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Protein signatures to identify the different genera within the Xanthomonadaceae family | SpringerLink

The Xanthomonadaceae family comprises the genera Xanthomonas and Xylella, which include plant pathogenic species that affect economically important crops. The family also includes the plant growth-promoting bacteria Pseudomonas geniculata and Stenotrophomonas rhizophila, and some other species with biotechnological, medical, and environmental relevance. Previous work identified molecular signatures that helped to understand the evolutionary placement of this family within gamma-proteobacteria. In the present study, we investigated whether insertions identified in highly conserved proteins may also be used as molecular markers for taxonomic classification and identification of members within the Xanthomonadaceae family. Four housekeeping proteins (DNA repair and replication-related and protein translation enzymes) were selected. The insertions allowed discriminating phytopathogenic and plant growth-promoting groups within this family, and also amino acid sequences of these insertions allowed distinguishing different genera and, eventually, species as well as pathovars. Moreover, insertions in the proteins MutS and DNA polymerase III (subunit alpha) are conserved in Xylella fastidiosa, but signatures in DNA ligase NAD-dependent and Valyl tRNA synthetase distinguish particular subspecies within the genus. The genus Stenotrophomonas and Pseudomonas geniculata could be distinguishable based on the insertions in MutS, DNA polymerase III (subunit alpha), and Valyl tRNA synthetase, although insertion in DNA ligase NAD-dependent discriminates these bacteria at the species level. All these insertions differentiate species and pathovars within Xanthomonas. Thus, the insertions presented support evolutionary demarcation within Xanthomonadaceae and provide tools for the fast identification in the field of these bacteria with agricultural, environmental, and economic relevance.
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Xanthomonas diversity, virulence and plant–pathogen interactions | Nature Rev Micro

Xanthomonas diversity, virulence and plant–pathogen interactions | Nature Rev Micro | Xanthomonas | Scoop.it
Xanthomonas spp. encompass a wide range of plant pathogens that use numerous virulence factors for pathogenicity and fitness in plant hosts. In this Review, we examine recent insights into host–pathogen co-evolution, diversity in Xanthomonas populations and host specificity of Xanthomonas spp. that have substantially improved our fundamental understanding of pathogen biology. We emphasize the virulence factors in xanthomonads, such as type III secreted effectors including transcription activator-like effectors, type II secretion systems, diversity resulting in host specificity, evolution of emerging strains, activation of susceptibility genes and strategies of host evasion. We summarize the genomic diversity in several Xanthomonas spp. and implications for disease outbreaks, management strategies and breeding for disease resistance. In this Review, Jones and colleagues describe the extremely diverse Xanthomonas spp. and how these plant pathogens use their extensive repertoire of effectors for virulence and immune evasion. Understanding these prototypical plant pathogens paves the way to combat disease.
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An Arabidopsis Secondary Metabolite Directly Targets Expression of the Bacterial Type III Secretion System to Inhibit Bacterial Virulence - Cell Host & Microbe

An Arabidopsis Secondary Metabolite Directly Targets Expression of the Bacterial Type III Secretion System to Inhibit Bacterial Virulence - Cell Host & Microbe | Xanthomonas | Scoop.it

Plants deploy a variety of secondary metabolites to fend off pathogen attack. Although defense compounds are generally considered toxic to microbes, the exact mechanisms are often unknown. Here, we show that the Arabidopsis defense compound sulforaphane (SFN) functions primarily by inhibiting Pseudomonas syringae type III secretion system (TTSS) genes, which are essential for pathogenesis. Plants lacking the aliphatic glucosinolate pathway, which do not accumulate SFN, were unable to attenuate TTSS gene expression and exhibited increased susceptibility to P. syringae strains that cannot detoxify SFN. Chemoproteomics analyses showed that SFN covalently modified the cysteine at position 209 of HrpS, a key transcription factor controlling TTSS gene expression. Site-directed mutagenesis and functional analyses further confirmed that Cys209 was responsible for bacterial sensitivity to SFN in vitro and sensitivity to plant defenses conferred by the aliphatic glucosinolate pathway. Collectively, these results illustrate a previously unknown mechanism by which plants disarm a pathogenic bacterium.

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Validation of an in vitro system for studies of pathogenicity mechanisms in Xanthomonas campestris | FEMS Microbiology Letters | Oxford Academic

Validation of an in vitro system for studies of pathogenicity mechanisms in Xanthomonas campestris | FEMS Microbiology Letters | Oxford Academic | Xanthomonas | Scoop.it

Several minimal media capable of inducing pathogenicity genes have been used to study plant–pathogen interactions. An in planta assay to study a closer interaction between the bacteria and the host was also developed and has been employed by our group. In order to determine whether growth medium could be improved to better approximate in planta conditions beyond that offered by the defined minimal medium XVM1, we compared the expression of 20 Xanthomonas campestris pv. campestris (Xcc) genes by quantitative reverse transcription - polymerase chain reaction (qRT-PCR) under in vivo (bacteria recovered from the plant) and in vitro (rich medium NYG, minimal medium XVM1 and XVM1 + leaf extract) growth systems. The results showed a higher expression level of the genes in the in planta system when compared to growth in culture media. In planta growth is closest to a real interaction condition and captures the complexity of the plant cell environment; however, this system has some limitations. The main finding of our work is that the addition of plant extract to XVM1 medium results in a gene expression profile that better matches the in planta profile, when compared with the XVM1 medium alone, giving support to the use of plant extract to study pathogenicity mechanisms in Xanthomonas.

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Mass-spectrometry-based draft of the Arabidopsis proteome

Mass-spectrometry-based draft of the Arabidopsis proteome | Xanthomonas | Scoop.it
Plants are essential for life and are extremely diverse organisms with unique molecular capabilities1. Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions. A quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana provides a valuable resource for plant research.
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Open Sesame! Uncovering a Hidden Key Used by Pathogenic Bacteria to Open the Doors Connecting Plant Cells | Plant Cell

Open Sesame! Uncovering a Hidden Key Used by Pathogenic Bacteria to Open the Doors Connecting Plant Cells | Plant Cell | Xanthomonas | Scoop.it
Pathogenic Bacteria Target Plant Plasmodesmata to Colonize and Invade Surrounding Tissues

A hallmark of multicellular organisms is their ability to maintain physiological homeostasis by communicating among cells, tissues, and organs. In plants, intercellular communication is largely dependent on plasmodesmata (PD), which are membrane-lined channels connecting adjacent plant cells. Upon immune stimulation, plants close PD as part of their immune responses. Here, we show that the bacterial pathogen Pseudomonas syringae deploys an effector protein, HopO1-1, that modulates PD function. HopO1-1 is required for P. syringae to spread locally to neighboring tissues during infection. Expression of HopO1-1 in Arabidopsis (Arabidopsis thaliana) increases the distance of PD-dependent molecular flux between neighboring plant cells. Being a putative ribosyltransferase, the catalytic activity of HopO1-1 is required for regulation of PD. HopO1-1 physically interacts with and destabilizes the plant PD-located protein PDLP7 and possibly PDLP5. Both PDLPs are involved in bacterial immunity. Our findings reveal that a pathogenic bacterium utilizes an effector to manipulate PD-mediated host intercellular communication for maximizing the spread of bacterial infection.
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A High-Throughput, Seedling Screen for Plant Immunity

A High-Throughput, Seedling Screen for Plant Immunity | Xanthomonas | Scoop.it
An understanding of how biological diversity affects plant–microbe interactions is becoming increasingly important, particularly with respect to components of the pathogen effector arsenal and the plant immune system. Although technological improvements have greatly advanced our ability to examine molecular sequences and interactions, relatively few advances have been made that facilitate high-throughput, in vivo pathology screens. Here, we present a high-throughput, microplate-based, nondestructive seedling pathology assay, and apply it to identify Arabidopsis thaliana effector-triggered immunity (ETI) responses against Pseudomonas syringae type III secreted effectors. The assay was carried out in a 48-well microplate format with spray inoculation, and disease symptoms were quantitatively recorded in a semiautomated manner, thereby greatly reducing both time and costs. The assay requires only slight modifications of common labware and uses no proprietary software. We validated the assay by recapitulating known ETI responses induced by P. syringae in Arabidopsis. We also demonstrated that we can quantitatively differentiate responses from a diversity of plant genotypes grown in the same microplate. Finally, we showed that the results obtained from our assay can be used to perform genome-wide association studies to identify host immunity genes, recapitulating results that have been independently obtained with mature plants.
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The pan-genome effector-triggered immunity landscape of a host-pathogen interaction | Science

The pan-genome effector-triggered immunity landscape of a host-pathogen interaction | Science | Xanthomonas | Scoop.it
Plant pathogens elicit an immune response through effector proteins. In turn, plant genomes encode genes that determine species-specific recognition of these effectors by a process known collectively as effector-triggered immunity (ETI). By examining a range of strains of the pathogen Pseudomonas syringae that infect the model plant Arabidopsis thaliana, Laflamme et al. generated a P. syringae Type III Effector Compendium (PsyTEC) and in turn identified the genes responsible for ETI in Arabidopsis. This pan-genome analysis revealed that relatively few A. thaliana genes are responsible for recognizing the majority of P. syringae effectors. These results provide insight into why most pathogenic microbes only infect specific plant species.
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Co-incidence of Damage and Microbial Patterns Controls Localized Immune Responses in Roots - ScienceDirect

Co-incidence of Damage and Microbial Patterns Controls Localized Immune Responses in Roots - ScienceDirect | Xanthomonas | Scoop.it

Highlights:

-Arabidopsisroots request cell damage to mount a strong, localized immune response

-Damaged cells upregulate pattern-recognition receptor expression in their neighbors

-Endodermal barriers compartmentalize immune responses in differentiated cell-types

-Damage-gating can minimize immune responses against non-pathogenic root colonizers

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The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol biosynthesis to suppress plant innate immunity | bioRxiv

The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol biosynthesis to suppress plant innate immunity | bioRxiv | Xanthomonas | Scoop.it

Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population38 related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is 39 largely under-studied. In this work, we show that the QS molecule DSF (cis-11-methyl-dodecenoic acid) 40 produced by Xanthomonas campestris pv. campestris can suppress pathogen-associated molecular 41 pattern (PAMP)-triggered immunity (PTI) in Arabidopsis thaliana, mediated by flagellin-induced activation 42 of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of 43 oligomerization states and endocytic internalization of plasma membrane FLS2. DSF altered the lipid 44 profile of Arabidopsis, with a particular increase of the phytosterol species, which impairs the general 45 endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF 46 effect on receptor dynamics and host immune responses could be entirely reversed by sterol removal. 47 Together, our results highlighted the importance of sterol homeostasis to plasma membrane organization 48 and demonstrate a novel mechanism by which pathogenic bacteria use their communicating molecule to 49 manipulate PAMP-triggered host immunity.

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Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes | Xanthomonas | Scoop.it
While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation. We determine that 1–3% of mutants in Streptococcus pneumoniae have a different fitness when grown in isolation and show how dTn-Seq can help identify leads for gene function, including those involved in hyper-competence, processing of alpha-1-acid glycoprotein, sensitivity against the human leukocyte elastase and microcolony formation. Additionally, we show dTn-Seq compatibility with microscopy, FACS and investigations of bacterial cell-to-cell and bacteria-host cell interactions. dTn-Seq reduces costs and retains the advantages of Tn-Seq, while expanding the method’s original applicability. Culturing transposon-mutant libraries in pools can mask complex phenotypes. Here the authors present microfluidics mediated droplet Tn-Seq, which encapsulates individual mutants, promotes isolated growth and enables cell-cell interaction analyses.
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Analysis of HrpG regulons and HrpG‐interacting proteins by ChIP‐seq and affinity proteomics in Xanthomonas campestris

Gamma‐proteobacteria Xanthomonas spp. cause at least 350 different plant diseases among important agricultural crops, which result in serious yield losses. Xanthomonas spp. rely mainly on the type III secretion system (T3SS) to infect their hosts and induce a hypersensitive response in nonhosts. HrpG, the master regulator of the T3SS, plays the dominant role in bacterial virulence. In this study, we used chromatin immunoprecipitation followed by sequencing (ChIP‐seq) and tandem affinity purification (TAP) to systematically characterize the HrpG regulon and HrpG interacting proteins in vivo. We obtained 186 candidate HrpG downstream genes from the ChIP‐seq analysis, which represented the genomic‐wide regulon spectrum. A consensus HrpG‐binding motif was obtained and three T3SS genes, hpa2, hrcU, and hrpE, were confirmed to be directly transcriptionally activated by HrpG in the inducing medium. A total of 273 putative HrpG interacting proteins were identified from the TAP data and the DNA‐binding histone‐like HU protein of Xanthomonas campestris pv. campestris (HUxcc) was proved to be involved in bacterial virulence by increasing the complexity and intelligence of the bacterial signalling pathways in the T3SS.

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MALDI TOF MS-profiling: Applications for bacterial and plant sample differentiation and biological variability assessment - ScienceDirect

MALDI TOF MS-profiling: Applications for bacterial and plant sample differentiation and biological variability assessment - ScienceDirect | Xanthomonas | Scoop.it
In this study, we evaluated the potential use of MALDI-TOF MS Profiling for the differentiation of biological samples submitted to different treatments. We compared the bacterium Xanthomonas campestris pv. campestris (Xcc), grown in culture medium and in vivo (recovered from the plant). Plant samples were also analyzed and included explants at different somatic embryogenesis (SE) stages, as well as leaves from Brassica oleracea and Arabidopsis thaliana inoculated with Xcc, at different time points. The results showed that bacteria and highly divergent plant samples, such as those from embryogenic stages, can be unequivocally differentiated and the clustering was in accordance with proteomic analysis performed by 2-DE. These results show an important application of MALDI-TOF MS Profiling to select and prioritize samples to be analyzed prior to more complex approaches including transcriptomics and proteomics. We also show that in plant-pathogen interactions, when more subtle differences are obtained, the main contribution of MALDI-TOF MS Profiling is in the assessment of experimental variability. This is relevant since reproducibility is a challenging issue when dealing with complex experimental conditions such as plant-pathogen interactions. We propose the use of MALDI-TOF MS Profiling to aid researchers in minimizing experimental variability unrelated to the condition being analyzed.
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Plant Immunity: Danger Perception and Signaling - Cell Review

Plant Immunity: Danger Perception and Signaling - Cell Review | Xanthomonas | Scoop.it

Plants employ numerous cell-surface and intracellular immune receptors to perceive a variety ofimmunogenic signals associated with pathogen infection and subsequently activate defenses. Im-mune signaling is potentiated by the major defense hormone salicylic acid (SA), which reprogramsthe transcriptome for defense. Here we highlight recent advances in understanding the mecha-nisms underlying activation of the main classes of immune receptors, summarize the current under-standing of their signaling mechanisms, and discuss an updated model for SA perception andsignaling. In addition, we discuss how different receptors are organized into networks and the im-plications of such networks in the integration of complex danger signals for appropriate defenseoutputs

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Identification of Molecular Integrators Shows that Nitrogen Actively Controls the Phosphate Starvation Response in Plants | Plant Cell

Identification of Molecular Integrators Shows that Nitrogen Actively Controls the Phosphate Starvation Response in Plants | Plant Cell | Xanthomonas | Scoop.it
Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other’s transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.
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Phosphorylation-Regulated Activation of the Arabidopsis RRS1-R/RPS4 Immune Receptor Complex Reveals Two Distinct Effector Recognition Mechanisms

Phosphorylation-Regulated Activation of the Arabidopsis RRS1-R/RPS4 Immune Receptor Complex Reveals Two Distinct Effector Recognition Mechanisms | Xanthomonas | Scoop.it
Highlights
 • Arabidopsis RRS1-R WRKY domain Thr phosphorylation is required for autoinhibition 
• Other RRS1-R C-terminal phosphorylation sites are required for PopP2 responsiveness 
• RRS1 derepression involves effector-enhanced proximity of TIR RRS1 to its C terminus 
• Enhanced TIR RRS1 and C terminus proximity relieves inhibition of TIR RRS1 on TIR RPS4
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Characterization, Phylogeny, and Genome Analyses of Nonpathogenic Xanthomonas campestris Strains Isolated from Brassica Seeds

Characterization, Phylogeny, and Genome Analyses of Nonpathogenic Xanthomonas campestris Strains Isolated from Brassica Seeds | Xanthomonas | Scoop.it
Xanthomonads were detected by using the Xan-D(CCF) medium from the brassica seeds, and their pathogenicity was determined by plant inoculation tests. It was found that some seed lots were infested with Xanthomonas campestris pv. campestris, some with X. campestris pv. raphani, and some with nonpathogenic xanthomonads. The nonpathogenic xanthomonad strains were identified as X. campestris, and the multilocus sequence analysis showed that the nonpathogenic X. campestris strains were grouped together with pathogenic X. campestris, but not with nonpathogenic strains of X. arboricola. In addition, all isolated X. campestris pv. campestris and X. campestris pv. raphani strains were positive in the hrpF-PCR, but the nonpathogenic strains were negative. It was further found that nonpathogenic X. campestris strain nE1 does not contain the entire pathogenicity island (hrp gene cluster; type III secretion system) and all type III effector protein genes based on the whole genome sequence analyses. The nonpathogenic X. campestris strain nE1 could acquire the entire pathogenicity island from the endemic X. campestris pv. campestris and X. campestris pv. raphani strains by conjugation, but type III effector genes were not cotransferred. The studies showed that the nonpathogenic X. campestris strains indeed exist on the brassica seeds, but it could be differentiated by the PCR assays on the hrp and type III effector genes. Nevertheless, the nonpathogenic X. campestris strains cannot be ignored because they may be potential gene resources to increase genetic diversity in the endemic pathogenic X. campestris pv. campestris and X. campestris pv. raphani strains.
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What is the role of putrescine accumulated under potassium deficiency? - Cui - - Plant, Cell & Environment

What is the role of putrescine accumulated under potassium deficiency? - Cui - - Plant, Cell & Environment | Xanthomonas | Scoop.it

Biomarker metabolites are of increasing interest in crops since they open avenues for precision agriculture, whereby nutritional needs and stresses can be monitored optimally. Putrescine has the potential to be a useful biomarker to reveal potassium (K+) deficiency. In fact, although this diamine has also been observed to increase during other stresses such as drought, cold or heavy metals, respective changes are comparably low. Due to its multifaceted biochemical properties, several roles for putrescine under K+ deficiency have been suggested, such as cation balance, antioxidant, reactive oxygen species mediated signalling, osmolyte or pH regulator. However, the specific association of putrescine build‐up with low K+ availability in plants remains poorly understood, and possible regulatory roles must be consistent with putrescine concentration found in plant tissues. We hypothesize that the massive increase of putrescine upon K+ starvation plays an adaptive role. A distinction of putrescine function from that of other polyamines (spermine, spermidine) may be based either on its specificity or (which is probably more relevant under K+ deficiency) on a very high attainable concentration of putrescine, which far exceeds those for spermidine and spermine. putrescine and its catabolites appear to possess a strong potential in controlling cellular K+ and Ca2+, and mitochondria and chloroplasts bioenergetics under K+ stress.

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Validation of suitable reference genes for qRT-PCR in cabbage ( Brassica oleracea L.) under different abiotic stress experimental conditions | SpringerLink

Validation of suitable reference genes for qRT-PCR in cabbage ( Brassica oleracea L.) under different abiotic stress experimental conditions | SpringerLink | Xanthomonas | Scoop.it
Cabbage (Brassica oleracea) is one of the most important vegetable crops worldwide. qRT-PCR is a sensitive technique for gene expression studies and choosing the appropriate reference gene is essential to obtain reliable results. In the present work, 22 candidate reference genes were evaluated under various experimental conditions, including NaCl, drought stress treatment, temperature treatments (cold and heat) and a set of hormones stress (6-BA, NAA, and ABA) treatments, across a range of tissue types and cultivars. Gene expression data taken from 45 cabbage samples was analyzed using two algorithms, geNorm and NormFinder. Suitable combinations of reference genes for qRT-PCR normalization should be applied according to different experimental conditions. In this study, the genes EF1a, GAPC2 and SAND were verified as the suitable reference genes across all tested samples. Additionally, each experimental condition had a unique set of reference genes best suited to samples within the particular condition. To validate the suitability of the candidate reference genes, the gene expression of WSD1, a gene that may be involved in biosynthesis pathway of wax esters in cabbage, was measured across all 45 samples and normalized using the three best reference gene candidates. WSD1 displayed variation in gene expression across different tissues and cultivars, and exhibited diverse up- or down- regulated expression patterns under various treatments, which indicate that BoWSD1 may play an important role in the response to abiotic stresses in cabbage. Our results provide the foundation for gene expression analysis in Brassica oleracea and other species of Brassica vegetables.
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A NanoLuc luciferase‐based assay enabling the real‐time analysis of protein secretion and injection by bacterial type III secretion systems - Westerhausen - - Molecular Microbiology

A NanoLuc luciferase‐based assay enabling the real‐time analysis of protein secretion and injection by bacterial type III secretion systems - Westerhausen - - Molecular Microbiology | Xanthomonas | Scoop.it

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate‐NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc‐based secretion assay features a very high signal‐to‐noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384‐well microplates. We further developed a split‐NanoLuc‐based assay that enables the real‐time monitoring of type III secretion‐dependent injection of effector‐HiBiT fusions into host cells stably expressing the complementing NanoLuc‐LgBiT.

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Extensive genomic rearrangements along with distinct mobilome and TALome is associated with extreme pathotypes of a rice pathogen | Genome Biology and Evolution | Oxford Academic

Extensive genomic rearrangements along with distinct mobilome and TALome is associated with extreme pathotypes of a rice pathogen | Genome Biology and Evolution | Oxford Academic | Xanthomonas | Scoop.it

Xanthomonas oryzae pv. oryzae (Xoo) is a serious pathogen of rice which displays tremendous inter-strain variation. The emergence of highly virulent strains of Xoo is a major threat to rice cultivation. Evolutionary insights into genome dynamics of highly virulent strains as compared to the less virulent ones are crucial for understanding the molecular basis of exceptional success of Xoo as a highly evolved plant pathogen. In the present study, we report a complete genome sequence of Xoo strains with extreme virulent pathotypes (XVPs) characterized based on their reaction towards ten resistance (Xa) genes. One strain, IXO1088 can overcome resistance mediated by all the ten resistance genes while the other strain IXO704 cannot overcome any of them. Interestingly, our investigation revealed that XVPs display dramatic variation in the genome structure with numerous rearrangements/inversions. Moreover, XPVs also possess distinct transposon content and prophage elements that may provide genomic flux required for the acquisition of novel gene cassettes and structural changes in the genome. Interestingly, analysis of transcription activator-like effector (TALE) proteins, which are major virulence determinants of Xanthomonas pathogen show marked variation in the TALE content and DNA binding domain of tal genes. Overall, the present study indicates the possible role of mobilomes and repetitive elements in major structural and sequence alterations, which may be leading to the emergence of novel and extreme pathotypes. The knowledge and resource of XVPs will be invaluable in the further systematic understanding of evolution and management of variant pathotypes of Xoo.

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Spatial metabolomics of in situ host–microbe interactions at the micrometre scale

Spatial metabolomics of in situ host–microbe interactions at the micrometre scale | Xanthomonas | Scoop.it
Spatial metabolomics describes the location and chemistry of small molecules involved in metabolic phenotypes, defence molecules and chemical interactions in natural communities. Most current techniques are unable to spatially link the genotype and metabolic phenotype of microorganisms in situ at a scale relevant to microbial interactions. Here, we present a spatial metabolomics pipeline (metaFISH) that combines fluorescence in situ hybridization (FISH) microscopy and high-resolution atmospheric-pressure matrix-assisted laser desorption/ionization mass spectrometry to image host–microbe symbioses and their metabolic interactions. The metaFISH pipeline aligns and integrates metabolite and fluorescent images at the micrometre scale to provide a spatial assignment of host and symbiont metabolites on the same tissue section. To illustrate the advantages of metaFISH, we mapped the spatial metabolome of a deep-sea mussel and its intracellular symbiotic bacteria at the scale of individual epithelial host cells. Our analytical pipeline revealed metabolic adaptations of the epithelial cells to the intracellular symbionts and variation in metabolic phenotypes within a single symbiont 16S rRNA phylotype, and enabled the discovery of specialized metabolites from the host–microbe interface. metaFISH provides a culture-independent approach to link metabolic phenotypes to community members in situ and is a powerful tool for microbiologists across fields. This work combines mass spectrometry imaging at high resolution with FISH for the visualization and identification of microorganisms. The authors develop a sample preparation and imaging pipeline called metaFISH to colocalize metabolite patterns with community members and apply it to a host–microbe symbiosis (mussel and its symbionts) to identify symbiosis-specific metabolites.
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Plant and Animal Innate Immunity Complexes: Fighting Different Enemies with Similar Weapons

Plant and Animal Innate Immunity Complexes: Fighting Different Enemies with Similar Weapons | Xanthomonas | Scoop.it

Both animals and plants express intracellular innate immunity receptors known as NLR (NOD-like receptors or nucleotide-binding domain and leucine-rich repeat receptors, respectively). For various mammalian systems, the specific formation of macromolecular
structures, such as inflammasomes by activated NLR receptors, has been extensively reported. However, for plant organisms, the formation of such structures was an open scientific question for many years. This year, the first plant ‘resistosome’ structure
was reported, revealing significant structural similarities to mammalian apoptosome and inflammasome structures.

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Xanthomonas campestris sensor kinase HpaS co‐opts the orphan response regulator VemR to form a branched two‐component system that regulates motility - Li - - Molecular Plant Pathology - Wiley Onlin...

Xanthomonas campestris sensor kinase HpaS co‐opts the orphan response regulator VemR to form a branched two‐component system that regulates motility - Li - - Molecular Plant Pathology - Wiley Onlin... | Xanthomonas | Scoop.it

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/ hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDSPAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection.

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Perception of damaged self in plants | Plant Physiology

Plants use specific receptor proteins on the cell surface to detect host13 derived danger signals released in response to attacks by pathogens or herbivores and activate 14 immune responses against them.
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