Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways
Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways
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DOI：
10.1111/j.1365-2958.2003.03932.x
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发表时间：
2004-03-01
影响因子：
3.6
通讯作者：
Dickman, MB
中科院分区：
文献类型：
--
作者：
Chen, CB;Dickman, MB
小G蛋白超家族是一组进化上保守的GTP酶，其调节多种信号传导途径，包括真核生物中的生长和发育途径。以前，我们发现，显性活性突变的独特Ras基因（DARas）的真菌植物病原体刺盘孢菌三叶草显示营养依赖性表型影响极性，生长和分化。在该突变体中，通过MAP激酶途径的信号传导也显著受损。在这里，我们描述的Rac（Ct-Rac 1），G蛋白的Rho家族的成员的克隆和功能特性。限制营养条件下DARas下调Ct-Rac 1表达。DARas与显性活性Rac（DARac）的共表达刺激MAPK活化并恢复野生型表型。MAPK活化的抑制抑制表型恢复，表明Rac介导的MAPK活化是负责回复到野生型表型。我们还研究了活性氧（ROS）在这些遗传背景中的作用。DARas突变株产生高水平的ROS，如通过DCFH-DA荧光测定的。与DNRac的共表达将ROS产生降低至野生型水平，并恢复正常的真菌生长和发育。用抗氧化剂或胞质磷脂酶A2抑制剂预处理DARas也恢复野生型表型。这些发现表明Ras介导的ROS产生依赖于Rac-cPLA（2）连接的信号通路。总之，这项研究提供了证据表明，Rac的功能，以恢复菌丝形态的DARas通过调节MAPK激活和细胞内ROS的产生。
The small G-protein superfamily is an evolutionarily conserved group of GTPases that regulate diverse signalling pathways including pathways for growth and development in eukaryotes. Previously, we showed that dominant active mutation in the unique Ras gene (DARas) of the fungal phytopathogen Colletotrichum trifolii displays a nutrient-dependent phenotype affecting polarity, growth and differentiation. Signalling via the MAP kinase pathway is significantly impaired in this mutant as well. Here we describe the cloning and functional characterization of Rac (Ct-Rac1), a member of the Rho family of G proteins. Ct-Rac1 expression is downregulated by DARas under limiting nutrition. Co-expression of DARas with dominant active Rac (DARac) stimulates MAPK activation and restores the wild-type phenotype. Inhibition of MAPK activation suppresses phenotypic restoration suggesting Rac-mediated MAPK activation is responsible for reversion to the wild-type phenotype. We also examined the role of reactive oxygen species (ROS) in these genetic backgrounds. The DARas mutant strain generates high levels of ROS as determined by DCFH-DA fluorescence. Co-expression with DNRac decreases ROS generation to wild-type levels and restores normal fungal growth and development. Pretreatment of DARas with antioxidants or a cytosolic phospholipase A2 inhibitor also restores the wild-type phenotype. These findings suggest that Ras-mediated ROS generation is dependent on a Rac-cPLA(2)-linked signalling pathway. Taken together, this study provides evidence that Rac functions to restore the hyphal morphology of DARas by regulating MAPK activation and intracellular ROS generation.