Transcription profiling by array of Arabidopsis after inoculation with Pseudomonas syringae pv. tomato
ABSTRACT: Pseudomonas syringae pv. tomato DC3000 (Pst) is a virulent pathogen, which causes disease on tomato and Arabidopsis. The type III secretion system (TTSS) plays a key role in pathogenesis by translocating virulence effectors from the bacteria into the plant host cell, while the phytotoxin coronatine (COR) contributes to virulence and disease symptom development. Recent studies suggest that both the TTSS and and COR are involved in the suppression of host basal defenses. However, little is known about the interplay between the host gene expression associated with basal defenses and the virulence activities of the TTSS and COR during infection. The global effects of the TTSS and COR on host gene expression associated with other host cellular processes during bacterial infection are also not well characterized. In this study, we used the Affymetrix full genome chip to determine the Arabidopsis transcriptome associated with basal defense to Pst DC3000 hrp mutants and the human pathogenic bacterium Escherichia coli O157:H7. We then used Pst DC3000 virulence mutants to characterize Arabidopsis transcriptional responses to the action of hrp-regulated virulence factors (e.g., TTSS and COR) during bacterial infection. Additionally, we used bacterial fliC mutants to assess the role of the PAMP flagellin in induction of basal defense-associated transcriptional responses. In total, our global gene expression analysis identified more than 5000 Arabidopsis genes that are reproducibly regulated more than 2-fold in three independent biological replicates of at least one type of comparison. Regulation of these genes provides a molecular signature for Arabidopsis basal defense to plant and human pathogenic bacteria, and illustrates both common and distinct global virulence effects of the TTSS, COR, and possibly other hrp-regulated virulence factors during Pst DC3000 infection. Experimenter name = William Underwood; Experimenter phone = 517-353-9182; Experimenter fax = 517-353-9168; Experimenter address = Michigan State University; Experimenter address = 222 Plant Biology Building; Experimenter address = 178 Wilson R.d. Experimenter address = East Lansing, MI; Experimenter zip/postal_code = 48824; Experimenter country = USA Experiment Overall Design: 40 samples were used in this experiment
Project description:Many bacterial pathogens of plants and humans cause infections by delivering effector proteins into host cells. Elucidation of how pathogen effector proteins function not only is critical for understanding bacterial pathogenesis, but also provides an important tool in discovering the functions of host genes. In this study, we characterized the Pseudomonas syringae pv. tomato DC3000 effector AvrE, the founding member of a widely distributed, yet functionally enigmatic, bacterial effector family. We show that AvrE is localized in the plasma membrane (PM) and PM-associated vesicle-like structures in the plant cell. AvrE contains two physically interacting domains, and the N terminal portion contains a plasma membrane localization signal. Genome-wide microarray analysis indicates that AvrE, as well as a functionally-redundant effector HopM1, down-regulates the expression of the NDR1/HIN1-Like 13 gene in Arabidopsis. Mutational analysis shows that NHL13 is required for plant immunity, as the nhl13 mutant plant displayed enhanced disease susceptibility. Our results defined the site of action of one of the most important bacterial virulence proteins in plants and the anti-bacterial immunity function of the NHL13 gene. To determine whether the type III effector AvrE’s virulence function is reflected by a possibly unique host gene expression signature, we conducted a genome-wide microarray analysis of Arabidopsis during Pst DC3000 infection. Because AvrE and another effector, HopM1, in Pst DC3000 are functionally redundant, the following bacterial strains were used to discern the shared and specific contributions of AvrE and HopM1 to host gene expression: Pst DC3000 (avrE+, hopM1+), the avrE mutant (hopM1+), the hopM1 mutant (avrE+) and the avrE hopM1 double mutant. Overall design: Leaves of 5-week-old Arabidopsis Col-0 plants were vacuum-infiltrated with Pst DC3000, the avrE mutant, the hopM1 mutant or the avrE hopM1 double mutant at 1x108 cfu/ml. Tissue samples were collected by snap-freezing in liquid nitrogen 7 h after bacterial infiltration and RNA was extracted using RNeasy plant mini kits (Qiagen). The Gene Chip Arabidopsis ATH1 genome array (Affymetrix) was used for microarray analysis in the Research Technology Support Facility at Michigan State University.
Project description:Pseudomonas syringae pv. tomato DC3000 is a model pathogen infecting tomato and Arabidopsis plants. Genes encoding the type III secretion system and substrate proteins (collectively called TTSS genes) of this bacterium are induced in plants and in minimal medium (MM). The induction of TTSS genes is mediated by HrpL, an alternative sigma factor recognizing the hrp box in the promoter of TTSS genes. The transcription of hrpL is activated by HrpR and HrpS, two homologous DNA binding proteins encoded by the hrpRS operon. Microarray analysis was conducted to evaluate the DC3000 genes regulated by hrpL and hrpRS in MM. The analysis identified a number of novel hrpLactivated genes with a putative TTSS-independent function. Genes regulated by hrpL were mostly regulated by hrpRS in the same manner, but a large number of genes regulated by hrpRS were hrpL-independent, indicating that hrpL represents one branch of the regulatory pathways downstream of hrpRS. The induction of the TTSS genes was associated with down-regulation of the house-keeping genes, indicating that the activation of the TTSS has a cost on the basic cellular activities. The novel genes/pathways identified by the microarray provide new insight into the bacterial functions coordinating with the TTSS. Overall design: Three samples (biological replicates) of each condition and mutation were analyzed, except for hrpRS-, which has two. 11 Samples analyzed.
Project description:Arabidopsis thaliana infiltrated with different strains of DC3000 were used to elicit PTI, ETI, or a disease response compared to a mock control. This experiment was carried out in both Col-0 wild-type plants as well as mutants deficient in non-sense mediated decay via disruption of the promoter of upf1 (genotype background upf1-5). To identify differentially expressed and alternatively spliced transcripts elicited by bacterial infection, the NMD-impaired and wild-type host transcriptomes were profiled using RNA-seq. Wild-type and upf1-5 mutant plants were independently challenged with pathogenic (DC3000 COR-) or non-pathogenic P. syringae strains (DC3000 COR- ΔhrpS and DC3000 COR- avrPphB).
Project description:The sfr6 mutant was identified on the basis of its failure to cold acclimate, and exhibits a marked deficiency in cold-and osmotic stress-inducible gene expression (Knight et al., 1999). We have demonstrated that genes of this type (so-called COR genes) are misregulated if they contain the DRE (drought-responsive element, or CRT; C-repeat) cis acting element in their promoter (Boyce et al., 2003). Micro-array analysis has allowed us to identify a number of COR genes misregulated in sfr6, all of which contain the DRE element. However, these experiments have indicated that other genes, not of the COR group, are also misregulated in the mutant and these do not contain the DRE element. We chose the three non-COR genes that were most clearly down-regulated in sfr6 on our previous micro-array, and identified each as of these as dark-inducible. We now seek to address two questions: (1) Can we discover more dark-regulated genes that are down-regulated in the mutant, and thus identify a second cis-acting element with which SFR6 may be interacting? (2) Do all of the non-COR genes that are misregulated in sfr6 fall into the category of dark-inducible, or is SFR6 likely to be interacting with three or more regulons?; For this micro-array experiment, we will subject sfr6 and wild type Arabidopsis, grown in a 16/8-h light/dark cycle, to darkness for 3h or 6h at ambient growth temperature. Under these conditions we see strong up-regulation of the three genes we have identified as dark-inducible, and we see clear down-regulation of expression in sfr6. References. Knight H, Veale E, Warren GJ, Knight MR (1999) The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif. Plant Cell 11: 875-886. Boyce JM, Knight H, Deyholos M, Openshaw MR, Galbraith DW, Warren G, Knight MR (2003). The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant Journal 34: 395-406. Experimenter name = Heather Knight; Experimenter phone = 01865 275023; Experimenter fax = 01865 275074; Experimenter institute = University of Oxford; Experimenter address = Department of Plant Sciences; Experimenter address = University of Oxford; Experimenter address = South Parks Road; Experimenter address = Oxford; Experimenter zip/postal_code = OX1 3RB; Experimenter country = UK Experiment Overall Design: 4 samples were used in this experiment
Project description:This study evaluates the transcriptome of Arabidopsis thaliana infected with the Pseudomonas syringae strain DC3000 cor- carrying the type three secretion system effector HopBB1 Overall design: Two-week-old Arabidopsis thaliana seedlings (Col-0 ecotype) were sprayed with a mock solution (10 mM MgCl2) or bacteria [Pto DC3000 (EV), Pto DC3000 cor- (EV); Pto DC3000 cor- (HopBB1); Pto DC3000 cor- (HopBB1G126D)] at OD600=0.2 with 10mM MgCl2 and 0.04% Silwet L-77. Samples were harvested 24 hours after infection. This experiment included three biological replicates, each one corresponding to approximately 30 seedlings grown in the same pot.
Project description:Innate immune responses of plant cells confer the first line of defence against pathogens. Signals generated by activated receptors are integrated inside the cell and converge on transcriptional programmes in the nucleus. In Arabidopsis, the CAMTA family of transcription factors plays a pivotal function in immunity. CAMTA binding motifs are highly enriched in the genes quickly induced during ETI and PTI. Using RNA-seq, we investigated the role of CAMTA TFs during the early ETI and PTI transcriptional responses. Overall design: We compared the expression changes between an Arabidopsis mutant line carrying the camta3-D (sr1-4d) dominant negative mutation and Col-0 wild-type plants following treatment with the PAMP flg22 (100 nM), and the avirulent bacterial strains Pst DC3000 AvrRpm1 (OD600=0.001) and Pst DC3000 AvrRps4 (OD600=0.001). The expression changes were analysed at one time point after each treatment (1 hour for flg22 and 4 hours for Pst DC3000 AvrRpm1 and Pst DC3000 AvrRps4 treatments).
Project description:In our laboratory we are interested in studying the functions of WRKY zinc finger type transcription factors. There are 74 members of this gene family in Arabidopsis. WRKY factors are key regulators of distinct plant defense responses and are involved in certain developmental programs e.g. trichome development and plant leaf senescence. We would like to determine the functions of a small sub-group (group II-a) of WRKY factors in response to bacterial infection. Our aim is to compare and contrast the gene expression profiles of 3-week-old wild type and WRKY T-DNA knockout mutants grown in a growth chamber under long day growth conditions and subsequently challenged for 6 hours with the virulent bacterial pathogen /Pseudomonas syringae/ DC3000. Experimenter name: Bekir Uelker; Experimenter phone: 49-221-5062-310; Experimenter fax: 49-221-5062-353; Experimenter department: Somsissich Lab; Experimenter institute: Max-Planck-Institute for Plant Breeding Research; Experimenter address: Department of Plant Microbe Interactions; Experimenter address: Carl-von-Linne-Weg 10; Experimenter address: Cologne; Experimenter zip/postal_code: 50829; Experimenter country: Germany Experiment Overall Design: 7 samples were used in this experiment
Project description:To characterize the PTI response of tomato and the effect of the delivery of a subset of effectors, we performed an RNA-seq analysis of tomato Rio Grande prf3 leaves challenged with either the flgII-28 peptide or the following bacterial strains: Agrobacterium tumefaciens GV2260, Pseudomonas fluorescens 55, Pseudomonas putida KT2440, Pseudomonas syringae pv. tomato (Pst) DC3000, Pst DC3000 deltahrcQ-U deltafliC and Pst DC3000 deltaavrPto deltaavrPtoB. NOTE: Samples in SRA were assigned the same sample accession. This is incorrect as there are different samples, hence “Source Name” was replaced with new values. Comment[ENA_SAMPLE] contains the original SRA sample accessions.
Project description:To reveal the underlying molecular mechanism of GH3.5 action in modulating the SA and auxin pathways, we performed transcriptional profiling of gh3.5-1D plants after infection with or without Pst DC3000(avrRpt2) on a global scale using the Affymetrix Arabidopsis ATH1 GeneChip Experiment Overall Design: Each 3 leaves of plants of 5-week-old wild type Columbia-0 and mutant gh3.5-1D (heterozygous) were inoculated with Pst DC3000(avrRpt2)at 105 cfu mL-1. Leaves were harvested at 0 h (uninoculated) and 48 hours after inoculation. Three biological repeats were performed on Columbia-0 (wild-type, Col-0) and gh3.5-1D (mutant) after infection with or without Pst DC3000(avrRpt2), respectively.
Project description:The aim of this BBSRC-funded project is to develop laser-capture microdissection (LCMD) to isolate small cell clusters in different regions of arabidopsis embryos at different stages of development; to develop RNA amplification procedures on dissected tissue sampes; and to use DNA microarray techniques to investigate global transcriptional differences between samples. Cryosectioned embryos of ecotype Col-O of globular, heart and torpedo stage were used to isolate cell clusters from the apical and basal regions, for RNA isolation and amplification. !Samples will be provided as T7-primed cDNA, with three biological replicates for each tissue to be analysed. Each replicate comprises cDNA from pooled tissue samples from ca. 15 embryos. The experimental details have been discussed with Sean May et al. at NASC. Experimenter name = Stuart Casson and Matthew Spencer Experimenter phone = 0191 374 7356 Experimenter fax = 0191 374 2417 Experimenter institute = Durham University Experimenter address = Integrative Cell Biology Laboratory Experimenter address = School of Biological Sciences Experimenter address = Durham University Experimenter address = South Road Experimenter address = Durham Experimenter zip/postal_code = DH1 3LE Experimenter country = UK Keywords: organism_part_comparison_design; development_or_differentiation_design Overall design: 27 samples were used in this experiment