ABSTRACT: A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves (Botrytis cinerea infection).
Project description:A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves (Dark Induced Senescence).
Project description:A network model has been generated that describes the immediate gene expression cascade surrounding three similar but distinct NAC transcription factors that have roles to play in leaf senescence and in many stress responses in Arabidopsis. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1 hybrid analysis and modeling using gene expression time course data has been used to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modeling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions.. Gene expression measurements in mutants of ANAC019 and ANAC055 at different times during leaf senescence have shown a distinctly different role for each of these genes. To assess mutant response to Botrytis cinerea infection, Col-0, myb2 and myb108 leaves were inoculated with 4-6 (depending on leaf size) evenly spaced 10μl droplets of B. cinerea spores. Infected leaves were harvested to provide 4 biological replicates. Samples for the comparison between myb108 and Col-0 were harvested at 26 and 30 hpi. Samples for the comparison between myb2 knockout line and Col-0 or myb2 knockout line and Col-0 were harvested at 26 and 30 hpi or 24 and 30 hpi respectively. Analysis of expression differences between Col-0 and myb2 and Col-0 and myb108 under each condition, using dye swaps, was performed using the R Bioconductor package limmaGUI (Wettenhall and Smyth, 2004).
Project description:A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves (expression between lines at timepoints).
Project description:Transcriptional profiling of Arabidopsis leaves comparing mock-treated leaves with Botrytis cinerea infected leaves over a time-course (12 and 24 hrs).
Project description:To screen Botrytis genes activated in infection process, we performed gene expression profiling analysis using data obtained from RNA-seq of Botrytis cinerea cultured in vitro or infecting Arabidopsis leaves.
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.
Project description:NACs (NAM/ATAF/CUC) proteins are plant-specific transcription factors with key roles in plant development and stress responses. Here, Vitis vinifera NAC61 was selected for its functional characterization as one of the key candidates of the grape post-harvest withering process. VviNAC61 is particularly expressed at veraison and post-veraison berry stages and also shows a high expression in lignified tissues such as roots, tendrils, seeds and rachis. In leaves, VviNAC61 achieves its highest expression at senescence stage. In line with this observation, a NAC family multispecies phylogenetic tree revealed that VviNAC61 clusters with VviNAC33 and AtANAC046, both involved in regulating senescence processes. VviNAC61 was transiently overexpressed in N. benthamiana leaves, showing wilting/water-soaking symptoms correlated to low stomatal conductance, high leaf temperature, increased loss of the membrane integrity and accumulation of hydrogen peroxide and water. Transcriptomic analysis of cultivar ‘Thompson Seedless’ leaves transiently overexpressing VviNAC61 and the search of TF-bound genes by DAP-seq was allowed us to narrow down a list of VviNAC61-regulated genes. The ‘secondary metabolism’, ‘transcription factor regulation’ and ‘oxidative stress’ functional categories were found to be the most enriched among the candidate target genes. More in detail, several genes encoding for the phenylpropanoid pathway-related enzymes, several MYBs and NAC genes (including VviNAC61), were bound in their promoter region by this TF and were among the upregulated genes. Direct regulation of the stilbene synthase regulator VviMYB14, the abiotic stress-related gene DHN1b, the Botrytis cinerea susceptibility gene VviWRKY52 and the VviNAC61 self-regulation were validated through dual luciferase assays. Lastly, the upstream regulation mechanism of VviNAC61 was further investigated demonstrating that VviNAC61 is controlled by and interacts with VviNAC60, a proposed master regulator of grapevine organ maturation.
Project description:Full transcriptomes of the Botrytis cinerea wild-type strain B0510 and the null-mutants deltaBcVEL1 and deltaBcLAE1, cultured onto solid grape juice medium with cellophane overlays , were compared to identify BcVEL1 or/and BcLAE1-dependent genes. The Botrytis cinerea wild-type strain and the null-mutants deltaBcVEL1 and BcLAE1 were cultured for 48h onto solid grape juice medium with cellophane overlays. 4 replicates were performed. The 12 total-RNA samples (3 strains* 4 replicates) 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: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).
