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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid-a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Imunidade Vegetal , Estômatos de Plantas/imunologia , Estômatos de Plantas/metabolismo , Ácido Abscísico/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
[Purpose] Compare four quick (approximately 60 s), reliable methods of assessing %body-fat (%BF) among young (Y, 18-34 years), middle-age (M, 35-59 years), and older (O, 60-88 years) healthy-adults. [Participants and Methods] One-hundred-eighty healthy males-and-females were equally (n=30) divided into Y, M, and O age groups to assess %BF. The %BF methods were: 1) Bioelectrical-impedance-Inbody770 (IB)-criterion reference; 2) Body-mass-index (BMI); 3) Abdominal-and-hip circumferences (CIR); and 4) Skinfold (SF). [Results] %BF were significantly different among the four body-fat methods and among the three age-groups for both males-and-females. %BF among IB,BMI,CIR, and SF were, respectively, 15.7 ± 4.7%, 19.6 ± 3.2%, 17.3 ± 3.5%, and 12.1 ± 4.1% for Y-males; 18.3 ± 5.7%, 22.8 ± 3.6%, 19.6 ± 3.6%, and 15.6 ± 4.5% for M-males; 24.4 ± 6.5%, 25.8 ± 3.3%, 24.0 ± 4.5%, and 20.0 ± 4.1% for O-males; 24.9 ± 6.9%, 28.9 ± 4.1%, 29.4 ± 4.6%, and 22.4 ± 6.3% for Y-females; 25.1 ± 7.0%, 31.4 ± 4.7%, 33.0 ± 4.5%, and 25.0 ± 4.5% for M-females; 35.1 ± 6.3%, 35.5 ± 4.3%, 38.4 ± 4.8%, and 26.4 ± 3.7% for O-females. [Conclusion]The most accurate %BF-methods to use in clinical settings are CIR for Y-and-M-males, CIR and BMI for O-males, SF for Y-and M-females, and BMI for O-females. The least accurate %BF methods are BMI and SF for Y-males, BMI for M-males, SF for O-males, BMI and CIR for Y-and M-females, and SF for O-females. While all 4-methods of assessing %BF can easily and quickly be employed in clinical settings, some methods significantly underestimate or overestimate %BF and yield different results among varying age groups and sex. These findings help identify people at early health risk of cardiometabolic disease, with O-males and O-females at higher risk.
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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.
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Mapeamento Cromossômico/métodos , Genes de Plantas/genética , Genoma de Planta , Mutação , Polimorfismo de Nucleotídeo Único , Software , Arabidopsis/genética , Cromossomos de Plantas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Homozigoto , Hordeum/genética , Análise de Sequência de DNA/métodos , Zea mays/genéticaRESUMO
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.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas Nucleares/metabolismo , Oomicetos/metabolismo , Imunidade Vegetal , Proteínas/metabolismo , ADP Ribose Transferases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Mutação/genética , Proteínas Nucleares/genética , Oomicetos/efeitos dos fármacos , Oomicetos/isolamento & purificação , Oomicetos/patogenicidade , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Plantas Geneticamente Modificadas , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Ácido Salicílico/farmacologia , Transdução de Sinais/efeitos da radiação , Transcrição Gênica/efeitos dos fármacos , Virulência/efeitos dos fármacosRESUMO
SUMMARY: Rapid technological advances have led to an explosion of biomedical data in recent years. The pace of change has inspired new collaborative approaches for sharing materials and resources to help train life scientists both in the use of cutting-edge bioinformatics tools and databases and in how to analyse and interpret large datasets. A prototype platform for sharing such training resources was recently created by the Bioinformatics Training Network (BTN). Building on this work, we have created a centralized portal for sharing training materials and courses, including a catalogue of trainers and course organizers, and an announcement service for training events. For course organizers, the portal provides opportunities to promote their training events; for trainers, the portal offers an environment for sharing materials, for gaining visibility for their work and promoting their skills; for trainees, it offers a convenient one-stop shop for finding suitable training resources and identifying relevant training events and activities locally and worldwide. AVAILABILITY AND IMPLEMENTATION: http://mygoblet.org/training-portal.
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Biologia Computacional/educação , Currículo , Sistemas de Gerenciamento de Base de Dados , Pesquisadores/educação , Ensino , Humanos , Linguagens de Programação , Design de SoftwareRESUMO
The activity of surface receptors is location specific, dependent upon the dynamic membrane trafficking network and receptor-mediated endocytosis (RME). Therefore, the spatio-temporal dynamics of RME are critical to receptor function. The plasma membrane receptor flagellin sensing2 (FLS2) confers immunity against bacterial infection through perception of flagellin (flg22). Following elicitation, FLS2 is internalized into vesicles. To resolve FLS2 trafficking, we exploited quantitative confocal imaging for colocalization studies and chemical interference. FLS2 localizes to bona fide endosomes via two distinct endocytic trafficking routes depending on its activation status. FLS2 receptors constitutively recycle in a Brefeldin A (BFA)-sensitive manner, while flg22-activated receptors traffic via ARA7/Rab F2b- and ARA6/Rab F1-positive endosomes insensitive to BFA. FLS2 endocytosis required a functional Rab5 GTPase pathway as revealed by dominant-negative ARA7/Rab F2b. Flg22-induced FLS2 endosomal numbers were increased by Concanamycin A treatment but reduced by Wortmannin, indicating that activated FLS2 receptors are targeted to late endosomes. RME inhibitors Tyrphostin A23 and Endosidin 1 altered but did not block induced FLS2 endocytosis. Additional inhibitor studies imply the involvement of the actin-myosin system in FLS2 internalization and trafficking. Altogether, we report a dynamic pattern of subcellular trafficking for FLS2 and reveal a defined framework for ligand-dependent endocytosis of this receptor.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endossomos/metabolismo , Proteínas Quinases/metabolismo , Androstadienos/farmacologia , Proteínas de Arabidopsis/análise , Transporte Biológico , Endocitose , Endossomos/efeitos dos fármacos , Macrolídeos/farmacologia , Proteínas Quinases/análise , Transporte Proteico , Tirfostinas/farmacologia , WortmaninaRESUMO
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.
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Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA/métodos , Regiões 3' não Traduzidas , Arabidopsis/genética , DNA Complementar/metabolismo , Regulação para Baixo , Biblioteca Gênica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Regulação para CimaRESUMO
MOTIVATION: Drawing genomic features in attractive and informative ways is a key task in visualization of genomics data. Scalable Vector Graphics (SVG) format is a modern and flexible open standard that provides advanced features including modular graphic design, advanced web interactivity and animation within a suitable client. SVGs do not suffer from loss of image quality on re-scaling and provide the ability to edit individual elements of a graphic on the whole object level independent of the whole image. These features make SVG a potentially useful format for the preparation of publication quality figures including genomic objects such as genes or sequencing coverage and for web applications that require rich user-interaction with the graphical elements. RESULTS: SVGenes is a Ruby-language library that uses SVG primitives to render typical genomic glyphs through a simple and flexible Ruby interface. The library implements a simple Page object that spaces and contains horizontal Track objects that in turn style, colour and positions features within them. Tracks are the level at which visual information is supplied providing the full styling capability of the SVG standard. Genomic entities like genes, transcripts and histograms are modelled in Glyph objects that are attached to a track and take advantage of SVG primitives to render the genomic features in a track as any of a selection of defined glyphs. The feature model within SVGenes is simple but flexible and not dependent on particular existing gene feature formats meaning graphics for any existing datasets can easily be created without need for conversion. AVAILABILITY: The library is provided as a Ruby Gem from https://rubygems.org/gems/bio-svgenes under the MIT license, and open source code is available at https://github.com/danmaclean/bioruby-svgenes also under the MIT License. CONTACT: dan.maclean@tsl.ac.uk.
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Gráficos por Computador , Genômica/métodos , Software , InternetRESUMO
Pest and pathogen losses jeopardise global food security and ever since the 19(th) century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.
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Genoma Fúngico , Phytophthora infestans/genética , Phytophthora infestans/patogenicidade , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Produtos Agrícolas/microbiologia , Variações do Número de Cópias de DNA , Perfilação da Expressão Gênica , Genes de Plantas , Interações Hospedeiro-Patógeno , Imunidade Inata , Proteínas de Plantas/genética , Polimorfismo Genético , Análise de Sequência de DNARESUMO
Given there are no known studies which have examined multiple lower extremity muscles between different ankle positions during bridging activities, the objective was to assess how employing two different ankle positions (PF versus DF) while performing five common bridging exercises (three bipedal and two unipedal) used in rehabilitation and athletic performance affect core and select lower extremity muscle EMG recruitment. Twenty healthy subjects performed a 5 s isometric hold during five two- and one-leg bridge exercises: (1) on right leg with left knee to chest (1LB-LFlex); (2) on right leg with left knee extended (1LB-LExt); (3) standard two-leg bridge (2LB); (4) two-leg bridge with resistance band around knees (2LB-ABD); and (5) two-leg bridge with ball between knees (2LB-ADD). Surface electromyographic (EMG) data were collected using a Noraxon Telemyo Direct Transmission System from fourteen muscles: (1) three superficial quadriceps (VM, VL, and RF); (2) three hip abductors (TFL, GMED, and GMAX); (3) medial hamstrings (ST) and lateral hamstrings (BF); (4) hip adductors (ADD); (5) erector spinae (ES); (6) latissimus dorsi (LATS); (7) upper rectus abdominis (RA); and (8) external oblique (EO) and internal oblique (IO). EMG data were normalized by maximum voluntary isometric contractions (MVICs). A paired t-test (p < 0.01) was used to assess differences in normalized mean EMG activities between DF and PF for each exercise. EMG activities were significantly greater in DF than PF for the (a) VM, VL, and RF during 1LB-LFlex; (b) ADD during 1LB-LFlex, 1LB-LExt; (c) EO during 1LB-LFlex; and (d) IO during 1LB-LFex. In contrast, EMG activities were significantly greater in PF than DF for ST and BF during all five bridge exercises. Bridging with PF (feet flat) was most effective in recruiting the hamstrings, while bridging with DF (feet up) was most effective in recruiting the quadriceps, hip adductors, and internal and external obliques.
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BACKGROUND: Wheat yellow (stripe) rust caused by Puccinia striiformis f. sp. tritici (PST) is one of the most devastating diseases of wheat worldwide. To design effective breeding strategies that maximize the potential for durable disease resistance it is important to understand the molecular basis of PST pathogenicity. In particular, the characterisation of the structure, function and evolutionary dynamics of secreted effector proteins that are detected by host immune receptors can help guide and prioritize breeding efforts. However, to date, our knowledge of the effector repertoire of cereal rust pathogens is limited. RESULTS: We re-sequenced genomes of four PST isolates from the US and UK to identify effector candidates and relate them to their distinct virulence profiles. First, we assessed SNP frequencies between all isolates, with heterokaryotic SNPs being over tenfold more frequent (5.29 ± 2.23 SNPs/kb) than homokaryotic SNPs (0.41 ± 0.28 SNPs/kb). Next, we implemented a bioinformatics pipeline to integrate genomics, transcriptomics, and effector-focused annotations to identify and classify effector candidates in PST. RNAseq analysis highlighted transcripts encoding secreted proteins that were significantly enriched in haustoria compared to infected tissue. The expression of 22 candidate effector genes was characterised using qRT-PCR, revealing distinct temporal expression patterns during infection in wheat. Lastly, we identified proteins that displayed non-synonymous substitutions specifically between the two UK isolates PST-87/7 and PST-08/21, which differ in virulence to two wheat varieties. By focusing on polymorphic variants enriched in haustoria, we identified five polymorphic effector candidates between PST-87/7 and PST-08/21 among 2,999 secreted proteins. These allelic variants are now a priority for functional validation as virulence/avirulence effectors in the corresponding wheat varieties. CONCLUSIONS: Integration of genomics, transcriptomics, and effector-directed annotation of PST isolates has enabled us to move beyond the single isolate-directed catalogues of effector proteins and develop a framework for mining effector proteins in closely related isolates and relate these back to their defined virulence profiles. This should ultimately lead to more comprehensive understanding of the PST pathogenesis system, an important first step towards developing more effective surveillance and management strategies for one of the most devastating pathogens of wheat.
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Basidiomycota/genética , Basidiomycota/patogenicidade , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Triticum/microbiologia , Proteínas Fúngicas/genética , Doenças das Plantas/genética , Polimorfismo Genético , VirulênciaRESUMO
Plant organs grow to characteristic sizes that are genetically controlled. In animals, signaling by mobile growth factors is thought to be an effective mechanism for measuring primordium size, yet how plants gauge organ size is unclear. Here, we identify the Arabidopsis cytochrome P450 KLUH (KLU)/CYP78A5 as a stimulator of plant organ growth. While klu loss-of-function mutants form smaller organs because of a premature arrest of cell proliferation, KLU overexpression leads to larger organs with more cells. KLU promotes organ growth in a non-cell-autonomous manner, yet it does not appear to modulate the levels of known phytohormones. We therefore propose that KLU is involved in generating a mobile growth signal distinct from the classical phytohormones. The expression dynamics of KLU suggest a model of how the arrest of cell proliferation is coupled to the attainment of a certain primordium size, implying a common principle of size measurement in plants and animals.
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Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/fisiologia , Componentes Aéreos da Planta/crescimento & desenvolvimento , Proliferação de Células , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Tamanho do Órgão , Fenótipo , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
SUMMARY: Scientists now use high-throughput sequencing technologies and short-read assembly methods to create draft genome assemblies in just days. Tools and pipelines like the assembler, and the workflow management environments make it easy for a non-specialist to implement complicated pipelines to produce genome assemblies and annotations very quickly. Such accessibility results in a proliferation of assemblies and associated files, often for many organisms. These assemblies get used as a working reference by lots of different workers, from a bioinformatician doing gene prediction or a bench scientist designing primers for PCR. Here we describe Gee Fu, a database tool for genomic assembly and feature data, including next-generation sequence alignments. Gee Fu is an instance of a Ruby-On-Rails web application on a feature database that provides web and console interfaces for input, visualization of feature data via AnnoJ, access to data through a web-service interface, an API for direct data access by Ruby scripts and access to feature data stored in BAM files. Gee Fu provides a platform for storing and sharing different versions of an assembly and associated features that can be accessed and updated by bench biologists and bioinformaticians in ways that are easy and useful for each. AVAILABILITY: http://tinyurl.com/geefu CONTACT: dan.maclean@tsl.ac.uk.
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Sequência de Bases , Bases de Dados Genéticas , Genômica , Armazenamento e Recuperação da Informação/métodos , Animais , Processamento Eletrônico de Dados , Genoma , Humanos , Internet , Análise em Microsséries , Alinhamento de Sequência , SoftwareRESUMO
The PhosPhAt database of Arabidopsis phosphorylation sites was initially launched in August 2007. Since then, along with 10-fold increase in database entries, functionality of PhosPhAt (phosphat.mpimp-golm.mpg.de) has been considerably upgraded and re-designed. PhosPhAt is now more of a web application with the inclusion of advanced search functions allowing combinatorial searches by Boolean terms. The results output now includes interactive visualization of annotated fragmentation spectra and the ability to export spectra and peptide sequences as text files for use in other applications. We have also implemented dynamic links to other web resources thus augmenting PhosPhAt-specific information with external protein-related data. For experimental phosphorylation sites with information about dynamic behavior in response to external stimuli, we display simple time-resolved diagrams. We have included predictions for pT and pY sites and updated pS predictions. Access to prediction algorithm now allows 'on-the-fly' prediction of phosphorylation of any user-uploaded protein sequence. Protein Pfam domain structures are now mapped onto the protein sequence display next to experimental and predicted phosphorylation sites. Finally, we have implemented functional annotation of proteins using MAPMAN ontology. These new developments make the PhosPhAt resource a useful and powerful tool for the scientific community as a whole beyond the plant sciences.
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Arabidopsis/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Proteínas , Fosfoproteínas/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Biologia Computacional/tendências , Genes de Plantas , Genoma de Planta , Armazenamento e Recuperação da Informação/métodos , Internet , Fosforilação , SoftwareRESUMO
A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho-oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Folhas de Planta/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/enzimologia , DNA Bacteriano/genética , Glucanos/metabolismo , Mutagênese Insercional , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Folhas de Planta/genética , RNA de Plantas/genéticaRESUMO
BACKGROUND: Next-generation sequencing technologies allow researchers to obtain millions of sequence reads in a single experiment. One important use of the technology is the sequencing of small non-coding regulatory RNAs and the identification of the genomic locales from which they originate. Currently, there is a paucity of methods for finding small RNA generative locales. RESULTS: We describe and implement an algorithm that can determine small RNA generative locales from high-throughput sequencing data. The algorithm creates a network, or graph, of the small RNAs by creating links between them depending on their proximity on the target genome. For each of the sub-networks in the resulting graph the clustering coefficient, a measure of the interconnectedness of the subnetwork, is used to identify the generative locales. We test the algorithm over a wide range of parameters using RFAM sequences as positive controls and demonstrate that the algorithm has good sensitivity and specificity in a range of Arabidopsis and mouse small RNA sequence sets and that the locales it generates are robust to differences in the choice of parameters. CONCLUSIONS: NiBLS is a fast, reliable and sensitive method for determining small RNA locales in high-throughput sequence data that is generally applicable to all classes of small RNA.
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Sequência de Bases , Biologia Computacional/métodos , MicroRNAs/química , Análise de Sequência de RNA/métodos , Algoritmos , Alinhamento de Sequência , SoftwareRESUMO
The Cucumber mosaic virus (CMV) 2b counter-defense protein disrupts plant antiviral mechanisms mediated by RNA silencing and salicylic acid (SA). We used microarrays to investigate defensive gene expression in 2b-transgenic Arabidopsis thaliana plants. Surprisingly, 2b inhibited expression of few SA-regulated genes and, in some instances, enhanced the effect of SA on certain genes. Strikingly, the 2b protein inhibited changes in the expression of 90% of genes regulated by jasmonic acid (JA). Consistent with this, infection of plants with CMV, but not the 2b gene-deletion mutant CMVDelta2b, strongly inhibited JA-inducible gene expression. JA levels were unaffected by infection with either CMV or CMVDelta2b. Although the CMV-Arabidopsis interaction is a compatible one, SA accumulation, usually considered to be an indicator of plant resistance, was increased in CMV-infected plants but not in CMVDelta2b-infected plants. Thus, the 2b protein inhibits JA signaling at a step downstream of JA biosynthesis but it primes induction of SA biosynthesis by another CMV gene product or by the process of infection itself. Like many plant viruses, CMV is aphid transmitted. JA is important in plant defense against insects. This raises the possibility that disruption of JA-mediated gene expression by the 2b protein may influence CMV transmission by aphids.
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Cucumovirus/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Interferência de RNA/fisiologia , RNA Viral/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cucumovirus/genética , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Plantas Geneticamente Modificadas , RNA Viral/genética , Ácido Salicílico/metabolismo , Transdução de Sinais/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Pseudomonas syringae is a widespread bacterial pathogen that causes disease on a broad range of economically important plant species. Pathogenicity of P. syringae strains is dependent on the type III secretion system, which secretes a suite of up to about thirty virulence 'effector' proteins into the host cytoplasm where they subvert the eukaryotic cell physiology and disrupt host defences. P. syringae pathovar tabaci naturally causes disease on wild tobacco, the model member of the Solanaceae, a family that includes many crop species as well as on soybean. RESULTS: We used the 'next-generation' Illumina sequencing platform and the Velvet short-read assembly program to generate a 145X deep 6,077,921 nucleotide draft genome sequence for P. syringae pathovar tabaci strain 11528. From our draft assembly, we predicted 5,300 potential genes encoding proteins of at least 100 amino acids long, of which 303 (5.72%) had no significant sequence similarity to those encoded by the three previously fully sequenced P. syringae genomes. Of the core set of Hrp Outer Proteins that are conserved in three previously fully sequenced P. syringae strains, most were also conserved in strain 11528, including AvrE1, HopAH2, HopAJ2, HopAK1, HopAN1, HopI, HopJ1, HopX1, HrpK1 and HrpW1. However, the hrpZ1 gene is partially deleted and hopAF1 is completely absent in 11528. The draft genome of strain 11528 also encodes close homologues of HopO1, HopT1, HopAH1, HopR1, HopV1, HopAG1, HopAS1, HopAE1, HopAR1, HopF1, and HopW1 and a degenerate HopM1'. Using a functional screen, we confirmed that hopO1, hopT1, hopAH1, hopM1', hopAE1, hopAR1, and hopAI1' are part of the virulence-associated HrpL regulon, though the hopAI1' and hopM1' sequences were degenerate with premature stop codons. We also discovered two additional HrpL-regulated effector candidates and an HrpL-regulated distant homologue of avrPto1. CONCLUSION: The draft genome sequence facilitates the continued development of P. syringae pathovar tabaci on wild tobacco as an attractive model system for studying bacterial disease on plants. The catalogue of effectors sheds further light on the evolution of pathogenicity and host-specificity as well as providing a set of molecular tools for the study of plant defence mechanisms. We also discovered several large genomic regions in Pta 11528 that do not share detectable nucleotide sequence similarity with previously sequenced Pseudomonas genomes. These regions may include horizontally acquired islands that possibly contribute to pathogenicity or epiphytic fitness of Pta 11528.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Pseudomonas syringae/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Hibridização Genômica Comparativa , Sequência Conservada , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Genômica/métodos , Pseudomonas syringae/patogenicidade , Regulon , Análise de Sequência de DNA , Fator sigma/genética , Fator sigma/metabolismo , VirulênciaRESUMO
Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.