rice cellular protein(s) that interacts with P2 by yeast two- hybrid screen using a ... as E3 ubiquitin ligases, which play important roles in plant metabolism and ...
Molecular Plant • Volume 7 • Number 6 • Pages 1057–1060 • June 2014
LETTER TO THE EDITOR
OsRFPH2-10, a RING-H2 Finger E3 Ubiquitin Ligase, Is Involved in Rice Antiviral Defense in the Early Stages of Rice dwarf virus Infection Dear Editor, Rice dwarf virus (RDV), a member of the Phytoreoviurs genus, is transmitted to rice (Oryza sativa) plants by leafhopper (Nephotettix cincticeps) in a propagative manner. Infection by RDV results in severe stunting growth phenotypes and a dramatic reduction in grain yield. The genome of RDV is composed of 12-segmented double-stranded RNAs (S1–S12 based on their migration rates in agarose gel electrophoresis). The 12 segments encode seven structural proteins (P1, P2, P3, P5, P7, P8, and P9 as the products of S1, S2, S3, S5, S7, S8, and S9, respectively) and five nonstructural proteins (Pns4, Pns6, Pns10, Pns11, and Pns12 as the products of S4, S6, S10, S11, and S12, respectively). The outer capsid protein P2 is essential for RDV infection of insects and thus influences transmission of RDV by the insect vector (Omura et al., 1998; Zhou et al., 2007). P2 also contributes to the dwarf phenotype of infected rice by interfering with gibberellic acid synthesis (Zhu et al., 2005). When RDV-infected rice plants were maintained via vegetative propagation for several years without insect transmission, they regained normal growth height due to loss of RDV P2 and Pns10 proteins (Pu et al., 2011). To gain insights into the functions of P2 in infected rice and to understand the possible mechanism(s) for its disappearance from the infected plants, we searched for potential rice cellular protein(s) that interacts with P2 by yeast twohybrid screen using a cDNA library prepared from 2-week-old rice seedlings. Among the 10 cDNA clones recovered from the screen, two were found to encode OsRFPH2-10 (referred to as H2-10), which contains a C3H2C3-type RING-finger motif in its N-terminus (Lim et al., 2013) (Supplemental Figure 1). To further verify and characterize the interaction between H2-10 and P2, we cloned the full-length cDNA of H2-10 and performed yeast two-hybrid and co-immunoprecipitation experiments. Results showed that the full-length H2-10 interacts with RDV P2 in yeast (Figure 1A) and plant cells (Figure 1B). RING (Really Interesting New Gene) finger proteins harbor special zinc-binding domains defined by the consensus sequence Cys–X2–Cys–X(9–39) –Cys–X(1–3) –His–X(2–3) –Cys/ His–X2–Cys–X(4–48) –Cys–X2–Cys, where X can be any amino acid. Depending on the fifth conserved amino acid, RING fingers are mainly classified as C3H2C3 (RING-H2)-type or C3HC4-type (RING-CH) (Freemont, 2000; Jackson et al., 2000; Lim et al., 2013). A number of RING proteins are identified
as E3 ubiquitin ligases, which play important roles in plant metabolism and development, and in responding to environmental stresses (Dreher and Callis, 2007; Alcaide-Loridan and Jupin, 2012; Chen and Hellmann, 2013). To test whether H2-10 possesses E3 ligase activities, we performed an in vitro E3 ubiquitination ligase assay. Both MBP and Ub immunoblot assays showed that poly-ubiquitinated products with higher molecular weight were detected only in the presence of all the components (E1, E2, and Ub) with MBP–H2-10 (Figure 1C), indicating that H2-10 is an active E3 ubiquitin ligase. Interaction tests between P2 and truncated versions of H2-10 in yeast indicated that RING-finger domain is unnecessary for H2-10 to interact with P2 (Supplemental Figure 2). RINGfinger domains in RING-finger proteins are mainly responsible for interacting with ubiquitin-conjugating enzyme E2 and not generally involved in substrate binding (Jackson et al., 2000). We speculated that P2 might be a substrate of H2-10mediated degradation. Direct experimental test would be an in vitro ubiquitination assay as described above with P2 as the substrate. However, P2 is a 130-kDa membrane protein and we were unable to obtain purified P2 for such an assay despite our efforts trying various expression systems and purification methods. Therefore, we decided first to investigate whether P2 can be degraded in planta in the presence of increasing amounts of H2-10. We found in the N. benthamiana transient expression system, the protein level of P2 decreased as the amount of FLAG–H2-10 increased (Figure 1D). Then we test P2 degradation rate in the absence or presence of H2-10 when Cyclohexamide (CHX) was added to inhibit translation in 293T cells. In the absence of H2-10, HA-P2 could still be detected 4 h after CHX was added. In the presence of H2-10, however, HA-P2 disappeared 2 h after CHX addition (Supplemental Figure 3). To test whether H2-10-mediated degradation of P2 protein took place via the ubiquitination pathway, the ubiquitinmediated degradation-specific inhibitor MG132 was used. As © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu007, Advance Access publication 30 January 2014 Received 13 November 2013; accepted 17 January 2014
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Figure 1. OsRFPH2-10 (Referred to as H2-10) Promotes RDV P2 Degradation and Plays an Important Antiviral Function at Early Stages of RDV Infection. (A) Yeast two-hybrid assay for AD-H2-10 and BD-P2 interaction. (B) Co-IP analysis of FLAG-H2-10 and HA-P2 in planta. (C) E3 ubiquitin ligase activity of H2-10. The numbers on the left denote the molecular masses of marker proteins in kilodaltons. The nickelhorseradish peroxidase was used to detect His-tagged ubiquitin (top panel) and the anti-MBP antibodies were used for detecting maltose fusion proteins (bottom panel). (D) Effects of H2-10 on the stability of P2 in plant cells. Numbers indicate the ratios of agrobacteria concentrations used in co-infiltration. (E) Effects of MG132 on the stability of P2 in plant cells. (F) White specks on the leaves of RDV-infected wild-type (WT) and H2-10-overexpression rice plants. The arrowheads indicate the specks on the leaves. (G) Detection of P2 and P8 accumulations in RDV-infected WT and H2-10 overexpression rice lines (H2-10 oe #1 and #5). H, mock-inoculated; R, RDV-infected. (H) Detection of the accumulation of RDV RNAs S2, S8, and S11 in RDV-infected WT and H2-10-overexpression rice lines with corresponding 32 P-labeled probes. (I) RDV infection rates. wpi, week post inoculation. * P
