Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of direct target genes of the Neurospora crassa essential plant biomass deconstruction transcription factors CLR-1, CLR-2 and XLR-1 (ChIP-Seq)

ABSTRACT: abstract: The plant cell wall is composed of many complex polymers, and its deconstruction requires an equally complex orchestration of a wide array of enzymes. In Neurospora crassa, clr-1, clr-2 and xlr-1 have been identified as the key transcription factors involved in cell wall breakdown. In order to define their regulons, we performed ChIPseq upon these three transcription factors. CLR-1, CLR-2 and XLR-1 each bind to the most highly and differentially expressed gene populations, which include the cellulases for the CLRs and the hemicellulases for XLR-1. CLR-1 also bound to its regulon under non-inducing conditions; however, this did not translate into gene expression. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest yeast homolog, GAL4. Co-immunoprecipitation studies were able to show that CLR-1 and CLR-2 act as homodimers. Finally, we report on a conserved XLR-1 point mutation that is sufficient to drive hemicellulase expression under non-inducing conditions. Understanding how these transcription factors work in concert to break down plant biomass can inform decisions on how to best engineer future fungal strains for decreased enzyme costs. RNAseq and ChIPseq upon knockout mutants and sild type growing on various carbon sources to determin the role of the transcription factors: CLR-1, CLR-2, and XLR-1 in plant cell wall degradation

ORGANISM(S): Neurospora crassa

SUBMITTER: James Craig

PROVIDER: E-GEOD-68516 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential feedstock for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs). Neurospora crassa has been shown to express and secrete plant cell wall associated enzymes. To better understand genes specifically associated with degradation of hemicellulose, we identified 353 genes by transcriptome analysis of N. crassa wild type strain grown on beechwood xylan. Exposure to xylan induces 9 of the 19 predicted hemicellulase genes. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of wild type showed that none were essential for growth on beechwood xylan. The transcription factor XlnR/Xyr1 in Aspergillus and Trichoderma species is considered to be the major transcriptional regulator of genes encoding both cellulases and hemicellulases. We identified a xlnR/xyr1 homolog in N. crassa, NCU06971, termed xlr-1 (xylanase regulator 1). Deletion of xlr-1 in N. crassa abolishes the growth on xylan and xylose, but growth on cellulose was indistinguishable from wild type. To determine regulatory mechanisms associated with hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose and xylanolytic versus cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in N. crassa and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. However, in N. crassa, xlr-1 is subject to non-CRE-1 mediated CCR. This systematic analysis provides the similarities and differences of hemicellulose degradation and regulation mechanisms used by N. crassa in comparison to other filamentous fungi. Four-condition experiments (minimal medium, xylan medium,xylose and Avicel medium) of mutant strain(xlr-1) compared to wild type strain; Cy3 and Cy5 dye swap

Project description:Epidemics of coffee leaf rust (CLR) lead to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of pesticide treatments by alternative solutions. A good protection strategy is to take advantage of the plant immune system by eliciting its constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation made by UFLA (Brazil) is, in addition to acibenzolar-S-methyl (ASM), showing promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica. In order to improve our understanding of the molecular mechanisms of the PRIs, proteomic (2DE-MALDI/TOF/TOF-MS/MS), physiological (leaf gas-exchange) and biochemical (enzymatic) analyses of coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were performed. Proteomic data showed metabolic adjustments mainly related with photosynthesis, protein metabolism and stress responses but, the proteins modulated by the two PRIs were different. Greenforce CuCa, on its own, increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. Upon H. vastratix infection, the Greenforce CuCa showed a higher protective effect on the leaf physiology than ASM. The enzymatic analyses indicated that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to increase the involvement of secondary metabolism. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The data also evidenced the existence of a link between the primary metabolism and defense responses. Furthermore, Greenforce CuCa emerged as a significant agent for CLR management. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programs.

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Identification of direct target genes of the Neurospora crassa essential plant biomass deconstruction transcription factors CLR-1, CLR-2 and XLR-1 (ChIP-Seq)

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