Plant pathogenic oomycetes deliver a troop of effector proteins into the nucleus of host cells to manipulate plant cellular immunity and promote colonization. Recently, researchers have focused on identifying how effectors are transferred into the host cell nucleus, as well as the identity of the nuclear targets. In this study, we found that the RxLR effector PvAVH53 from the grapevine (Vitis vinifera) oomycete pathogen Plasmopara viticola physically interacts with grapevine nuclear import factor importin alphas (VvImpα and VvImpα4), localizes to the nucleus and triggers cell death when transiently expressed in tobacco (Nicotiana benthamiana) cells. Deletion of a nuclear localization signal (NLS) sequence from PvAVH53 or addition of a nuclear export signal (NES) sequence disrupted the nuclear localization of PvAVH53 and attenuated its ability to trigger cell death. Suppression of two tobacco importin-α genes, namely, NbImp-α1 and NbImp-α2, by virus-induced gene silencing (VIGS) also disrupted the nuclear localization and ability of PvAVH53 to induce cell death. Likewise, we transiently silenced the expression of VvImpα/α4 in grape through CRISPR/Cas13a, which has been reported to target RNA in vivo. Finally, we found that attenuating the expression of the Importin-αs genes resulted in increased susceptibility to the oomycete pathogen Phytophthora capsici in N. benthamiana and P. viticola in V. vinifera. Our results demonstrate that importin-αs are required for the nuclear localization and function of PvAVH53 and are essential for host innate immunity. The findings provide insight into the functions of importin-αs in grapevine against downy mildew.

中文翻译：

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葡萄球菌效应器 PvAVH53 的核定位和功能需要 Importin-αs

植物致病性卵菌将一组效应蛋白传递到宿主细胞的细胞核中，以操纵植物细胞免疫并促进定殖。最近，研究人员专注于确定效应子如何转移到宿主细胞核中，以及核目标的身份。在这项研究中，我们发现来自葡萄藤的 RxLR 效应子 PvAVH53 (葡萄) 卵菌病原体葡萄单胞菌与葡萄核输入因子importin alphas（VvImpα和VvImpα4）物理相互作用，定位于细胞核并在烟草中瞬时表达时引发细胞死亡。本氏烟草） 细胞。从 PvAVH53 中删除核定位信号 (NLS) 序列或添加核输出信号 (NES) 序列会破坏 PvAVH53 的核定位并减弱其触发细胞死亡的能力。通过病毒诱导的基因沉默 (VIGS) 抑制两种烟草输入蛋白-α 基因，即 NbImp-α1 和 NbImp-α2，也破坏了 PvAVH53 的核定位和诱导细胞死亡的能力。同样，我们通过 CRISPR/Cas13a 暂时沉默了葡萄中 VvImpα/α4 的表达，据报道其在体内靶向 RNA。最后，我们发现减弱 Importin-αs 基因的表达会导致对卵菌病原体的易感性增加辣椒疫霉在本塞姆氏烟草和P. viticola在五、葡萄. 我们的研究结果表明，importin-αs 是 PvAVH53 的核定位和功能所必需的，并且对于宿主先天免疫至关重要。这些发现提供了对葡萄藤中importin-αs对抗霜霉病的功能的深入了解。