Insight Into Function and Subcellular Localization of Plasmopara viticola Putative RxLR Effectors

Abstract

Grapevine downy mildew, caused by oomycete fungus Plasmopara viticola, is one of the most devastating diseases of grapes across the major production regions of the world. Although many putative effector molecules have been identified from this pathogen, the functions of the majority of these are still unknown. In this study, we analyzed the potential function of 26 P. viticola effectors from the highly virulent strain YL. Using transient expression in leaf cells of the tobacco Nicotiana benthamiana, we found that the majority of the effectors could suppress cell death triggered by BAX and INF1, while seven could induce cell death. The subcellular localization of effectors in N. benthamiana was consistent with their localization in cells of Vitis vinifera. Those effectors that localized to the nucleus (17/26) showed a variety of subnuclear localization. Ten of the effectors localized predominantly to the nucleolus, whereas the remaining seven localized to nucleoplasm. Interestingly, five of the effectors were strongly related in sequence and showed identical subcellular localization, but had different functions in N. benthamiana leaves and expression patterns in grapevine in response to P. viticola. This study highlights the potential functional diversity of P. viticola effectors.

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

“…Previously, we identified several P. viticola RxLR effectors capable of triggering cell death when expressed transiently in N. benthamiana cells. Our preliminary results suggested that among these effectors, PvAVH53 and PvAVH54804 induced cell death the most rapidly and at the lowest apparent concentration 33 . Imaging of N. benthamiana protoplasts transiently expressing PvAVH53-GFP or PvAVH54804-GFP showed that PvAVH53-GFP localizes to the nucleus, whereas PvAVH54804-GFP accumulates in both the nucleus and cytoplasm (Fig.…”

“…Previous experiments have indicated that the P. viticola effectors PvAVH53 and PvAVH54804 can induce cell death in N. benthamiana and are localized strictly in the nucleus or both in the nucleus and cytoplasm, respectively 33 . Our experiments further showed that PvAVH53 and PvAVH54804 induce cell death in N. benthamiana.…”

“…In previous preliminary work, we observed that the P. viticola effector PvAVH53 localized to the nucleus and triggered cell death in N. benthamiana 33 . In this study, we report that PvAVH53 physically interacts with importinαs (VvImpα/VvImpα4, and NbImpα1/NbImpα2) from V. vinifera and N. benthamiana and that these interactions are crucial for the function of the effector PvAVH53.…”

“…We previously found that PvAVH53 localizes to the nucleus when expressed transiently in N. benthamiana cells 33 . To identify potential interaction partners of PvAVH53 in V. vinifera, we conducted a yeast two-hybrid assay using PvAVH53 as bait in combination with a V. vinifera cDNA expression library.…”

“…3a). Finally, we used the yeast two-hybrid assay to evaluate the interaction between VvImpa4 and a panel of 10 P. viticola RxLR-type effectors 33 previously identified in our laboratory (the details of the effectors are provided in the supplementary material in Table S1). With two exceptions (PvAVH1 and PvAVH54804), all effectors showed interactions with VvImpα4 ( Fig.…”