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First published online October 30, 2009; 10.1104/pp.109.147983 Plant Physiology 152:255-266 (2010) © 2010 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Host RNA Helicase-Like Protein, AtRH8, Interacts with the Potyviral Genome-Linked Protein, VPg, Associates with the Virus Accumulation Complex, and Is Essential for Infection1,[C],[W],[OA]Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, Ontario N5V 4T3, Canada (T.-S.H., T.W., A.W.); Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada (T.-S.H., T.W., A.W.); and Institut National de la Recherche Scientifique, Institut Armand-Frappier, Laval, Quebec H7V 1B7, Canada (J.-F.L.)
The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.
Plant viruses are obligate intracellular parasites that infect many agriculturally important crops and cause severe losses each year. One of the common characteristics of plant viruses is their relatively small genome that encodes a limited number of viral proteins, making them dependent on host factors to fulfill their infection cycles (Maule et al., 2002  ; Whitham and Wang, 2004; Nelson and Citovsky, 2005; Decroocq et al., 2006). In order to establish a successful infection, the invading virus must recruit an array of host proteins (host factors) to translate and replicate its genome and to move locally from cell to cell via the plasmodesmata and systemically via the vascular system. It has been suggested that down-regulation or mutation of some of the required host factors may result in recessively inherited resistance to viruses (Kang et al., 2005b).
Potyviruses, belonging to the genus Potyvirus in the family Potyviradae, constitute the largest group of plant viruses (Rajamäki et al., 2004
Previous studies have shown that VPg and its precursor NIa interact with several host proteins, including three essential components of the host protein translation apparatus (Thivierge et al., 2008 In this study, we describe the identification of an Arabidopsis DEAD-box RNA helicase (DDX), AtRH8, and a peach (Prunus persica) DDX-like protein, PpDDXL, both interacting with the potyviral VPg protein. Using the atrh8 mutant, we demonstrate that AtRH8 is not required for plant growth and development in Arabidopsis but is necessary for infection by two plant potyviruses, PPV and TuMV. Furthermore, we present evidence that AtRH8 colocalizes with the virus accumulation complex in potyvirus-infected leaf tissues, which reveals a possible role of AtRH8 in virus infection. Finally, we have identified the VPg-binding region (VPg-BR) of AtRH8 and PpDDX and show that overexpression of the VPg-BR either from AtRH8 or PpDDXL suppresses virus accumulation.
Identification of a VPg-Interacting DEAD Box RNA Helicase-Like Protein from Peach and Arabidopsis
To identify VPg-interacting host proteins in plants, a yeast two-hybrid cDNA library screen was carried out. The library was constructed from PPV-infected peach leaf tissues in order to search VPg-interacting host candidates in its natural host during virus infection. A total of 1.3 x 106 transformed cDNA clones were tested against the PPV VPg as bait. The resulting positive clones were rescued and isolated for sequencing. Based on the results of BLASTX searches (E value The full-length cDNA of PpDDXL was obtained using RACE PCR techniques and deposited into GenBank (accession no. GQ865547). The interactions between the partial or full-length PpDDXL proteins and the PPV VPg were confirmed in yeast (Fig. 1 ). The full-length cDNA of PpDDXL is 1,692 bp, with a 5' untranslated region of 293 bp, an ORF of 1,242 bp, and a 3' untranslated region of 157 bp (Supplemental Fig. S1). It encodes a polypeptide of 413 amino acids with a predicted molecular mass of 47 kD and a pI of 5.48. The corresponding genomic DNA sequence of PpDDXL was obtained by PCR amplification of genomic DNA (GenBank accession no. GQ865548). Alignment of the cDNA and genomic sequence of PpDDXL indicated that PpDDXL contains three introns and four exons (Supplemental Fig. S1A). Domain analyses using the Pfam program (http://pfam.sanger.ac.uk/) identified the DEAD/DEAH box (amino acids 64–230) signature and helicase-conserved C-terminal (amino acids 298–374) domains (Supplemental Fig. S1, B and C).
Due to the unavailability of an efficient genetic transformation protocol for the characterization of gene functions in Prunus species, Arabidopsis was selected as a model host for exploring the roles of PpDDXL and related RNA helicases in potyvirus infections. BLAST searches against the Arabidopsis database revealed 10 Arabidopsis DDXs that shared high sequence similarity to PpDDXL (Supplemental Fig. S2A). Although the three Arabidopsis eIF4As and two putative eIF4As (AT3G13920, AT1G54270, AT1G72730, AT3G1960, and AT1G51380, respectively) are most similar to PpDDXL, there were no corresponding homozygous knockout T-DNA lines available. Extensive screening of progeny plants from the eight heterozygous eIF4A T-DNA lines (SALK_038072, SALK_072655, SALK_107633, SALK_123728, SALK_135778, SALK_107633, SAIL_755_B08, and WiscDsLox254D02) failed to recover any homozygous plants. These data suggest a possible detrimental effect to the plant when silencing these eIF4As. Indeed, even the heterozygous T-DNA seedlings showed abnormal phenotypes in the number and length of root hair (Supplemental Fig. 2B). Thus, AtRH8 (AT4G00660), the next most related candidate to PpDDXL, was selected for functional characterization. The ORF of AtRH8 was obtained from Arabidopsis wild-type Columbia cDNA using reverse transcription (RT)-PCR with gene-specific primers. The ORF of AtRH8 consists of 1,518 nucleotides encoding a 505-amino acid protein. A yeast two-hybrid assay confirmed a positive interaction between the PPV VPg and AtRH8 (Fig. 1).
To study if AtRH8 and VPg colocalize in planta, transient expression vectors encoding AtRH8-cyan fluorescent protein (CFP) fusion (AtRH8-CFP) and VPg-yellow fluorescent protein (YFP) fusion (VPg-YFP) were constructed. Transient expression of these chimeric genes was achieved through agroinfiltration. As a control, AtRH8 was expressed alone (Fig. 2A
) or coexpressed with YFP (Fig. 2B). The distribution of AtRH8 was found in the cytoplasm (Fig. 2, A and B), whereas YFP was in the cytoplasm and in the nucleus (due to diffusion; Fig. 2B). In addition, AtRH8 also formed some punctate structures in the cytoplasm (Fig. 2, A and B). In Nicotiana benthamiana epidermal cells coexpressing AtRH8-CFP and VPg-YFP, the two proteins colocalized in the nucleus and in the cytoplasm (Fig. 2C). Previously, VPg-YFP was reported to localize mainly in the nucleus when expressed alone (Wei and Wang, 2008
Requirement of AtRH8 for Potyviral Infection To investigate the functional role of AtRH8 in virus infection, a homozygous T-DNA line of AtRH8, SALK_016830, with a T-DNA insertion in the promoter region was acquired from the Arabidopsis Biological Resource Center (http://www.biosci.ohio-state.edu/pcmb/Facilities/abrc/abrchome.htm). The T-DNA PCR screen on genomic DNA as well as genetic analysis confirmed that there is only one T-DNA insertion (data not shown). RT-PCR analysis of total RNA isolated from leaf tissues of this mutant line and the wild type revealed no expression of AtRH8 in the homozygous T-DNA line (Supplemental Fig. S4). Thus, this line (atrh8) is a true knockout mutant of AtRH8.
To test if AtRH8 is required for PPV infection, the atrh8 mutant and wild-type plants were mechanically inoculated with a Canadian PPV-D isolate. Total RNA was extracted from the upper newly emerged leaves 14 d post infection (dpi). RT-PCR assays were used to monitor the accumulation of the viral RNA. The PPV genomic RNA was detected only in the wild type (Fig. 3A
). Mild disease symptoms such as slight growth retardation were found in the infected wild-type plants, consistent with our previous observation (Babu et al., 2008
To test if AtRH8 is also needed by another potyvirus during infection, the wild-type and atrh8 plants were agroinfiltrated with a GFP-tagged TuMV infectious clone (TuMV-GFP). Diagnosis of these plants 14 d post agroinfiltration (dpa) with RT-PCR revealed the presence of the TuMV genomic RNA in the wild-type plants but not in the atrh8 mutants (Fig. 3B). Consistent with our PCR results, a strong emission of GFP fluorescence was observed in the newly emerged leaves of the infiltrated wild-type plants but not the atrh8 plants (Fig. 3C). Phenotypes of the wild-type plants and atrh8 mutants agroinfiltrated with TuMV-GFP or mock infiltrated were closely examined. Under the normal growth conditions without TuMV infiltration, atrh8 mutants showed no developmental differences from wild-type plants (see mock-inoculated wild type and atrh8; Fig. 3, D and E) and displayed normal vegetative growth, flowering development, and seed production. At 3 dpa, the wild-type plants agroinfiltrated with TuMV-GFP began exhibiting yellowing on the surface of the leaves and slight growth stunting. In contrast, no difference was observed between TuMV-infiltrated atrh8 mutants and mock-infiltrated wild-type or atrh8 plants (Fig. 3D). At the later infection stage (14 dpa), the infected wild-type plant displayed the full spectrum of disease symptoms, including mosaic and necrosis on leaves, severe growth retardation, reduced apical dominance, curled bolts, and the typical inflorescence stunting (Fig. 3E), similar to previous descriptions (Lellis et al., 2002 ). The TuMV-infiltrated atrh8 mutants, however, showed normal growth and fertility with no signs of infection symptoms. When the TuMV-infected plants were exposed under UV light at 19 dpa, the stunted wild-type plants exhibited bright green fluorescent emission from the tagged GFP, but no GFP was exhibited in TuMV-infiltrated atrh8 plants (Fig. 3F). Taken together, these results indicate that AtRH8 is required for both PPV and TuMV infections.
To explore a possible role of AtRH8 in virus infection, the subcellular localization of AtRH8 was examined in planta in the presence of virus infection. Potyviral 6K2 protein is an integral membrane protein that induces the formation of the endoplasmic reticulum-derived vesicles (Schaad et al., 1997
Determination of VPg-BR in AtRH8 and PpDDXL
To determine the VPg-BR of AtRH8, a series of deletions were conducted on AtRH8. Initially, the protein was truncated into two moieties, with the N-terminal portion containing 250 amino acids and the C-terminal portion containing 257 amino acids (Fig. 5, A and B
). The truncated protein was fused into the pAD-GAL plasmid of the yeast two-hybrid system. The interaction assay was conducted using the PPV VPg as the interaction partner cloned into the pBD-GAL plasmid. Growth of the yeast transformants on selective medium showed that VPg positively interacted exclusively with the N-terminal fragment of AtRH8 and not the C-terminal portion, suggesting that the interaction site resides within the first 250 amino acids of AtRH8. The N-terminal 250 amino acids were subjected to additional sequential deletions (Fig. 5, A and B). Based on the deletion analyses, the VPg-BR of AtRH8 consists of 50 amino acids (amino acids 201–250; Fig. 5, A and B). Protein sequence comparison of the VPg-BR (amino acids 140–189) of PpDDXL and that of AtRH8 indicated a 74% similarity (Fig. 5C). The protein predictor SSpro version 4.5 program from the ExPasy Proteomics Server (http://www.expasy.ch) identified an
In order to verify the interaction between the PPV VPg and the VPg-BR in planta, BiFC assays between the PPV VPg and the VPg-BR of AtRH8 or that of PpDDXL were conducted in N. benthamiana plants. Three-week-old plants were agroinfiltrated to coexpress the BR-YN (the VPg-BR of AtRH8 or PpDDXL attached to the N-terminal fragment of YFP) and the VPg-YC (the PPV VPg fused to the C-terminal fragment of YFP) as well as the reverse combinations. Infiltrated leaf tissues were observed by confocal microscopy 2 dpa. A positive interaction was observed between the VPg-BR of AtRH8 and the PPV VPg mainly in the nucleus (Fig. 5D). The interaction of the VPg-BR of PpDDXL and the PPV VPg was found in the nucleus and in the cytoplasm (Fig. 5E). These data illustrate that the VPg-BR of AtRH8 and PpDDXL is responsible for protein-protein interaction with the PPV VPg.
As described above, potyvirus infection requires the presence of AtRH8. To determine if overexpression of AtRH8 affects potyvirus infection, N. benthamiana leaves were coinfiltrated with the TuMV-GFP clone and an empty vector (as a control) or with TuMV-GFP and a plant AtRH8 expression vector. TuMV infection was assessed by real-time PCR analyses 2 dpa and visualized by confocal observation 3 dpa. In comparison with the control leaves (infiltrated with TuMV-GFP and an empty vector), leaves expressing AtRH8 and infected by TuMV-GFP displayed a much stronger green fluorescence intensity (Fig. 6A ). Quantification of TuMV using real-time PCR revealed a 1.6-fold increase in virus accumulation in the leaves overexpressing AtRH8 (Fig. 6B).
To assess the effect of transient overexpression of the VPg-BR of AtRH8 on potyvirus infection, the TuMV-GFP infectious clone was coinfiltrated into N. benthamiana with an expression plasmid containing the VPg-BR of AtRH8. Coexpression of the VPg-BR led to a reduction of the virus accumulation relative to the control (coinfiltration of TuMV-GFP clone with an empty vector; Fig. 6A). Quantitative analysis of the viral RNA indicated that viral RNA in the leaves expressing the VPg-BR of AtRH8 accumulated about 31% of that in the control (Fig. 6B). Furthermore, we tested the effect of the VPg-BR of AtRH8 on the infection of Tobacco etch virus (TEV), another member of the Potyvirus genus. Since the GFP-tagged TEV infectious clone was incompatible with the agroinfiltration system (Schaad et al., 1997 ), mechanical inoculation of TEV was used for infection. The accumulation of TEV in the leaves expressing the VPg-BR of AtRH8 was approximately 34% of that in the control (Supplemental Fig. S5). Confocal visualization of the inoculated tissues revealed a consistent reduction of green fluorescence emission as quantified by real-time PCR results (Supplemental Fig. S5). Similarly, real-time PCR analyses of TEV and TuMV infections in the presence of the VPg-BR of PpDDXL revealed a decrease of virus accumulation by approximately 5- and 3-fold in comparison with the respective controls (Supplemental Fig. S6). These data suggest that overexpression of the VPg-BR of AtRH8 or that of PpDDXL exerts a suppression effect on potyvirus infections.
In this study, we have reported the identification of two VPg-interacting plant DDX proteins, AtRH8 from Arabidopsis and PpDDXL from peach (Figs. 1 and 2). These DDX proteins share sequence homology with eIF4A, a component of the eIF4F multiprotein complex. We used the Arabidopsis AtRH8 homozygous T-DNA insertion lines to functionally characterize the requirement of AtRH8 in potyvirus infection. We found that AtRH8 knockout plants (atrh8 mutants) grew and developed as the wild-type plants, indicating that AtRH8 is dispensable for the normal plant growth and development (Fig. 3). But these mutants were unable to support PPV and TuMV infections, suggesting that AtRH8 is required for virus infections (Fig. 3). Therefore, AtRH8 is a host factor that plays an essential role in the virus infection cycle. To our knowledge, this report is the first showing that a plant DDX protein is required for virus infection in plants.
RNA helicases represent a large family of proteins implicated in almost every step of RNA metabolism (de la Cruz et al., 1999
The presence of AtRH8 in the virus accumulation complex but not in the nucleus in infected cells (Fig. 4) is consistent with the recent finding that eIF(iso)4E, also a VPg-interacting translation initiation factor, is localized to the TuMV replication complex (Cotton et al., 2009
As discussed above, both PpDDXL and AtRH8 appear to be RNA helicases by sequence comparison (Supplemental Fig. S1). Interestingly, the potyviral CI also contains an RNA-binding domain and has ATPase and RNA helicase activities (Laín et al., 1990
The result in this study showing that atrh8 mutants were resistant to both PPV and TuMV (Fig. 3) is in concordance with the properties of recessive resistance. Recently, Kang et al. (2007)
Yeast Two-Hybrid Screen
The yeast two-hybrid screen was conducted using the Matchmaker Library Construction and Screening Kits (Clontech) following the supplier's instruction manual. The VPg of a PPV-D strain was cloned into the bait vector, pGBKT7, encoding the binding domain to generate plasmid pGBKT7-VPg. The peach (Prunus persica) cDNA library was prepared by inserting cDNA derived from PPV-infected peach leaf tissues into the prey vector pGADT7-rec, encoding the activation domain. Positive clones were isolated and transformed into the Escherichia coli DH5
To obtain the 5' terminus of the PpDDXL gene, 5' RACE was performed using the FirstChoice RLM-RACE kit (Ambion) following the manufacturer's instructions. The 5' RACE PpDDXL outer primer, 5' RACE PpDDXL inner primer, 3' RACE PpDDXL outer primer, and 3' RACE PpDDXL inner primer (listed in Supplemental Table S1) were used to obtain the full-length PpDDXL cDNA. All PCRs were performed using the Phusion High-Fidelity DNA polymerase (New England Biolabs) at an annealing temperature of 60°C for 30 cycles. The PCR product was cloned with the Zero Blunt TOPO Cloning Kit (Invitrogen) and sequenced. Multiple sequence alignment to homolog proteins in different plant species was done using SeqMan from DNAStar version 6 and ClustalW alignment programs.
All the T-DNA insertion lines used in this study were obtained from the Arabidopsis Biological Resource Center. The T-DNA insertion site of the atrh8 mutant was confirmed by PCR using the T-DNA left border-specific primer (LBa1) and AtRH8-specific primers (LP16830 and RP16830). The position of the T-DNA insertion in the AtRH8 gene mutant allele was verified by DNA sequencing of the PCR products. The single T-DNA insertion was confirmed by DNA gel-blot analyses. The expression of AtRH8 was verified by RT-PCR with AtRH8-specific primers (AtRH8-F and AtRH8-R) to confirm the T-DNA line as a true knockout mutant.
cDNA encoding the full-length AtRH8 was amplified by PCR with primers AtRH8-F and AtRH8-BamHI-R using cDNA derived from the wild-type Arabidopsis (Arabidopsis thaliana) plants. The PCR products were inserted into the pGADT7 vector to generate plasmid pGAD-T7-AtRH8. AtRH8 deletion analysis fragments were obtained using primers listed in Supplemental Table S1. All constructs were confirmed by sequencing.
To construct the TuMV''6K-YFP infectious clone, the infectious clone TuMV''6K-GFP (or pCambiaTunos/6KGFP; Cotton et al., 2009
Gateway technology (Invitrogen) was used to generate plasmids for expression in plants as described previously (Wei and Wang, 2008
The binary vectors were introduced into Agrobacterium tumefaciens strain GV3101 or EHA105 by electroporation. The Agrobacterium culture was prepared according to Sparkes et al. (2006)
Mechanical inoculation of Arabidopsis with PPV-D and TuMV-GFP was as described (Babu et al., 2008
All RT-PCR and real-time PCR analyses were performed with three biological replicates. Total RNA was prepared following the instructions of the RNeasy Plant Mini Kit (Qiagen). The first-strand synthesis and subsequent PCR amplification of both the internal standard and target gene fragments were performed using the SuperScript two-step RT-PCR system (Invitrogen). For AtRH8 expression analysis, the Arabidopsis Actin2 gene was used as the internal control of RT-PCR using Actin2-specific primers At-actin-F and At-actin-R. For virus detection, newly emerged leaves of virus-inoculated plants were harvested at 14 dpi or 14 dpa. The infection of PPV and TuMV was diagnosed by RT-PCR with PPV-specific primers PPVcp-F and PPVcp-R and TuMV-specific primers HC-Pro-F and HC-Pro-R, respectively.
Real-time PCR preparations were carried out using the LightCycler480 Probes Master Kit (Roche) on a LightCycler480 real-time PCR system (Roche) following the manufacturer's instructions. Three pairs of primers, TEVcp-F and TEVcp-R, TuHC-F and TuHC-R, and NbEF-1 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers GQ865547 and GQ865548.
The following materials are available in the online version of this article.
We are indebted to Jim Carrington (Oregon State University) for providing plasmid TEV-GFP, Yuhai Cui (Agriculture and Agri-Food Canada [AAFC]) for providing a modified BiFC vector, Lorne Stobbs and Antonet Svircev (AAFC) for assistance in PPV infection assays, Alex Molnar (AAFC) for photography, Jamie McNeil (AAFC) for expert technical assistance, and Mark Bernards (University of Western Ontario) for helpful discussion and suggestions. We also acknowledge two anonymous reviewers for their very helpful comments in improving the manuscript. Received September 23, 2009; accepted October 27, 2009; published October 30, 2009.
1 This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and Agriculture and Agri-Food Canada to A.W. and by scholarships (Ontario Graduate Scholarship and Ontario Graduate Scholarship in Science and Technology) to T.-S.H.  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: Aiming Wang (aiming.wang{at}agr.gc.ca).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.147983 * Corresponding author; e-mail aiming.wang{at}agr.gc.ca.
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ABSTRACT
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1 x 10–10), a total of 85 peach proteins were identified. Of these positive clones, five contained a stretch of the same cDNA sequence. The predicted peptide from the longest clone shares 96% to 98% sequence similarity to a number of proteins, including the ATP-dependent RNA helicase and eIF4A, that belong to the DDX family. Thus, this gene is designated PpDDXL. Based on the multiple occurrences of PpDDXL in the screen, the fact that RNA helicase is part of the eIF4F translation complex, and the assumption that host RNA helicases may be involved in viral genome replication, PpDDXL was chosen for further molecular and functional characterizations. 
-Gal and incubated at 28°C for 4 d. [See online article for color version of this figure.]
