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First published online November 18, 2005; 10.1104/pp.105.072306 Plant Physiology 139:1935-1945 (2005) © 2005 American Society of Plant Biologists The Rice Dwarf Virus P2 Protein Interacts with ent-Kaurene Oxidases in Vivo, Leading to Reduced Biosynthesis of Gibberellins and Rice Dwarf Symptoms1Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Science, Peking University, Beijing 100871, China
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.
Knowledge of viral disease mechanisms has fundamental importance in understanding the evolution of virus-host interactions, basic cellular functions, and engineering of host resistance. For plant viruses, viral symptom determinants have been mapped to specific viral proteins (Brigneti et al., 1998  ; Kong et al., 2000; Chellappan et al., 2005; Padmanabhan et al., 2005; Shepherd et al., 2005; Wang and Metzlaff, 2005) and DNA/RNA sequences (Rodriguez-Cerezo et al., 1991; Fernandez et al., 1999; Dai et al., 2004). Changes in gene expression of infected plants (Whitham and Wang, 2004; Wang and Metzlaff, 2005) and altered metabolism (Roger, 2002) have also been reported. Recent studies suggest that viral suppressors of RNA silencing may also alter host microRNA metabolism that contributes to symptom development (Kasschau et al., 2003; Chen et al., 2004). In general, however, there is a lack of mechanistic insights about how a viral protein interacts directly with a specific host factor(s), thereby altering the function of a cellular pathway leading to disease development.
We use Rice dwarf virus (RDV) infection as a model system to address molecular mechanisms of viral diseases that have vital economic importance. RDV is a member of the genus Phytoreovirus, family Reoviridae (Boccardo and Milne, 1984
RDV encodes at least seven structural proteins and five nonstructural proteins. The seven structural proteins, namely, P1, P2, P3, P5, P7, P8, and P9, are products of segments S1, S2, S3, S5, S7, S8, and S9, respectively (Mao et al., 1998
The P2 protein was previously determined to be essential for RDV infection in its insect vectors and subsequent transmission to its host plants from these vectors (Yan et al., 1996
The yeast two-hybrid technology is a proven tool for identifying protein-protein interactions that lead to an understanding of the function of the protein of interest (Chien et al., 1991
Identification of an ent-Kaurene Oxidase-Like Protein That Interacts with RDV P2 To identify rice proteins that interact with the RDV P2 protein, we used a yeast two-hybrid system with RDV P2 as the bait to screen prey plasmids representing a rice cDNA library. Seven positive colonies were identified among the approximately 2 x 106 cDNA clones that were screened. The cDNA fragments from all the seven colonies encode an identical polypeptide containing 66 amino acid residues (Fig. 1). Sequence analysis showed that the polypeptide shares a high degree of identity with rice ent-kaurene oxidases (OsKO1, BAD54595, 75% identity; and OsKO2, BAD54598, 75% identity), rice ent-kaurene oxidase-like proteins (OsKOL4, BAD54592, 100% identity; and OsKOL5, BAD54586, 84% identity), Arabidopsis (Arabidopsis thaliana) ent-kaurene oxidase (AtKO1, AAC39507, 53% identity), pumpkin (Cucurbita pepo) ent-kaurene oxidase (CmKO1, AAG41776, 58% identity), and pea (Pisum sativum) ent-kaurene oxidase (PsKO1, AAP69988, 60% identity).
Cloning of the Full-Length ORF Genes Encoding Rice ent-Kaurene Oxidases or ent-Kaurene Oxidase-Like Proteins The full-length ORF of the rice ent-kaurene oxidase-like gene was cloned from rice through reverse transcription (RT)-PCR and primers designed based on sequence information available in the rice genome database (http://btn.genomics.org.cn/rice). Sequence analysis of the coding region indicated that the ORF of the rice ent-kaurene oxidase-like gene contains 1,530 nucleotides and encodes a protein of 510 amino acids (Fig. 1). The cloned gene was subsequently named OsKOS1 (GenBank accession no. AY579214).
The deduced amino acid sequence of OsKOS1 was used to search the protein database for similar sequences using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST). The BLAST results show that there are at least four genes encoding ent-kaurene oxidases or ent-kaurene oxidase-like proteins in the rice genome. To determine whether P2 could interact with other ent-kaurene oxidases or ent-kaurene oxidase-like proteins, the cDNA fragments containing the full-length ORFs of rice cDNAs (GenBank accession nos. AK071743, AK066285, and AK100964) were cloned from rice by RT-PCR and named OsKOS2 (GenBank accession no. AY660664), OsKOS3 (GenBank accession no. AY660665), and OsKOS4 (GenBank accession no. AY660666). We compared the sequences of our cDNA fragments with the genes encoding ent-kaurene oxidases or ent-kaurene oxidase-like proteins cloned and analyzed previously (Itoh et al., 2004
To test whether P2 can interact with the four full-length rice ent-kaurene oxidases and ent-kaurene oxidase-like proteins, the cDNAs encoding the protein of interest were inserted in frame into the GAL4 DNA binding domain vector pGBKT7 or GAL4 activation domain vector pGADT7 (CLONTECH), respectively. pGAD-OsKO1, pGAD-OsKO2, pGAD-OsKOL4, and pGAD-OsKOL5 encoding the fusions between the GAL4 activation domain and the respective ent-kaurene oxidases or ent-kaurene oxidase-like proteins were cotransformed with pGBKS2 into yeast. Yeast cells cotransformed with constructs pGBKT7/pGADT7, pGBKS2/pGADT7, pGBKT7/pGAD-OsKO1, pGBKS8/pGADT7, pGBKS8/pGAD-OsKO1, pGBKT7/pGAD-OsKO2, pGBKT7/pGAD-OsKOL4, and pGBKT7/pGAD-OsKOL5 served as negative controls. The pGBKS8 construct encodes one of the RDV outer capsid proteins. Only the yeast cells cotransformed with pGBKS2/pGAD-OsKO1, pGBKS2/pGAD-OsKO2, pGBKS2/pGAD-OsKOL4, and pGBKS2/pGAD-OsKOL5 were able to grow on the selective media (Fig. 2). These results established that P2 interacts specifically with OsKO1, OsKO2, OsKOL4, and OsKOL5 in yeast cells.
P2 Interacts with Rice ent-Kaurene Oxidases and ent-Kaurene Oxidase-Like Proteins in Plant Cells Specific interaction of P2 with rice ent-kaurene oxidase and ent-kaurene oxidase-like proteins in yeast suggests functional significance. To test this further, we used coimmunoprecipitation to determine whether such interaction occurs in plant cells. As shown in Figure 3, the hemagglutinin (HA)-epitope-tagged P2 coimmunoprecipitated with the FLAG (synthetic octapeptide)-OsKO2 or FLAG-OsKOL4 after Agrobacterium-mediated transient expression in Nicotiana benthamiana. This interaction was confirmed with the reciprocal experiments, in which FLAG-OsKO2 and FLAG-OsKOL4 coimmunoprecipitated with the HA-P2, respectively (Fig. 3). These results provided evidence that P2 interacts with OsKO2 or OsKOL4 in plant cells. Furthermore, similar assays demonstrated that P2 interacts with OsKO1 or OsKOL5 (data not shown).
Decrease of GA1 in RDV-Infected Rice Plants
ent-Kaurene oxidases play an essential role in GA biosynthesis (Helliwell et al., 1998
To further test whether the reduced accumulation of GAs in RDV-infected plants contributed to the disease symptoms, we asked whether symptoms caused by RDV infection could be rescued by exogenously supplied GA. As shown in Figure 5, RDV-infected rice plants sprayed with GA3 grew almost as tall as the uninfected rice plants. Significantly, application of indole-3-acetic acid (IAA) to the RDV-infected plants failed to restore the height of infected plants (Fig. 5). These results provided compelling evidence that the reduced endogenous GA level in RDV-infected plants is specifically responsible for the development of growth stunting symptoms.
RDV Infection Resulted in Down-Regulation of OsKO1, OsKO2, OsKOL4, and OsKOL5 Expression Levels The reduced GA levels in RDV-infected plants could be attributed to inhibited activity and/or expression of ent-kaurene oxidases or ent-kaurene oxidase-like proteins as a result of P2 interactions. While testing the activity of these enzymes awaits development of biochemical assay systems, their expression levels could be investigated by standard molecular methods. To this end, we determined the accumulation levels of OsKO and OsKOL mRNAs by quantitative real-time RT-PCR. Such analysis showed that the overall accumulation levels of OsKO or OsKOL transcripts in leaves of RDV-infected rice were 50% of that in healthy rice plants (Fig. 6).
Semiquantitative RT-PCR was used to analyze the accumulation levels of OsKO1, OsKO2, OsKOL4, and OsKOL5 mRNAs in RDV-infected and healthy rice plants using specific primers of longer lengths (>500 bp), respectively. The results showed that the accumulation levels of OsKOL4 and OsKOL5 were reduced in RDV-infected rice plants compared with that from healthy plants (Fig. 7). The transcription of OsKO1 and OsKO2 was also down-regulated in RDV-infected rice plants (data not shown).
Plant virus infections often lead to alterations in physiological, biochemical, and metabolic processes, resulting in symptoms such as plant stunting and leaf mottling and/or wrinkling (Jameson and Clarke, 2002 ). For many virus diseases, alterations in plant growth were thought to be the result of cytopathic effects leading to changes in plant hormone metabolism upon virus infection (Jameson and Clarke, 2002). Despite extensive research efforts, the host factors that are primary targets for viral proteins in disease formation remain largely elusive. RDV infection causes growth stunting and leaf darkening in rice, which are also typical of GA-deficiency symptoms. P2 has been implicated as a pathogenicity determinant of RDV (Tomaru et al., 1997; T. Omura, personal communication). In this study, we have identified rice ent-kaurene oxidases as the primary targets of the RDV P2 protein for symptom expression. First, P2 interacts with these enzymes in yeast (Fig. 2). More importantly, this interaction occurs in plant cells (Fig. 3). Second, the growth stunting and leaf darkening symptoms of RDV-infected plants are correlated with reduced levels of GAs (Fig. 4). Such symptoms were alleviated with exogenously supplied GAs but not IAA (Fig. 5). This observation indicates that GA perception and signal transduction pathways appeared to be functional in RDV-infected rice plants and there was a correlation between GA1 content and dwarf phenotype. Third, RDV-infected plants exhibit down-regulated expression of ent-kaurene oxidases (Fig. 6). These observations are fully consistent with the established role of ent-kaurene oxidases in GA biosynthesis and with the established role of GAs in plant growth and development. It is significant to note that the phenotypes of RDV-infected plants are strikingly similar to those exhibited by the ent-kaurene oxidase mutant rice plant d35Tan-Ginbozu, which also showed GA deficiency (Kobayashi et al., 1989; Ogawa et al., 1996; Itoh et al., 2004). Taking all data together, we propose that in an RDV-infected plant, the RDV P2 interacts with ent-kaurene oxidases. The reduced expressions and/or activities of these enzymes, as a result of P2 interactions, compromise the biosynthesis of GAs. The reduced accumulation of GAs contributes directly to the abnormal functions of the GA-regulated cellular processes that lead to symptom expression.
Our findings have broad significance in studying the mechanisms of GA metabolism in plant growth and development and in plant-viral interactions. The GAs are a large family of tetracyclic diterpenoid plant regulators that are involved in a number of plant growth and developmental processes, including seed germination, stem elongation, flowering, fruit development, apical dominance, and regulation of gene expression in the cereal aleurone layer (Hooley, 1994
Decreases in GA1 level were reported in two uninfected dwarf rice cultivars (Tan-Ginbozu and Waito-C; Kobayashi et al., 1989
Although the P2 protein plays a role in inhibiting plant growth, other RDV factors may also be involved in this process. Kimura et al. (1987)
Our yeast two-hybrid and coimmunoprecipitation assays showed that P2 also interacted with rice ent-kaurene oxidase-like proteins (OsKOL4 and OsKOL5; Figs. 2 and 3). The interaction of P2 with the two ent-kaurene oxidase-like proteins (OsKOL4 and OsKOL5) suggests other possibilities of functional significance. The expression of OsKOL4 and OsKOL5 was also reduced upon RDV infection (Figs. 6 and 7). Itoh et al. (2004) In summary, our study suggests that, during RDV infection, the P2 interacts with ent-kaurene oxidases, resulting in inhibition of their activities and/or transcription. This further leads to reduced GA levels that contribute to growth stunting and the associated symptoms. The interaction of P2 with ent-kaurene oxidase-like proteins may have important roles that remain to be understood. Our findings establish a foundation to further investigate the molecular mechanisms of how a viral protein interacts with critical cellular factors that lead to altered cellular functions to cause disease symptoms.
PCR Primer Sequences The oligonucleotides used in this study are listed in Table I.
Plasmid Construction
RDV gene segment S2 (GenBank accession no. AY847464) containing the S2 ORF was ligated into the NcoI/EcoRI site (Promega) within the GAL4 DNA binding domain vector pGBKT7 (CLONTECH). RDV gene segment S8 (GenBank accession no. U36565) was also cloned and placed into the pGBKT7 vector. Briefly, the pGBKT7 vector was digested with NcoI, the ends made blunt with T4 DNA polymerase (Promega), and then digested with BglII. PCR products of S8 amplified with primer pairs F1 and R1 were made blunt and digested with BglII, followed by ligation into the pGBKT7 vector. The recombinant plasmid containing the RDV S2 or S8 segment was designated as pGBKS2 or pGBKS8. The cDNA fragment identified during the yeast two-hybrid screening had sequence identity with the C terminus of CYP701A8 (japonica cultivar group), a putative gene encoding a rice (Oryza sativa) ent-kaurene oxidase-like protein (http://drnelson.utmem.edu/rice.color.dec29.html), designated as OsKOL4 presently. The full-length ORF of the gene was cloned using RNA extracted from rice seedlings (cv Xiu shui 11 japonica). First-strand cDNA was prepared from the total RNA using Superscript II RT (Invitrogen) and primer R2 specific for the OsKOL4. The cDNA was then subjected to PCR using primers R2 and F2. A 1.5-kb PCR product containing the ORF of OsKOL4 was cloned directly into the pENTR/D-TOPO vector as described (Invitrogen). The three other genes encoding rice ent-kaurene oxidases or ent-kaurene oxidase-like proteins were also cloned from rice seedlings with primer pairs F3/R3, F4/R4, and F5/R5, which were designed according to rice cDNA sequences (GenBank accession nos. AK071743, AK066285, and AK100964). The specific fragments were cloned into pENTR/D-TOPO vector, respectively. Constructs containing OsKO1 or OsKO2 ORF were digested with BglII and EcoRI, and fragments were ligated into BglII/EcoRI-linearized pGADT7 vector. Constructs containing OsKOL4 or OsKOL5 ORF were digested with NcoI and SalI, and fragment was ligated into NcoI- and SalI-digested pGADT7 vector. The recombinant plasmids were designated as pGAD-OsKO1, pGAD-OsKO2, pGAD-OsKOL4, and pGAD-OsKOL5, respectively. HA- and FLAG-epitope tags were added to S2 or OsKO (OsKOL) constructs by PCR with gene-specific primers (F6 and R6, F7 and R7, F8 and R7, F9 and R8, and F10 and R9). Restriction fragments containing ORFs of HA-S2, FLAG-OsKOL4, or FLAG-OsKOL5 were ligated into a cauliflower mosaic virus 35S-based pRTL2 transient-expression vector digested with NcoI and BamHI (Restrepo et al., 1990
A rice seedling two-hybrid cDNA library from rice cv Xiu shui 11 was constructed with CLONTECH protocols. The titer of the library was determined after amplification and was approximately 6 x 108 cfu/mL. The cDNA encoding full-length P2 protein was inserted in frame into the GAL4 DNA binding domain vector pGBKT7 (CLONTECH). The rice cDNA library in GAL4 activation domain vector pGADT7 was screened and the isolation of positive clones was performed using MATCHMAKER GAL4 Two-Hybrid System 3 and Libraries (CLONTECH).
Agrobacterium strain EHA105 carrying the gene of interest expressed from a binary vector was infiltrated into leaves of Nicotiana benthamiana essentially as described previously (Tai et al., 1999
After Agrobacterium-mediated transient expression for 24 h, N. benthamiana leaves (approximately 0.3 g) were harvested and ground to a powder in liquid nitrogen. Ground tissues were resuspended in 3.0 mL of IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Nonidet P-40, 5 mM dithiothreitol, and 1.5x Complete Protease Inhibitor [Roche]; Leister et al., 2005
Protein samples were separated by SDS-PAGE on 8% polyacrylamide gels and transferred by electroblotting to nitrocellulose membranes. Membranes were probed with anti-HA horseradish peroxidase (Roche) or anti-FLAG peroxidase (Sigma-Aldrich) to detect HA- and FLAG-epitope-tagged proteins, respectively. All immunoprecipitation experiments were repeated at least three times, and the identical results were obtained.
Procedure used to purify GAs from RDV-infected or healthy rice plant was as described previously (Weiler, 1986
The responses of RDV-infected rice plants to gibberellin (GA3) and IAA were examined with RDV-infected rice plants of 30 d post-RDV infection, which were inoculated at the stage of five leaves. RDV-infected rice seedlings of similar size were chosen and divided into four groups. The seedlings were sprayed once every 5 d with 10 mL of GA3 (50 mg/L), IAA (30 mg/L), or water (Fukazawa et al., 2000
Total RNA was extracted from rice leaves using TriReagent (Sigma) and was treated with RQ1 RNase-free DNase (Promega). Five micrograms of total RNA was reverse transcribed by SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) with random hexamers according to the manufacturer's instructions. The real-time PCR reaction was performed using DyNAmo SYBR Green qPCR kits (FINNZYMES) following the manufacturer's instructions. EF1
Semiquantitative RT-PCR was used to analyze the transcription level of OsKO1, OsKO2, OsKOL4, and OsKOL5 with specific primers in RDV-infected and healthy rice plants, respectively. Due to the high nucleotide sequence similarity among these four genes, the upstream primers used were F13 for OsKO1 and OsKO2, F14 for OsKOL4, and F15 for OsKOL5, and the downstream primer used for all reactions was R12. Total RNA was extracted from frozen rice leaves using TriReagent (Sigma) as described above. The RT condition was 42°C for 50 min. Each PCR reaction (25 µL) contained 2 µL of cDNA template, and PCR was conducted for 25 cycles for Actin 1 and 35 cycles for OsKO1, OsKO2, OsKOL4, or OsKOL5 at 94°C, 5 min; 94°C for 50 s; 55°C for 1 min; 72°C for 90 s; and 72°C for 10 min. Transcription level of Actin 1 gene in these rice samples was analyzed using primers F16 and R13 as a control. The PCR products were visualized in 1.0% agarose gel after staining with ethidium bromide. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AY579214, AY660664, AY660665, and AY660666.
We thank James C. Carrington for providing pRTL2; Hongbing Shu for assistance with the coimmunoprecipitation experiments; and Biao Ding, Richard S. Nelson, Xingshun Ding, Yuxian Zhu, and Sara Berg for critical comments on the manuscript. Received October 6, 2005; returned for revision October 6, 2005; accepted October 19, 2005.
1 This work was supported by grants of the National Outstanding Youth Grant (contract no. 30125004) and the National Science Foundation of China (to Y.L.), and by the National Key Basic Research Program (973; contract no. G6200016201 to C.H.W.). 
2 Present address: Plant Protection Department, Zhejiang University, Hua Jia Chi, Hangzhou 310029, China. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yi Li (liyi{at}pku.edu.cn). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.072306. * Corresponding author; e-mail liyi{at}pku.edu.cn; fax 86–10–6275–4427.
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was used as an internal control. Quantifications of each cDNA sample were made in triplicate, and the consistent results from at least five separately prepared RNA samples were used. Purified plasmids or cDNAs representing OsKOs (OsKOLs) and EF1
