Cucumber Ribosomal Protein CsRPS21 Interacts With P22 Protein of Cucurbit Chlorotic Yellows Virus

Abstract

Cucurbit chlorotic yellows virus (CCYV) is a cucurbit-infecting crinivirus. RNA silencing can be initiated as a plant defense against viruses. Viruses encode various RNA silencing suppressors to counteract antiviral silencing. P22 protein encoded by RNA1 of CCYV is a silencing suppressor, but its mechanism of action remains unclear. In this study, the cucumber ribosomal-like protein CsRPS21 was found to interact with P22 protein in vitro and in vivo. A conserved CsRPS21 domain was indispensable for its nuclear localization and interaction with P22. Transient expression of CsRPS21 in Nicotiana benthamiana leaves interfered with P22 accumulation and inhibited P22 silencing suppressor activity. CsRPS21 expression in N. benthamiana protoplasts inhibited CCYV accumulation. Increasing numbers of ribosomal proteins are being found to be involved in viral infections of plants. We identified a P22-interacting ribosomal protein, CsRPS21, and uncovered its role in early viral replication and silencing suppressor activity. Our study increases knowledge of the function of ribosomal proteins during viral infection.

Keywords: P22; cucurbit chlorotic yellows virus; ribosomal protein; silencing suppressor; virus accumulation.

FIGURE 1

Identification of the interaction between P22 and CsRPS21. (A) Growth of Y2HGold yeast cells co-transformed with ADCsRPS21 and BDP22 on low-(SD/–Trp/–Leu) and high-(SD/–Trp/–Leu/–His/–Ade/Aba/X-α-gal) stringency selection media. ADT + BDP53 and ADT + BDLam served as positive and negative controls, respectively. (B) Visualization of the interaction between P22 and CsRPS21 in N. benthamiana epidermal cells using BiFC. P22 fused with the N-terminal portion of YFP (P22-nYFP) was transiently co-expressed with CsRPS21 fused with the C-terminal portion of YFP (CsRPS21-cYFP). Bar represents 10 μm. Photos were taken at 2 dpi using a Zeiss LSM710 laser scanning microscope. (C) Localization of CsRPS21 in N. benthamiana epidermal cells. YFP-tagged CsRPS21 (YFP-CsRPS21) was expressed in planta. Confocal images were taken at 2 dpi. Nuclear localization protein DAB-CFP was used as a nucleus indicator. Bar represents 10 μm. (D) Localization of P22 in N. benthamiana epidermal cells. YFP-tagged P22 (YFP-P22) was expressed in planta. Confocal images were taken at 2 dpi. Bar represents 10 μm.

FIGURE 2

Identification of the interaction between CsRPS21 and CCYV proteins (A,B) ADCsRPS21 was co-transformed with CCYV proteins, as P4.9, P1-6, P2-6, P9, P26, CP, CPm, P59, and HSP70, into Y2HGold yeast cells and plated on a low-(SD/–Trp/–Leu) and high-(SD/–Trp/–Leu/–His/–Ade/Aba/X-α-gal) stringency selection media. ADT + BDP53 and ADT + BDLam served as positive and negative controls, respectively.

FIGURE 3

Nuclear localization signal determination and its involvement in the interaction. (A) The full amino acid sequence of CsRPS21. Amino acids constituting the nuclear localization signal predicted using cNLS mapper are shown in red. The conserved ribosomal protein S21 domain predicted using the SMART protein prediction tool is underlined. (B) Three YFP-tagged deletion mutants of CsRPS21 (YFP-CsRPS21_1–127, YFP-CsRPS21_128–183, and YFP-CsRPS21_91–145) were transiently expressed in N. benthamiana leaves. Bar represents 20 μm. Photos were taken at 2 dpi using a Zeiss LSM710 laser scanning microscope. (C) Left panel: schematic representation of the CsRPS21 deletion mutants. Six CsPRS21 deletion mutants were used: RP1 (residues 91–183), RP2 (residues 1–145), RP3 (residues 128–183), RP4 (residues 1–127), RP5 (residues 91–145), and RP6 (residues 1–90). Right panel: Interaction between BDP22 and the CsRPS21 deletion mutants. Growth of Y2HGold yeast cells co-transformed with BDP22 and ADRP1, ADRP2, ADRP3, ADRP4, ADRP5, or ADRP6 on high-stringency selection medium (SD/–Leu/–Trp/–His/–Ade/Aba/X-α-gal). (D) Interaction between P22 and CsRPS21_1–145 (RP2) in N. benthamiana epidermal cells using BiFC. P22 fused with the N-terminal portion of YFP (P22-nYFP) was transiently co-expressed with CsRPS21_1–145 fused with the C-terminal portion of YFP (CsRPS21_1–145-cYFP). Bar represents 20 μm. Photos were taken at 3 dpi using a Zeiss LSM710 laser scanning microscope.

FIGURE 4

CsRPS21 negatively regulates P22 silencing suppressor activity (A–C) Silencing suppression ability of P22 was tested in 16c N. benthamiana plants, with transient co-expression of GFP and CsRPS21 or GUS (as a control protein). GFP fluorescence was revealed by UV illumination at 5 dpi with Agrobacterium constructs. Western blotting (B) and northern blotting (C) were used to detect the accumulation of GFP. (D) N. benthamiana leaves were infiltrated with a mixture of three Agrobacterium cultures carrying pGDGFP, pGDFlag-P22, pGDMyc-GUS, or pGDMyc-CsRPS21 and photographed at 3 dpi. Images of GFP fluorescence from agroinfiltrated leaves were taken under a Nikon ECLIPSE Ti-S fluorescence microscope. Bar represents 200 μm. (E) Western blotting analysis of protein extracted from the N. benthamiana leaves indicated above. GFP, CsRPS21 and P22 expression were confirmed using anti-GFP, anti-Myc and anti-Flag antibody. Coomassie Brilliant Blue (CBB) staining of the large subunit of Rubisco served as a loading control (Supplementary Figure 5).

FIGURE 5

Transient expression of CsRPS21 in N. benthamiana protoplasts inhibited CCYV accumulation. (A,B) Accumulation of CCYV RNA1 of transfected protoplasts was measured using RT-qPCR and northern blotting with the probe of CCYV RNA1 at 24 hpi. (C) Protoplasts isolated from YFP-CsRPS21 and YFP agroinfiltrated N. benthamiana leaves were transfected with CCYV RNA1 and RNA2. Accumulation of CsRPS21 was measured using RT-qPCR at 24 hpi. Data were pooled across experiments and statistically analyzed using t-tests. Bars represents the mean ± standard deviation (SD). Single and double asterisks indicate significant differences at ^∗p < 0.05 and ^∗∗p < 0.01, respectively.