Project description:The primary cell walls of land plants are composed principally of a load bearing cellulose microfibril-hemicellulose network embedded in a matrix of pectic polysaccharides. The pectic matrix is multifunctional and in additional to a directly structural role it is central to many fundamental plant processes including cell expansion, defence and cell signalling. The sequencing of the Arabidopsis genome has revealed the massive investment made by plants in modulating the pectic matrix in response to local functional requirements but despite concerted biochemical-based efforts over many years none of the genes involved in pectin biosynthesis/pectic matrix assembly have so far been identified. The pectin matrix contains some of the most complex polysaccharides found in nature and based on linkage analysis it is known that at least 53 glycosyltransferases must be involved in its construction. Our proposal to identify genes involved in pectin biosynthesis and matrix assembly exploits the well characterised phenomenon that many plants and cultured plant cells that are exposed to treatments that disrupt the synthesis of one cell wall component are capable of a compensatory increases in other components - including pectin. Specifically suspension cultured cells that are incrementally exposed to increasing concentrations of the herbicide 26-dichlorobenzonitrile (DCB) which specifically inhibits cellulose synthesis compensate for the resulting almost complete loss of cellulose from their cell walls by constructing walls made predominantly of pectin. We believe that the significant up-regulation of pectin biosynthesis in this system offers an opportunity to identify genes that function in the assembly of the pectic matrix by microarray comparison of transcripts of DCB-treated Arabidopsis cells with untreated cells. The use of Arabidopsis suspension-cultured cells rather than plants or seedlings offers the significant advantage that extracted RNA would be derived from only one cell type. It is anticipated therefore that the output from a transcriptome analysis of this system will indicate a number of genes of unknown function and lead to the identification of genes involved in pectin biosynthesis and the assembly of the pectin matrix. Experiment Overall Design: 6 samples
Project description:The primary cell walls of land plants are composed principally of a load bearing cellulose microfibril-hemicellulose network embedded in a matrix of pectic polysaccharides. The pectic matrix is multifunctional and in additional to a directly structural role it is central to many fundamental plant processes including cell expansion, defence and cell signalling. The sequencing of the Arabidopsis genome has revealed the massive investment made by plants in modulating the pectic matrix in response to local functional requirements but despite concerted biochemical-based efforts over many years none of the genes involved in pectin biosynthesis/pectic matrix assembly have so far been identified. The pectin matrix contains some of the most complex polysaccharides found in nature and based on linkage analysis it is known that at least 53 glycosyltransferases must be involved in its construction. Our proposal to identify genes involved in pectin biosynthesis and matrix assembly exploits the well characterised phenomenon that many plants and cultured plant cells that are exposed to treatments that disrupt the synthesis of one cell wall component are capable of a compensatory increases in other components - including pectin. Specifically suspension cultured cells that are incrementally exposed to increasing concentrations of the herbicide 26-dichlorobenzonitrile (DCB) which specifically inhibits cellulose synthesis compensate for the resulting almost complete loss of cellulose from their cell walls by constructing walls made predominantly of pectin. We believe that the significant up-regulation of pectin biosynthesis in this system offers an opportunity to identify genes that function in the assembly of the pectic matrix by microarray comparison of transcripts of DCB-treated Arabidopsis cells with untreated cells. The use of Arabidopsis suspension-cultured cells rather than plants or seedlings offers the significant advantage that extracted RNA would be derived from only one cell type. It is anticipated therefore that the output from a transcriptome analysis of this system will indicate a number of genes of unknown function and lead to the identification of genes involved in pectin biosynthesis and the assembly of the pectin matrix. Keywords: compound_treatment_design
Project description:A. thaliana lines with reduced lignin content through down-regulation of the lignin biosynthesis enzymes cinnamoyl CoA reductase (CCR) exhibit extensive cell wall remodeling which results in the release of a mixture of pectic oligosaccharide elicitors of pathogenesis-related (PR) protein gene expression through the salicylic acid signaling pathway. Loss of function of FERONIA, a CrRLK1-like subfamily receptor-like kinase resulted in loss of PR-1, -2 and- 5 gene activation in stems of the ccr1/fer-4 double mutant.
Project description:In this study we show that the Arabidopsis transcription factor MYB46, previously described to regulate secondary cell wall biosynthesis in the vascular tissue of the stem, is pivotal for mediating disease susceptibility to the fungal pathogen Botrytis cinerea. We identified MYB46 by its ability to bind to a new cis element located in the 5´ promoter region of the pathogen-induced Ep5C gene which encodes a type III cell wall-bound peroxidase. We present genetic and molecular evidence indicating that MYB46 modulates the magnitude of Ep5C gene induction following pathogenic insults. Moreover, we demonstrate that different myb46 knock-down mutant plants exhibit increased disease resistance to B. cinerea, a phenotype that is accompanied by selective transcriptional reprogramming of a set of genes encoding cell wall proteins and enzymes, of which extracellular type III peroxidases are conspicuous. In essence our results substantiates that defense-related signaling pathways and cell wall integrity are interconnected, and MYB46 likely functions as a disease susceptibility modulator to B. cinerea through the integration of cell wall remodeling and downstream activation of secondary lines of defense.
Project description:Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a co-expression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.