Importin-αs are required for the nuclear localization and function of the Plasmopara viticola effector PvAVH53

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 (VvImp alpha and VvImp alpha 4), 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-alpha genes, namely, NbImp-alpha 1 and NbImp-alpha 2, 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 VvImp alpha/alpha 4 in grape through CRISPR/Cas13a, which has been reported to target RNA in vivo. Finally, we found that attenuating the expression of the Importin-alpha s 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-alpha s 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-alpha s in grapevine against downy mildew.