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A conserved signalling network regulates Epichloë festucae cell-cell fusion and the mutualistic symbiotic interaction between E. festucae and Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
Epichloё festucae is a filamentous fungus that forms a mutually beneficial symbiotic
association with Lolium perenne. The NADPH oxidase complex components noxA, noxR and
racA, the transcription factor proA, and the cell wall integrity (CWI) MAP kinases, mkkA and
mpkA, are required for mutualistic E. festucae-L. perenne associations and cell-cell fusion.
Homologues of these genes in Neurospora crassa, Sordaria macrospora and Podospora
anserina are required for cell-cell fusion and sexual fruiting body maturation, thereby
establishing a link between self signalling and hyphal network formation in the E. festucae-L.
perenne symbiosis. In Podospora anserina, IDC2 and IDC3 are required for cell-cell fusion,
crippled growth and fruiting body formation. In S. macrospora and N. crassa, components of
the STRIPAK complex regulate cell-cell fusion and fruiting body formation. The aim of this
project was to test if E. festucae homologues of IDC2 and IDC3, and the STRIPAK complex
protein MOB3, named SymB, SymC and MobC, respectively, are also required for cell-cell
fusion and plant symbiosis. Gel shift assays showed the promoters of symB and symC are targets
for the transcription factor ProA. In culture, the frequency of cell-cell fusion of ΔmobC was
reduced, but in ΔsymB and ΔsymC mutants, totally abolished. All three mutants hyperconidiated
and formed intra-hyphal hyphae. Plants infected with these mutants were severely stunted and
hyphae exhibited proliferative growth and increased colonisation of the intercellular spaces and
vascular bundles. Expressoria formation, structures allowing colonisation of the leaf surface,
was reduced in ΔmobC, and abolished in ΔsymB and ΔsymC mutants. Microscopy analyses
showed SymB-GFP and SymC-mRFP1 co-localise to the plasma membrane and septa. SymC
also localised to highly dynamic punctate structures. Although ΔsymB and ΔsymC phenotypes
are identical to ΔmpkA, and the E. festucae pheromone response pathway scaffold ΔidcA
mutants, MpkA and MpkB phosphorylation and cellular localisation was unchanged compared
to wild-type. Using yeast-two-hybrid assays, an interaction between SymC and the STRIPAK
complex associated protein GPI1 was demonstrated. Collectively these results show that MobC,
SymB and SymC are required for E. festucae cell-cell fusion and host symbiosis. It is proposed
that SymB and SymC interact to form a sensor complex at the cell wall which regulates cell-cell
fusion in culture and hyphal network development in planta.
Content redacted from thesis due to copyright reasons: Figure 1.1 Page 4
Schardl, C. L. (2001) Epichloë festucae and related mutualistic symbionts of grasses. Fungal Genet. Biol., 33, 69-82.
Schardl, C. L., Young, C. A., Hesse, U., Amyotte, S. G., Andreeva, K., Calie, P. J., et al . (2013) Plant-symbiotic
fungi as chemical engineers: Multigenome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. Plos
Genetics, 9(2), e1003323.
Christensen, M. J., Bennett, R. J., Ansari, H. A., Koga, H., Johnson, R. D., Bryan, G. T., et al. (2008) Epichloë
endophytes grow by intercalary hyphal extension in elongating grass leaves. Fungal Genet. Biol., 45, 84-93.
Becker, M., Becker, Y., Green, K. & Scott, B. (2016) The endophytic symbiont Epichloë festucae establishes an
epiphyllous net on the surface of Lolium perenne leaves by development of an expressorium, an appressorium-like leaf
exit structure. New Phytol., 211, 240-254.
Figure 1.2 Page 6
Scott, B. (2015) Conservation of fungal and animal nicotinamide adenine dinucleotide phosphate oxidase complexes. Mol
Microbiol. 95(6):910-3. doi: 10.1111/mmi.12946.
Figure 1.3 Page 9
Haruo Saito & Kazuo Tatebayashi (2004) Regulation of the Osmoregulatory HOG MAPK Cascade in Yeast. The
Journal of Biochemistry. 136 (3): 267-272.
Figure 1.4 Page 10
Eaton, C., Mitic, M., Scott., B (2012) Signalling in the Epichloë festucae: Perennial Ryegrass Mutualistic Symbiotic
Interaction. Signaling and Communication in Plant Symbiosis, pp.143-181. DOI: 10.1007/978-3-642-20966-6_7.
Figure 1.5 Page 14
Kück, U., Pöggeler, S., Nowrousian, M., Nolting, N., Engh, I., (2009) Sordaria macrospora, a model system for fungal
development. In: Anke, T., Weber, D. (Eds.), The Mycota XV, Physiology and Genetics. Springer, Berlin/Heidelberg, pp.
Figure 1.6 Page 16
Coppin, E., Debuchy, R., S. Arnaise, & Picard, M. (1997) Mating types and sexual development in filamentous
ascomycetes. Microbiology and Molecular Biology Reviews, 61, 411-428.
Aldabbous, M. S., Roca, M. G., Stout, A., Huang, I. C., Read, N. D. & Free, S. J. (2010) The ham-5, rcm-1 and rco-1
genes regulate hyphal fusion in Neurospora crassa. Microbiology, 156, 2621- 2629.
Silar, P., Lalucque, H., & Vierny, C. (2001) Cell degeneration in the model system Podospora anserina.
Biogerontology, 2, 1–17.
Jamet-Vierny, C., Debuchy, R., Prigent, M. & Silar, P. (2007) IDC1, a pezizomycotina-specific gene that belongs to
the PaMpk1 MAP kinase transduction cascade of the filamentous fungus Podospora anserina. Fungal Genet. Biol., 44,
Figure 1.7 page 20
Fleißner, A., Leeder, A. C., Roca, M. G., Read, N. D. & Glass, N. L. (2009) Oscillatory recruitment of signaling
proteins to cell tips promotes coordinated behavior during cell fusion. Proc. Natl. Acad. Sci. USA, 106, 19387-19392.
Figure 1.8 page 22
Kück, U., Beier, A. M. & Teichert, I. (2016) The composition and function of the striatin-interacting phosphatases and
kinases (STRIPAK) complex in fungi. Fungal Genet. Biol., 90, 31-38.
Figure 4.8 page 143
Frey, S., Lahmann, Y., Hartmann, T., Seiler, S. & Pöggeler, S. (2015) Deletion of Smgpi1 encoding a GPI-anchored
protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora. Mol.
Microbiol., 97, 676-697.