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, 139 (4), 1935-45

The Rice Dwarf Virus P2 Protein Interacts With Ent-Kaurene Oxidases in Vivo, Leading to Reduced Biosynthesis of Gibberellins and Rice Dwarf Symptoms

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The Rice Dwarf Virus P2 Protein Interacts With Ent-Kaurene Oxidases in Vivo, Leading to Reduced Biosynthesis of Gibberellins and Rice Dwarf Symptoms

Shifeng Zhu et al. Plant Physiol.

Abstract

The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development.

Figures

Figure 1.
Figure 1.
Alignment of ent-kaurene oxidases or ent-kaurene oxidase-like proteins from rice, Arabidopsis, pumpkin, and pea. The alignment was made using DNAMAN (4.0). Identical (*) and conservatively substituted (.) amino acid residues are shown. The 66 amino acid residues identified in the yeast two-hybrid screening are underlined. The additional 17 amino acid residues in OsKOS1 but absent from OsKOL4 are indicated in the open box. The proteins can also be identified by their GenBank accession numbers as follows: AAT46567 (OsKOS1), BAD54592 (OsKOL4), AAT81229 (OsKOS2), BAD54586 (OsKOL5), AAT81230 (OsKOS3), BAD54598 (OsKO2), AAT91065 (OsKOS4), BAD54595 (OsKO1), AAC39507 (AtKO1), AAG41776 (CmKO1), and AAP69988 (PsKO1), respectively.
Figure 2.
Figure 2.
Interaction of P2 with rice ent-kaurene oxidases or ent-kaurene oxidase-like proteins in yeast. a, P2 interacts with OsKO1. b, P2 interacts with OsKO2, OsKOL4, and OsKOL5. The transformants were plated on a SD/−Leu/−Trp/−His/−Ade medium. A and G, pGBKT7 and pGADT7; B and H, pGBKS2 and pGADT7; C, pGBKT7 and pGAD-OsKO1; D, pGBKS2 and pGAD-OsKO1; E, pGBKS8 and pGADT7; F, pGBKS8 and pGAD-OsKO1; I, pGBKT7 and pGAD-OsKO2; J, pGBKS2 and pGAD-OsKO2; K, pGBKT7 and pGAD-OsKOL4; L, pGBKS2 and pGAD-OsKOL4; M, pGBKT7 and pGAD-OsKOL5; N, pGBKS2 and pGAD-OsKOL5.
Figure 3.
Figure 3.
P2 associates with OsKO2 (OsKOL4) in plant cells. A, Immunoblot showing P2 coimmunoprecipitated with OsKO2. The total proteins were isolated from Agrobacterium-infiltrated N. benthamiana leaves expressing HA:P2 and the FLAG:OsKO2, and immunoprecipitated with anti-HA (top) and anti-FLAG (bottom) antibodies. B, P2 (HA:P2) coimmunoprecipitates with FLAG:OsKOL4.
Figure 4.
Figure 4.
GA1 content in RDV-infected rice plants. Each column represents the mean ± se of measurement from four plants. g fw, Gram fresh weight. Extract residues were dissolved in phosphate-buffered saline (0.01 m, pH 7.4) to determine GA content. The content of GA1 was measured by an indirect ELISA technique with anti-GA3 antibodies. Healthy means uninfected healthy rice seedlings. RDV means RDV-infected rice seedlings.
Figure 5.
Figure 5.
Restoration of the dwarf phenotype of RDV-infected rice plants by application of GA3. A, GA3 restored the phenotype of RDV-infected rice plants. Rice plants at 30 d postinfection, inoculation at the stage of five leaves, were treated with GA3 (50 mg/L), IAA (30 mg/L), or water. The height of RDV-infected plants was measured before and after treatment. The RDV-infected plants were sprayed with water, GA3, or IAA once every 5 d. The spray volume was about 10 mL. Ten days after the second spraying, the height of the treated plants was measured. In contrast to RDV-infected rice plants, wild-type plants were only treated with water and were measured correspondingly. Each group had more than 10 plants and the experiments were repeated three times. B, Statistical analysis of plant height treated with GA3. Each column represents the mean ± se of 30 wild-type (white bars) or RDV-infected plants (gray bars). The sds are also indicated. Healthy means uninfected healthy rice seedlings. RDV means RDV-infected rice seedlings.
Figure 6.
Figure 6.
The relative accumulation levels of OsKO (OsKOL) mRNAs in leaves of healthy and RDV-infected rice plants by quantitative real-time RT-PCR. Data represent means plus sds based on results from five replicate experiments.
Figure 7.
Figure 7.
The expression levels of OsKOL4 and OsKOL5 in healthy and RDV-infected rice plants. RT-PCR was used to detect the transcriptional levels of OsKOL4 and OsKOL5. A, PCR analysis of primer specificity for OsKOL4 and OsKOL5; B, OsKOL4; C, OsKOL5; Healthy, RNA was extracted from uninfected healthy rice plants; RDV, RNA was extracted from RDV-infected rice plants. As an internal standard, the rice Actin 1 gene was amplified using Actin 1-specific primers.

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