Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex
Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex
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DOI：
10.1073/pnas.1017309108
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发表时间：
2011-01
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影响因子：
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通讯作者：
D. Takemoto;S. Kamakura;Sanjay Saikia;Y. Becker;Ruth E. Wrenn;A. Tanaka;H. Sumimoto;B. Scott
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文献类型：
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作者：
D. Takemoto;S. Kamakura;Sanjay Saikia;Y. Becker;Ruth E. Wrenn;A. Tanaka;H. Sumimoto;B. Scott
膜结合真菌 NADPH 氧化酶 (Nox) 调节活性氧 (ROS) 的合成在真菌形态发生、生长和发育中发挥着关键作用。植物共生真菌 Epichloë festucae 产生活性氧 (ROS) 需要由 NoxA、调节成分 NoxR 和小 GTP 酶 RacA 组成的多亚基复合物进行功能性组装。然而，该复合物在质膜上的组装和激活机制尚不清楚。我们通过酵母双杂交和免疫共沉淀测定发现 E. festucae NoxR 与酵母极性蛋白 Bem1 和 Cdc24 的同源物相互作用，并且在这些蛋白质中发现的 Phox 和 Bem1 (PB1) 蛋白结构域对于这些相互作用至关重要。 BemA、Cdc24 和 NoxR 的 GFP 融合体优先定位于活跃生长的菌丝尖端和隔膜。如双分子荧光互补测定所示，这些蛋白质在体内这些相同的细胞位点上彼此相互作用。 NoxR 的 PB1 结构域对于定位到菌丝尖端至关重要。羊茅 ΔbemA 突变体在菌丝形态发生以及培养物和植物中的生长方面存在缺陷。植物中真菌生长的变化导致了共生相互作用表型的缺陷。我们无法分离出 Δcdc24 突变体，这表明该基因是必需的。这些结果表明 BemA 和 Cdc24 在将 NoxR 蛋白定位到真菌菌丝形态发生和生长位点方面发挥着关键作用。我们的研究结果确定了真菌极性建立所需的蛋白质机制与控制细胞分化的 Nox 复合物之间存在潜在的共同祖先联系。
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.