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

Abstract

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.

Fig. 1. PvAVH53 requires nuclear localization to trigger cell death but PvAVH54804 does not.

a Transient expression of PvAVH53 with a nuclear export signal (PvAVH53^NES) or nuclear localization signal (NLS) deletion (PvAVH53^ΔNLS) cotransformed with an NLS-mCherry marker in Nicotiana benthamiana protoplasts, showing that both mutants of PvAVH53 failed to stabilize nuclear localization. b Transient expression of the PvAVH53 mutants in N. benthamiana leaves by agroinfiltration showed that PvAVH53 mutants failed to trigger cell death. c Transient expression of PvAVH54804 with a nuclear export signal (NES) or nuclear localization signal (NLS) cotransformed with an NLS-mCherry marker in N. benthamiana protoplasts. d Transient expression of the PvAVH54804 mutants in N. benthamiana leaves by agroinfiltration showed that PvAVH54804^NLS failed to trigger cell death. Photos were captured at 6 d post infiltration. Ion leakage from the infiltrated leaf discs was measured as a percentage of leakage from boiled discs to quantify cell death. Scale bar = 5–10 μm. The data are the means ± SEs based on three independent replicates (Student’s t-test, P < 0.01)

Fig. 2. Y2H assay showing that PvAVH53 interacts with Vitisvinifera VvImpα/α4.

The short segment of PvAVH53 containing the NLS (124–134 amino acids) interacts with the two arm domains of VvImp α4, as evidenced by growth on DDO (minimal medium, double dropout: SDLeu/-Trp) and QDO/A/X (minimal medium, quadruple dropout: SD-Ade/-His/-Leu/-Trp) in the presence of Aba and Xα-Gal. Left panel: Schematic diagram of the full length and deletion constructs of VvImpα4 and PvAVH53. Right panel: Confirmation of the interaction by cotransformation of different plasmids

Fig. 3. PvAVH53 and VvImpαs localize to and interact in the Vitis vinifera cell nucleus.

a Subcellular localization of V. vinifera nuclear import factors VvImpα4/VvImpα, PvAVH53, and GFP as controls cotransformed with NLS-mCherry (as the nuclear localization marker) in V. vinifera protoplasts. b A BiFC assay confirmed that VvImpαs interacted with PvAVH53. Photos were taken after protoplasts were incubated for 20–24 h under weak lighting at 25 °C. Scale bar = 5–10 μm

Fig. 4. PvAVH53 interacts with Nicotiana benthamiana NbImpαs.

a Phylogenetic analyses of the VvImpαs (VvImpα XP_002274422.1, VvImpα2 XP_002282816.1, VvImpα4 XP_002281670.1, VvImpα6-like XP_010646172.1, VvImpα5 XP_002281591.1), and NbImpαs (NbImpα1: EF137253.1 and NbImpα2: EF137254.1) were conducted with MEGA5 software. b Sequence analysis of the VvImpα/α4 and NbImpα1/2. The sequence alignment of the NbImpαs and VvImpαs indicated that VvImpα and VvImpα4 showed high sequence identity with the two NbImpαs. c NbImpα1/2 interact with PvAVH53 in yeast Y2H Gold cells. The yeast cells grew and turned blue on DDO (SD-Leu/-Trp) and QDO/A/X (SD-Ade/-His/-Leu/-Trp) in the presence of Aba and X-α-Gal

Fig. 5. NbImpα1/2 silencing reduces plant growth and disturbs the nuclear localization of PvAVH53.

a Morphology of N. benthamiana plants with TRV:GFP (control) and TRV:Impα1/2. b Relative quantification of the expression of NbImpα1/2 with TRV constructs using qRT-PCR. The NbEF1α gene was used as an internal control. (c) Subcellular localization in leaves of control (TRV:GFP) and Impα1/2-silenced plants analyzed by confocal microscopy. Photos were taken after protoplasts were incubated for 20–24 h under weak lighting at 25 °C. Scale bar = 10 μm. The experiments were repeated three times with similar results, and at least two protoplasts were observed each time

Fig. 6. NbImpα1/2 silencing decreases the cell death triggered by PvAVH53 but PvAVH54804 does not.

a Leaves of control (TRV:GFP) and Impα1/2-silenced plants were agroinfiltrated with PvAVH53/PvAVH54804 expression plasmids. Proteins were extracted from infiltrated spots to analyze the expression (spot number: 1, INF1; 2, GFP; 3, PvAVH54804; 4, PvAVH53). b Ion leakage (%) measurement at infiltration sites. The data are the means ± SEs based on three independent replicates (Student’s t-test: **P < 0.01)

Fig. 7. VvImpα/α4 silencing changes the sublocalization of PvAVH53 and promotes the susceptibility of V. vinifera to P. viticola.

a Subcellular localization of PvAVH53 on leaves of control (pCR11) and pCR11-VvImpα/α4-silenced grape leaves was analyzed by confocal microscopy. Scale bar = 5–7.5 μm. b Phenotype of control (pCR11) and pCR11-VvImpα/α4-silenced grape leaves infected with P. viticola. Leaf discs were photographed at 2 days and 5 days post infection. Western blotting detected Cas13a protein expression in control and VvImpα/α4-silenced grape leaves. c P. viticola development at inoculation sites in leaf discs of control and VvImpα/α4-silenced grape leaves was revealed by aniline blue staining. d Relative quantification of the expression of VvImpα/α4 in control and VvImpα/α4-silenced grape leaves via qRT-PCR. The VvActin (AY680701) gene was used as an internal reference. The data are the means ± SEs based on three independent replicates (Student’s t-test: **P < 0.01)