title: Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex.
abstract: Regulated synthesis of reactive oxygen species (ROS) by membrane-bound fungal NADPH oxidases (Nox) plays a key role in fungal morphogenesis, growth, and development. Generation of reactive oxygen species (ROS) by the plant symbiotic fungus, Epichloë festucae, requires functional assembly of a multisubunit complex composed of NoxA, a regulatory component, NoxR, and the small GTPase RacA. However, the mechanism for assembly and activation of this complex at the plasma membrane is unknown. We found by yeast two-hybrid and coimmunoprecipitation assays that E. festucae NoxR interacts with homologs of the yeast polarity proteins, Bem1 and Cdc24, and that the Phox and Bem1 (PB1) protein domains found in these proteins are essential for these interactions. GFP fusions of BemA, Cdc24, and NoxR preferentially localized to actively growing hyphal tips and to septa. These proteins interact with each other in vivo at these same cellular sites as shown by bimolecular fluorescent complementation assays. The PB1 domain of NoxR is essential for localization to the hyphal tip. An E. festucae ΔbemA mutant was defective in hyphal morphogenesis and growth in culture and in planta. The changes in fungal growth in planta resulted in a defective symbiotic interaction phenotype. Our inability to isolate a Δcdc24 mutant suggests this gene is essential. These results demonstrate that BemA and Cdc24 play a critical role in localizing NoxR protein to sites of fungal hyphal morphogenesis and growth. Our findings identify a potential shared ancestral link between the protein machinery required for fungal polarity establishment and the Nox complex controlling cellular differentiation.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
authors.forename: Daigo
authors.initials: D
authors.lastname: Takemoto
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kamakura
authors.forename: Sanjay
authors.initials: S
authors.lastname: Saikia
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Becker
authors.forename: Ruth
authors.initials: R
authors.lastname: Wrenn
authors.forename: Aiko
authors.initials: A
authors.lastname: Tanaka
authors.forename: Hideki
authors.initials: H
authors.lastname: Sumimoto
authors.forename: Barry
authors.initials: B
authors.lastname: Scott
mesh_terms: D001483:Base Sequence; D019295:Computational Biology; D055329:Epichloe / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D025301:Hyphae / Q000254:growth & development* / Q000378:metabolism*; D047468:Immunoprecipitation; D008129:Lolium / Q000382:microbiology*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000235:genetics* / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013559:Symbiosis; D020798:Two-Hybrid System Techniques
chemical_list: D005656:Fungal Proteins; D046912:Multiprotein Complexes; D017382:Reactive Oxygen Species; D049452:Green Fluorescent Proteins; D019255:NADPH Oxidases