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 Infection

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein 2 involved in viral genome translation and replication. Previous studies have shown that 3 both eIF4E and eIF4G or their respective isoforms from the eIF4F complex, which 4 modulates the initiation of protein translation, selectively interact with VPg and are 5 required for potyvirus infection. Here, we report the identification of two DEAD-box 6 RNA helicase-like proteins, PpDDXL and AtRH8 from Prunus persica and Arabidopsis 7 thaliana , respectively, both interacting with VPg. We show that AtRH8 is dispensable for 8 plant growth and development but necessary for potyvirus infection. In potyvirus- 9 infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast- 10 bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome 11 translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding 12 region. Comparison of this region and the corresponding region of PpDDXL suggests 13 that they are highly conserved and share the same secondary structure. Moreover, 14 overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses 15 potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together these data 16 demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus 17 infection process and both AtRH8 and PpDDXL may be manipulated for the development 18 of genetic resistance against potyvirus infections. analysis of PPV and TuMV accumulation, 20 respectively. Wild type (WT) plants and atrh8 mutants were mechanically inoculated and 21 agro-infiltrated with PPV and TuMV, respectively. Total RNA extracted from upper newly emerged leaves two weeks post inoculation was used for cDNA synthesis. The cDNA was amplified using PPV coat protein (CP)-specific primers and TuMV HC-Pro specific-primers for the corresponding assay. Actin2 selected as the endogenous 25 gene to serve as an internal control of RT-PCR.


INTRODUCTION 1
Plant viruses are obligate intracellular parasites that infect many agriculturally 2 important crops and cause severe losses each year. One of the common characteristics of 3 plant viruses is their relatively small genome that encodes a limited number of viral 4 proteins, making them dependent on host factors to fulfill their infection cycles (Maule et 5 al., 2002;Whitham and Wang, 2004;Nelson and Citovsky, 2005;Decroocq et al., 2006). 6 In order to establish a successful infection, the invading virus must recruit an array of 7 host proteins (host factors) to translate and replicate its genome, and to move locally from 8 cell to cell via the plasmodesmata and systemically via the vascular system. It has been 9 suggested that downregulation or mutation of some of the required host factors may 10 result in recessively inherited resistance to viruses (Kang et al., 2005b). 11 Potyviruses, belonging to the genus Potyvirus in the family Potyviradae, 12 constitute the largest group of plant viruses (Rajamäki et al., 2004). Potyviruses have a 13 single positive-strand RNA genome approximately 10 kilobases (kb) in length, with a 14 viral genome-linked protein (VPg) covalently attached to the 5' end and a poly(A) tail at 15 the 3' end (Urcuqui-Inchima et al., 2001;Rajamäki et al., 2004). The viral genome 16 contains a single open reading frame (ORF) that translates into a polypeptide with a 17 molecular mass of approximately 350 kDa, which is cleaved into 10 mature proteins by 18 viral proteases (Urcuqui-Inchima et al., 2001). Recently, a novel viral protein resulting 19 from a frameshift in the P3 cistron has been reported (Chung et al., 2008). Of the 11 viral 20 proteins, VPg is a multifunctional protein and the only other viral protein present in the 21 viral particles (virions) besides the coat protein (CP) and the cyclindrical inclusion 22 protein (CI) (Oruetxebarria et al., 2001;Puustinen et al., 2002;Gabrenaite-Verkhovskaya 23 et al., 2008). The non-structural protein is linked to the viral RNA by a phophodiester 24 bond between the 5' terminal uridine residue of the RNA and the O 4 -hydroxyl group of 25 amino acid tyrosine (Murphy et al., 1996;Oruetxebarria et al., 2001;Puustinen et al., 26 2002). Mutation of the tyrosine residue that links VPg to the viral RNA abolishes virus 27 infectivity completely (Murphy et al., 1996). In infected cells, VPg and it precursor NIa 28 are present in the nucleus and in the membrane-associated virus replication vesicles in the 29 cytoplasm (Carrington et al., 1993;Rajamäki and Valkonen, 2003;Cotton et al., 2009

persica and A. thaliana 23
To identify VPg-interacting host proteins in plants, a yeast two-hybrid cDNA 24 library screen was carried out. The library was constructed from PPV-infected P. persica 25 leaf tissues in order to search VPg-interacting host candidates in its natural host during 26 virus infection. A total of 1.3 x 10 6 transformed cDNA clones were tested against the 27 PPV VPg as bait. The resulting positive clones were rescued and isolated for sequencing. 28 Based on the results of BLASTX searches (E value <1x10 -10 ), a total of 85 P. persica 29 proteins were identified. Of these positive clones, five contained a stretch of the same 30 cDNA sequence. The predicted peptide from the longest clone shares 96 to 98% of 31 sequence similarity to a number of proteins including the ATP-dependent RNA helicase 1 and eIF4A that belong to the DDX family. Thus, this gene is designated PpDDXL 2 (Prunus persica DDX-like). Based on the multiple occurrences of PpDDXL in the screen, 3 the fact that RNA helicase is part of the eIF4F translation complex, and the assumption 4 that host RNA helicases may be involved in viral genome replication, PpDDEL was 5 chosen for further molecular and functional characterizations. 6 The full-length cDNA of PpDDXL was obtained using RACE PCR techniques 7 and deposited into GenBank (accession number GQ865547). The interactions between 8 the partial or full-length PpDDXL proteins with the PPV VPg were confirmed in yeast 9 ( Fig. 1) number and length of root hair (Supplemental Fig. 2B). Thus, AtRH8 (AT4G00660), the 1 next most related candidate to PpDDXL, was selected for functional characterization. The 2 ORF of AtRH8 was obtained from A. thaliana wild-type Col-0 cDNA using RT-PCR 3 with gene specific primers. The ORF of AtRH8 consists of 1518 nucleotides (nt) 4 encoding a 505 aa protein. A yeast two-hybrid assay confirmed a positive interaction 5 between the PPV VPg and AtRH8 (Fig. 1). 6 To study if AtRH8 and VPg colocalize in planta, transient expression vectors 7 encoding AtRH8-cyan fluorescent protein (CFP) fusion (AtRH8-CFP) and VPg-yellow 8 fluorescent protein (YFP) fusion (VPg-YFP) were constructed. Transient expression of 9 these chimeric genes was achieved through agro-infiltration. As a control, AtRH8 was 10 expressed alone ( Fig. 2A) or coexpressed with YFP (Fig. 2B). The distribution of AtRH8 11 was found in the cytoplasm ( Fig. 2 A and B), whereas YFP was in the cytoplasm and in 12 the nucleus (due to diffusion) (Fig. 2B). In addition, AtRH8 also formed some punctate 13 structures in the cytoplasm (Fig. 2 A and B). In N. benthamiana epidermal cells 14 coexpressing AtRH8-CFP and VPg-YFP, the two proteins colocalized in the nucleus and 15 in the cytoplasm (Fig. 2C). Previously, VPg-YFP was reported to localize mainly in the 16 nucleus when expressed alone (Wei and Wang, 2008). Thus, the VPg-YFP interfered in 17 the distribution of AtRH8-CFP. To further investigate the interaction between VPg and 18 AtRH8 in planta, a bimolecular fluorescence complementation (BiFC) assay was carried 19 out. Several BiFC negative control combinations were set up to ensure the validity of the 20 BiFC results. These combinations included the N-terminal (YN) and C-terminal (YC) 21 fragments of YFP, and YN 22 and VPg-YC (Supplemental Fig. S3). When AtRH8-YN was coexpressed with VPg-YC 23 in N. bethamiana plants, a strong emission of YFP fluorescence was observed in the 24 cytoplasm and in the nucleus as early as two days post agro-infiltration (Fig. 2D) state.edu/pcmb/Facilities/abrc/abrchome.htm). The T-DNA PCR screen on genomic 1 DNA as well as genetic analysis confirmed that there is only one T-DNA insertion (not 2 shown). RT-PCR analysis of total RNA isolated from leaf tissues of this mutant line and 3 wild-type revealed no expression of AtRH8 in the homozygous T-DNA line 4 (Supplemental Fig. S4). Thus, this line (atrh8) is a true knockout mutant of AtRH8. 5 To test if AtRH8 is required for PPV infection, the atrh8 mutant and wild-type 6 plants were mechanically inoculated with a Canadian PPV-D isolate. Total RNA was 7 extracted from the upper newly emerged leaves 14 days post infection (dpi). RT-PCR 8 assays were used to monitor the accumulation of the viral RNA. The PPV genomic RNA 9 was detected only in the wild-type (Fig. 3A). Mild disease symptoms such as slight 10 growth retardation were found in the infected wild-type plants, consistent with our 11 previous observation (Babu et al., 2008). In contrast, the atrh8 mutant plants inoculated 12 with PPV did not show any disease symptoms and no PPV was detectable in these 13 inoculated mutant plants (Fig. 3A). Taken together, these data suggest atrh8 mutants are 14 resistant to PPV. 15 To test if AtRH8 is also needed by another potyvirus during infection, the wild- flowering development and seed production. At 3 dpa, the wild-type plants agro-27 infiltrated with TuMV-GFP began exhibiting yellowing on the surface of the leaves and 28 slight growth stunting. In contrast, no difference was observed between TuMV-infiltrated 29 atrh8 mutants and mock-infiltrated wild-type or atrh8 plants (Fig. 3D). At the later 30 infection stage (14 dpa), the infected wild-type plant displayed the full spectrum of 31 disease symptoms including mosaic and necrosis on leaves, severe growth retardation, 1 reduced apical dominance, curled bolts, and the typical inflorescence stunting (Fig. 3E) 2 similar to previous descriptions (Lellis et al., 2002). The TuMV-infiltrated atrh8 mutants, 3 however, showed normal growth and fertility with no signs of infection symptoms. When 4 the TuMV-infected plants were exposed under the ultraviolet light 19 dpa, the stunted 5 wild-type plants exhibited bright green fluorescent emission from the tagged GFP but no 6 GFP was exhibited in TuMV-infiltrated atrh8 plants (Fig. 3F). Taken together, these 7 results indicate that AtRH8 is required for both PPV and TuMV infections.

Determination of the VPg-binding region (VPg-BR) in AtRH8 and PpDDXL 29
To determine the VPg-BR of AtRH8, a series of deletions were conducted on 30 AtRH8. Initially, the protein was truncated into two moieties with the N-terminal portion 31 containing 250 aa and the C-terminal portion containing 257 aa ( Fig. 5 A and B). The 1 truncated protein was fused into the pAD-GAL plasmid of the yeast two-hybrid system. 2 The interaction assay was conducted using the PPV VPg as the interaction partner cloned 3 into the pBD-GAL plasmid. Growth of the yeast transformants on selective media 4 showed VPg positively interacted exclusively with the N-terminal fragment of AtRH8 5 and not the C-terminal portion, suggesting the interaction site resides within the first 250 6 aa of AtRH8. The N-terminal 250 aa was subjected to additional sequential deletions 7 were coinfiltrated with the TuMV-GFP clone and an empty vector (as a control) or with 30 TuMV-GFP and a plant AtRH8 expression vector. TuMV infection was assessed by real-31 time PCR analyses 2 dpa and visualized by confocal observation 3 dpa. In comparison to 1 the control leaves (infiltrated with TuMV-GFP and an empty vector), leaves expressing 2 AtRH8 and infected by TuMV-GFP displayed a much stronger green fluorescence 3 intensity (Fig. 6A). Quantification of TuMV using real-time PCR revealed a 1.6-fold 4 increase in virus accumulation in the leaves overexpressing AtRH8 (Fig. 6B). 5 To assess the effect of transient overexpression of the VPg-BR of AtRH8 on 6 potyvirus infection, the TuMV-GFP infectious clone was coinfiltrated into N. 7 benthamiana with an expression plasmid containing the VPg-BR of AtRH8. 8 Coexpression of the VPg-BR led to a reduction of the virus accumulation relative to the 9 control (coinfiltration of TuMV-GFP clone with an empty vector) (Fig. 6A). Quantitative 10 analysis of the viral RNA indicated that viral RNA in the leaves expressing the VPg-BR 11 of AtRH8 accumulated about 31% of that in the control (Fig. 6B). Furthermore, we tested In this study, we have reported the identification of two VPg-interacting plant 27 DDX proteins, i.e., AtRH8 from A. thaliana and PpDDXL from P. persica (Figs. 1 and  28 2). These DDX proteins share sequence homology with eIF4A, a component of the eIF4F 29 multiprotein complex. We used the A. thaliana AtRH8 homozygous T-DNA insertion 30 lines to functionally characterize the requirement of AtRH8 in potyvirus infection. We 31 found that AtRH8 knockout plants (atrh8 mutants) grew and developed as the wild-type 1 plants, indicating that AtRH8 is dispensable for the normal plant growth and development 2 (Fig. 3). But these mutants were unable to support PPV and TuMV infections, suggesting 3 AtRH8 is required for virus infections (Fig. 3). Therefore, AtRH8 is a host factor that 4 plays an essential role in the virus infection cycle. To our knowledge, this report was the 5 first showing a plant DDX protein is required for virus infection in plants. 6 RNA helicases (RH) represent a large family of proteins implicated in almost 7 every step of RNA metabolism (de la Cruz et al., 1999;Tanner and Linder, 2001;Lorsch, 8 2002;Mohr et al., 2002). During the virus infection process, RHs have been suggested to 9 be involved in (i)  infected cells (Fig. 4) The presence of AtRH8 in the virus accumulation complex but not in the nucleus 7 in infected cells (Fig. 4) is consistent with the recent finding that eIF(iso)4E, also an 8 VPg-interacting translation initiation factor, is localized to the TuMV replication 9 complex (Cotton et al., 2009). In infected cells, NIa is the major form of VPg, which, as a 10 viral RNA genome-linked protein, is present in the cytoplasm or, as a nuclear localization 11 signal-containing protein, is translocated into the nucleus (Restrepo-Hartwig and 12 Carrington, 1992;Carrington et al., 1993;Rajamäki andValkonen, 2003, 2009). It is 13 puzzling that AtRH8 and eIF(iso)4E were mainly found in the cytoplasm but not in the 14 nucleus. One possible explanation is that in the early infection stage, NIa or VPg modified forms have different binding abilities to AtRH8. Further determination of their 1 subcellular localization over the infection course and analysis of their ability to interact 2 with AtRH8 may help understand the mechanism underlying the recruitment of AtRH8 to 3 the virus accumulation complex. 4 As discussed above, both PpDDXL and AtRH8 appear to be RNA helicases by 5 sequence comparison (Supplemental Fig. S1). Interestingly, the potyviral CI also contains 6 an RNA-binding domain and has ATPase and RNA helicase activities (Laín et al., 1990(Laín et al., , 7 1991Eagles et al., 1994). A genetic study on the CI using a TEV infectious clone 8 revealed that CI plays essential dual roles in TEV replication and cell-to-cell movement 9 (Carrington et al., 1998). In agreement with this finding, the potyviral CI has been shown 10 to associate with the TuMV replication complex that contains host factors such as 11 eIF (iso) The result in this study showing that atrh8 mutants were resistant to both PPV and 30 TuMV (Fig. 3)

MATERIALS AND METHODS 24
Yeast two-hybrid screen 25 The yeast two-hybrid screen was conducted using the Matchmaker Library 26 Construction & Screening Kits (Clontech) following the supplier's instruction manual. 27 The VPg of a PPV-D strain was cloned into the bait vector, pGBKT7 encoding the 28 binding domain (BD) to generate plasmid pGBKT7-VPg. The peach cDNA library was 29 prepared by inserting cDNA derived from PPV-infected P. persica leaf tissues into the 30 prey vector pGADT7-rec encoding the activation domain (AD To obtain the 5´ terminus of the PpDDXL gene, the 5´ rapid amplification of 5 cDNA ends (RACE) was performed using the FirstChoice RLM-RACE kit (Ambion) 6 following the manufacturer's instructions. The 5´ RACE PpDDXL outer primer, 5´ 7 RACE PpDDXL inner primer, 3´ RACE PpDDXL outer primer, and 3´ RACE PpDDXL 8 inner primer (listed in Supplemental Tab. S1) were used to obtain the full-length 9 PpDDXL cDNA. All PCR reactions were performed using the PhusionTM High- AtRH8-F, and AtRH8-BamHI-R using cDNA derived from the wild-type A. thaliana 27 plants. The PCR products were inserted into the pGADT7 vector to generate plasmid 28 pGAD-T7-AtRH8. AtRH8 deletion analysis fragments were obtained using primers 29 listed in Supplemental Tab. S1. All constructs were confirmed by sequencing.

Agro-infiltration and confocal microscopy 24
The binary vectors were introduced into Agrobacterium tumefaciens strain 25 GV3101 or EHA105 by electroporation. The Agrobacterium culture was prepared 26 according to Sparkes et al. (2006). Real-time PCR reaction preparations were carried out using the LightCycler®480 20 Probes Master kit (Roche) on a LightCycler®480 real-time PCR system (Roche) 21 following the manufacturer's instructions. Three pairs of primers, i.e., TEVcp-F and 22 TEVcp-R, TuHC-F and TuHC-R, and NbEF-1α-F and NbEF-1α-R were used for 23 quantification analyses of TEV, TuMV, and N. benthamiana elongation factor-1α 24 (NbEF-1α), respectively. NbEF-1α served as the internal reference control. The 25 hydrolysis probe designs were based on TaqMan® Probe design tutorial guidelines by 26 Beacon DesignerTM & AlleleID®. The corresponding hydrolysis probes to TEV, TuMV 27 and NbEF1α are listed in Supplemental Tab. S1. Standard curve of each sample was 28 generated to achieve an efficiency of 2.0 prior to the relative quantification analysis. 29 Each sample was assayed in triplicate 20 μ L volumes and data were analyzed using the 30 LightCycler®480 software SW1.5 (Roche). The RNA level was calculated using the 31  and PpDDXL from peach (P. persica) or AtRH8 from A. thaliana. Yeast co-3 transformants were grown on the selective medium SD/-Ade/-His/-Leu/-Trp plus X-α-4 Gal and incubated at 28ºC for four days. 5