1. Cell wall biogenesis in Neurospora crassa 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 Glucan synthase 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 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. OCH-1 is required for galactomannan synthesis Galactomannan

8. 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 Δoch-1 has severe morphological defects

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

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 Δ och-1 cell extract Δ och-1 secreted Δ mnn-9 cell extract Δ mnn-9 secreted Δ ktr-1 secreted WT secretedΔ Ktr-1 cell extract WT cell extract Δmnn-9 is 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 glycoproteins to the glucan/chitin matrix. The N. crassa genome 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. Δdfg5, Δdcw1 double mutant resembles Δoch-1 phenotype. DFG5 and DCW1 function to incorporate glycoproteins into the cell wall

14. The Δdfg5, Δdcw1 double mutant is defective in incorporating cell wall proteins into the wall. DFG5 and DCW1 function to incorporate glycoproteins into the cell wall Western blot analysis of ACW-1 cell wall protein WT secreted Δ dcw1 secreted Δ dfg5 secreted Δ Dcw1, Δ dfg5 secreted

15. 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 Conclusions 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 Crytococcus neoformans. 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 – Mol. Weight markers 1.WT cell extract 2.WT secreted protein 3.Δcps-1 cell extract 4.Δcps-1 secreted protein M 1 2 3 4

18. Δcps-1 releases cell wall protein into the medium 1 2 3 4 1.Δcps-1 secreted protein 2.Δcps-1 cell extract 3.WT secreted protein 4.WT cell extract Western blot analysis of ACW-1 cell wall protein 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 Abhiram Maddi – 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