Project description:Botrytis cinerea, the causal agent of the gray mold, is one of the most important phytopathogenic fungi due to its ability to affect more than 1400 cultivable vegetal species. Signal transduction cascades mediate the dialogue between the environment, the plant and the fungus, which is essential in the infection process of B. cinerea. In this context, surface proteins (surfactome) have an important role as the first receptors in the signaling cascades, connecting the fungus response to the environment changes. Moreover, the relevant role of surfactome in infection process of pathogenic microorganism has been described, but nothing has been reported in B. cinerea. Therefore, in order to unravel new proteins and to complete the whole view of signal transduction cascades during infection of B. cinerea, surfactome of this fungus has been analyzed under two plant-based elicitors previously validated by the research group: glucose (GLU) as a constitutive stage and deproteinized tomato cell wall (TCW) as a virulence inductor. To identify the B. cinerea surfactome, the trypsin shaving of intact living cells approach has been optimized. Subsequently, all the peptide mixture obtained in surfactome isolation was analyzed using LC-MS/MS to identify their components. Our results shows that none of the used protocols disturbs the hyphae integrity, obtaining the best when PBS buffer plus 30% sucrose was used. In conclusion, in this work it has been carried out for the first time the optimization of a surface protein extraction protocol in Botrytis cinerea giving rise to a new subproteome; the surfactome. Integration of surfactome data inside the previous proteomics data collected by the research group has improved the complete view of signal transduction cascades during infection of B. cinerea, helping to unravel key points in the regulation of this process and therefore to further develop new and better discriminatory fungicides from a molecular approach.
Project description:Tomato fruit ripening is associated with a dramatic increase in susceptibility to the fungal pathogen Botrytis cinerea, the causal agent of gray mold. Mature green fruit, prior to ripening, are largely resistant to B. cinerea, whereas red fruit, at the end of ripening, are susceptible to B. cinerea infection. We used microarrays to detail the gene expression changes that are induced by B. cinerea when tomato fruit at unripe and ripe stages are infected. Keywords: plant responses to pathogens
Project description:Tomato fruit ripening is associated with a dramatic increase in susceptibility to the fungal pathogen Botrytis cinerea, the causal agent of gray mold. Mature green fruit, prior to ripening, are largely resistant to B. cinerea, whereas red fruit, at the end of ripening, are susceptible to B. cinerea infection. We used microarrays to detail the gene expression changes that are induced by B. cinerea when tomato fruit at unripe and ripe stages are infected. Experiment Overall Design: Tomato fruit at mature green and red ripe stages were wound inoculated with a water suspension of B. cinerea conidia. Twenty four hours post inoculation fruit pericarp and epicarp tissue around and including the inoculation sites was collected and the total RNA extracted. Total RNA was also collected from healthy and mock inoculated fruit.
Project description:Gray mold disease affects fruits, vegetables and ornemental plants around the world, causing considerable losses every year. Its causing agent, the fungus Botrytis cinerea, produces infection cushions (IC) that are complex appressorial structure dedicated to the penetration of the plant tissues. A microarray analysis identified 1,231 up-regulated genes in IC-enriched mycelium, among which several genes coding for putative secreted proteins. These data were supported by qPCR analysis in vitro and in planta. They highlight a secretion of proteins involved in virulence like proteases, plant cell wall degrading enzymes and necrosis inducers. To validate these transcriptomic data, a proteomic analysis of the IC exo-proteome is required.
Project description:The VELVET complex in the gray mold fungus Botrytis cinerea: impact of BcLAE1 on differentiation, secondary metabolism and virulence
Project description:Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infectious process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus development. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes up-regulated during infection of MB are enriched in functional categories related to necrotrophy such as degradation of plant cell wall, proteolysis, membrane transport, reactive oxygen species generation and detoxification. Quantitative-PCR on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but stops at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathways switch during berry ripening. In response to B. cinerea infection, VB activated a burst of reactive oxygen species (ROS), the salicylate (SA)-dependent defense pathway, the synthesis of the resveratrol phytoalexin and cell-wall strengthening. In opposite, infected MB activated the jasmonate (JA)-dependent pathway which does not stop the fungal necrotrophic process. Grapevine berries at veraison (VB) and harvesting stages (MB) were inoculated with Botrytis cinerea B05-10 and samples were taken at 24h and 48h post-inoculation. An additional uninfected control sample taken at 0h post-inoculation was included in the experimental design. 3 replicates per sample were performed. The total-RNA samples were labeled and used for hybridization on NimbleGen 12plex Vitis vinifera gene expression array.
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: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.
Project description:Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infectious process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus development. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes up-regulated during infection of MB are enriched in functional categories related to necrotrophy such as degradation of plant cell wall, proteolysis, membrane transport, reactive oxygen species generation and detoxification. Quantitative-PCR on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but stops at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathways switch during berry ripening. In response to B. cinerea infection, VB activated a burst of reactive oxygen species (ROS), the salicylate (SA)-dependent defense pathway, the synthesis of the resveratrol phytoalexin and cell-wall strengthening. In opposite, infected MB activated the jasmonate (JA)-dependent pathway which does not stop the fungal necrotrophic process.
Project description:Botrytis cinerea causes grey mold disease in leading crop plants. The disease develops only at cool temperatures, but the fungus remains viable in warm climates and can survive periods of extreme heat. We discovered a strong heat priming effect in which the exposure of B. cinerea to moderately high temperatures greatly improves its ability to cope with subsequent, potentially lethal temperature conditions. We showed that priming promotes protein solubility during heat stress and discovered a group of priming-induced serine-type peptidases. Several lines of evidence, including transcriptomics, proteomics, pharmacology and mutagenesis data, link these peptidases to the B. cinerea priming response, highlighting their important roles in regulating priming-mediated heat adaptation. By imposing a series of sub-lethal temperature pulses that subverted the priming effect, we managed to eliminate the fungus and prevent disease development, demonstrating the potential for developing temperature-based plant protection methods by targeting the fungal heat priming response.