Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway

As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.


Introduction
The S-phase kinase-associated protein 1 (SKP1) is a core component of the SKP1/Cullin/Fbox (SCF) complex, an E3 ubiquitin ligase that triggers protein degradation through the 26S proteasome [1,2]. Within the SCF complexes, the scaffold-like Cullin binds to Rbx1  (GID2) in rice are key players involved in the GA signaling pathway [44]. GA perception is mediated by a soluble receptor, GID1 [45]. Interaction of bioactive GA with GID1 triggers a conformational change of GID1, which allows the nuclear growth inhibitors DELLAs to bind to GID1 by its DELLA/TVHYNP domains [46][47][48]. The GA-GID1-DELLA complex facilitates the binding of DELLA to the E3 ligase SCF SLY1/GID2 , promoting the ubiquitylation and subsequent destruction of DELLA through the 26S proteasome [13,[49][50][51]. Thus, GA facilitates plant growth by controlling the degradation of DELLA proteins in a proteasome-dependent manner. It has been shown that the F-box protein GID2 interacts with OSK1, OSK13, OSK20 and OSK25 [13,52], and acts as a component of the SCF complex in rice [13]. In the GA signaling pathway, GID2 acts as a positive regulator that interacts with the phosphorylated DELLA protein Slender Rice 1 (SLR1) and triggers degradation of SLR1 through the ubiquitin/proteasome pathway [13]. Mutant plants lacking GA show a dwarf phenotype [51]. RBSDV causes dwarfed growth abnormality in infected rice and maize plants. It is reported that concentration of GA 3 is lower in RBSDV-infected plants than in healthy plants [53]. Participation of plant hormones JA and BR in RBSDV infection was recently described [54]; however, the role that GA plays during RBSDV infection remains to be elucidated. It is not known whether RBSDV P7-2 forms a SCF complex. In this study, we further explored the interactions between P7-2 and 25 type I OSKs. We found that P7-2 could bind eight OSK members using yeast two-hybrid assays, and interactions of P7-2 with OSK1, OSK5 and OSK20 were further confirmed by co-immunoprecipitation (co-IP). A yeast three-hybrid assay showed that P7-2, OSK1 and O. sativa Cullin-1 (OsCUL1) were able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with GID2 from rice and maize plants. Further study showed that the N-terminal region of P7-2 was responsible for the interaction with GID2.

Plant materials and growth conditions
The N. benthamiana plants used in this study were grown and maintained at 24˚C with 16 h light and 8 h darkness.
In vivo co-IP P7-2 was inserted into the vector pMDC32 [56] containing 3xFlag at the C-terminus, and GFP was introduced into the vector pGD-3xFlag, a modified binary vector pGD with 3xFlag at its C-terminus [57]. OSK1, OSK5, OSK20 and GFP were cloned into pGD-6xMyc that contained 6xMyc tag at the C-terminus [58]. Primers used for constructing the recombinant plasmids mentioned above are shown in S1 Table. The 35S:P7-2-3xFlag construct was transiently expressed with 35S:OSK1-6xMyc, 35S:OSK5-6xMyc and 35S:OSK20-6xMyc, respectively, together with 35S:P19 [59] in N. benthamiana leaves by Agrobacterium-mediated infiltration method as described previously [55]. GFP-6xMyc and GFP-3xFlag served as negative controls. In vivo co-IP was performed [60]. Briefly, 72 h after infiltration, 3 g of the infiltrated leaves were collected and ground in liquid nitrogen, suspended in an equal volume (w/v) of extraction buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 10 mM Dithiothreitol, 1 mM Ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 2% w/v polyvinylpolypyrrolidone, 1 × proteinase inhibitor cocktail and 0.1% Triton-X 100). After vigorous vortexing, the suspension was placed on ice for 30-60 min. A 200-mesh nylon net was used to filtrate the suspension to exclude the cell walls and other impurities and then centrifuged at 12,000 rpm for 15 min. The resulting supernatant was homogenized on a mute mixer at 4˚C for 4 h with anti-Flag beads that had been balanced in IP buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol and 0.1% Triton-X 100) and blocked with Bovine serum albumin. The precipitated samples were extensively washed nine times with IP buffer, and then boiled with 2 × sodium dodecyl sulfate (SDS) sample buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.2% bromophenol blue and 5% β-mercaptoethanol added before use) for 10 min.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis SDS-PAGE and western blot analysis were conducted as described [36,55]. Briefly, samples prepared from co-IP were separated by 12.5% SDS polyacrylamide gel electrophoresis (PAGE) and western blot analysis was performed by adding an anti-Flag polyclonal antibody (1:5000; Abmart, Berkeley Heights, NJ, USA) or an anti-c-Myc polyclonal antibody (1:5000; Abmart) followed by a goat anti-mouse horseradish peroxidase secondary polyclonal antibody (1:3000; Bio-Rad, Hercules, CA, USA) or an goat anti-rabbit horseradish peroxidase secondary polyclonal antibody (1:3000; Bio-Rad). Signals were detected with an enhanced chemiluminescence detection kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions.

Yeast three-hybrid assay
The P7-2 and OSK1/OSK5/OSK20 were cloned into the pBridge vector (Clontech) to produce fusions with the GAL4 DNA-binding domain (BD) and Met promoter, respectively, and were transformed into yeast strain Y187. OsCUL1 was inserted into the pGADT7 vector (Clontech) to generate pGAD-OsCUL1 and was transformed into yeast strain AH109. Double transformants were selected on dropout meida (SD/-Met/-Leu/-Trp). Protein interactions were confirmed by growth on selective media (SD/-Met/-Leu/-Trp/-His/-Ade, X-α-gal). Serial dilution analysis was performed as described previously. S1 Table shows the primers used for construction of the aforementioned recombinant plasmids.

P7-2 interacted with GID2 from rice and maize plants
It has been reported that RBSDV caused stunting in rice and maize plants, and a decrease of GA 3 contents was observed in RBSDV-infected plants [53]. To test whether P7-2 interacted with OsGID1, OsSLR1 and OsGID2, which are key proteins involved in GA signaling pathway [44], a yeast two-hybrid assay was conducted. Total RNA was extracted from 4-week-old rice plants, and OsGID1, OsGID2 and OsSLR1 were cloned by RT-PCR and inserted into pGADT7 to gain AD-OsGID1, AD-OsGID2 and AD-OsSLR1, respectively. We found that P7-2 interacted with OsGID2 (Fig 4A and 4B), but did not bind OsGID1 and OsSLR1 in yeast (S1 Fig). GID2 from maize plants was also cloned by RT-PCR and introduced into pGADT7 to test the interaction between P7-2 and ZeaGID2. The yeast two-hybrid assay showed that P7-2 also interacted with ZeaGID2 protein (Fig 5).

Discussion
In this study, we investigated the interaction between P7-2 and 25 type I OSKs using yeast two-hybrid assays. The results showed that P7-2 interacted with eight OSKs with different binding affinity (Fig 1). Co-IP further validated the interactions of P7-2 with OSK1, OSK5 and OSK20 (Fig 2). It has been demonstrated that some F-box proteins can bind one or more Arabidopsis ASK proteins [61]. Wang reported that P7-2 interacted with OSK1 [36]. Our study showed that P7-2 not only interacted with OSK1, but could also bind OSK4, OSK5, OSK6, Rice black streaked dwarf virus P7-2 formed SCF complex, and interacted with GID2 protein OSK9, OSK10, OSK20 and OSK25 (Fig 1). Binding to P7-2 may render OSKs inaccessible for other host proteins and thus disturb some physiological processes in plants. It was previously found that the 21 Arabidopsis ASK proteins exhibit considerable differences in binding capabilities to to various F-box proteins [62]. Here, we found that varied OSKs interacted with P7-2 with different binding abilities (Fig 1). P7-2 interacted strongly with OSK1, OSK5, OSK6, OSK20 and OSK25, but bound OSK4, OSK9 and OSK10 with low affinity (Fig 1). Sequence alignment of OSK proteins interacting with P7-2 indicated that different binding affinity among these OSKs towards P7-2 might arise from key amino acid changes (S2 Fig). There are 26 key amino-acids in the human SKP1 (Hs-SKP1) that have been reported to connect the collected and adjusted to OD 600 = 1.0, and diluted to 1, 10 −1 , 10 −2 and 10 −3 . Plasmids expressing only the DNA-activation domain or DNA-binding domain were used as negative controls. Numbers indicate a.a. residues of P7-2 fused to BD. SD/WL, -Trp-Leu; SD/AHWL/X-α-gal, -Ade-His-Trp-Leu containing X-α-gal.
https://doi.org/10.1371/journal.pone.0177518.g004 Rice black streaked dwarf virus P7-2 formed SCF complex, and interacted with GID2 protein human SKP2 F-box protein [7,63]. These amino acid residues closely related to the interaction between SKP1 and F-box proteins are also conserved among SKP1 proteins from plant species [52,64]. In terms of sequence alignment, four out of the 26 crucial amino-acids in OSK10 were changed compared with the OSKs that interacted strongly with P7-2 (S2 Fig). Specifically, the polar amino acids Q/N/K/R at site 142 (referring to OSK10) were changed to the nonpolar amino acid I, the amino acid C at site 165 was changed to A, the nonpolar amino acid F at site 190 was changed to the polar amino acid H, and the amino acid N at site 202 was changed to Y compared with the OSKs that showed high binding affinity to P7-2 (S2 Fig). Alterations in these four crucial amino acid residues might result in low binding affinity of OSK10 to P7-2. It was reported that Hs-SKP1 contains eight helices (H1-H8) [63], and these helices are also conserved among other SKP1 proteins [52]. Helices H5-H8 of Hs-SKP1 form the human SKP2 Fbox protein-binding site [63,65]. The 106 th residue of OSK4 in H5 was changed from polar amino acids D/N/E to nonpolar amino acid G (S2 Fig). Similar to OSK4, the 98 th residue of OSK9 in H5 was changed from the polar amino acids D/N/E to the nonpolar amino acid G, and a Glu (E) residue of OSK9 in H8 was missing compared with the OSKs that interacted strongly with P7-2 (S2 Fig). These amino acid changes in H5 and/or H8 of OSK4 and OSK9 might contribute to their weak interaction with P7-2. The OSKs show different expression patterns in various growth stages of rice. OSK1, OSK8, OSK11, OSK20 and OSK23 are widely and strongly expressed [52], suggesting their involvement in diverse developmental processes. Particularly, OSK1 is the most strongly and widely expressed OSK gene. However, most OSKs are expressed in flowers [52]. Kahlou et al. showed that OSK1 and OSK20 bind to a majority of the nine F-box proteins examined in a yeast two-hybrid assay [52], suggesting that OSK1 and OSK20 have a role in forming various SCF complexes. In accordance, P7-2 had a high affinity for OSK1 and OSK20 in yeast (Fig 1).
The SCF complex is an E3 ubiquitin ligase, mediating protein degradation through the 26S proteasome [1,2]. Here, we demonstrated that P7-2 associated with OSK1 and OsCUL1 to form a SCF complex (Fig 3), suggesting the involvement of P7-2 in ubiquitin-mediated degradation of cellular proteins in rice. Plant viruses were found to act as a F-box protein and interact with SKP1 to target important host factors [15]. For example, P0 proteins encoded by the poleroviruses Cucurbit aphid-borne yellows virus and Beet western yellows virus interact with Arabidopsis homologs of SKP1, Arabidopsis SKP1-like 1 (ASK1) and ASK2 through their F-box motif [17] to target ARGONAUTE1 (AGO1) [66,67], the core subunit of the RNAinduced silencing complex functioning in the RNA silencing pathway [68]. In addition, the Fbox protein CLINK (Cell cycle link) from the nanovirus Faba bean necrotic yellow virus interacts with SKP1 and the retinoblastoma tumor-suppressor protein pRB. Through inactivation of pRB, the virus is able to affect cell cycling and thus facilitates its replication [16].
In the current study, yeast two-hybrid assays showed that P7-2 interacted with both OsGID2 and ZeaGID2 (Figs 4 and 5). In addition, we found that the N-terminal region of P7-2 was essential for the interaction with GID2 (Figs 4 and 5). GID2 is involved in the GA signaling pathway and functions as a component of the SCF complex that specifically interacts with the phosphorylated DELLA protein SLR1 and triggers the ubiquitin-mediated degradation of SLR1 in rice [13]. It is possible that binding of P7-2 to GID2 might impair the interaction between GID2 and SLR1 by hijacking GID2, which might result in increased accumulation of SLR1 protein in RBSDV-infected plants or plants overexpressing P7-2. It was reported that the F-box protein GID2 interacted with OSK1, OSK13, OSK20 and OSK25 [13,52]. Our results also implied that P7-2 might interfere with the interaction between GID2 and OSKs by hijacking GID2 and OSKs. The capacity of P7-2 to bind both GID2 and OSKs also offered the possibility that P7-2 promoted the ubiquitin-mediated degradation of GID2. It is reported that the P2 protein encoded by Rice dwarf virus interacts with ent-kaurene oxidases, which play an essential role in synthesis of GAs, leading to lower concentration of GA and dwarf symptoms in rice [69]. RBSDV infection causes dwarf symptoms in rice and maize plants, and the endogenous GA concentration is reduced in response to RBSDV infection [53]. However, whether P7-2 affects the accumulation of GID2 remains to be investigated. Participation of plant hormones in RBSDV infection was described recently. It was revealed that genes in the JA pathway were induced, whereas the genes involved in the BR pathway were down-regulated in RBSDV-infected rice plants. It was further demonstrated that JA suppresses RBSDV infection and BR mediates susceptibility to RBSDV infection through infection assay using JA-insensitive mutant coi1-13 and BR-insensitive mutant Go [54]. However, the relationship between GA and RBSDV infection is poorly understood. Our results showed that P7-2 interacted with GID2, the crucial protein functioning in the GA signaling in rice, suggesting that GA might play a role in RBSDV infection.
In conclusion, our results demonstrated that RBSDV P7-2 interacted with eight OSKs by yeast two-hybrid assays, and the capacity of P7-2 to bind OSK1, OSK5 and OSK20 was further validated by co-IP. Yeast three-hybrid assay revealed that P7-2 associated with OSK1 and OsCUL1 to form the SCF complex. In addition, yeast two-hybrid assays showed that P7-2 interacted with OsGID2 and ZeaGID2 through its N-terminal region. These results demonstrated that RBSDV P7-2 served as a component of the SCF complex in rice, and suggested that the GA signaling pathway may play some role during RBSDV infection.