Analysis of Arabidopsis gene expression after priming with BABA and infection with Pseudomonas syringae pv tomato DC3000
ABSTRACT: Arabidopsis thaliana (Col-0) plants were treated with beta-aminobutyric acid (BABA), and gene expression differences to control plants were monitored after infection with Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Keywords: transcript profiling, response to BABA-induced priming and infection 2 independent replicates for each condition were analyzed by two color co-hybridizations. Leaf RNA from Pseudomonas-infected control plants (Cy3-labeled cDNA) cohybridized with leaf RNA from Pseudomonas-infected BABA-pretreated plants (Cy5-labeled cDNA). Samples were collected 22 hours after bacterial inoculation. BABA pretreatment was performed two days before bacterial inoculation.
Project description:Arabidopsis thaliana (Col-0) plants were treated with BABA and gene expression differences to control plants were monitored after dip-inoculation with Pseudomonas syringae pv tomato DC3000. Keywords: transcript profiling, response to BABA-induced priming and infection Overall design: 3 independant replicates were analyzed by two color co-hybridizations. Leaf RNA from Pseudomonas infected control plants (Cy3 labeled cDNA) was cohybridized with leaf RNA from Pseudomonas infected BABA pretreated plants (Cy5 labeled cDNA). Samples were collected 22 hours after bacterial inoculation. BABA pretreatment was performed two days before bacterial inoculation. To assess the effect of BABA alone on gene expression, leaf RNA from BABA treated plants (Cy5 labeled cDNA) was cohybridized with leaf RNA (Cy3 labeled cDNA) from water treated plants.
Project description:Pathogen invasion in plants is associated with transcriptional reprogramming. Enigmatically, plants induce similar transcriptome responses upon infection by virulent or avirulent pathogens. This renders the importance of transcriptional reprogramming for immunity obscure. Here, using RNA-seq, we generate time-series transcriptome data coupled with genetic perturbations to reveal temporal dynamics upon infection by virulent or avirulent strains of a bacterial pathogen, Pseudomonas syringae, in Arabidopsis thaliana. Fast and sustained transcriptional reprogramming occurs upon infection with avirulent strains while virulent strain infection leads to a slower response with comparable gene expression patterns and magnitudes. Importantly, transcriptome analysis of resistant and susceptible mutants responding to avirulent strains links delayed transcriptional reprogramming to compromised immunity. Taken together, our results pinpoint the early critical time window of transcriptional reprogramming for establishing effective immunity against the bacterial pathogen. Overall design: Leaves of Col-0 and all the single, double, triple and quadruple mutants of dde2-2, ein2-1, pad4-1, sid2-2 were syringe-infiltrated with mock (water) or suspensions of Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) carrying an empty vector (pLAFR), Pto DC3000 carrying AvrRpt2, or Pto DC3000 carrying AvrRpm1 at the OD600 of 0.001. Similarly, leaves of the rpm1-3 rps2-101C mutant plants were inoculated with mock, Pto DC3000 carrying AvrRpt2 or Pto DC3000 carrying AvrRpm1. Three fully-expanded leaves (leaves 7-9) from three different plants were harvested as a single biological replicate at 1, 2, 3, 4, 6, 9, 12, 16, 20, 24, 36, 48 hours post inoculation (hpi). To generate three biological replicates, three independent experimental trials were carried out, in which plant positions within pots and growth chambers were randomized in order to avoid undesirable systematic effects. For the statistcal analysis, 348 samples (M001-M348) were used.
Project description:This study investigates extent and functional significance of alternative splicing in Arabidopsis thaliana defense against the bacterial pathogen Pseudomonas syringae pv tomato (Pst). We have provided a detailed characterization of the Arabidopsis thaliana transcriptional response to Pseudomonas syringae infection in both susceptible and resistant hosts. We carried out two independent inoculation experiments (biological replicates) for each treatment. Col-0 is susceptible to virulent Pst DC3000 but has a functional RPS4 resistance gene effective against DC3000 expressing AvrRps4
Project description:In order to study the transcriptome changes in tomato during Pto/Prf-mediated ETI, we infiltrated tomato Rio Grande (RG)-PtoR resistant plants (plants that have a functional Pto/Prf signaling pathway, Pto/Pto, Prf/Prf) and two different susceptible plants: RG-prf3 and RG-prf19 (Pto/Pto, prf/prf), with Pseudomonas syringae pv. tomato DC3000 (DC3000). The susceptible lines have a non-functional Prf gene due to a 1.1 kb deletion or a G-insertion at position 2,584 (which causes a frameshift), respectively. We collected leaf tissue at 4 and 6 h after inoculation (hai) to assess early changes in host gene expression after translocation of DC3000 effectors AvrPto and AvrPtoB, which occur at about 3 hai. The plants were then maintained in the same conditions to observe signs of disease. As expected, RG-PtoR plants did not develop speck disease whereas the RG-prf plants did. 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:We used three different strains of Pseudomonas syringae pv tomato DC3000 to investigate systemic responses to infection in Arabidopsis and the development of SAR. Wildtype DC3000, the hrpA mutant and DC3000 carrying the avirulence gene avrRpm1 were syringe infiltrated into 4-week-old plants at a concentration of 10e8 cfu/ml. At least 5 leaves per plant were infiltrated and at least 10 plants were pooled for each sample. Systemic, uninfected tissue was then harvested at 8, 12 and 21h after inoculation. Three independent experiments were carried out to give three biological replicates for each timepoint. Overall design: 27 samples were used in this experiment.
Project description:We used three different strains of Pseudomonas syringae pv tomato DC3000 to investigate systemic responses to infection in Arabidopsis and the development of SAR. Wildtype DC3000, the hrpA mutant and DC3000 carrying the avirulence gene avrRpm1 were syringe infiltrated into 4-week-old plants at a concentration of 10e8 cfu/ml. At least 5 leaves per plant were infiltrated and at least 10 plants were pooled for each sample. Systemic, uninfected tissue was then harvested at 8, 12 and 21h after inoculation. Three independent experiments were carried out to give three biological replicates for each timepoint. 27 samples were used in this experiment.
Project description:Plant pathogens can cause serious diseases that impact global agriculture1. Understanding how the plant immune system naturally restricts pathogen infection holds a key to sustainable disease control in modern agricultural practices. However, despite extensive studies into the molecular and genetic basis of plant defense against pathogens since the 1950s2,3, one of the most fundamental questions in plant pathology remains unanswered: how resistant plants halt pathogen growth during immune activation. In the case of bacterial infections, a major bottleneck is an inability to determine the global bacterial transcriptome and metabolic responses in planta. Here, we developed an innovative pipeline that allows for in planta high-resolution bacterial transcriptome analysis with RNA-Seq, using the model plant Arabidopsis thaliana and the foliar bacterial pathogen Pseudomonas syringae. We examined a total of 27 combinations of plant immunity and bacterial virulence mutants to gain an unprecedented insight into the bacterial transcriptomic responses during plant immunity. We were able to identify specific bacterial transcriptomic signatures that are linked to bacterial inhibition during two major forms of plant immunity: pattern-triggered immunity and effector-triggered immunity. Among them, regulation of a P. syringae sigma factor gene, involved in iron regulation and an unknown process(es), was found to play a causative role in bacterial restriction during plant immunity. This study unlocked the enigmatic mechanisms of bacterial growth inhibition during plant immunity; results have broad basic and practical implications for future study of plant diseases. Overall design: Two leaves from three four-and-one-half week-old A. thaliana plants were infiltrated with either 0.005% dimethyl sulfoxide (DMSO, mock) or 500 nM flg22 using a needleless syringe. Twenty hours post-infiltration, plants were inoculated with a bacterial suspension at an OD600 of 0.75 (10^9 CFU ml-1) of Pto DC3000. Seven hours after Pto DC3000 inoculation, leaves were collected for RNA extraction. Three independent replicates (including 3 technical replicates for flg22 treated samples) were taken.
Project description:Natural variation within plant species is an important resource for discovery of genes controlling biological traits. Gene-expression profiling of natural variation is increasingly used to identify genes affecting a trait. Here, we explored variation among Arabidopsis thaliana accessions with respect to defense against Pseudomonas syringae pv. tomato DC3000 (Pst), with a focus on R-gene mediated resistance triggered by the Pst type III effector protein AvrRpt2. We explored variation at two phenotypic levels: growth of the bacteria and hypersensitive response (HR) measured by electrolyte leakage. Considerable variation among accessions was found at both phenotypic levels. The genetic variation among accessions affected both growth of Pst with (Pst avrRpt2) and without (Pst) the AvrRpt2 effector, with some variation being specific for the bacterial strains, and other variation affecting both strains in a similar manner. Variation in HR was not correlated with variation in bacterial growth. Additionally we examine variation in gene-expression profiles after mock- and Pst avrRpt2-inoculated plants, obtained using a dedicated microarray Gene-expression profiling at 6 h post inoculation identified clusters of genes from which expression levels are correlated with bacterial growth and electrolyte leakage. The expression levels of some of these clusters correlate with more than one phenotypic characteristic, such as growth of both Pst and Pst avrRpt2, whereas other clusters were correlated with just one biological parameter. Thus we demonstrate that variation in gene-expression profiles among Arabidopsis accessions is correlated with variation in phenotypic responses. Keywords: Comparisons of Arabidopsis thaliana accessions with respect to inoculation with Pseudomonas syringae pv tomato avrRpt2. Overall design: We compared gene-expression six hours after mock- or Pst avrRtp2-inoculation of nine Arabidopsis thaliana accessions (Col-0, Cvi-1, Est-1, Kas-1, Kin-0, Ler-0, Mt-0, Tsu-1, Ws-2), using three replicates per accession-treatment combination.
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:The goal of the microarray experiment was to determine the induction kinetics of transcriptome changes in the Arabidopsis mutant Atelp2, npr1 and wild type in response to infection of the avirulent bacterial pathogen Pst DC3000/avrRpt2. Results indicated that Atelp2 exhibited slower kinetcis of transcriptional changes than the wild type after Pst DC3000/avrRpt2 infection, whereas npr1 did not show significant alteration in the induction kinetics. Three biological replicates with leaves from 8 plants per sample were collected at 0, 4, 8, and 12 hours after inoculation with the avirulent bacterial pathogen Pst DC3000/avrRpt2. After extraction, RNA concentration was determined on a NanoDrop Spectrophotometer (Thermofisher Scientific, Waltham, MA) and sample quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Each replicate was used as one sample for microarray analysis.