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. 2021 Apr 17;10(4):934.
doi: 10.3390/cells10040934.

The p23 of Citrus Tristeza Virus Interacts with Host FKBP-Type Peptidyl-Prolylcis-Trans Isomerase 17-2 and Is Involved in the Intracellular Movement of the Viral Coat Protein

Zuokun Yang  1   2 , Yongle Zhang  1   2 , Guoping Wang  1   2 , Shaohua Wen  1   3 , Yanxiang Wang  1   2 , Liu Li  1   2 , Feng Xiao  1   2 , Ni Hong  1   2
Affiliations

The p23 of Citrus Tristeza Virus Interacts with Host FKBP-Type Peptidyl-Prolylcis-Trans Isomerase 17-2 and Is Involved in the Intracellular Movement of the Viral Coat Protein

Zuokun Yang et al. Cells. .

Abstract

Citrus tristeza virus is a member of the genus Closterovirus in the family Closteroviridae. The p23 of citrus tristeza virus (CTV) is a multifunctional protein and RNA silencing suppressor. In this study, we identified a p23 interacting partner, FK506-binding protein (FKBP) 17-2, from Citrus aurantifolia (CaFKBP17-2), a susceptible host, and Nicotiana benthamiana (NbFKBP17-2), an experimental host for CTV. The interaction of p23 with CaFKBP17-2 and NbFKBP17-2 were individually confirmed by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Subcellular localization tests showed that the viral p23 translocated FKBP17-2 from chloroplasts to the plasmodesmata of epidermal cells of N. benthamiana leaves. The knocked-down expression level of NbFKBP17-2 mRNA resulted in a decreased CTV titer in N. benthamiana plants. Further, BiFC and Y2H assays showed that NbFKBP17-2 also interacted with the coat protein (CP) of CTV, and the complexes of CP/NbFKBP17-2 rapidly moved in the cytoplasm. Moreover, p23 guided the CP/NbFKBP17-2 complexes to move along the cell wall. To the best of our knowledge, this is the first report of viral proteins interacting with FKBP17-2 encoded by plants. Our results provide insights for further revealing the mechanism of the CTV CP protein movement.

Keywords: FK506-binding protein; citrus tristeza virus; coat protein; p23; protein-protein interaction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Interactions between citrus tristeza virus (CTV) p23 and FKBP17-2. (A) Alignment of deduced amino acid sequences of CaFKBP17-2 and NbFKBP17-2. Shaded letters indicate the conserved amino acid residues. The FK506 binding domain (FKBd) was indicated by a red box. (B) Split-ubiquitin yeast two-hybrid assays. p23 was fused to the C-terminal half of ubiquitin (Cub). CaFKBP17-2 and NbFKBP17-2 were respectively fused to the N-terminal half of ubiquitin (NubG). The yeast cell of co-transformed with p20-Cub and NubG-p20 was used as a positive control, and the yeast cell with p23-Cub and NubG was used as a negative control, respectively. A series of dilutions (1, 10−1, 10−2, and 10−3) were shown at the top of the figure. (C) Bimolecular-fluorescence complementation assays. The p23 fused to YFP C-terminal (p23-YC) was separately co-expressed with CaFKBP17-2 and NbFKBP17-2 fused to YFP N-terminal (CaFKBP17-2-YN and NbFKBP17-2-YN) by agroinfiltration on N. benthamiana leaves. Aniline blue stained callose at plasmodesmata (PD) was used as a marker. Arrow heads pointed to PD. Confocal images were taken at 48 hpi. Bars, 20 μm.
Figure 2
Figure 2
Subcellular localization analyses of citrus tristeza virus (CTV) p23 and NbFKBP17-2 in Nicotiana benthamiana cells. (A) Localization of CTV p23 and chloroplasts. The red signals represented the autofluorescent signals of chlorophyll in chloroplasts. (B) Localization of NbFKBP17-2 and chloroplasts. Confocal images were taken at 48 hpi. Bars, 20 μm. (C) The co-localization was further analyzed by overlapping fluorescence spectra, and areas indicated with white dotted lines in enlarged sections of (A,B) were used for this analysis.
Figure 3
Figure 3
Subcellular co-localization analyses of p23-eCFP and NbFKBP17-2-eYFP in Nicotiana benthamiana epidermal cells. (A,B) represented two different visual fields showing the co-localization of p23 and NbFKBP17-2 at plasmodesmata (PD) (A) and partial fluorescent signals of NbFKBP17-2 on chloroplasts (B), respectively. The white boxes indicated areas showing a magnified view. Arrows point to PD. The blue signals represented the chlorophyll autofluorescence in chloroplasts. Confocal images were taken at 48 hpi. Bars, 20 μm. (C,D) showed overlapping fluorescence spectra of eCFP, eYFP and chloroplast signals along the white dots depicted in (A,B), respectively.
Figure 4
Figure 4
NbFKBP17-2 interacted and co-moved with citrus tristeza virus (CTV) CP in Nicotiana benthamiana cells. (A) Bimolecular-fluorescence complementation assays. The CP fused to YFP C-terminal (CP-YC) was co-expressed with NbFKBP17-2 fused to YFP N-terminal (NbFKBP17-2-YN) by agroinfiltration on N. benthamiana leaves. Blue fluorescence represented chlorophyll autofluorescence. (B) Split-ubiquitin yeast two-hybrid assays. CP was fused to the C-terminal half of ubiquitin (Cub), NbFKBP17-2 was fused to the N-terminal half of ubiquitin (NubG). Yeast cell of co-transformed with p20-Cub and NubG-p20 was used as a positive control and p20-Cub and NubG was used as a negative control, respectively. A series of dilutions (1, 10−1, 10−2 and 10−3) were shown at the top of the figure. (C) Time-lapse images of CP/NbFKBP17-2 interaction and CP self-interaction. Two panels of each interaction combination were the captured signals in two fields. Red bodies were chloroplasts with chlorophyll autofluorescence signals. (D) Co-localization of CP/NbFKBP17-2 interaction complexes with endoplasmic reticulum marker mCherry-HDEL. The arrow heads pointed to CP/NbFKBP17-2 interaction complexes. Confocal images were taken at 48 hpi. Bars, 20 μm.
Figure 5
Figure 5
Co-localization (A) and integrated time-lapse movement (B) of CP/NbFKBP17-2 interaction complexes with p23-eCFP. Arrow heads pointed to p23/CP/NbFKBP17-2 complexes in different channels. Confocal images were taken at 48 hpi. Bars, 20 μm.
Figure 6
Figure 6
The phenotypes and relative expression levels of NbFKBP17-2 and citrus tristeza virus (CTV) genome in NbFKBP17-2 silenced Nicotiana benthamiana plants. (A) Phenotypes of TRV: 00 and TRV: NbFKBP17-2 pre-inoculated plants at 14 dpi of mock and CTV. (B) RT-qPCR analysis of NbFKBP17-2 mRNA expression in N. benthamiana leaves. The expression levels of NbFKBP17-2 mRNA were normalized to NbActin mRNA levels. (C) RT-qPCR analysis of relative expression levels of CTV genome in N. benthamiana leaves. Values were means ±SD from three independent experiments. Asterisks denoted significant differences in Student’s t-test between the two treatments (two-sided, ** p < 0.01).
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