Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
Mol Plant Pathol. 2013 Jun;14(5):470-82. doi: 10.1111/mpp.12020. Epub 2013 Feb 5.
Endocytosis is an essential cellular process in eukaryotic cells that involves concordant functions of clathrin and adaptor proteins, various protein and lipid kinases, phosphatases and the actin cytoskeleton. In Saccharomyces cerevisiae, Ark1p is a member of the serine/threonine protein kinase (SPK) family that affects profoundly the organization of the cortical actin cytoskeleton. To study the function of MoArk1, an Ark1p homologue identified in Magnaporthe oryzae, we disrupted the MoARK1 gene and characterized the ΔMoark1 mutant strain. The ΔMoark1 mutant exhibited various defects ranging from mycelial growth and conidial formation to appressorium-mediated host infection. The ΔMoark1 mutant also exhibited decreased appressorium turgor pressure and attenuated virulence on rice and barley. In addition, the ΔMoark1 mutant displayed defects in endocytosis and formation of the Spitzenkörper, and was hyposensitive to exogenous oxidative stress. Moreover, a MoArk1-green fluorescent protein (MoArk1-GFP) fusion protein showed an actin-like localization pattern by localizing to the apical regions of hyphae. This pattern of localization appeared to be regulated by the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins MoSec22 and MoVam7. Finally, detailed analysis revealed that the proline-rich region within the MoArk1 serine/threonine kinase (S_TKc) domain was critical for endocytosis, subcellular localization and pathogenicity. These results collectively suggest that MoArk1 exhibits conserved functions in endocytosis and actin cytoskeleton organization, which may underlie growth, cell wall integrity and virulence of the fungus.
内吞作用是真核细胞中的一种基本细胞过程，涉及网格蛋白和衔接蛋白的协调功能、各种蛋白和脂质激酶、磷酸酶以及肌动蛋白细胞骨架。在酿酒酵母中，Ark1p 是丝氨酸/苏氨酸蛋白激酶 (SPK) 家族的成员，它对皮质肌动蛋白细胞骨架的组织有深远的影响。为了研究 MoArk1 的功能，我们鉴定了 Magnaporthe oryzae 中的 Ark1p 同源物，敲除了 MoARK1 基因并对 ΔMoark1 突变株进行了表征。ΔMoark1 突变体表现出各种缺陷，从菌丝生长和分生孢子形成到附着胞介导的宿主感染。ΔMoark1 突变体还表现出附着胞膨压降低和对水稻和大麦的毒力减弱。此外，ΔMoark1 突变体在胞吞作用和 Spitzenkörper 的形成中存在缺陷，对外源氧化应激的敏感性降低。此外，MoArk1-绿色荧光蛋白 (MoArk1-GFP) 融合蛋白通过定位于菌丝顶端区域显示出肌动蛋白样定位模式。这种定位模式似乎受到 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 蛋白 MoSec22 和 MoVam7 的调节。最后，详细分析表明，MoArk1 丝氨酸/苏氨酸激酶 (S_TKc) 结构域内的富含脯氨酸区域对于内吞作用、亚细胞定位和致病性至关重要。这些结果共同表明，MoArk1 在胞吞作用和肌动蛋白细胞骨架组织中表现出保守功能，这可能是真菌生长、细胞壁完整性和毒力的基础。
