Project description:The goal of the microarray experiment was to identify defense genes that were differentially expressed in the Arabidopsis mutant elp2 and wild type in response to infection of the necrotrophic fungal pathogen Botrytis cinerea. Results indicated that, compared with the wild type, the WRKY33/OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59)/ETHYLENE RESPONSE FACTOR1 transcriptional cascades are down-regulated, whereas the MYC2 transcriptional cascade is up-regulated in the elp2 mutant. Three biological replicates with leaves from 8-12 plants per sample were collected at 0, 3, 6, 12, 24, and 48 hours after inoculation with the necrotrophic fungal pathogen Botrytis cinerea. 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). Equal amount of RNA from the 3 biological replicates were used for microarray analysis. The channels of the dual-channel arrays were analyzed independently.
Project description:Full transcriptomes of the Botrytis cinerea wild-type strain B0510 inoculated on mature grapevine berries (Marselan cultivar) at 16h, 24h, 48h, and in vitro were compared to identify B. cinerea genes diffentially expressed during the infection stages. Grapevine berries were inoculated with Botrytis cinerea conidia and samples were taken at 16h, 24h and 48 hours post-inoculation. An additional control sample corresponding to mycelium growing on static liquid minimal medium was included in the experimental design in order to identify genes that would be differentially expressed between in vitro and in planta growth conditions. 3-4 replicates were performed. The 13 total-RNA samples were used for hybridization on NimbleGen 4plex gene expression arrays (20,885 gene models from Botrytis cinerea with three 60-mer probes per gene).
Project description:Arabidopsis thaliana exhibits differential susceptibility to the fungal pathogen Botrytis cinerea depending on the time of day that infection occurs. We hypothesised that this is driven by teh circadian clock and that differences in the amplitude or speed of the plant defence response will underlie the difference in susceptiblity. A major component of the defence response is transcriptional reprogramming, hence we investigated whether the transcriptional response to B. cinerea infection differs following inoculation at subjective dawn or night (the points of greatest difference in susceptiblity) under constant light conditions. Overall design: Arabidopsis thaliana (ecotype Col-0) plants were grown for four weeks under 16h:8h light:dark conditions and then moved into constant light 24 hours prior to infection. Detached leaves were inoculated with Botrytis cinerea spores (5 x 104 spores mL-1) or mock-inoculated at subjective dawn or subjective night. Leaves were harvested for transcriptome profiling at 18 and 22 hours after inoculation. Four individual leaves were pooled for one sample. Two technical replicates were carried out for each sample. Gene expression was analysed using NimbleGen 12 x 135K microarrays, designed for the TAIR10 genome. Data was normalised using the Robust Multichip Averaging algorithm.
Project description:Microbial infections in plant leaves remain a major challenge in agriculture. Hence an understanding of disease mechanisms at the molecular level is of paramount importance for identifying possible intervention points for their control. Whole-transcriptome changes during early disease stages in susceptible plant species are less well-documented than those of resistant ones. This study focuses on the differential transcriptional changes at 24?hours post inoculation (hpi) in tomato leaflets affected by three pathogens: (1) Phytophthora infestans, (2) Botrytis cinerea, and (3) Oidium neolycopersici. Grey mould (B. cinerea) was the disease that had progressed the most by 24?hpi, both in terms of visible symptoms as well as differential gene expression. By means of RNA-seq, we identified 50 differentially expressed tomato genes specifically induced by B. cinerea infection and 18 specifically induced by P. infestans infection at 24?hpi. Additionally, a set of 63 genes were differentially expressed during all three diseases when compared by a Bayesian approach to their respective mock infections. And Gene expression patterns were found to also depend on the inoculation technique. These findings suggest a specific and distinct transcriptional response in plant leaf tissue in reaction to B. cinerea and P. infestans invasion at 24?hpi, indicating that plants may recognize the attacking pathogen.
Project description:Plant nitrogen (N) fertilization is known to affect disease; however, the underlying mechanisms remain mostly unknown. We investigated the impact of N supply on the Arabidopsis thaliana-Botrytis cinerea interaction. A. thaliana plants grown in low nitrate were more tolerant to all wild-type B. cinerea strains tested. We determined leaf nitrate concentrations and showed that they had a limited impact on B. cinerea growth in vitro. For the first time, we performed a dual RNA-Seq of infected leaves of plants grown with different nitrate concentrations. Transcriptome analysis showed that plant and fungal transcriptomes were marginally affected by plant nitrate supply. Indeed, only a limited set of plant (182) and fungal (22) genes displayed expression profiles altered by nitrate supply. The expression of selected genes was confirmed by quantitative reverse transcription PCR at 6 hr postinfection (hpi) and analysed at a later time point (24 hpi). We selected three of the 22 B. cinerea genes identified for further analysis. B. cinerea mutants affected in these genes were less aggressive than the wild-type strain. We also showed that plants grown in ammonium were more tolerant to B. cinerea. Furthermore, expression of the selected B. cinerea genes in planta was altered when plants were grown with ammonium instead of nitrate, demonstrating an impact of the nature of N supplied to plants on the interaction. Identification of B. cinerea genes expressed differentially in planta according to plant N supply unveils two novel virulence functions required for full virulence in A. thaliana: a secondary metabolite (SM) and an acidic protease (AP).
Project description:BACKGROUND: In a previous study we have shown that wounding of Arabidopsis thaliana leaves induces a strong and transient immunity to Botrytis cinerea, the causal agent of grey mould. Reactive oxygen species (ROS) are formed within minutes after wounding and are required for wound-induced resistance to B. cinerea. RESULTS: In this study, we have further explored ROS and resistance to B. cinerea in leaves of A. thaliana exposed to a soft form of mechanical stimulation without overt tissue damage. After gentle mechanical sweeping of leaf surfaces, a strong resistance to B. cinerea was observed. This was preceded by a rapid change in calcium concentration and a release of ROS, accompanied by changes in cuticle permeability, induction of the expression of genes typically associated with mechanical stress and release of biologically active diffusates from the surface. This reaction to soft mechanical stress (SMS) was fully independent of jasmonate (JA signaling). In addition, leaves exposed soft mechanical stress released a biologically active product capable of inducing resistance to B. cinerea in wild type control leaves. CONCLUSION: Arabidopsis can detect and convert gentle forms of mechanical stimulation into a strong activation of defense against the virulent fungus B. cinerea.
Project description:Transcriptional profiling of Arabidopsis leaves comparing mock-treated leaves with Botrytis cinerea infected leaves over a time-course (12 and 24 hrs). Overall design: Two-condition experiment, Mock-treated vs infected leaves. Biological replicates: 5 Mock-treated, 5 infected, independently grown and harvested. One replicate per array.
Project description:Many eukaryotic RNAs have been considered non-coding as they only contain short open reading frames (sORFs). There is increasing evidence for the translation of these sORFs into bioactive peptides. Yet only a few small peptides are annotated in the model organism Arabidopsis thaliana. To aid the functional annotation of small peptides, we have developed ARA-PEPs, a repository and webserver of putative peptides encoded by sORFs in the Arabidopsis genome from in house Tiling arrays, RNA sequencing and from publicly available datasets. In order to identify novel oxidative stress-induced peptides in Arabidopsis thaliana a tiling array analysis (GeneChip® Arabidopsis Tiling 1.0R Arrays ) was performed on mRNA extracted from leaves inoculated with Botrytis cinerea (BC). Normalized log signals were obtained using the Affymetrix Tiling Analysis Software - Version 1.1, Build 2. ON and OFF probes were selected using a threshold, based on positive controls. Next, groups of 4-13 successive ON probes were combined into short TARs and a selection was made of TARs having an average signal intensity at least 2.6-fold higher after BC treatment compared to the control treatment, resulting in 195 BC induced TARs. Overall design: Botrytis cinerea treated A. thaliana leaves (2 replicates) vs control (water) treated A. thaliana leaves (2 replicates), (4 samples in total)
Project description:The Arabidopsis thaliana mutant wrky33 is highly susceptible to the necrotrophic fungus Botrytis cinerea. Comparing the expression profiles of B. cinerea-infected wrky33 and WT plants we identified 2765 differentially expressed genes dependent on WRKY33, of which 1675 were up-regulated in the mutant (termed WRKY33-repressed genes) and 1090 were down-regulated in the mutant. Combined with ChIP-seq data 318 genes were identified as direct functional targets of WRKY33 at 14 h post inoculation with spores of Botrytis cinerea 2100. Comparison of altered gene expression in Arabidopsis WT and wrky33 mutant plants 14 hours post inoculation with Botrytis cinerea 2100.
Project description:Transcriptional profiling of Arabidopsis leaves comparing mock-treated leaves with Botrytis cinerea infected leaves over a time-course (12 and 24 hrs). Two-condition experiment, Mock-treated vs infected leaves. Biological replicates: 5 Mock-treated, 5 infected, independently grown and harvested. One replicate per array.