, remains understudied. RESULTS: In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17 Xanthomonas strains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-borne hrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. CONCLUSIONS: The diversity in the HrpG* regulon suggests that HrpG-dependent virulence in Xanthomonas might be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS.
Subject(s)
Bacterial Proteins , Gene Expression Regulation, Bacterial , Regulon , Xanthomonas , Xanthomonas/pathogenicity , Xanthomonas/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Virulence/genetics , Transcriptome , Type III Secretion Systems/genetics , Type III Secretion Systems/metabolism , Gene Expression Profiling , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain-of-function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.
ABSTRACT
Molecular mechanisms underlying quantitative variations of pathogenicity remain elusive. Here, we identified the Xanthomonas campestris XopJ6 effector that triggers disease resistance in cauliflower and Arabidopsis thaliana. XopJ6 is a close homolog of the Ralstoniapseudosolanacearum PopP2 YopJ family acetyltransferase. XopJ6 is recognized by the RRS1-R/RPS4 NLR pair that integrates a WRKY decoy domain mimicking effector targets. We identified a XopJ6 natural variant carrying a single residue substitution in XopJ6 WRKY-binding site that disrupts interaction with WRKY proteins. This mutation allows XopJ6 to evade immune perception while retaining some XopJ6 virulence functions. Interestingly, xopJ6 resides in a Tn3-family transposon likely contributing to xopJ6 copy number variation (CNV). Using synthetic biology, we demonstrate that xopJ6 CNV tunes pathogen virulence on Arabidopsis through gene dosage-mediated modulation of xopJ6 expression. Together, our findings highlight how sequence and structural genetic variations restricted at a particular effector gene contribute to bacterial host adaptation.
ABSTRACT
Pseudomonas simiae WCS417 is a plant growth-promoting rhizobacterium that improves plant health and development. In this study, we investigate the early leaf responses of Arabidopsis thaliana to WCS417 exposure and the possible involvement of formate dehydrogenase (FDH) in such responses. In vitro-grown A. thaliana seedlings expressing an FDH::GUS reporter show a significant increase in FDH promoter activity in their roots and shoots after 7 days of indirect exposure (without contact) to WCS417. After root exposure to WCS417, the leaves of FDH::GUS plants grown in the soil also show an increased FDH promoter activity in hydathodes. To elucidate early foliar responses to WCS417 as well as FDH involvement, the roots of A. thaliana wild-type Col and atfdh1-5 knock-out mutant plants grown in soil were exposed to WCS417, and proteins from rosette leaves were subjected to proteomic analysis. The results reveal that chloroplasts, in particular several components of the photosystems PSI and PSII, as well as members of the glutathione S-transferase family, are among the early targets of the metabolic changes induced by WCS417. Taken together, the alterations in the foliar proteome, as observed in the atfdh1-5 mutant, especially after exposure to WCS417 and involving stress-responsive genes, suggest that FDH is a node in the early events triggered by the interactions between A. thaliana and the rhizobacterium WCS417. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Proteome/metabolism , Proteomics , Plant Roots/microbiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Soil , Gene Expression Regulation, PlantABSTRACT
Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.
Subject(s)
Oryza , Xanthomonas , Transcription Activator-Like Effectors/genetics , Xanthomonas/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Virulence/genetics , Biological Transport , Plant Diseases/microbiology , Oryza/geneticsABSTRACT
Bacteria inject effector proteins into host cells to manipulate cellular processes that promote disease. Since bacteria deliver minuscule amounts of effectors only into targeted host cells, it is technically challenging to capture effector-dependent cellular changes from bulk-infected host tissues. Here, we report a new technique called effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), which facilitates affinity-based purification of nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors. Analysis of purified nuclei reveals that the Xanthomonas effector XopD manipulates the expression of Arabidopsis abscisic acid signalling-related genes and activates OSCA1.1, a gene encoding a calcium-permeable channel required for stomatal closure in response to osmotic stress. The loss of OSCA1.1 causes leaf wilting and reduced bacterial growth in infected leaves, suggesting that OSCA1.1 promotes host susceptibility. eINTACT allows us to uncover that XopD exploits host OSCA1.1/abscisic acid osmosignalling-mediated stomatal closure to create a humid habitat that favours bacterial growth and opens up a new avenue for accurately elucidating functions of effectors from numerous gram-negative plant bacteria in native infection contexts.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Xanthomonas , Arabidopsis/metabolism , Virulence , Abscisic Acid/metabolism , Xanthomonas/physiology , Arabidopsis Proteins/metabolism , Calcium Channels/metabolism , Plant Diseases/microbiology , Bacterial Proteins/geneticsABSTRACT
Competition assays are a simple phenotyping strategy that confront two bacterial strains to evaluate their relative fitness. Because they are more accurate than single-strain growth assays, competition assays can be used to highlight slight differences that would not otherwise be detectable. In the frame of host-pathogens interactions, they can be very useful to study the contribution of individual bacterial genes to bacterial fitness and lead to the identification of new adaptive traits. Here, we describe how to perform such competition assays by taking the example of the model phytopathogenic bacterium Xanthomonas campestris pv. campestris during infection of the mesophyll of its cauliflower host. This phenotypic assay is based on the use of a Competitive Index (CI) that compares the relative abundance of co-inoculated strains before and after inoculation. Since multiplication is a direct proxy for bacterial fitness, the evolution of the ratio between both strains in the mixed population is a direct way to assess differences in fitness in a given environment. In this protocol, we exploit the blue staining of GUS-expressing bacteria to count blue vs. white colonies on plates and estimate the competitiveness of the strains of interest in plant mesophyll.
ABSTRACT
Bellenot et al. introduce hydathodes, an oft-overlooked plant organ that acts as a pressure valve to expel excess guttation sap at the leaf margin, typically visible at dawn.
Subject(s)
Plant LeavesABSTRACT
Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization, especially at early infection stages. Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogen Xanthomonas campestris pv campestris (Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site of Xcc, in xylem sap and in synthetic media. Xcc did not face a strong bottleneck during hydathode infection. In total, 181 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism but only few genes previously known to be involved in virulence. The biological relevance of 12 genes was independently confirmed by phenotyping single mutants. Notably, we show that XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence of Xcc possibly through c-di-GMP-mediated regulation. This study revealed yet unsuspected social behaviors adopted by Xcc individuals when confined inside hydathodes at early infection stages.
Subject(s)
Brassica , Xanthomonas campestris , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Brassica/microbiology , Plant Diseases/microbiology , Virulence/genetics , Xylem/metabolismABSTRACT
Xanthomonas campestris pv. campestris is a group of phytopathogenic bacteria causing black rot disease on Brassicaceae crops. Here, we report on draft genome sequences of 17 strains representing eight of nine known races of this pathogen, including the pathotype strain CFBP 6865.
ABSTRACT
Pathovars of Xanthomonas campestris cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of X. campestris strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available X. campestris genomes revealed a correlation between pathovar and genomic relatedness and provide support for X. campestris pv. barbareae, the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of Matthiola incana, and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene cbsA with coding sequence for a signal peptide was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse Xanthomonas species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to cbsA influence tissue specificity of X. campestris pathovars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Subject(s)
Xanthomonas campestris , Xanthomonas , Genomics , Organ Specificity , Protein Sorting Signals , Xanthomonas/genetics , Xanthomonas campestris/geneticsABSTRACT
Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted vascular pathogen causing black rot disease on cultivated and wild Brassicaceae. Xcc enters the plant tissues preferentially via hydathodes, which are organs localized at leaf margins. To decipher both physiological and virulence strategies deployed by Xcc during early stages of infection, the transcriptomic profile of Xcc was analysed 3 days after entry into cauliflower hydathodes. Despite the absence of visible plant tissue alterations and despite a biotrophic lifestyle, 18% of Xcc genes were differentially expressed, including a striking repression of chemotaxis and motility functions. The Xcc full repertoire of virulence factors had not yet been activated but the expression of the HrpG regulon composed of 95 genes, including genes coding for the type III secretion machinery important for suppression of plant immunity, was induced. The expression of genes involved in metabolic adaptations such as catabolism of plant compounds, transport functions, sulphur and phosphate metabolism was upregulated while limited stress responses were observed 3 days postinfection. We confirmed experimentally that high-affinity phosphate transport is needed for bacterial fitness inside hydathodes. This analysis provides information about the nutritional and stress status of bacteria during the early biotrophic infection stages and helps to decipher the adaptive strategy of Xcc to the hydathode environment.
Subject(s)
Brassica , Xanthomonas campestris , Xanthomonas , Adaptation, Physiological/genetics , Bacterial Proteins/metabolism , Brassica/genetics , Gene Expression Regulation, Bacterial , Plant Diseases/genetics , Transcriptome/genetics , Virulence/genetics , Xanthomonas/metabolism , Xanthomonas campestris/geneticsABSTRACT
Pathogens deploy effector proteins that interact with host proteins to manipulate the host physiology to the pathogen's own benefit. However, effectors can also be recognized by host immune proteins, leading to the activation of defence responses. Effectors are thus essential components in determining the outcome of plant-pathogen interactions. Despite major efforts to decipher effector functions, our current knowledge on effector biology is scattered and often limited. In this study, we conducted two systematic large-scale yeast two-hybrid screenings to detect interactions between Arabidopsis thaliana proteins and effectors from two vascular bacterial pathogens: Ralstonia pseudosolanacearum and Xanthomonas campestris. We then constructed an interactomic network focused on Arabidopsis and effector proteins from a wide variety of bacterial, oomycete, fungal, and invertebrate pathogens. This network contains our experimental data and protein-protein interactions from 2,035 peer-reviewed publications (48,200 Arabidopsis-Arabidopsis and 1,300 Arabidopsis-effector protein interactions). Our results show that effectors from different species interact with both common and specific Arabidopsis interactors, suggesting dual roles as modulators of generic and adaptive host processes. Network analyses revealed that effector interactors, particularly "effector hubs" and bacterial core effector interactors, occupy important positions for network organization, as shown by their larger number of protein interactions and centrality. These interactomic data were incorporated in EffectorK, a new graph-oriented knowledge database that allows users to navigate the network, search for homology, or find possible paths between host and/or effector proteins. EffectorK is available at www.effectork.org and allows users to submit their own interactomic data.
Subject(s)
Arabidopsis , Databases, Chemical , Disease Resistance , Protein Interaction Maps , Arabidopsis/metabolism , Arabidopsis/microbiology , Arabidopsis Proteins/metabolism , Bacterial Proteins/metabolism , Disease Resistance/physiology , Host-Pathogen Interactions , Plant Diseases/microbiology , Proteome/metabolism , Ralstonia/metabolism , Software , Virulence Factors/metabolism , Xanthomonas/metabolism , Xanthomonas campestris/metabolismABSTRACT
Vascular plant pathogens travel long distances through host veins, leading to life-threatening, systemic infections. In contrast, nonvascular pathogens remain restricted to infection sites, triggering localized symptom development. The contrasting features of vascular and nonvascular diseases suggest distinct etiologies, but the basis for each remains unclear. Here, we show that the hydrolase CbsA acts as a phenotypic switch between vascular and nonvascular plant pathogenesis. cbsA was enriched in genomes of vascular phytopathogenic bacteria in the family Xanthomonadaceae and absent in most nonvascular species. CbsA expression allowed nonvascular Xanthomonas to cause vascular blight, while cbsA mutagenesis resulted in reduction of vascular or enhanced nonvascular symptom development. Phylogenetic hypothesis testing further revealed that cbsA was lost in multiple nonvascular lineages and more recently gained by some vascular subgroups, suggesting that vascular pathogenesis is ancestral. Our results overall demonstrate how the gain and loss of single loci can facilitate the evolution of complex ecological traits.
Subject(s)
Xanthomonas , Bacteria , Hydrolases , Phylogeny , Plants/genetics , Xanthomonas/geneticsABSTRACT
Hydathode is a plant organ responsible for guttation in vascular plants, i.e. the release of droplets at leaf margin or surface. Because this organ connects the plant vasculature to the external environment, it is also a known entry site for several vascular pathogens. In this study, we present a detailed microscopic examination of leaf apical hydathodes in monocots for three crops (maize, rice and sugarcane) and the model plant Brachypodium distachyon. Our study highlights both similarities and specificities of those epithemal hydathodes. These observations will serve as a foundation for future studies on the physiology and the immunity of hydathodes in monocots.
Subject(s)
Brachypodium/ultrastructure , Crops, Agricultural/ultrastructure , Oryza/ultrastructure , Plant Leaves/ultrastructure , Saccharum/ultrastructure , Zea mays/ultrastructureABSTRACT
Hydathodes are organs found on aerial parts of a wide range of plant species that provide almost direct access for several pathogenic microbes to the plant vascular system. Hydathodes are better known as the site of guttation, which is the release of droplets of plant apoplastic fluid to the outer leaf surface. Because these organs are only described through sporadic allusions in the literature, this review aims to provide a comprehensive view of hydathode development, physiology, and immunity by compiling a historic and contemporary bibliography. In particular, we refine the definition of hydathodes.We illustrate their important roles in the maintenance of plant osmotic balance, nutrient retrieval, and exclusion of deleterious chemicals from the xylem sap. Finally, we present our current understanding of the infection of hydathodes by adapted vascular pathogens and the associated plant immune responses.
Subject(s)
Plant Leaves , XylemABSTRACT
Xanthomonas transcription activator-like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE-encoding (tal) genes were thought to be absent from Brassicaceae-infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences. We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near-complete description of the TALEs found in Xc (Xc TALome). The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis. This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.
Subject(s)
Host-Pathogen Interactions/genetics , Transcription Activator-Like Effectors/genetics , Xanthomonas campestris/genetics , Xanthomonas campestris/pathogenicity , Brassica/microbiology , Genome, Bacterial , Phylogeny , Plant Diseases/microbiologyABSTRACT
Hydathodes are water pores found on leaves of a wide range of vascular plants and are the sites of guttation. We report here on the detailed anatomy of cauliflower (Brassicaoleracea) and Arabidopsis (Arabidopsis thaliana) hydathodes. Hydathode surface presents pores resembling stomata giving access to large cavities. Beneath, the epithem is composed of a lacunar and highly vascularized parenchyma offering a direct connection between leaf surface and xylem vessels. Arabidopsis hydathode pores were responsive to ABA and light similar to stomata. The flg22 flagellin peptide, a well-characterized elicitor of plant basal immunity, did not induce closure of hydathode pores in contrast to stomata. Because hydathodes are natural infection routes for several pathogens, we investigated hydathode infection by the adapted vascular phytopathogenic bacterium Xanthomonas campestris pv campestris (Xcc), the causal agent of black rot disease of Brassicaceae. Microscopic observations of hydathodes six days postinoculation indicated a digestion of the epithem cells and a high bacterial multiplication. Postinvasive immunity was shown to limit pathogen growth in the epithem and is actively suppressed by the type III secretion system and its effector proteins. Altogether, these results give a detailed anatomic description of Brassicaceae hydathodes and highlight the efficient use of this tissue as an initial niche for subsequent vascular systemic dissemination of Xcc in distant plant tissues.