Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 22
Filter
1.
Eur Urol Oncol ; 5(4): 412-419, 2022 08.
Article in English | MEDLINE | ID: mdl-35450835

ABSTRACT

BACKGROUND: Bacteria play a suspected role in the development of several cancer types, and associations between the presence of particular bacteria and prostate cancer have been reported. OBJECTIVE: To provide improved characterisation of the prostate and urine microbiome and to investigate the prognostic potential of the bacteria present. DESIGN, SETTING, AND PARTICIPANTS: Microbiome profiles were interrogated in sample collections of patient urine (sediment microscopy: n = 318, 16S ribosomal amplicon sequencing: n = 46; and extracellular vesicle RNA-seq: n = 40) and cancer tissue (n = 204). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Microbiomes were assessed using anaerobic culture, population-level 16S analysis, RNA-seq, and whole genome DNA sequencing. RESULTS AND LIMITATIONS: We demonstrate an association between the presence of bacteria in urine sediments and higher D'Amico risk prostate cancer (discovery, n = 215 patients, p < 0.001; validation, n = 103, p < 0.001, χ2 test for trend). Characterisation of the bacterial community led to the (1) identification of four novel bacteria (Porphyromonas sp. nov., Varibaculum sp. nov., Peptoniphilus sp. nov., and Fenollaria sp. nov.) that were frequently found in patient urine, and (2) definition of a patient subgroup associated with metastasis development (p = 0.015, log-rank test). The presence of five specific anaerobic genera, which includes three of the novel isolates, was associated with cancer risk group, in urine sediment (p = 0.045, log-rank test), urine extracellular vesicles (p = 0.039), and cancer tissue (p = 0.035), with a meta-analysis hazard ratio for disease progression of 2.60 (95% confidence interval: 1.39-4.85; p = 0.003; Cox regression). A limitation is that functional links to cancer development are not yet established. CONCLUSIONS: This study characterises prostate and urine microbiomes, and indicates that specific anaerobic bacteria genera have prognostic potential. PATIENT SUMMARY: In this study, we investigated the presence of bacteria in patient urine and the prostate. We identified four novel bacteria and suggest a potential prognostic utility for the microbiome in prostate cancer.


Subject(s)
Microbiota , Prostatic Neoplasms , Bacteria/genetics , Humans , Male , Microbiota/genetics , Prostate/pathology , Prostatic Neoplasms/pathology , RNA, Ribosomal, 16S/genetics
2.
Genome Biol ; 20(1): 208, 2019 10 22.
Article in English | MEDLINE | ID: mdl-31639030

ABSTRACT

BACKGROUND: Human tissue is increasingly being whole genome sequenced as we transition into an era of genomic medicine. With this arises the potential to detect sequences originating from microorganisms, including pathogens amid the plethora of human sequencing reads. In cancer research, the tumorigenic ability of pathogens is being recognized, for example, Helicobacter pylori and human papillomavirus in the cases of gastric non-cardia and cervical carcinomas, respectively. As of yet, no benchmark has been carried out on the performance of computational approaches for bacterial and viral detection within host-dominated sequence data. RESULTS: We present the results of benchmarking over 70 distinct combinations of tools and parameters on 100 simulated cancer datasets spiked with realistic proportions of bacteria. mOTUs2 and Kraken are the highest performing individual tools achieving median genus-level F1 scores of 0.90 and 0.91, respectively. mOTUs2 demonstrates a high performance in estimating bacterial proportions. Employing Kraken on unassembled sequencing reads produces a good but variable performance depending on post-classification filtering parameters. These approaches are investigated on a selection of cervical and gastric cancer whole genome sequences where Alphapapillomavirus and Helicobacter are detected in addition to a variety of other interesting genera. CONCLUSIONS: We provide the top-performing pipelines from this benchmark in a unifying tool called SEPATH, which is amenable to high throughput sequencing studies across a range of high-performance computing clusters. SEPATH provides a benchmarked and convenient approach to detect pathogens in tissue sequence data helping to determine the relationship between metagenomics and disease.


Subject(s)
Metagenomics/methods , Neoplasms/microbiology , Whole Genome Sequencing , Alphapapillomavirus/isolation & purification , Benchmarking , Helicobacter/isolation & purification , Humans
3.
BMC Bioinformatics ; 20(1): 9, 2019 Jan 07.
Article in English | MEDLINE | ID: mdl-30616525

ABSTRACT

BACKGROUND: Traditional Map based Cloning approaches, used for the identification of desirable alleles, are extremely labour intensive and years can elapse between the mutagenesis and the detection of the polymorphism. High throughput sequencing based Mapping-by-sequencing approach requires an ordered genome assembly and cannot be used with fragmented, un-scaffolded draft genomes, limiting its application to model species and precluding many important organisms. RESULTS: We addressed this gap in resource and presented a computational method and software implementations called CHERIPIC (Computing Homozygosity Enriched Regions In genomes to Prioritise Identification of Candidate variants). We have successfully validated implementation of CHERIPIC using three different types of bulk segregant sequence data from Arabidopsis, maize and barley, respectively. CONCLUSIONS: CHERIPIC allows users to rapidly analyse bulk segregant sequence data and we have made it available as a pre-packaged binary with all dependencies for Linux and MacOS and as Galaxy tool.


Subject(s)
Chromosome Mapping/methods , Genes, Plant/genetics , Genome, Plant , Mutation , Polymorphism, Single Nucleotide , Software , Arabidopsis/genetics , Chromosomes, Plant , High-Throughput Nucleotide Sequencing/methods , Homozygote , Hordeum/genetics , Sequence Analysis, DNA/methods , Zea mays/genetics
4.
Wellcome Open Res ; 4: 155, 2019.
Article in English | MEDLINE | ID: mdl-32055707

ABSTRACT

The identification of microbiological infection is usually a diagnostic investigation, a complex process that is firstly initiated by clinical suspicion. With the emergence of high-throughput sequencing (HTS) technologies, metagenomic analysis has unveiled the power to identify microbial DNA/RNA from a diverse range of clinical samples (1). Metagenomic analysis of whole human genomes at the clinical/research interface bypasses the steps of clinical scrutiny and targeted testing and has the potential to generate unexpected findings relating to infectious and sometimes transmissible disease. There is no doubt that microbial findings that may have a significant impact on a patient's treatment and their close contacts should be reported to those with clinical responsibility for the sample-donating patient. There are no clear recommendations on how such findings that are incidental, or outside the original investigation, should be handled. Here we aim to provide an informed protocol for the management of incidental microbial findings as part of the 100,000 Genomes Project which may have broader application in this emerging field. As with any other clinical information, we aim to prioritise the reporting of data that are most likely to be of benefit to the patient and their close contacts. We also set out to minimize risks, costs and potential anxiety associated with the reporting of results that are unlikely to be of clinical significance. Our recommendations aim to support the practice of microbial metagenomics by providing a simplified pathway that can be applied to reporting the identification of potential pathogens from metagenomic datasets. Given that the ambition for UK sequenced human genomes over the next 5 years has been set to reach 5 million and the field of metagenomics is rapidly evolving, the guidance will be regularly reviewed and will likely adapt over time as experience develops.

5.
New Phytol ; 220(1): 232-248, 2018 10.
Article in English | MEDLINE | ID: mdl-30156022

ABSTRACT

The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)-induced defense genes and alters plant growth responses to light. HaRxL106-mediated suppression of immunity is abolished in RCD1 loss-of-function mutants. We report that RCD1-type proteins are phosphorylated, and we identified Mut9-like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1-interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared with wild-type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/immunology , Arabidopsis/microbiology , Nuclear Proteins/metabolism , Oomycetes/metabolism , Plant Immunity , Proteins/metabolism , ADP Ribose Transferases/metabolism , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant/radiation effects , Mutation/genetics , Nuclear Proteins/genetics , Oomycetes/drug effects , Oomycetes/isolation & purification , Oomycetes/pathogenicity , Plant Diseases/microbiology , Plant Immunity/drug effects , Plants, Genetically Modified , Protein Domains , Protein Multimerization/drug effects , Salicylic Acid/pharmacology , Signal Transduction/radiation effects , Transcription, Genetic/drug effects , Virulence/drug effects
6.
PLoS Genet ; 13(5): e1006639, 2017 May.
Article in English | MEDLINE | ID: mdl-28472137

ABSTRACT

Plant immunity protects plants from numerous potentially pathogenic microbes. The biological network that controls plant inducible immunity must function effectively even when network components are targeted and disabled by pathogen effectors. Network buffering could confer this resilience by allowing different parts of the network to compensate for loss of one another's functions. Networks rich in buffering rely on interactions within the network, but these mechanisms are difficult to study by simple genetic means. Through a network reconstitution strategy, in which we disassemble and stepwise reassemble the plant immune network that mediates Pattern-Triggered-Immunity, we have resolved systems-level regulatory mechanisms underlying the Arabidopsis transcriptome response to the immune stimulant flagellin-22 (flg22). These mechanisms show widespread evidence of interactions among major sub-networks-we call these sectors-in the flg22-responsive transcriptome. Many of these interactions result in network buffering. Resolved regulatory mechanisms show unexpected patterns for how the jasmonate (JA), ethylene (ET), phytoalexin-deficient 4 (PAD4), and salicylate (SA) signaling sectors control the transcriptional response to flg22. We demonstrate that many of the regulatory mechanisms we resolved are not detectable by the traditional genetic approach of single-gene null-mutant analysis. Similar to potential pathogenic perturbations, null-mutant effects on immune signaling can be buffered by the network.


Subject(s)
Arabidopsis Proteins/genetics , Carboxylic Ester Hydrolases/genetics , Flagellin/genetics , Host-Pathogen Interactions/genetics , Plant Immunity/genetics , Transcriptome/genetics , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis Proteins/immunology , Carboxylic Ester Hydrolases/immunology , Cyclopentanes/immunology , Cyclopentanes/metabolism , Ethylenes/immunology , Ethylenes/metabolism , Flagellin/immunology , Gene Expression Regulation, Plant , Gene Regulatory Networks/immunology , Host-Pathogen Interactions/immunology , Oxylipins/immunology , Oxylipins/metabolism , Plant Diseases/genetics , Plant Diseases/immunology , Salicylic Acid/immunology , Salicylic Acid/metabolism , Signal Transduction , Transcriptome/immunology
7.
BMC Biol ; 15(1): 20, 2017 03 20.
Article in English | MEDLINE | ID: mdl-28320402

ABSTRACT

BACKGROUND: Plants are exposed to diverse pathogens and pests, yet most plants are resistant to most plant pathogens. Non-host resistance describes the ability of all members of a plant species to successfully prevent colonization by any given member of a pathogen species. White blister rust caused by Albugo species can overcome non-host resistance and enable secondary infection and reproduction of usually non-virulent pathogens, including the potato late blight pathogen Phytophthora infestans on Arabidopsis thaliana. However, the molecular basis of host defense suppression in this complex plant-microbe interaction is unclear. Here, we investigate specific defense mechanisms in Arabidopsis that are suppressed by Albugo infection. RESULTS: Gene expression profiling revealed that two species of Albugo upregulate genes associated with tryptophan-derived antimicrobial metabolites in Arabidopsis. Albugo laibachii-infected tissue has altered levels of these metabolites, with lower indol-3-yl methylglucosinolate and higher camalexin accumulation than uninfected tissue. We investigated the contribution of these Albugo-imposed phenotypes to suppression of non-host resistance to P. infestans. Absence of tryptophan-derived antimicrobial compounds enables P. infestans colonization of Arabidopsis, although to a lesser extent than Albugo-infected tissue. A. laibachii also suppresses a subset of genes regulated by salicylic acid; however, salicylic acid plays only a minor role in non-host resistance to P. infestans. CONCLUSIONS: Albugo sp. alter tryptophan-derived metabolites and suppress elements of the responses to salicylic acid in Arabidopsis. Albugo sp. imposed alterations in tryptophan-derived metabolites may play a role in Arabidopsis non-host resistance to P. infestans. Understanding the basis of non-host resistance to pathogens such as P. infestans could assist in development of strategies to elevate food security.


Subject(s)
Anti-Infective Agents/metabolism , Arabidopsis/immunology , Arabidopsis/microbiology , Biosynthetic Pathways , Disease Resistance/immunology , Phytophthora infestans/physiology , Plant Diseases/microbiology , Tryptophan/metabolism , Arabidopsis/drug effects , Arabidopsis/genetics , Biomass , Biosynthetic Pathways/drug effects , Biosynthetic Pathways/genetics , Brassica/microbiology , Disease Resistance/drug effects , Disease Susceptibility , Gene Expression Profiling , Gene Expression Regulation, Plant/drug effects , Gene Ontology , Genes, Plant , Glucosinolates/metabolism , Indoles/metabolism , Metabolic Networks and Pathways/drug effects , Mutation/genetics , Plant Diseases/immunology , Plant Immunity/drug effects , Plant Leaves/drug effects , Plant Leaves/microbiology , Reproducibility of Results , Salicylic Acid/pharmacology , Signal Transduction/drug effects , Thiazoles/metabolism , Up-Regulation/drug effects
9.
Nat Commun ; 6: 8635, 2015 Oct 26.
Article in English | MEDLINE | ID: mdl-26497596

ABSTRACT

Phenylpropanoids comprise an important class of plant secondary metabolites. A number of transcription factors have been used to upregulate-specific branches of phenylpropanoid metabolism, but by far the most effective has been the fruit-specific expression of AtMYB12 in tomato, which resulted in as much as 10% of fruit dry weight accumulating as flavonols and hydroxycinnamates. We show that AtMYB12 not only increases the demand of flavonoid biosynthesis but also increases the supply of carbon from primary metabolism, energy and reducing power, which may fuel the shikimate and phenylalanine biosynthetic pathways to supply more aromatic amino acids for secondary metabolism. AtMYB12 directly binds promoters of genes encoding enzymes of primary metabolism. The enhanced supply of precursors, energy and reducing power achieved by AtMYB12 expression can be harnessed to engineer high levels of novel phenylpropanoids in tomato fruit, offering an effective production system for bioactives and other high value ingredients.


Subject(s)
Flavonoids/biosynthesis , Plants, Genetically Modified/metabolism , Solanum lycopersicum/genetics , Solanum lycopersicum/metabolism , Biosynthetic Pathways/genetics , Flavonoids/analysis , Fruit/chemistry , Fruit/genetics , Fruit/metabolism , Gene Expression Regulation, Plant , Solanum lycopersicum/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/chemistry , Plants, Genetically Modified/genetics
10.
Elife ; 4: e07460, 2015 Jul 29.
Article in English | MEDLINE | ID: mdl-26219214

ABSTRACT

In 2013, in response to an epidemic of ash dieback disease in England the previous year, we launched a Facebook-based game called Fraxinus to enable non-scientists to contribute to genomics studies of the pathogen that causes the disease and the ash trees that are devastated by it. Over a period of 51 weeks players were able to match computational alignments of genetic sequences in 78% of cases, and to improve them in 15% of cases. We also found that most players were only transiently interested in the game, and that the majority of the work done was performed by a small group of dedicated players. Based on our experiences we have built a linear model for the length of time that contributors are likely to donate to a crowd-sourced citizen science project. This model could serve a guide for the design and implementation of future crowd-sourced citizen science initiatives.


Subject(s)
Community Participation , Computational Biology/methods , DNA, Fungal/genetics , Fraxinus/microbiology , Plant Diseases/microbiology , Sequence Alignment/methods , DNA, Fungal/chemistry , England , Sequence Analysis, DNA
11.
Plant J ; 83(4): 686-704, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26108615

ABSTRACT

Given the potential health benefits of polyphenolic compounds in the diet, there is a growing interest in the generation of food crops enriched with health-protective flavonoids. We undertook a series of metabolite analyses of tomatoes ectopically expressing the Delila and Rosea1 transcription factor genes from snapdragon (Antirrhinum majus), paying particular attention to changes in phenylpropanoids compared to controls. These analyses revealed multiple changes, including depletion of rutin and naringenin chalcone, and enhanced levels of anthocyanins and phenylacylated flavonol derivatives. We isolated and characterized the chemical structures of the two most abundant anthocyanins, which were shown by NMR spectroscopy to be delphinidin-3-(4'''-O-trans-p-coumaroyl)-rutinoside-5-O-glucoside and petunidin-3-(4'''-O-trans-p-coumaroyl)-rutinoside-5-O-glucoside. By performing RNA sequencing on both purple fruit and wild-type fruit, we obtained important information concerning the relative expression of both structural and transcription factor genes. Integrative analysis of the transcript and metabolite datasets provided compelling evidence of the nature of all anthocyanin biosynthetic genes, including those encoding species-specific anthocyanin decoration enzymes. One gene, SlFdAT1 (Solyc12g088170), predicted to encode a flavonoid-3-O-rutinoside-4'''-phenylacyltransferase, was characterized by assays of recombinant protein and over-expression assays in tobacco. The combined data are discussed in the context of both our current understanding of phenylpropanoid metabolism in Solanaceous species, and evolution of flavonoid decorating enzymes and their transcriptional networks in various plant species.


Subject(s)
Anthocyanins/metabolism , Plant Proteins/metabolism , Solanum lycopersicum/metabolism , Transcription Factors/metabolism , Gene Expression Regulation, Plant , Solanum lycopersicum/genetics , Molecular Sequence Data , Plant Proteins/genetics , Transcription Factors/genetics
12.
Cell ; 161(5): 1089-1100, 2015 May 21.
Article in English | MEDLINE | ID: mdl-26000484

ABSTRACT

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/immunology , Plant Proteins/metabolism , Amino Acid Sequence , Arabidopsis/microbiology , Arabidopsis Proteins/chemistry , Bacterial Proteins/immunology , Immunity, Innate , Molecular Sequence Data , Protein Structure, Tertiary , Pseudomonas fluorescens/metabolism , Pseudomonas fluorescens/pathogenicity , Pseudomonas syringae/immunology , Pseudomonas syringae/metabolism , Nicotiana/immunology , Nicotiana/microbiology
13.
Plant J ; 81(1): 40-52, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25284001

ABSTRACT

Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/metabolism , Karyopherins/physiology , Active Transport, Cell Nucleus , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Conserved Sequence , Escherichia coli/genetics , Host-Pathogen Interactions , Karyopherins/chemistry , Karyopherins/genetics , Karyopherins/metabolism , Models, Molecular , Oomycetes/genetics , Protein Structure, Tertiary
14.
PLoS Pathog ; 10(10): e1004443, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25329884

ABSTRACT

Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.


Subject(s)
Arabidopsis/drug effects , Arabidopsis/genetics , Host-Pathogen Interactions/immunology , Oomycetes/drug effects , Plant Diseases/immunology , Plant Immunity/immunology , Salicylic Acid/pharmacology , Arabidopsis Proteins/genetics , Base Sequence/genetics , Gene Expression Regulation, Plant , Host-Pathogen Interactions/drug effects , Oomycetes/genetics , Plant Diseases/genetics , Plant Diseases/microbiology , Salicylic Acid/metabolism
15.
PLoS Genet ; 10(10): e1004655, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25340333

ABSTRACT

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific "avirulent" pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.


Subject(s)
Arabidopsis Proteins/genetics , Disease Resistance/genetics , Plant Diseases/genetics , Plant Proteins/genetics , Amino Acid Motifs , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Cell Nucleus , Gene Expression Regulation, Plant , Mutation , Plant Diseases/immunology , Plant Immunity/genetics , Pseudomonas syringae/pathogenicity , Ralstonia solanacearum/pathogenicity
16.
BMC Genomics ; 15: 341, 2014 May 06.
Article in English | MEDLINE | ID: mdl-24884414

ABSTRACT

BACKGROUND: Next Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10-20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5' ends of mRNA or use of RNA ligations. RESULTS: We have developed EXpression Profiling through Randomly Sheared cDNA tag Sequencing (EXPRSS) that employs acoustic waves to randomly shear cDNA and generate sequence tags at a relatively defined position (~150-200 bp) from the 3' end of each mRNA. Implementation of the method was verified through comparative analysis of expression data generated from EXPRSS, NlaIII-DGE and Affymetrix microarray and through qPCR quantification of selected genes. EXPRSS is a strand specific and restriction enzyme independent tag sequencing method that does not require cDNA length-based data transformations. EXPRSS is highly reproducible, is high-throughput and it also reveals alternative polyadenylation and polyadenylated antisense transcripts. It is cost-effective using barcoded multiplexing, avoids the biases of existing SAGE and derivative methods and can reveal polyadenylation position from paired-end sequencing. CONCLUSIONS: EXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis.


Subject(s)
Computational Biology/methods , Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing , Sequence Analysis, RNA/methods , 3' Untranslated Regions , Arabidopsis/genetics , DNA, Complementary/metabolism , Down-Regulation , Gene Library , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Up-Regulation
17.
PLoS Biol ; 11(12): e1001732, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24339748

ABSTRACT

Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis (Hpa), and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a), resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa. Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET) signalling in the presence of HaRxL44, and also 3 d after infection with Hpa. Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA)-triggered immunity (SATI) in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a. Using a PR1::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression.


Subject(s)
Arabidopsis/physiology , Host-Pathogen Interactions/physiology , Peronospora/immunology , Plant Diseases/microbiology , Plant Growth Regulators/physiology , Plant Immunity/physiology , Salicylic Acid/metabolism , Arabidopsis Proteins/physiology , Host-Pathogen Interactions/immunology , Mediator Complex/physiology , Plant Diseases/immunology
18.
Mol Plant Microbe Interact ; 26(7): 745-57, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23734779

ABSTRACT

The genome of the pathogenic oomycete Hyaloperonospora arabidopsidis is predicted to encode at least 134 high-confidence effectors (HaRxL) carrying the RxLR motif implicated in their translocation into plant cells. However, only four avirulence genes (ATR1, ATR13, ATR5, and ATR39) have been isolated. This indicates that identification of HaRxL effectors based on avirulence is low throughput. We aimed at rapidly identifying H. arabidopsidis effectors that contribute to virulence by developing methods to detect and quantify multiple candidates in bacterial mixed infections using either Illumina sequencing or capillary electrophoresis. In these assays, referred to here as in planta effector competition assays, we estimate the contribution to virulence of individual effectors by calculating the abundance of each HaRxL in the bacterial population recovered from leaves 3 days after inoculation relative to abundance in the initial mixed inoculum. We identified HaRxL that enhance Pseudomonas syringae pv. tomato DC3000 growth in some but not all Arabidopsis accessions. Further analysis showed that HaRxLL464, HaRxL75, HaRxL22, HaRxLL441, and HaRxL89 suppress pathogen-associated molecular pattern-triggered immunity (PTI) and localize to different subcellular compartments in Nicotiana benthamiana, providing evidence for a multilayered suppression of PTI by pathogenic oomycetes and molecular probes for the dissection of PTI.


Subject(s)
Arabidopsis/parasitology , Oomycetes/genetics , Plant Diseases/immunology , Pseudomonas syringae/growth & development , Amino Acid Motifs , Antibiosis , Arabidopsis/cytology , Arabidopsis/immunology , Arabidopsis/microbiology , Electrophoresis, Capillary , High-Throughput Nucleotide Sequencing , Host-Pathogen Interactions , Oomycetes/pathogenicity , Plant Diseases/microbiology , Plant Diseases/parasitology , Plant Leaves/cytology , Plant Leaves/immunology , Plant Leaves/microbiology , Plant Leaves/parasitology , Protein Transport , Proteins/genetics , Proteins/metabolism , Pseudomonas syringae/genetics , Pseudomonas syringae/metabolism , Pseudomonas syringae/pathogenicity , Recombinant Fusion Proteins , Sequence Analysis, DNA , Nicotiana/cytology , Nicotiana/immunology , Nicotiana/metabolism , Nicotiana/microbiology , Virulence/genetics
19.
Nat Biotechnol ; 28(4): 365-9, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20231819

ABSTRACT

Plant diseases cause massive losses in agriculture. Increasing the natural defenses of plants may reduce the impact of phytopathogens on agricultural productivity. Pattern-recognition receptors (PRRs) detect microbes by recognizing conserved pathogen-associated molecular patterns (PAMPs). Although the overall importance of PAMP-triggered immunity for plant defense is established, it has not been used to confer disease resistance in crops. We report that activity of a PRR is retained after its transfer between two plant families. Expression of EFR (ref. 4), a PRR from the cruciferous plant Arabidopsis thaliana, confers responsiveness to bacterial elongation factor Tu in the solanaceous plants Nicotiana benthamiana and tomato (Solanum lycopersicum), making them more resistant to a range of phytopathogenic bacteria from different genera. Our results in controlled laboratory conditions suggest that heterologous expression of PAMP recognition systems could be used to engineer broad-spectrum disease resistance to important bacterial pathogens, potentially enabling more durable and sustainable resistance in the field.


Subject(s)
Bacterial Physiological Phenomena , Immunity, Innate/physiology , Plant Diseases/immunology , Plant Diseases/microbiology , Plants, Genetically Modified/microbiology , Plants, Genetically Modified/physiology , Receptors, Pattern Recognition/metabolism , Plant Diseases/prevention & control , Receptors, Pattern Recognition/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...