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Cell wall biogenesis in Neurospora crassa

Cell wall biogenesis in Neurospora crassa. Stephen Free Department of Biological Sciences SUNY University at Buffalo. The Cell Wall is a critical organelle for the fungi. The cell wall protects the cell from desiccation, and other environmental stresses.

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Cell wall biogenesis in Neurospora crassa

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  1. Cell wall biogenesis in Neurosporacrassa Stephen Free Department of Biological Sciences SUNY University at Buffalo

  2. The Cell Wall is a critical organelle for the fungi. • The cell wall protects the cell from desiccation, and other environmental stresses. • The cell wall protects the fungus from other microorganisms. • The cell wall provides protection to fungal pathogen from host defensives. • The cell wall components allow the cell to assess it’s environment and act as the “upstream most” element for signal transduction pathways. • The cell wall is necessary for polarized growth and morphology. • Many antifungal agents target steps in cell wall biogenesis.

  3. Fungal Cell Wall Structure

  4. Key Points • The N-linked galactomannan is used to cross-link proteins into the cell wall. • DFG5 and DCW1 are cell wall α-1,6-mannanases/ mannantransferases which cross-link proteins into the cell wall. • The CPS-1 polysaccharide synthase synthesizes a polysaccharide that is required for cross-linking proteins into the cell wall.

  5. Cell Wall Polysaccharide Biogenesis • Glucansynthase and chitin synthase are plasma membrane-associated, multi-pass transmembrane proteins. • As they are synthesized, the linear glucan and chitin polymers are extruded into the cell wall space. • Glucan and chitin are cross-linked to other polysaccharides and proteins in the cell wall matrix. • The Gel1, Crh1, and Bgl2 glycosylhydrolase/ glycosyltransferase families are involved in creating a glucan/chitin matrix.

  6. Cell Wall Protein Biosynthesis

  7. OCH-1 is required for galactomannan synthesis • Och-1 encodes a Golgi-associated α-1,6-mannosyltransferase. • In N. crassa, OCH-1 adds the initial α-1,6-mannose of the galactomannan onto N-linked oligosaccharides. Galactomannan

  8. Δoch-1 has severe morphological defects Δoch-1 mutant characteristics: • Tight colonial phenotype • Slow growth • Defective in aerial hyphae • Abnormal conidiation Microscopic examination: • Short, globular hyphae • Abnormal branching pattern • Tight colonial growth

  9. OCH-1 is required for protein cross-linking into the cell wall Western blot analysis of ACW-1 cell wall protein Δoch-1 cell extract Δoch-1 secreted WT cell extract Δoch-1 secreted WT secreted WT secreted • Δoch-1 is defective in incorporating cell wall proteins into the cell wall.

  10. MNN-9 and KTR-1 catalyze galactomannan synthesis MNN-9 functions to synthesize the α-1,6-mannose backbone and the KTR-1 functions to add the α-1,2-mannose side chains of the galactomannan. Mnn-9 Ktr-1

  11. MNN-9 and KTR-1 are needed for the effective incorporation of protein into the cell wall. Western blot analysis of ACW-1 cell wall protein Δoch-1 cell extract Δmnn-9 cell extract ΔKtr-1 cell extract WT cell extract Δmnn-9 secreted Δoch-1 secreted WT secreted Δktr-1 secreted Δmnn-9is defective in incorporating ACW-1 into the wall Δktr-1 is less efficient than the wild type in incorporating ACW-1 into the cell wall

  12. Identifying cross-linking enzymes that recognize the galactomannan • α-1,6-mannanases are likely to cross-link glycoproteinsto the glucan/chitin matrix. • The N. crassagenome contains nine genes in the gh76 (α-1,6-mannanase) gene family and the single gene deletion library contains mutants for all nine genes. • Deletion mutants for all nine genes were tested and two of them were identified as having a defect in cell wall biogenesis.

  13. DFG5 and DCW1 function to incorporate glycoproteins into the cell wall • Δdfg5, Δdcw1 double mutant resembles Δoch-1 phenotype.

  14. DFG5 and DCW1 function to incorporate glycoproteins into the cell wall • The Δdfg5, Δdcw1 double mutant is defective in incorporating cell wall proteins into the wall. Western blot analysis of ACW-1 cell wall protein ΔDcw1,Δdfg5 secreted Δdcw1 secreted Δdfg5 secreted WT secreted

  15. Conclusions • The DFG5 and DCW1 α-1,6-mannanases recognize the N-linked galactomannan, cleave the α-1,6-mannan backbone, and cross-link the N-linked galactomannan into the glucan/chitin matrix α-1,6-mannan backbone

  16. Characterization of the Δcps-1 mutant • The cps-1 gene encodes a 511 amino acid protein related to the capsid polysaccharide synthase from Crytococcusneoformans. • In screening the single deletion library for female developmental mutants we noted that the Δcps-1 (polysaccharide synthase) mutant had a semi-colonial growth phenotype. • Δcps-1 is sensitive to cell wall perturbation reagents. • RIP experiments demonstrate that the deletion of cps-1 is responsible for the mutant phenotype.

  17. CPS-1 is needed for the incorporation of cell wall proteins into the wall M 1 2 3 4 M – Mol. Weight markers WT cell extract WT secreted protein Δcps-1 cell extract Δcps-1 secreted protein

  18. Δcps-1 releases cell wall protein into the medium Western blot analysis of ACW-1 cell wall protein 1 2 3 4 Δcps-1 secreted protein Δcps-1 cell extract WT secreted protein WT cell extract CPS-1 is required for the incorporation of cell wall proteins into the cell wall.

  19. Model for incorporating cell wall protein into the cell wall • The CPS-1 generated polysaccharide is synthesized and extruded (reducing end first) into the cell wall space, and cross-linked into the glucan/chitin matrix. • DFG5 and DCW1 binds to the reducing end of the CPS-1 polymer and places the reducing end of CPS-1 in its enzymatic active site. • DFG5 and DCW1 then binds the N-linked galactomannan and uses the reducing end of cps-1 generated polysaccharide to attack its α-1,6-backbone. • The terminal part of the galactomannan is released as a new glycosidic bond is generated between the cps-1 polysaccharide and the α-1,6-mannose backbone, which effectively cross-links the protein into the cell wall matrix.

  20. Key Points • The N-linked galactomannan is used to cross-link proteins into the cell wall. • DFG5 and DCW1 are cell wall α-1,6-mannanases/ mannantransferases which cross-link proteins into the cell wall. • The CPS-1 polysaccharide synthase synthesizes a polysaccharide that is used in cross-linking proteins into the cell wall.

  21. Acknowledgements AbhiramMaddi – och-1, dfg-5, and dcw-1 Eleanor Sokolow – cps-1 Ci Fu – cps-1 Funding by NIH grants R01 078589 and R03 AI103897 and the UB Foundation

  22. α-1,3-glucan is a cell-type cell wall glucan Wild type Δags-1 Δags-1 has a distinctive phenotype, affecting the production of conidia

  23. Δags-1 is affected in conidia production

  24. α-1,3-glucan is found in the conidial cell wall.

  25. The ags-1 5’regulatory DNA directs expression in aerial hyphae and developing conidia

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