1

RESEARCH ARTICLE 1 2

The Nucleolar Fibrillarin Protein is Required for Helper 3 Virus-Independent Long-Distance Trafficking of a Subviral Satellite 4 RNA in Plants 5

6 Chih-Hao Changa,b, Fu-Chen Hsub, Shu-Chuan Leeb, Yih-Shan Lob, Jiun-Da Wangb, 7 Jane Shawc, Michael Talianskyc, Ban-Yang Changd, Yau-Heiu Hsue, and Na-Sheng 8 Lina,b,1 9

10 aInstitute of Plant Biology, National Taiwan University, Taipei, Taiwan 11106 11 bInstitute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 11529 12 cThe James Hutton Institute, Invergowrie, Dundee, United Kingdom DD2 5DA 13 dDepartment of Biochemistry, National Chung Hsing University, Taichung, Taiwan 40227 14 eGraduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 15 40227 16 1Corresponding author: nslin@sinica.edu.tw 17

18 Short title: Subviral RNA long-distance trafficking 19

20 The author responsible for distribution of materials integral to the findings presented in this 21 article in accordance with the policy described in the Instructions for Authors 22 (www.plantcell.org) is: Na-Sheng Lin (nslin@sinica.edu.tw) 23

24 One sentence summary: Bamboo mosaic virus satellite RNA can move autonomously in a 25 fibrillarin-dependent manner in the absence of its helper virus in Nicotiana benthamiana. 26

27 28 29

Plant Cell Advance Publication. Published on October 4, 2016, doi:10.1105/tpc.16.00071

©2016 American Society of Plant Biologists. All Rights Reserved

2

ABSTRACT 30 RNA trafficking plays pivotal roles in regulating plant development, gene silencing, and 31

adaptation to environmental stress. Satellite RNAs (satRNAs), parasites of viruses, depend on 32 their helper viruses (HVs) for replication, encapsidation, and efficient spread. However, it 33 remains largely unknown how satRNAs interact with viruses and the cellular machinery to 34 undergo trafficking. Here, we show that the P20 protein of Bamboo mosaic potexvirus 35 satRNA (satBaMV) can functionally complement in trans the systemic trafficking of 36 P20-defective satBaMV in infected Nicotiana benthamiana. The transgene-derived satBaMV, 37 uncoupled from HV replication, was able to move autonomously across a graft union 38 identified by RT-qPCR, northern blot and in situ RT-PCR analyses. Co-immunoprecipitation 39 experiments revealed that the major nucleolar protein fibrillarin is co-precipitated in the P20 40 protein complex. Notably, silencing fibrillarin suppressed satBaMV-, but not HV-, 41 phloem-based movement following grafting or co-inoculation with HV. Confocal microscopy 42 revealed that the P20 protein co-localized with fibrillarin in the nucleoli and formed punctate 43 structures associated with plasmodesmata. The mobile satBaMV RNA appears to exist as 44 ribonucleoprotein (RNP) complex composed of P20 and fibrillarin, whereas BaMV 45 movement proteins, capsid protein, and BaMV RNA are recruited with HV co-infection. 46 Taken together, our findings provide insight into movement of satBaMV via the fibrillarin–47 satBaMV–P20 RNP complex in phloem-mediated systemic trafficking. 48

49

INTRODUCTION 50

RNA trafficking is essential for plant development, nutrient allocation, gene 51

silencing, and stress responses (Lucas et al., 2001; Kehr and Buhtz, 2008; Turgeon 52

and Wolf, 2009; Ursache et al., 2014). For efficient trafficking, plants have evolved 53

complex networks of regulatory components that enable local and long-distance 54

communication (Middleton et al., 2012). While systemic trafficking is enabled by 55

phloem transport, local cell-to-cell communication relies on microchannels that 56

traverse plant cell walls, known as plasmodesmata (PD) (Lucas et al., 2009; Kragler, 57

2013). Companion cell-sieve element PDs mediate the selective trafficking of RNAs 58

through the phloem translocation stream (Aoki et al., 2005; Kehr and Buhtz, 2008; 59

Turgeon and Wolf, 2009; Ursache et al., 2014). Recent findings on the movement of 60

3

small RNAs, including microRNAs and small interfering RNAs (siRNAs) (Dunoyer 61

et al., 2010; Molnar et al., 2010), have greatly advanced our understanding of the 62

intercellular signaling that coordinates gene expression during development. 63

RNAs can assemble with various proteins into ribonucleoprotein (RNP) complexes 64

during cell-to-cell or long-distance trafficking through the phloem (Haywood et al., 65

2005; Bailey-Serres et al., 2009; Ham et al., 2009; Pallas and Gomez, 2013). 66

Long-distance trafficking has been shown to be mediated by the binding of RNA to 67

the RNA-interacting domains of phloem-mobile RNA-binding proteins (RBPs) (Ham 68

et al., 2009; Pallas and Gomez, 2013). For example, phloem RBP50 binds to the 69

polypyrimidine-tract binding motif of GA-INSENSITIVE PHLOEM RNA (Ham et al., 70

2009) for phloem-mediated trafficking. In addition, phloem RBPs can selectively bind 71

to small RNAs, as well as mRNAs or viral RNAs, to mediate their trafficking (Aoki et 72

al., 2005; Kehr and Buhtz, 2008; Ham et al., 2009; Hipper et al., 2013). Thus, phloem 73

RBPs are translocated with plant RNAs and are likely to be important determinants of 74

plant RNA vascular trafficking (Lucas et al., 2001; Aoki et al., 2005; Kehr and Buhtz, 75

2008; Ham et al., 2009; Turgeon and Wolf, 2009; Pallas and Gomez, 2013). 76

Viral spread from infected cells to neighboring cells requires that the PD size 77

exclusion limits be increased through the action of viral movement protein (MP) 78

(Gopinath and Kao, 2007; Canetta et al., 2008; Harries et al., 2009; Hipper et al., 79

2013). Changes in PD permeability are also thought to enable movement into the 80

vascular system during systemic phloem-mediated trafficking. Thus, viruses must 81

have evolved various strategies to interact with cellular factors to be loaded into and 82

unloaded from the vascular system (Chen et al., 2000; Kim et al., 2007; Harries et al., 83

2009; Raffaele et al., 2009; Taliansky et al., 2010; Semashko et al., 2012; Hipper et al., 84

2013). Host factors such as myosin are required for MP targeting to and virus 85

4

movement through the PD (Amari et al., 2014; Harries et al., 2009); intercellular and 86

long-distance trafficking of Tobacco mosaic virus (TMV) (Chen et al., 2000) and 87

Groundnut rosette virus (GRV) (Kim et al., 2007; Taliansky et al., 2010; Semashko et 88

al., 2012) were substantially delayed in plants silenced for PECTIN 89

METHYLESTERASE and fibrillarin. However, remorin, a Solanaceae protein resident 90

in membrane rafts and plasmodesmata, can interact physically with the MP from 91

Potato virus X (PVX) and negatively regulates PVX intercellular and 92

phloem-mediated trafficking (Raffaele et al., 2009). Some viral RNAs and viroids 93

also contain important 3D RNA motifs required for intercellular movement (Takeda 94

et al., 2011). 95

Satellite RNAs (satRNAs) are parasites of RNA viruses that are almost exclusively 96

associated with plant viruses. These entities lack appreciable sequence similarity to 97

the genomes of their helper viruses (HVs), but depend on HV-encoded proteins for 98

replication and encapsidation (Hu et al., 2009). The mechanisms by which satRNAs 99

undergo intracellular or intercellular trafficking and whether satRNA transport 100

depends on HV replication remain unknown. 101

Bamboo mosaic virus (BaMV)-associated satRNA (satBaMV) has a single-stranded 102

positive-sense RNA genome of ~835 nt (Lin and Hsu, 1994). SatBaMV encodes a 20 103

kDa nonstructural RNA-binding protein (P20) that is dispensable for replication (Lin 104

et al., 1996) but is required for long-distance satBaMV transport in HV co-infected 105

Nicotiana benthamiana (Palani et al., 2006; Vijayapalani et al., 2012). The P20 106

protein has several MP features, including RNA-binding activity (Tsai et al., 1999), 107

strong self-interactions, and efficient cell-to-cell movement (Palani et al., 2006). P20 108

accumulates in the cytoplasm and nuclei of HV and satBaMV co-infected cells 109

(Palani et al., 2009) and can be phosphorylated to negatively regulate the formation of 110

5

satBaMV RNP complexes (Vijayapalani et al., 2012). BaMV HV is a member of the 111

Potexvirus genus that contains a single-stranded, positive-sense RNA genome with 112

five open reading frames (ORFs). These ORFs encode a replicase, three MPs (TGBp1, 113

TGBp2, TGBp3, encoded by a triple gene block), and a capsid protein (CP) (Lin et al., 114

1994), each required for cell-to-cell movement (Lin et al., 2004; Lin et al., 2006; Lan 115

et al., 2010; Lee et al., 2011; Wu et al., 2011; Chou et al., 2013). However, the factors 116

involved in long-distance trafficking of BaMV remain to be determined. 117

Here we provide the evidence that trafficking of P20-defective satBaMV can be 118

restored in transgenic plants expressing P20 protein. Grafting experiments performed 119

to uncouple the replication and trafficking events of satBaMV showed that satBaMV 120

RNA alone can move systemically across a graft union into non-satBaMV-expressing 121

tissue. Co-immunoprecipitation (co-IP) analysis revealed that a nucleolar protein, 122

fibrillarin, was associated with the P20 protein complex. Thus it can be suggested that 123

movement of satBaMV requires P20 interaction with fibrillarin to form mobile 124

satBaMV RNP complexes. TGBp1, TGBp2, TGBp3, and CP were also present in the 125

fibrillarin–satBaMV–P20 RNP complex after co-infection with satBaMV and HV, 126

indicating that viral proteins also play important roles in satRNP complex trafficking. 127

128

RESULTS 129

The P20 Protein Plays a Crucial Role in SatBaMV Systemic Movement in N. 130

benthamiana 131

Previously, we showed that the trafficking of P20-defective satBaMV exhibits low 132

efficiency in N. benthamiana co-infected with BaMV HV (Lin et al., 1996; Palani et 133

al., 2006; Vijayapalani et al., 2012). To provide further insights into the role of P20 in 134

the systemic movement of satBaMV, we generated transgenic N. benthamiana 135

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expressing the P20 protein under the phloem companion cell–specific SUC2 promoter 136

(Fig. 1A). Immunoblot analysis confirmed that P20 was expressed in all 20 transgenic 137

lines; we selected homozygous lines 1-29 and 3-1 for this study (Fig. 1A). No 138

abnormal phenotypes were observed for SUC2 promoter-driven P20 transgenic N. 139

benthamiana plants. To verify the localization of SUC2 driven P20, we created the 140

construct SUC2pro:P20:eGFP for transient assays. Confocal microscopy clearly 141

showed that P20 expressed from the SUC2pro:P20:eGFP construct was present in the 142

phloem along the leaf vein of agro-infiltrated leaves whereas it localized to almost all 143

cell types when it was expressed from 35Spro:P20:eGFP (Figure 1B). 144

For complementation assays, we first generated P20-defective satBaMV. The P20 145

gene of pCBSF4, the full-length cDNA clone of satBaMV (Lin et al., 2004), was 146

replaced with GFP to generate a P20-defective satBaMV reporter plasmid 147

(pCBSGFP); plants were mechanically inoculated with this plasmid and the BaMV 148

infectious cDNA clone, pCB (Lin et al., 2004). BSGFP RNA accumulation was 149

observed in the inoculated leaves of both wild type (WT) and P20-transgenic lines, 150

but BSGFP RNA was not detected in the upper uninoculated leaves of WT plants (Fig. 151

1C, lane 4; Fig. 1C and Supplemental Figure 1A). Notably, a significant amount of 152

BSGFP RNA (about 50% compared to WT BSF4 satBaMV in the WT inoculated 153

leaves) was detected in the upper uninoculated leaves of two independent P20 154

transgenic lines at 20 days post-inoculation (DPI) from 4 independent experiments, 155

each involving 4 plants (Fig. 1C, lane 8; Fig. 1D and Supplemental Figure 1A). To 156

further confirm the accumulation of BSGFP RNA in BaMV co-infected WT or P20 157

transgenic line 1-29, we performed confocal microscopy to visualize the accumulation 158

of BSGFP protein after co-infection of N. benthamiana with pCB. Long-distance 159

trafficking of BSGFP RNA from inoculated leaves to the systemic leaves was rescued 160

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in the P20 transgenic line 1-29 (Supplemental Figure 1B-d), which suggests that P20 161

in trans significantly contributes to long-distance trafficking of BSGFP RNA. 162

HV-Independent Long-Distance Trafficking of SatBaMV in N. benthamiana 163

SatBaMV depends entirely on HV for replication, encapsidation, and efficient 164

spread in plants (Lin and Hsu, 1994). To determine whether satBaMV can move 165

systemically in the absence of HV, we generated five 35S promoter-driven satBaMV 166

transgenic N. benthamiana lines. The satBaMV RNA of the transgene was expressed 167

in all N. benthamiana tissues, including root, stem, leaves and flowers, in lines 2-6 168

and 9-2 (Fig. 2A). No abnormal phenotypes were observed in the 35S 169

promoter-driven satBaMV transgenic N. benthamiana plants. 170

To determine whether satBaMV RNA alone can move systemically, we grafted 171

satBaMV-transgenic N. benthamiana onto WT N. benthamiana and vice versa via 172

cleft grafting (Figs. 2B to 2D; Supplemental Figure 2). As shown in Figure 2B, the 173

leaves L7 and L8 were detached before grafting, whereas leaves L6, L9, and L10 174

were harvested at 12 days after grafting (DAG) and L11 at 15 DAG. SatBaMV was 175

detected in transgenic and WT leaves from chimeric grafts regardless of whether the 176

WT plants served as scions or stocks (Fig. 2B; Supplemental Figure 2A). When 177

satBaMV transgenic lines served as scions and WT as stocks, satBaMV RNA and P20 178

protein were detected in all assayed leaves from L6 to L11; however, satBaMV RNA 179

and P20 protein were detected only in L6 and L9 when satBaMV transgenic lines 180

served as stocks and WT as scions (Fig. 2B). Quantitative analyses of four 181

independent experiments revealed that satBaMV RNA was undetectable in L10 and 182

L11 by RNA gel blot (Supplemental Figure 2A). Moreover, quantitative RT-PCR 183

(RT-qPCR) detected that accumulation of satBaMV progressively decreased in stem, 184

petiole and leaf L9 while satBaMV trafficked from satBaMV transgenic stocks to WT 185

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scion with levels about 82%, 41% and 14%, respectively, of those in L6 of the stocks 186

(Supplemental Figure 3A). Our grafting experiments indeed showed that satBaMV 187

RNA can move long distance alone across the graft union. 188

To investigate satBaMV long-distance trafficking in the presence of HV, L6 or L9 189

leaves near the graft union were co-agroinfiltrated with the HV infectious cDNA 190

clone pKB (Liou et al., 2013) at 9 DAG. As expected, satBaMV RNA accumulated in 191

the leaves of both scions and stocks of satBaMV transgenic and WT plants after 192

grafting with HV infection at 12 DAG (Supplemental Figure 2C, lanes 5-8). 193

To further determine whether satBaMV can undergo trafficking without HV in 194

response to source-to-sink strength, we detached mature and young leaves (L7-L11) 195

and left only the newly emerged youngest leaf L12 after grafting onto the scions (Fig. 196

2C). RNA gel blot (Fig. 2C), RT-PCR (Supplemental Figure 2B) and RT-qPCR 197

(Supplemental Figure 3B) analyses revealed no satBaMV RNA accumulation in L12 198

of WT grafted onto satBaMV transgenic lines, nor was there P20 protein 199

accumulation based on immunoblot analysis (Fig. 2C). However, satBaMV RNA and 200

P20 protein were detected in L6 of WT stocks supported by satBaMV transgenic 201

scions from 4 independent experiments (Fig. 2C; Supplemental Fig. 2B; Supplemental 202

Figure 3B). 203

Since satBaMV could not move to the L12 (Fig. 2C), we examined whether dark 204

treatment could enhance satBaMV long-distance trafficking, because previous studies 205

have indicated that dark treatment may alter the source–sink relationship and increase 206

virus susceptibility (Lemonie et al., 2013; Helms and McIntyre, 1967). Scions were 207

subjected to dark treatment for 3 days before the leaves and stems were harvested for 208

analysis (i.e., dark treatment started at 12 DAG), and RNA or protein was then 209

extracted from L6 to L14 and shoot apex (SA) for analysis by RT-PCR or 210

9

immunoblot, respectively; stem tissues were blotted onto nitrocellulose membrane by 211

tissue blotting (Lin et al., 1990) at 15 DAG. RT-PCR or immunoblot analysis detected 212

satBaMV or P20 protein accumulation in L12, L13, and SA after dark treatment but 213

not in leaves without dark treatment (Fig. 2D, upper panel). Stem tissues were 214

harvested between L6 and L12 at 15 DAG (Fig. 2D, lower panel). Tissue blotting 215

further confirmed satBaMV RNA accumulation in WT scion stems under dark 216

treatment, even in young stems close to L12. By contrast, only stem samples derived 217

near L6 or L9 showed positive signals for satBaMV; satBaMV RNA failed to be 218

detected in the upper stems near L10~L12 without dark treatment (Fig. 2D, lower 219

panel; Supplemental Figure 3C). Therefore, dark treatment increased the 220

long-distance trafficking of satBaMV to young leaves and stems, as well as to the SAs 221

of WT plants (Fig. 2D; Supplemental Figure 3C). 222

To determine whether other RNA is also mobile, we grafted WT plants onto 223

transgenic N. tabacum plants expressing the satRNA of Cucumber mosaic virus 224

(satCMV), or N. benthamiana expressing BaMV CP or GFP (16c) (Brigneti et al., 225

1998) as controls. However, none of these transgene RNAs was detectable in WT 226

scions at 15 DAG (Fig. 2E). These results indicate that satBaMV differs from 227

satCMV in being dispensable for HV in systemic trafficking. 228

In Situ Localization of SatBaMV 229

To verify the phloem transportation and cell-to-cell movement of satBaMV, we 230

used a modified method of in situ RT-PCR (Yoo et al., 2004). In situ RT-PCR allows 231

the fluorescence associated with satBaMV to be visualized under confocal 232

microscopy (Fig. 3A-L). We carried out the grafting experiment as for Figure 2B. 233

Fresh sections were obtained from grafting stems, petioles and leaves of WT scions at 234

12 DAG. In situ RT-PCR revealed strong fluorescence in the stem (Fig. 3A-B), 235

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petiole (Fig. 3C-D) and leaf (Fig. 3E-F) of satBaMV transgenic stock, including pith, 236

phloem, cortex, epidermis, parenchyma, and mesophyll cells, with relatively lower 237

fluorescence in xylem. The fluorescence associated with satBaMV was mainly 238

detected in phloem and some in the cortex of the WT scion stems (Fig. 3G-H). In WT 239

scion petioles, fluorescence was particularly located in internal phloem, external 240

phloem and parenchyma cells (Fig. 3I-J). In addition, strong fluorescence was 241

detected in phloem and parenchyma cells of the major veins of WT scion leaves, with 242

little in the mesophyll and xylem (Fig. 3K-L), indicating that satBaMV was able to 243

move cell-to-cell from phloem and parenchyma to mesophyll cells in WT scion leaves. 244

SIEVE ELEMENT OCCLUSION 1 (SEO1) mRNA was detected as an internal control 245

for in situ RT-PCR and was found only in phloem (Fig. 3M-N), which is consistent 246

with previous promoter assay findings (Ernst et al., 2012). Furthermore, we used 247

another control, TOBACCO POLYPHENOL OXIDASE 1 (TobP1), established as a 248

flower-specific gene (Goldman et al, 1998); TobP1 mRNA was not detected in N. 249

benthamiana stem tissues (Fig. 3O-P). The absence of signal within stem and tissues 250

in experiments with reverse transcriptase omitted from the reaction for satBaMV 251

established the specificity of the protocol used in these studies (Fig. 3Q-R). Taken 252

together, these studies confirm that satBaMV can move from cell to cell and long 253

distance via phloem. 254

255

Host Factors Immunoprecipitated with P20 Protein 256

Since satBaMV is always associated with BaMV in the natural environment, we 257

examined the host factors involved in movement of satBaMV in the presence of HV. 258

Therefore, we used HV and satBaMV co-infected systemic leaves at 7 DPI to search 259

for possible host factors involved in trafficking of satBaMV. 260

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We performed co-IP experiments and analyzed the precipitated products by gel 261

electrophoresis followed by liquid chromatography tandem mass spectrometry 262

(LC-MS/MS). To avoid interference from Rubisco, the most abundant protein in 263

leaves, we prepared Rubisco-depleted fractions and then incubated those with 264

anti-P20 or pre-immune IgG, followed by Protein A Sepharose CL-4B. After washing, 265

bound proteins were eluted and fractionated by gel electrophoresis. In 266

mock-inoculated samples or in controls with pre-immune IgG, only small numbers of 267

non-specific proteins were detected in silver-stained gels (Fig. 4B). By contrast, 268

numerous proteins were resolved after anti-P20 IgG precipitation (Fig. 4B). 269

LC-MS/MS analysis revealed several peptide sequences associated with anti-P20 270

co-immunoprecipitates after HV and satBaMV co-infection (Table 1). In three 271

independent experiments, P20 was observed in band 4, which migrated slightly 272

slower than the 17-kDa standard; this band was absent in co-IP complexes from total 273

proteins of co-infected leaves treated with pre-immune IgG (Fig. 4B), which indicates 274

the specificity of the anti-P20 IgG preparation in the co-IP experiments. As shown in 275

Table 1, the most abundant viral CP was detected in band 3, whereas TGBp1 was in 276

band 2. With the exception of Rubisco, which remained enriched in band 1, the most 277

abundant host protein precipitating with anti-P20 IgG was a nucleolar protein, 278

fibrillarin, which was detected in band 2 in three independent experiments (Table 1). 279

Fibrillarin is known to interact with viral MPs and is important for long-distance 280

trafficking of several RNA viruses (Kim et al., 2007; Taliansky et al., 2010; 281

Semashko et al., 2012; Zheng et al., 2015). The LC-MS/MS results were confirmed 282

by immunoblot analysis, revealing the presence of P20 and fibrillarin in the P20 co-IP 283

complex in the HV co-infected tissues (Figs. 4C and 4D). 284

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As shown in Figure 2B-D, satBaMV systemic trafficking could be HV-independent; 285

therefore, we also verified the satBaMV movement complex in the absence of HV. 286

The WT scion N. benthamiana, grafted onto satBaMV transgenic line 2-6 (Fig. 4E), 287

was harvested at 15 DAG and proteins were precipitated with anti-P20 IgG. In three 288

independent experiments, P20 was detected in band 8 and fibrillarin was also detected 289

in band 7 independent of HV by LC-MS/MS (Table 2). In summary, with or without 290

HV, P20 and fibrillarin may form a complex in vivo. 291

Fibrillarin Silencing Suppresses Long-Distance Trafficking of SatBaMV 292

To examine whether fibrillarin has a role in satBaMV trafficking in the absence of 293

HV, we used virus-induced gene silencing (VIGS) with a Tobacco rattle virus (TRV) 294

vector (Ratcliff et al., 2001) to reduce fibrillarin expression in N. benthamiana plants. 295

PHYTOENE DESATURASE (PDS), the silencing of which is reflected by 296

photobleaching of leaves, was used as a control. Agroinfection with pTRV-NbFib 297

carrying a fragment of the fibrillarin gene from N. benthamiana (Kim et al., 2007) 298

resulted in 60% reduction in levels of both fibrillarin mRNA and protein in VIGS 299

plants (Fig. 5A). RNA gel blot analysis revealed that the satBaMV RNA was no 300

longer detectable in fibrillarin-silenced scion plants after grafting onto the satBaMV 301

transgenic line at 9 DAG (Fig. 5A). 302

To further confirm that satBaMV systemic movement without HV depends on 303

fibrillarin, we grafted the N. benthamiana fibrillarin knockdown line (Shaw et al., 304

2014) onto satBaMV transgenic plants. RNAi knockdown of coilin, encoding one of 305

the main components of Cajal bodies (Ogg and Lamond, 2002), served as a control. 306

SatBaMV did not move into fibrillarin RNAi scions, but substantial movement was 307

evident in the coilin RNAi scions at 9 DAG (Fig. 5B); quantitative analysis revealed 308

that satBaMV mRNA was greatly reduced in fibrillarin RNAi scions but not coilin 309

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RNAi scions (Fig. 5C). Thus, this result supports the crucial role of fibrillarin in 310

satBaMV long-distance transport without HV. 311

To examine the effect of fibrillarin on satBaMV co-infection with HV, we 312

agroinfiltrated N. benthamiana plants with pKB (Liou et al., 2013) and pKF4 (Liou et 313

al., 2013), carrying the infectious cDNA clones of BaMV and satBaMV, respectively. 314

Although mRNA expression of coilin and fibrillarin was greatly suppressed in 315

infiltrated and upper uninfiltrated leaves of RNAi N. benthamiana plants (Fig. 5B and 316

data not shown), the inoculated leaves of WT, coilin, or fibrillarin RNAi plants 317

contained similar levels of BaMV or satBaMV RNA accumulation after infiltration 318

with BaMV or co-infiltration with HV+satBaMV at 5 DPI (Fig. 5D). Notably, 319

satBaMV RNA was greatly decreased in the upper leaves of fibrillarin RNAi plants 320

(Fig. 5E, lanes 21-24 vs 5-8) but not coilin RNAi plants (Fig. 5E, lanes 13-16 vs 5-8; 321

Supplemental Figure 4B) co-agroinfiltrated with pKB and pKF4. Quantitative 322

analyses of three independent experiments revealed that satBaMV accumulation in the 323

upper, non-infiltrated leaf L4 of fibrillarin RNAi plants was about 20% of that of the 324

WT or coilin RNAi plants but undetectable in L5 of fibrillarin RNAi plants 325

(Supplemental Figure 4B). SatBaMV accumulation in L4 or L5 of WT and coilin 326

RNAi plants did not differ (Supplemental Figure 4B). However, BaMV accumulation 327

was not greatly affected in the upper non-infiltrated leaves (L4-L7) among the WT, 328

fibrillarin RNAi, or coilin RNAi plants after BaMV or satBaMV co-infection at 15 329

DPI (Fig. 5E; Supplemental Figure 4A). Therefore, fibrillarin silencing impaired only 330

the long-distance trafficking of satBaMV in HV and satBaMV co-infected plants. 331

Fibrillarin and P20 Form Complexes with SatBaMV RNA 332

The previous results suggest that fibrillarin may be important for long-distance 333

trafficking of satBaMV during BaMV co-infection. Therefore, we performed co-IP 334

14

assays to determine the nature of the putative protein–protein interactions. Total 335

proteins were extracted from the leaves of N. benthamiana plants infected with HV or 336

co-infected with satBaMV, and co-IP assays were then performed with anti-P20 or 337

anti-fibrillarin IgG. Input proteins and co-IP fractions were confirmed by immunoblot 338

analysis (Fig. 6). 339

With anti-P20 IgG, protein samples extracted from leaves co-infected with HV and 340

satBaMV revealed signals for P20, fibrillarin, TGBp1, TGBp2, and CP (Fig. 6A, lane 341

6). These proteins were not detected in samples from plants infected with HV alone or 342

in healthy control leaves (Fig. 6A, lanes 2 and 4). Likewise, co-IP assay with 343

anti-fibrillarin IgG revealed equivalent signals for P20, fibrillarin, TGBp1, TGBp2, 344

and CP in HV and satBaMV co-infected leaves (Fig. 6B, lane 6), indicating that 345

fibrillarin interacted directly or indirectly with P20, TGBp1, TGBp2, and CP. To 346

determine whether fibrillarin interacts with P20 directly, we generated fusions of 347

activation domain (AD) or binding domain (BD) with fibrillarin or P20 for yeast 348

two-hybrid assays. We observed strong self-interactions for both P20 and fibrillarin 349

and also a direct interaction between fibrillarin and P20, regardless of which protein 350

was fused to AD (Supplemental Figure 6). This interaction is consistent with the 351

results obtained by co-IP assay with anti-P20 or anti-fibrillarin IgG. 352

Like potexviruses, all three TGBps and CP are required for BaMV cell-to-cell and 353

systemic movement (Lin et al., 2004; Lin et al., 2006; Lan et al., 2010; Lee et al., 354

2011; Wu et al., 2011; Chou et al., 2013). To examine the interaction of TGBp3 with 355

P20 and/or fibrillarin, we inoculated N. benthamiana leaves with a 35S 356

promoter-driven HV derivative (pCB-P3HA) carrying a TGBp3::HA fusion (Fig. 6C, 357

Chou et al., 2013) with or without the satBaMV pCBSF4 plasmid (Lin et al., 2004). 358

Co-IP assays with an HA antibody precipitated TGBp3HA, TGBp1, TGBp2, and CP 359

15

but not P20 or fibrillarin in fractions, from pCB-P3HA-infected plants (Fig. 6C, lane 360

4). The HA antibody additionally precipitated fibrillarin and P20 from protein extracts 361

of plants co-inoculated with pCB–P3HA and pCBSF4 (Fig. 6C, lane 5) but not from 362

uninoculated WT, pCB-inoculated, or pCB and pCBSF4 co-inoculated plants. 363

However, co-IP assays with anti-P20 IgG (Fig. 6D) or anti-fibrillarin IgG (Fig. 6E) 364

revealed positive signals for TGBp3HA in HV and satBaMV co-infected leaves (Figs. 365

6D and Fig. 6E, lane 6). Hence, consistent with observations in other potexviruses 366

(Park et al., 2013), BaMV TGBps and CP may interact with each other, and P20 may 367

form protein complexes with fibrillarin, BaMV TGB proteins and CP in plant tissues 368

co-infected with HV and satBaMV. 369

To further confirm that the P20–fibrillarin protein complexes are RNP complexes, 370

we extracted RNA from total sap and co-IP fractions after incubation with anti-P20 or 371

anti-fibrillarin IgG. In plants co-infected with HV and satBaMV, both HV and 372

satBaMV RNAs were present in the total sap and co-IP fractions, as shown by using 373

anti-P20 (Fig. 6F, lanes 5-6 and 11-12) or anti-fibrillarin IgG (Fig. 6G, lanes 5-6 and 374

11-12). In the negative control (immunoprecipitation of total sap of HV-infected 375

plants by using anti-P20 IgG), HV RNA was detected in only total sap from 376

HV-infected plants but not in co-IP fractions with anti-P20 or anti-fibrillarin IgG 377

(Figs. 6F and 6G, lanes 3-4). These results support the conclusion that P20 can form 378

RNP complexes with fibrillarin and also complexes with HV proteins as well as HV 379

and satBaMV RNAs in satBaMV co-infected plants. 380

However, WT scions grafted onto the satBaMV transgenic stock, 2-6 line, without 381

HV, showed satBaMV RNA in co-IP fractions with anti-P20 or anti-fibrillarin IgG for 382

the formation of a satBaMV-P20-fibrillarin RNP complex (Fig. 6H). 383

16

The Fibrillarin–SatBaMV–P20 RNP Complex is Absent from the Fibrillarin 384

RNAi Transgenic Line 385

To verify whether the composition of the satBaMV–P20 RNP complex with or 386

without HV was changed by reduced fibrillarin level, the fibrillarin RNAi plants were 387

grafted onto satBaMV transgenic lines 2-6 (Supplemental Figure 5A). Fibrillarin 388

RNAi scion tissues were harvested and proteins were precipitated with anti-P20 IgG. 389

In three independent experiments, P20 was detected in band 7; other detected proteins 390

were chloroplast, ribosomal and helicase proteins (Supplemental Table 1). Total 391

proteins were extracted from the leaves of fibrillarin RNAi plants coinfected with HV 392

and satBaMV, and co-IP assays were performed with anti-P20 IgG at 15 DPI 393

(Supplemental Figure 5B). The most abundant P20 was detected in band 9, whereas 394

viral CP and TGBp1 co-migrated into band 8 (Supplemental Table 2), and ATP 395

synthase, pyrophosphorylase 2, glycine decarboxylase P-protein 2, and ribosomal and 396

helicase proteins were detected in three independent experiments. These data show 397

that the composition of the satBaMV–P20 RNP complex with and without HV was 398

distinct in plants with reduced fibrillarin levels. Furthermore, no fibrillarin was 399

detected in the satBaMV–P20 RNP complex in fibrillarin RNAi transgenic line 400

(Supplemental Table 1 and 2), suggesting that the residual fibrillarin produced in the 401

RNAi line is completely or nearly completely used for its primary function in 402

ribosome biogenesis. 403

P20 Co-localizes with Fibrillarin and Forms Punctate Structures at the Cell 404

Periphery 405

To reveal the subcellular localization of P20, we constructed an 406

Agrobacterium-compatible plasmid, pBin-P20-eGFP, for transient expression of 407

GFP-tagged P20. P20-eGFP was located in the nucleus and also at the cell periphery, 408

17

where it formed punctate structures (Fig. 7A-a), as described previously (Palani et al., 409

2006; 2009). At higher magnification, P20-eGFP could be seen to form one or two 410

foci in the nucleus (Fig. 7B-a to c). To determine whether P20-eGFP co-localized with 411

fibrillarin (FIB2), we agro-infiltrated leaves with pBin-mCherry-NbFIB2 alone or 412

with pBin-P20-eGFP. Transiently expressed mCherry-NbFIB2 was mainly localized 413

in the nucleolus (Fig. 7B-d to f). When P20-eGFP was co-expressed with 414

mCherry-NbFIB2, these two proteins exhibited perfect co-localization in the 415

nucleolus (Fig. 7B-g to i), indicating that P20 can be imported into the nucleus and 416

become enriched in the nucleolus. 417

P20 protein was previously found to move cell-to-cell autonomously (Palani et al., 418

2006); accordingly, we examined whether the P20 peripheral punctae were associated 419

with the PD. We used both DsRed-tagged TMV MP (Fig. 7C-a to f) and aniline blue 420

staining (Fig. 7C-g to l) as PD markers. Regardless of which PD marker was used, 421

most, if not all, of the P20-eGFP co-localized with labeled PD. 422

To further determine whether the P20-eGFP localized in the PD channel, we used 423

leaves expressing P20-eGFP and TMVMP-DsRed plasmolyzed with 1 M NaCl. 424

Nearly half of the leaf cells were plasmolyzed after NaCl treatment. A large amount 425

of P20-eGFP localized along the shrunken cell membrane. However, substantial 426

P20-eGFP still remained in the cell wall and co-localized with the PD marker 427

TMVMP-DsRed after plasmolysis (Fig. 7D). Taken together, these findings suggest 428

that P20 can localize to the nucleolus with fibrillarin and form punctate structures at 429

the cell periphery with integration into the PD channel. 430

DISCUSSION 431

Although satRNAs are subviral agents that require HVs for efficient replication and 432

long-distance trafficking in co-infected plants, our data clearly show that transgenic 433

18

satBaMV alone can move systemically across a graft union in N. benthamiana. We 434

also demonstrated that expression of P20 in phloem (Fig. 1B) can complement 435

long-distance trafficking of P20-defective satBaMV in P20-transgenic N. 436

benthamiana (Figs. 1C and 1D; Supplemental Figure 1). In addition, autonomous 437

satBaMV trafficking into WT scions is fibrillarin-dependent (Fig. 5). In the absence 438

of HV, satBaMV RNA appears to exist as an RNP complex composed of P20 and 439

fibrillarin (Fig. 4E and Table 2), whereas in the presence of HV, viral MPs and CP are 440

also recruited into the mobile RNP complex for efficient trafficking (Fig. 6). 441

Therefore, satBaMV trafficking appears not to require prior replication of satBaMV 442

with HV or encapsidation by HV CP (Lin and Hsu, 1994), and the mobility of 443

satBaMV can be independent of replication. This observation implies that satBaMV 444

may have an advantage for survival in nature because its capacity for autonomous 445

trafficking to the distal leaves may enhance its chances to encounter HV for further 446

amplification and spread once satBaMV and HV initially infect different cells. 447

Fibrillarin is required for only satBaMV, but not HV long-distance trafficking, which 448

suggests that HV and satRNA may have evolved distinct methods for trafficking. A 449

previous study also found a differential requirement for the host factor heat shock 450

protein 90 in the replication of HV and satBaMV (Huang et al., 2012). 451

Viroids do not encode any translatable products and are not encapsidated. Viroid 452

RNA alone can replicate and traffic efficiently without the need for an HV in host 453

plants (Flores et al., 2009). However, differentiating between replication and 454

movement of viroids through their interactions with cellular factors is problematic. 455

Although replication-independent long-distance trafficking of Brome mosaic virus 456

RNA3 can be recapitulated by agroinfection of individual cDNA components into 457

different expression sites in N. benthamiana, the detection of the movement signal of 458

19

RNA3 requires the subsequent replication of RNA1 and RNA2 (Gopinath and Kao, 459

2007). Thus, uncoupling of satBaMV replication from trafficking will provide an 460

opportunity to gain greater molecular insights into satRNA systemic movement in 461

planta. 462

Unlike most satRNA-encoded proteins required for satRNA replication (Hu et al., 463

2009), the satBaMV-encoded P20 non-structural protein assists satBaMV 464

long-distance trafficking in plants (Lin et al., 1996; Palani et al., 2006; Vijayapalani et 465

al., 2012) (Fig. 1). Previously, we showed that P20 can preferentially bind to the 5’- 466

and 3’- UTRs of satBaMV to form satBaMV–P20 RNP complexes (Tsai et al., 1999; 467

Vijayapalani et al., 2012), and that formation of the complexes is negatively regulated 468

by P20 phosphorylation (Vijayapalani et al., 2012). Our current results show that P20 469

can form punctate structures localized at PD (Fig. 7) and the satBaMV–P20 RNP 470

complexes can traffic autonomously through the phloem in satBaMV-transgenic 471

stocks or scions (Figs. 2B-D). In situ RT-PCR experiments also provided strong 472

support that satBaMV moves within the functional phloem system. On grafting, the 473

satBaMV is present in phloem of the stem, petiole and major vein of leaves in WT 474

scions (Fig. 3). The accumulation pattern of visualized satBaMV within vascular 475

tissues in N. benthamiana phloem resembles that of SEO1 in N. tabacum (Ernst et al., 476

2012). Thus, evidence in support of the satBaMV phloem trafficking was provided by 477

the combination of grafting, RNA gel blot analysis, in situ RT-PCR and tissue 478

blotting techniques. With these experimental approaches, we can conclude that the 479

satBaMV is translocated through satBaMV transgenic stock into the WT scion via the 480

phloem. This finding agrees with other recent studies of cellular or viral RNAs 481

indicating that there are systemic recombination signals, that small RNAs alone can 482

undergo systemic transport across a graft union (Turgeon and Wolf, 2009; Dunoyer et 483

20

al., 2010), and that grafting is able to determine the specificity and efficiency of RNA 484

trafficking (Kehr and Buhtz, 2008). We found that GFP RNA, BaMV CP RNA, and 485

CMV satRNA in transgenic lines were all restricted to stocks (Fig. 2E). Therefore, 486

satBaMV may contain long-distance trafficking determinants that involve specific 487

RNA sequences or structural elements, and the P20 protein may interact with host 488

factors. 489

Using a combination of co-IP and LC-MS/MS along with VIGS assays or the 490

fibrillarin RNAi transgenic line, we further determined that the HV-independent or 491

-dependent systemic movement of the satBaMV RNP complexes depends on 492

fibrillarin (Fig. 5; Supplemental Figure 5; Supplemental Tables 1-2). Fibrillarin is 493

required for ribosomal RNA processing (Barneche et al., 2000; Saez-Vasquez et al., 494

2004) and is a nucleolar-localized RBP required for systemic infection of GRV (Kim 495

et al., 2007; Canetta et al., 2008), Potato leafroll virus (Haupt et al., 2005), and Rice 496

stripe tenuivirus (Zheng et al., 2015). Nucleolar co-localization of fibrillarin and P20 497

protein (Fig. 7), along with detection of fibrillarin, P20 protein, and satBaMV RNA in 498

co-IP complexes (Fig. 4 and 6) but not in the fibrillarin RNAi transgenic line 499

(Supplemental Figure 5; Supplemental Table 1-2), is consistent with the evidence for 500

interaction of fibrillarin with satBaMV–P20 RNP complexes (Figure 4E and Table 2). 501

Experiments with transgenic RNAi lines reinforced our findings that fibrillarin is 502

crucial for systemic movement of satBaMV but not BaMV trafficking (Fig. 5E). 503

Hence, fibrillarin is the identified host factor that is differentially required for satRNA 504

and HV long-distance trafficking. Our results also suggest that satBaMV and BaMV 505

may move independently by interacting with distinct cellular factors. Increasing 506

evidence suggests that interactions between viral MPs and nuclear-localized proteins 507

may represent an essential step for virus systemic trafficking (Solovyev and Savenkov, 508

21

2014), but determining where and how such interactions occur requires additional 509

experimentation. Mobile satBaMV RNA complexes containing P20, fibrillarin, 510

satBaMV RNA, TGBp1-3, and CP are also present with HV co-infection (Fig. 6). In 511

this way, BaMV TGBps may participate in PD gating (Howard et al., 2004) or by 512

interaction with fibrillarin–satBaMV–P20 RNP complex to help with satBaMV 513

movement. Taken together, our findings for the fibrillarin–satBaMV–P20 RNP 514

complex suggest that the nuclear/nucleolar-localized fibrillarin activity may involve 515

cytosolic interactions that require a high degree of coordination of the P20 protein of 516

satBaMV during phloem-mediated trafficking. 517

Phloem-mobile RNP complexes can move in the translocation stream from source 518

to sink tissues (Ursache et al., 2014). This RNP complex transport pathway can be 519

regulated when plants respond to environmental cues or pathogen attack (Pallas and 520

Gomez, 2013; Ursache et al., 2014). Dark treatment may alter the source–sink 521

relationship and increase virus susceptibility (Lemonie et al., 2013; Helms and 522

McIntyre, 1967). Our results demonstrate that dark treatment facilitates long-distance 523

trafficking of fibrillarin-based satBaMV RNP complexes, presumably via a change in 524

photoassimilate allocation. Several studies have also indicated that fibrillarin can exit 525

the nucleoli and move to other cell compartments during exposure to biotic or abiotic 526

stresses, such as actinomycin D (Chen and Jiang, 2004), mercury treatment (Chen et 527

al., 2002), or GRV infection (Kim et al., 2007). Hence, fibrillarin may function in 528

defense responses under stress conditions. However, whether P20 co-moves with 529

fibrillarin or whether the nucleolar activity of P20 results in the relocalization of 530

fibrillarin to the cytoplasm during satBaMV transport through the phloem remains to 531

be determined. 532

22

In addition to identifying fibrillarin, we identified other host proteins, such as 533

histone H3, which is crucial for trafficking of a Geminivirus DNA through the nuclear 534

pore complex and PD (Zhou et al., 2011), in our co-IP experiments (Table 1). These 535

proteins may also interact with P20, directly or indirectly, to form fibrillarin-based 536

satBaMV RNP complexes. Whether the phloem-mobile satBaMV RNP complexes 537

contain any phloem proteins or RNA-specific chaperones that could modify the 538

satBaMV RNA structure, thereby facilitating systemic spread, requires further 539

investigation. 540

541

METHODS 542

Construction of Plasmids 543

The gene encoding the satBaMV P20 protein was placed under the control of the 544

SUC2 promoter in companion cells (Haywood et al., 2005) by inserting the SUC2 545

promoter at the HindIII/XbaI sites of p1390 (Haywood et al., 2005) to generate 546

p1390-SUC2pro. The P20 gene was then inserted at the XbaI/BamHI sites of 547

p1390-SUC2pro vector to generate SUC2pro:P20 for transformation. To generate 548

SUC2pro:P20-eGFP for transient expression P20, the P20-eGFP DNA fragment was 549

amplified from pCass-P20-EGFP (Palani et al., 2006) with the primers 550

P20-eGFP-XbaI-F and P20-eGFP-BamHI-R (Supplemental Table 3), then cloned in 551

the p1390-SUC2:P20 plasmid at the XbaI/BamHI sites. 552

To express P20-eGFP in N. benthamiana, we constructed pBIN-P20-eGFP. The 553

P20-eGFP DNA fragment was amplified from pCass-P20-EGFP (Palani et al., 2006) 554

using primers Tf-XmaI-F and Tf-XmaI-R (Supplemental Table 3), and then cloned 555

into the pBIN61 plasmid at the XmaI site to generate pBIN-P20-eGFP. For transient 556

expression of mCherry-NbFIB2 in N. benthamiana, we constructed 557

23

pBIN-mCherry-NbFIB2. The mCherry DNA fragment was first amplified from 558

pBA-mCh-p1 (Chou et al., 2013) using primers mCherry-SmaI-F and 559

mCherry-KpnI-R (Supplemental Table 3), and then cloned into the pCass plasmid 560

(Ding et al., 1995) at the SmaI and KpnI sites to generate pCass-mCherry. Then, the 561

FIB2 coding region was amplified from N. benthamiana cDNA using primers 562

NbFIB2-KpnI-F and NbFIB2-EcoRI-R (Supplemental Table 3). The amplified FIB2 563

fragment was then cloned into pCass-mCherry to generate pCass-mCherry-NbFIB2. 564

Finally, pBIN-mCherry-NbFIB2 was generated by amplifying the mCherry-NbFIB2 565

fragment from pCass-mCherry-NbFIB2 using primers Tf-XmaI-F and Tf-XmaI-R 566

(Supplemental Table 3), and then cloning it into the pBIN61 vector at the XmaI site. 567

To construct the pBIN-TMVMP-DsRed plasmid required as a PD marker, we first 568

amplified the DsRed DNA fragment from pdNR (Addgene) using primers DsRed 569

KpnI-F and DsRed EcoRI-R (Supplemental Table 3); the fragment was then cloned 570

into the pCass plasmid at the KpnI and EcoRI sites to generate pCass-DsRed. The 571

DNA of TMVMP was amplified from pTMV-ΔCP using primers TMV-MP-StuI-F 572

and TMV-MP-KpnI-F (Supplemental Table 3), and then cloned into the pCass-DsRed 573

plasmid at the StuI and KpnI sites to generate pCass-TMVMP-DsRed. Finally, the 574

DNA of TMVMP-DsRed was amplified from pCass-TMVMP-DsRed using primers 575

Tf-XmaI-F and Tf-XmaI-R, and cloned into the pBIN61 plasmid at the XmaI site to 576

generate pBIN-TMVMP-DsRed. 577

Generation of Transgenic Plants 578

The procedures for transformation and regeneration of transgenic SUC2pro:P20 N. 579

benthamiana plants were previously described (Lin et al., 2013). The transgenic lines 580

were selected based on their resistance to 50 mg/L hygromycin on MS agar medium 581

(Sigma-Aldrich Co. LLC). Seedlings exhibiting resistance to hygromycin were 582

24

examined for P20 gene expression by RT-PCR using primers P20-EcoRI-F and 583

P20-EcoRI-R (Supplemental Table 3). In total, we obtained 20 independent 584

homozygous lines after segregation analysis. The homozygous lines 1-29 and 3-1 585

were used in this study. Transgenic N. benthamiana lines expressing satBaMV driven 586

by the 35S promoter were a gift from Dr. Yau-Heiu Hsu (National Chung Hsing 587

University, Taichung, Taiwan). Five homozygous lines were obtained and lines 2-6 588

and 9-2 were used for grafting experiments. The transgenic N. tabacum line 589

expressing satCMV driven by the 35S promoter was a gift from Dr. Yau-Heiu Hsu 590

(National Chung Hsing University, Taichung, Taiwan). 591

Fibrillarin RNAi transgenic N. benthamiana plants were generated by 592

transformation with A. tumefaciens LBA4404 carrying the plasmid 593

pFGC5941.Fibrillarin3’, as described for coilin RNAi plants (Shaw et al., 2014). The 594

plasmid consisted of a 321-bp fragment (region 501-821) of the N. benthamiana 595

fibrillarin gene (AM269909) cloned in opposite orientations flanking the 596

CHALCONE SYNTHASE intron. Three independent N. benthamiana fibrillarin RNAi 597

lines were selected and were found to elicit an approximately 60% reduction in 598

fibrillarin expression. None of the lines exhibited obvious phenotype alterations, and 599

Line #1 was used in this study. 600

Coilin RNAi transgenic N. benthamiana plants were previously characterized 601

(Shaw et al., 2014). Transformation and regeneration of fibrillarin and coilin RNAi 602

transgenic plants were as previously described (Taliansky et al., 2004). To avoid the 603

possibility of “off-target” silencing, we confirmed that the fibrillarin and coilin 604

fragments used in the RNAi constructs did not contain any 21-nt stretches showing 605

similarity to other genes by using the siRNA scan website 606

(http://bioinfo2.noble.org/RNAiScan.htm). 607

25

Protein Analysis 608

Leaves of SUC2pro:P20 transgenic plants, or those of BaMV- and 609

satBaMV-infected plants, were ground in liquid nitrogen and resuspended in 610

extraction buffer (50 mM Tris-HCl, pH 8, 10% glycerol, 1 mM EDTA, 100 mM NaCl, 611

1 mM PMSF). For co-IP, 20-fold diluted protein samples were used for immunoblot 612

analysis with anti-P20, anti-TGBp1, anti-TGBp2, anti-HA (Chou et al., 2013), rabbit 613

anti-actin (1/5000 dilution, provided by Y.-Y. Hsu) or anti-CP antibodies (Lin et al., 614

1992; Chou et al., 2013) together with anti-fibrillarin IgG, H-140 (sc-25397) (Santa 615

Cruz Biotech), followed by staining with goat anti-rabbit IgG HRP secondary 616

antibody (Abcam). 617

Plant Growth, Inoculation, and Grafting 618

All WT and transgenic N. benthamiana or N. tabacum plants were grown at 28℃ 619

in a walk-in plant growth chamber under a 16 h light/8 h dark cycle with a white light 620

(Philips TLD 36W/840 ns) intensity of 185~222 μmol m-2 s-1 at the leaf surface. For 621

each set of experiments, we used 4-week-old plants for inoculation. The methods for 622

inoculation by Agrobacterium expressing BaMV or satBaMV were described 623

previously (Liou et al., 2013). 624

WT or grafted N. benthamiana plants were agroinfected with Agrobacterium 625

expressing full-length infectious cDNA clones of BaMV, pKB (Liou et al., 2013) or 626

satBaMV, pKF4 (Liou et al., 2013) in pKYLX7 (Schardl et al., 1987) binary vector. 627

For complementation assays, WT and SUC2pro:P20 transgenic plants were 628

inoculated with 0.5 μg pCB alone, or co-inoculated with 0.5 μg pCBSF4 or pCBSGFP 629

(Lin et al., 2004; Vijayapalani et al., 2012). 630

Grafting was performed as described (Turnbull et al., 2002) except that 631

approximately 40-day-old N. benthamiana or N. tabacum plants were used for cleft 632

26

grafting. Each grafting experiment was repeated at 4 times, each including 4 plants. 633

Agrobacterium Culture, Infiltration, and Virus-Induced Gene Silencing (VIGS) 634

Plasmids (pKB or pKF4) for protein transient expression were transformed into 635

Agrobacterium C58C1 by electroporation. Agrobacterium cultures were grown as 636

described (Liou et al., 2013) and diluted to an optical density of 0.4-1.0 at 600 nm for 637

infiltration into leaves of N. benthamiana plants. For co-expression of two plasmids, 638

two solutions of Agrobacterium, each harboring a specific plasmid, were mixed in a 639

1:1 ratio prior to agro-infiltration. Agrobacterium was infiltrated into the intercellular 640

space of N. benthamiana leaves. 641

For VIGS assays, 4-week-old plants of N. benthamiana were infiltrated with 642

mixture of A. tumefaciens LBA4404 harboring either the TRV1 or TRV2 RNA2 643

vector (p0704) containing the fibrillarin fragment (Kim et al., 2007). In parallel, 644

silencing of the PHYTOENE DESATURASE (PDS) leading to a photobleached 645

phenotype was used as a marker for monitoring the effectiveness of VIGS. At 7 days 646

after agroinfiltration, we harvested systemically infected leaves for RNA 647

accumulation assays. 648

RNA Analysis 649

Total RNA was extracted from N. benthamiana or N. tabacum tissues using Tripure 650

in accordance with the manufacturer's instructions (Roche). RNA gel blot analysis 651

was carried out as described previously (Lin et al., 1996). BaMV and satBaMV 652

accumulation was analyzed using 32P-labeled RNA probes specific for the BaMV 3’ 653

end generated from HindIII-linearized pBaHB (Lin et al., 1993) and specific for 654

full-length satBaMV generated from EcoRI-linearized pBSHE (Lin et al., 2013), 655

respectively. The satCMV probe was transcribed from HindIII-linearized pGEM4 656

(provided by Dr. Yau-Heiu Hsu) using SP6 RNA polymerase. The GFP probe was 657

27

transcribed from EcoRI-linearized pGEM-T Easy GFP using SP6 RNA polymerase 658

(New England Biolabs) (Vijayapalani et al., 2012). Accumulation of fibrillarin, coilin 659

and actin mRNA was conducted as described by Kim et al. (2007). Four independent 660

replicates were performed for each experiment. 661

For RT-qPCR, 2 μg total RNA extracted from plants was reverse-transcribed into 662

poly d(T) cDNA by using SuperScript III reverse transcriptase (Invitrogen) in 663

triplicates with the GeneAmp 9700 sequence detection Real-Time PCR system (Life 664

Technologies) and SYBR Green I core reagent (Life Technologies). Normalization of 665

satBaMV accumulation was to the 18S gene. Primers for satBaMV (satBaMVrt-F and 666

satBaMVrt-R) and 18S (18S-F and 18S-R) are in Supplemental Table 3. 667

To analyze BaMV or satBaMV RNA in the co-IP fractions, we extracted total RNA 668

from the anti-P20 or anti-fibrillarin co-IP fractions followed by RT-PCR. Primer set 669

BaMV-F and BaMV-R (Supplemental Table 3) was used to amplify a 729-nt BaMV 670

cDNA 3’ fragment (produced after 25 cycles of PCR). Similarly, primers satBaMV-F 671

and satBaMV-R (Supplemental Table 3) were used to amplify full-length satBaMV 672

cDNA. 673

Tissue Blotting 674

Sections were cut from fresh stem tissues by hand with a new razor blade. Tissue 675

blots were made by pressing the newly cut surface onto a membrane; a 32P-labeled 676

RNA probe specific for full-length satBaMV was generated from EcoRI-linearized 677

pBSHE (Lin et al., 2013), and then used to detect the distribution of satBaMV in the 678

tissues (Lin et al., 1990). 679

In Situ RT-PCR 680

The localization of satBaMV was determined by using established protocols for 681

in situ RT-PCR (Lee et al., 2004). A reverse-transcriptase cocktail (containing 682

28

SuperScript III reverse-transcriptase components, and satBaMV-R primer 683

[Supplemental Table 3] at 75 μM) was prepared immediately before use. Fresh 684

200-μm thick sections were obtained using a D.S.K. Microslicer DTK-1000. Sections 685

were placed onto a glass slide, covered by a 25 μL aliquot of the above cocktail and 686

sealed with amplicover discs and clips. The reverse-transcription step was performed 687

at 50°C for 60 min. For the PCR step, the satBaMV-F primer of 75 μM (Supplemental 688

Table 3) and ChromaTide Alexa Fluor 488-5-dUTP (20 μM; Molecular Probes, 689

Eugene, OR) were added; dTTP was reduced to 10 μM. The amplification protocol 690

consisted of 10 cycles; 30 s at 94°C, 30 sat 55°C and 60 s at 72°C. A MJ research 691

PTC-200 Peltier Thermal cycler was used for these experiments. 692

After this reaction series, sections were incubated (1 min) in absolute ethanol, 693

followed by rinsing in 1 mM EDTA, and then overnight washing (16 h), at 22°C, in 694

this EDTA solution. 695

Co-Immunoprecipitation (co-IP) and LC-MS/MS 696

Plant total proteins were extracted using Tris-HCl buffer (Dharmasiri et al., 2005) 697

from systemically infected leaves of N. benthamiana agro-infiltrated with pKB at 7 698

DPI or together with pKF4. Plant Rubisco was removed using Seppro® RuBisCO 699

Spin Columns (Catalog Number SEP070, Sigma-Aldrich Co. LLC) in accordance 700

with the manufacturer’s instructions. 701

In vivo co-IP experiments with anti-P20 (Palani et al., 2009) and anti-fibrillarin IgG, 702

H-140 (sc-25397) (Santa Cruz Biotech) were performed as described (Dharmasiri et 703

al., 2005). Briefly, plant extracts containing 1 mg protein were incubated with 704

anti-P20 IgG (1:150 v/v) for 1 h at 4°C on a rotary shaker. Then, 20 μL Protein A 705

agarose beads (GE Healthcare Life Science, Sweden) were added, and the mixture 706

was incubated for 3 h at 4°C. After washing with Tris-HCl buffer (Dharmasiri et al., 707

29

2005), the immunoprecipitate resuspended in 2X sample buffer (4% SDS, 20% 708

Glycerol, 0.12 M Tris pH 6.8, and 10% β-mercaptoethanol) and separated on a 709

NuPAGE Novex 4-12% Bis-Tris protein gel (Invitrogen Life Science Technologies). 710

Gels were silver-stained, and protein bands were then excised and digested with 711

trypsin for analysis by LC-MS/MS (Lo et al., 2011). LC-MS/MS fragmented ions 712

were used with Mascot (http://www.matrixscience.com/search_form_select.html) to 713

search against the most recent Arabidopsis, BaMV, and satBaMV databases in the 714

National Center for Biotechnology Information (NCBI). 715

Yeast Two-Hybrid Assay 716

Yeast two-hybrid assays were performed as recommended by the manufacturers of 717

the GAL4 Two-Hybrid Phagemid Vector kits (Agilent Technologies, Inc. 2011). The 718

full-length coding sequence of fibrillarin was generated using NbFIB2-EcoRI-F and 719

NbFIB2-EcoRI-R, and P20 was generated using P20-EcoRI-F and P20-EcoRI-R 720

(Supplemental Table 3). Fibrillarin or P20 was cloned into downstream of the GAL4 721

activation domain [AD] at EcoRI site or downstream of the GAL4 DNA binding 722

domain [BD] at the EcoRI site. The rich medium yeast extract, peptone, dextrose 723

(YPD) is most commonly used for growing yeast under nonselective conditions. To 724

test interactions between fibrillarin and P20, we coexpressed the constructs in YRG-2 725

yeast cells, and selected by incubation in leucine-tryptophan-histidine medium at 28℃ 726

for 2-3 days until colonies appeared. Each experiment was repeated three times. 727

Confocal microscopy 728

To visualize satBaMV accumulation from in situ RT-PCR (Fig. 3), fresh tissues 729

were examined under a Zeiss LSM880 laser scanning microscope with a 40x/1.2 W 730

Korr UV-VIS-IR objective lens. Images were captured by using the ZEN software 731

with Ex/Em: 488 nm/505-550 nm. 732

30

To visualize PD in leaf epidermal cells, we infiltrated aniline blue fluorochrome 733

(Biosupplies) (0.1 mg/ml in water) into agro-infected leaves of N. benthamiana, and 734

then immediately examined the leaves using a Zeiss LSM510 laser scanning 735

microscope with a 40x/1.2 W Korr UV-VIS-IR objective lens (Fig. 7). Images were 736

captured using the LSM510 software with filters for aniline blue fluorochrome 737

(Ex/Em: 405 nm/480-510 nm), GFP ((Ex/Em: 488 nm/505-575 nm), and 738

mCherry/DsRed ((Ex/Em: 543 nm/560–615 nm). All images were processed and 739

cropped using Zeiss LSM Image Browser and Photoshop CS5 (Adobe). 740

The agro-infiltrated leaves expressing P20-eGFP and TMVMP-DsRed at 2 days after 741

inoculation were used for plasmolysis studies (Fig. 7D). The leaf discs were treated 742

with 1 M NaCl for 20 min before observation. Samples were scanned under a Zeiss 743

LSM880 laser scanning microscope with a C-Apochromat 40x/1.2 W Korr FCS M27 744

objective lens. Images were captured by using ZEN2 software with the filters of GFP 745

(EX/Em: 488 / 500-550 nm) and DsRed (EX/Em: 561 / 570-619 nm). 746

747

Accession Numbers 748

Sequence data from this article can be found in the GenBank/EMBL databases under 749

accession numbers shown in Tables 1 and 2, and Supplemental Tables 1 and 2. 750

751

Supplemental Data 752

Supplemental Figure 1. Trans-Complementation of the Systemic Movement of 753

P20-defective Satellite RNA of Bamboo Mosaic Virus (satBaMV) in P20 transgenic N. 754

benthamiana. 755

Supplemental Figure 2. Systemic movement of SatBaMV with and without HV. 756

Supplemental Figure 3. RT-qPCR analysis of satBaMV mRNA accumulation in N. 757

31

benthamiana grafting experiments. 758

Supplemental Figure 4. BaMV and SatBaMV accumulation in coilin or fibrillarin 759

RNAi transgenic lines and fibrillarin accumulation in fibrillarin VIGS plants. 760

Supplemental Figure 5. Identification of P20-interacting protein complex from 761

grafting N. benthamiana fibrillarin RNAi leaves by co-immunoprecipitation (co-IP). 762

Supplemental Figure 6. Yeast two-hybrid analysis of interactions between fibrillarin 763

and P20. 764

Supplemental Table 1. Proteins identified by LC-MS/MS after the 765

immunoprecipitation of P20 IgG from fibrillarin RNAi scions grafted onto satBaMV 766

transgenic stock. 767

Supplemental Table 2. Proteins identified by LC-MS/MS after the 768

immunoprecipitation of P20 IgG from BaMV and satBaMV co-infected fibrillarin 769

RNAi plants. 770

Supplemental Table 3. List of primer sequences used in this study. 771

772

ACKNOWLEDGEMENTS 773

We are grateful to Dr. Andy Jackson (Plant and Microbial Biology Department, 774

University of California-Berkeley, Berkeley, California) for editing the manuscript. 775

We thank M.-Z. Fang for sequencing, Dr. W.-N. Jane for in situ RT-PCR, and 776

technical assistance from the Proteomics Core Lab, Transgenic Plant Core Lab, and 777

Plant Cell Biology Core Lab at the Institute of Plant and Microbial Biology, Academia 778

Sinica, Taiwan. This research was supported by grants from the Ministry of Science 779

and Technology (NSC 1002313B001002MY3) and Academia Sinica Investigator 780

Award to NSL, Taiwan. The work of J.S. and M.T. was funded by the Scottish 781

Government Rural and Environmental Science and Analytical Services Division. 782

32

AUTHOR CONTRIBUTIONS 783

C.-H.C., N.-S.L., and Y.-H.H. designed the project; C.-H.C. and Y.-S.L. conducted 784

most of the experiments; S.-C.L. and J.-D.W. obtained confocal microscope images; 785

J.S., M.T., B.-Y.C., and Y.-H.H designed the viral constructs and characterized the 786

transgenic lines; C.-H.C., F.-C.H, Y.-H.H, M.T., and N.-S.L. analyzed data; and 787

C.-H.C., F.-C.H., M.T., and N.-S.L. wrote the paper. 788

789

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792

793

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795

796

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800

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806

807

33

FIGURE LEGENDS 808

Figure 1. Trans-Complementation of the Systemic Movement of P20-Defective 809

Satellite RNA of Bamboo Mosaic Virus (satBaMV BSGFP) in P20 Transgenic N. 810

benthamiana. 811

(A) Physical map of a SUC2 promoter-driven P20 expression plasmid and 812

immunoblot analysis of P20 accumulation in leaves of SUC2pro:P20 transgenic N. 813

benthamiana (lines 1-29 and 3-1). WT: wild-type plant. rP20: recombinant P20 814

protein purified from E. coli. CBB: Coomassie Brilliant Blue staining. 815

(B) P20-eGFP localization under SUC2 promoter- and 35S promoter-driven 816

expression in WT N. benthamiana leaves at 3 days post-inoculation (DPI). 817

Arrowheads represent the leaf midrib. 818

(C) Schematic maps of satBaMV infectious clones and RNA accumulation of WT 819

satBaMV (BSF4) and P20-defective satBaMV (BSGFP) in WT and P20 transgenic N. 820

benthamiana (line 1-29). Leaves of WT and P20-transgenic N. benthamiana were 821

co-inoculated with pCB (Lin et al., 2004) and pCBSF4 (Lin et al., 2004) or pCBSGFP. 822

Inoculated leaves (IL) were harvested at 10 DPI and uninoculated upper leaves (UL) 823

at 20 DPI for RNA gel blot analysis of BaMV and satBaMV RNA accumulation. 824

BaMV and satBaMV accumulation was detected by using 32P-labeled RNA probes 825

specific for the BaMV 3’ end and satBaMV 3’-UTR, respectively. 826

(D) Quantitative analysis of satBaMV accumulation in WT and P20-transgenic line 827

1-29 from four independent biological samples, each involving four plants. Values are 828

normalized against BSF4 satBaMV in inoculated leaves of WT plants. Data are 829

mean±SD from four experiments and were analyzed by Student t test. Different letters 830

indicate significant difference (p < 0.05). 831

832

34

Figure 2. HV-Independent Systemic Movement of SatBaMV. 833

(A) Physical map of pKF4 for generating satBaMV transgenic plants and satBaMV 834

RNA accumulation in transgenic N. benthamiana lines 2-6 and 9-2 by RNA gel blot. r: 835

root; s: stem; L1: 1st leaf; L2: 2nd leaf; L3: 3rd leaf; f: flower. rRNA of EtBr served as 836

loading control. Four independent biological samples, each involving four plants, 837

generated similar results. 838

(B, C) Illustrations of grafting experiments with 40-day-old WT and 839

satBaMV-transgenic N. benthamiana (sat). SatBaMV RNA and P20 protein 840

accumulation were examined by RNA gel blot and immunoblot analysis, respectively. 841

rRNA and Coomassie blue staining were used for loading controls. Four independent 842

biological samples, each involving four plants, generated similar results. (B) Leaf 7 843

(L7) and leaf 8 (L8) were detached before grafting. L6, L9, and L10 near the graft 844

union were harvested at 12 days after grafting (DAG) and L11 at 15 DAG. (C) Leaves 845

L7 to L11 were detached immediately after grafting; L6 and L12 were harvested at 15 846

DAG. 847

(D) RT-PCR and tissue blot detection of satBaMV RNA in grafting scions after dark 848

treatment. The dark treatment of scions started at 12 DAG for 3 days. RNA and 849

protein extracted from L6, L12-L14, and shoot apex (SA) were sampled at 15 DAG 850

and examined by RT-PCR and immunoblot analysis, respectively. Plants without dark 851

treatment (w/o dark treatment) were used as controls. rRNA and Coomassie blue 852

staining were used for loading controls. The tissue blots from left to right were 853

prepared from grafting stem tissues between L6 to L12, followed by hybridization 854

with satBaMV-specific probe. Four independent biological samples, each involving 855

four plants, generated similar results. 856

(E) Detection of transgene RNA in transgenic stocks and WT scions after grafting. 857

35

WT plants were grafted onto transgenic N. benthamiana expressing GFP (GFP) or 858

BaMV capsid protein (CP) or transgenic N. tabacum expressing Cucumber mosaic 859

virus satRNA (satCMV). RT-PCR analysis of mRNA level at 15 DAG. Four 860

independent biological samples, each involving four plants, generated similar results. 861

Figure 3. In situ RT-PCR Detection of SatBaMV. 862

The grafting experiment was illustrated as in Fig. 2B. Stems and petioles between 863

L6 and L9 were harvested at 12 DAG for in situ RT-PCR detection. 864

(A-F) Presence of satBaMV RNA in the stem (A, B), petiole (C, D) and leaf midrib 865

(E, F) of satBaMV transgenic N. benthamiana stock. 866

(G-L) Presence of satBaMV RNA in the stem (G, H), petiole (I, J) and leaf midrib 867

(K, L) of WT scion. 868

(M, N) Detection of SIEVE ELEMENT OCCLUSION 1 (SEO1) mRNA in the WT 869

scion stem. SEO1 mRNA was restricted to the sieve element (Ernst et al., 2012). 870

Arrows in (M) indicate sieve element. 871

(O, P) Detection of TOBACCO POLYPHENOL OXIDASE 1 mRNA in the WT 872

scion stem, which was exclusively present in flower organs (Goldman et al., 1998), as 873

a negative control. 874

(Q, R) Detection of satBaMV RNA without reverse transcriptase in the reaction in 875

the WT scion stem as control for non-specific amplification during RT-PCR. 876

The results of in situ RT-PCR were observed by confocal laser scanning microscopy. 877

Green signal represents incorporation of Alexa Fluor 488-labeled nucleotides during 878

specific amplification of target genes indicated beside images and the respective 879

merged image, with bright-field image in the right panel. Four independent biological 880

samples, each involving four plants, of in situ RT-PCR detection generated similar 881

36

results. Scale bars: 100 μm. Pi, pith; Xy, xylem; Co, cortex; Ep, epidermis; Ph, 882

phloem; IP, internal phloem; EP, external phloem; Pa, parenchyma; Me, mesophyll. 883

Figure 4. Identification of P20-interacting Protein Complex from Grafting N. 884

benthamiana Leaves with HV-dependent or -independent SatBaMV Infection by 885

Co-immunoprecipitation (co-IP). 886

(A) Coomassie blue staining of total protein extracted from leaves of healthy or 887

BaMV (pKB) + satBaMV (pKF4) (Liou et al., 2013) co-infected N. benthamiana at 7 888

DPI. 889

(B) Co-IP protein complexes by pre-immune IgG (PIS) or anti-P20 IgG (P20) were 890

separated by SDS-PAGE. Protein bands were visualized by silver staining; frames 891

indicate protein bands excised for LC-MS/MS protein identification. The gel is 892

representative of 3 independent experiments. 893

(C-D) Detection of P20 and fibrillarin in co-IP complex from anti-P20 IgG or PIS 894

antibody. Input (-) and complex were separated by SDS-PAGE followed by 895

immunoblot analyses with anti-P20 (C) or anti-FIB IgG (D). 896

(E) HV-independent grafting experiments are illustrated in the left as in Fig. 2B. 897

Total protein was extracted from WT scion leaves after grafting at 15 DAG. Co-IP and 898

protein analysis were performed as in (B). 899

Figure 5. Fibrillarin Silencing Suppresses SatBaMV Trafficking. 900

(A) Accumulation of satBaMV RNA and fibrillarin mRNA and protein in N. 901

benthamiana leaves from satBaMV transgenic stock and grafted WT and 902

TRV-induced fibrillarin silenced (Fib-s) scions. Plants were first infiltrated with A. 903

tumefaciens strain LBA4404 carrying binary vectors expressing pTRV1 and 904

pTRV2-fibrillarin (Fib-s); 7 days later, a fib-s scion was grafted onto satBaMV 905

transgenic line 2-6 (sat). At 9 DAG, stock and scion leaves were harvested for RNA 906

37

gel blot and immunoblot analyses. CBB: Coomassie Brilliant Blue staining. Four 907

independent biological samples, each involving four plants, generated similar results. 908

Actin and CBB were used as loading controls. 909

(B) Accumulation of satBaMV RNA, coilin and fibrillarin mRNA and fibrillarin 910

protein in N. benthamiana leaves from satBaMV transgenic stock and grafted WT, 911

fibrillarin and coilin RNAi (Shaw et al., 2014) transgenic scions. RNA and protein 912

analysis were performed at 9 DAG as described in (A). Four independent biological 913

samples, each involving four plants generated similar results. 914

(C) Statistical analysis of satBaMV accumulation in grafted coilin or fibrillarin 915

RNAi transgenic scions. Values are normalized against the WT sample. Data are mean 916

± SD from four independent biological samples, each involving four plants and were 917

analyzed by Student t test. The asterisk represents significant difference between WT 918

and fibrillarin RNAi lines (* = P < 0.001, ns = not significant). 919

(D, E) Accumulation of BaMV and satBaMV in WT, coilin, and fibrillarin RNAi 920

transgenic plants. RNA gel blot analyses of BaMV and satBaMV RNAs in WT, coilin 921

and fibrillarin RNAi transgenic plants agroinfected with pKB (B) or pKB + pKF4 922

(B+S) in inoculated leaves (IL) harvested at 5 DPI (D) and uninoculated upper leaves 923

(UL) at 15 DPI (E). Four independent biological samples, each involving four plants 924

generated similar results. “−“: uninoculated (healthy) plant control. rRNA: loading 925

control. 926

Figure 6. Mobile SatBaMV–P20 Complexes Contain Fibrillarin (FIB), TGBp1, 927

TGBp2, TGBp3, CP, BaMV, and SatBaMV RNAs in N. benthamiana Agroinfected 928

with pKB and pKF4. 929

(A, B) WT N. benthamiana plants were agroinfected with pKB (B) or pKB + pKF4 930

(B+S). Healthy (H) leaves were used as a control. Total proteins were extracted from 931

38

uninoculated upper leaves of healthy plants or agroinfected plants at 15 DPI, and 932

co-IP was performed with anti-P20 (A) or anti-fibrillarin (FIB) (B) IgG followed by 933

protein A agarose immunoprecipitation. Input (−) and eluted (+) proteins were 934

separated by SDS-PAGE followed by immunoblot analyses with anti-P20, anti-FIB, 935

anti-TGBp1, anti-TGBp2 or anti-CP IgG. Lane 6 was loaded in 20-fold dilution. CBB 936

indicates the input protein before co-IP. Four independent biological samples 937

generated similar results. 938

(C) Genomic map of BaMV infectious clone pCB-P3HA (Chou et al., 2013). N. 939

benthamiana plants were inoculated with WT pCB (B), pCB + pCBSF4 (B+S) or 940

pCB-P3HA (B-P3HA), or pCB-P3HA + pCBSF4 (B-P3HA+S). Co-IP and 941

immunoblot analyses were as described in (A, B), except that the HA antibody was 942

used for immunoprecipitation. Input proteins before co-IP were detected by 943

immunoblot against actin and CBB staining. Four independent biological samples 944

generated similar results. 945

(D, E) Immunoblot analysis of TGBp3HA in co-IP complex. Co-IP with anti-P20 946

(D) or anti-FIB (E) IgG followed by protein A agarose. Input (−) and eluted (+) 947

proteins were separated by SDS-PAGE followed by immunoblot analyses with 948

anti-HA antibody. Lane 6 was loaded in 20-fold dilution. Four independent biological 949

samples were examined. 950

(F, G) RT-PCR detection of BaMV (left panels) and satBaMV RNA (right panels) 951

in co-IP fractions. RNA was extracted from anti-P20 (F) or anti-FIB (G) IgG co-IP 952

fractions from healthy (H) or agroinfected with pKB (B) or pKB + pKF4 (B+S) N. 953

benthamiana leaves. RT-PCR was used to detect the presence of BaMV and satBaMV 954

RNAs. Amplified products were separated with agarose gel and the expected sizes of 955

the BaMV (0.8 kb) and satBaMV (0.8 kb) fragments were shown. Four independent 956

39

biological samples generated similar results. 957

(H) RT-PCR detection of satBaMV RNA in co-IP fractions from non-grafted or 958

grafted plants. The grafting experiment was illustrated in Fig. 2B. Total protein from 959

non-grafted WT (lane 1) or grafted L6 (lane 2) and L9 (lane 3) leaves was extracted 960

and used for co-IP at 15 DAG. RNA was extracted and detected by RT-PCR from 961

anti-P20 or anti-FIB IgG co-IP fractions. Four independent biological samples were 962

examined. 963

Figure 7. Subcellular Localization of satBaMV-Encoded Protein P20 and 964

Fibrillarin in N. benthamiana Epidermal Cells. Proteins were fused to eGFP or 965

DsRed/mCherry and expressed in N. benthamiana leaves by agro-infiltration. Two 966

days after agro-infiltration, leaf sections were examined under a confocal laser 967

scanning microscope. 968

(A) P20-eGFP localizes to the nucleus and cell periphery as punctate structures (a). 969

The cell periphery is shown through the merging of the fluorescent signal with the 970

bright field (b). (B) Localization of P20-eGFP and mCherry-NbFIB2 in nucleus and 971

nucleolus. Epidermal cells expressing P20-eGFP (a-c) or mCherry-NbFIB2 (d-f), or 972

co-expressing both proteins (g-i) were examined. The nuclear area is indicated by the 973

dashed circle. 974

(C) Localization of P20-eGFP peripheral punctate adjacent to plasmodesmata. 975

P20-eGFP co-expressed with TMVMP-DsRed (a-f) or callose deposition (as stained 976

with aniline blue) (g-l). The images in the dashed square areas of (a-c) and (g-i) are 977

shown magnified in (d-f) and (j-l), respectively. 978

(D) Localization of P20-eGFP after plasmolysis. The leaf tissues were plasmolyzed 979

with 1 M NaCl before confocal microscopy. Images show localization of P20-eGFP (a) 980

and TMVMP-DsRed (b) and merged images (c and d) in the plasmolyzed cell. The 981

40

plasmolyzed region is labeled (Plasmolysis). Green, red and blue represent eGFP, 982

DsRed/mCherry and aniline blue signals, respectively. Arrows and arrowheads 983

indicate nucleus and nucleolus, respectively. Open triangles indicate the 984

plasmodesmata. Scale bars: 40 μm in (A), 5 μm in (B) and (D), 10 μm in (C, a-c and 985

g-i) and 2 μm in (C, d-f and j-l). Individual and merged images were edited using 986

Photoshop CS5. 987

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1242

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Table 1. Proteins identified by LC-MS/MS after immunoprecipitation of P20 IgG from BaMV and 1243 satBaMV co-infected N. benthamiana. 1244

Band Accession Protein name Mass (kDa) Score

1 ATCG00490.1 RBCL ribulose-bisphosphate carboxylase 53.4 59

2 AT4G25630.1 FIB2, ATFIB2 fibrillarin 2 33.8 42

2 *gi|345134903|dbj|BAK64672.1| *BaMV TGBp1 27.7 43

3 AT5G01530.1 Light harvesting complex photosystem II 25.2 130

3 *gi|2407623|gb|AAB70566.1| *BaMV CP 25.5 818

4 AT3G16640.1 TCTP translationally controlled tumor

protein

21.3 55

4 *gi|37782235|gb|AAP31339.1| *SatBaMV P20 19.9 26

5 AT1G77300.1 Histone H3 17.0 25

6 ATCG00580.1 PSBE photosystem II reaction center protein 10.0 36

*: BaMV- or satBaMV-encoded proteins 1245 Score is parameter characterizing identification reliability of a certain protein. In general, at score value > 23, identification is 1246 considered as reliable (p<0.05). 1247 1248 1249

49

Table 2. Proteins identified by 1D LC-MS/MS after immunoprecipitation of P20 IgG from wild-type 1250 scion grafted onto satBaMV transgenic stock. 1251

Band Accession Protein name Mass (kDa) Score

1 AT5G04140.2 FD-GOGAT glutamate synthase 1 181.2 76

2 AT2G26080.1 GLDP2 glycine decarboxylase P-protein 2 114.6 246

3 AT3G09440.1 Heat shock protein 70 (Hsp 70) family protein 71.5 181

4 AT1G55490.1 LEN1 chaperonin 60 beta 64.1 85

5 AT4G38970.1 FBA2 fructose-bisphosphate aldolase 2 43.1 486

6 ATCG00020.1 photosystem II reaction center protein A 39.0 218

7 AT4G25630.1 FIB2, ATFIB2 fibrillarin 2 33.8 28

8 *gi|37782235|gb|AAP31339.1| *SatBaMV P20 19.9 27

8 AT2G36160.1 Ribosomal protein S11 family protein 16.3 56

*: satBaMV-encoded proteins 1252 Score is parameter characterizing identification reliability of a certain protein. In general, at score value > 23, identification is 1253 considered as reliable (p<0.05). 1254 1255

1256

Figure 1. Trans-Complementation of the Systemic Movement of P20-Defective Satellite RNA of Bamboo Mosaic Virus (satBaMV BSGFP) in P20 Transgenic N. benthamiana.

(A) Physical map of a SUC2 promoter-driven P20 expression plasmid and immunoblot analysis of P20 accumulation in leaves of SUC2pro:P20 transgenic N. benthamiana (lines 1-29 and 3-1). WT: wild-type plant. rP20: recombinant P20 protein purified from E. coli. CBB: Coomassie Brilliant Blue staining.

(B) P20-eGFP localization under SUC2 promoter- and 35S promoter-driven expression in WT N. benthamiana leaves at 3 days post-inoculation (DPI). Arrowheads represent the leaf midrib.

(C) Schematic maps of satBaMV infectious clones and RNA accumulation of WT satBaMV (BSF4) and P20-defective satBaMV (BSGFP) in WT and P20 transgenic N. benthamiana (line 1-29). Leaves of WT and P20-transgenic N. benthamiana were co-inoculated with pCB (Lin et al., 2004) and pCBSF4 (Lin et al., 2004) or pCBSGFP. Inoculated leaves (IL) were harvested at 10 DPI and uninoculated upper leaves (UL) at 20 DPI for RNA gel blot analysis of BaMV and satBaMV RNA accumulation. BaMV and satBaMV accumulation was detected by using 32P-labeled RNA probes specific for the BaMV 3’ end and satBaMV 3’-UTR, respectively.

(D) Quantitative analysis of satBaMV accumulation in WT and P20-transgenic line 1-29 from four independent biological samples, each involving four plants. Values are normalized against BSF4 satBaMV in inoculated leaves of WT plants. Data are mean±SD from four experiments and were analyzed by Student t test. Different letters indicate significant difference (p < 0.05).

Figure 2. HV-Independent

Systemic Movement of

SatBaMV. (A) Physical map of pKF4 for

generating satBaMV transgenic

plants and satBaMV RNA

accumulation in transgenic N.

benthamiana lines 2-6 and 9-2

by RNA gel blot. r: root; s: stem;

L1: 1st leaf; L2: 2nd leaf; L3: 3rd

leaf; f: flower. rRNA of EtBr

served as loading control. Four

independent biological samples,

each involving four plants,

generated similar results.

(B, C) Illustrations of grafting

experiments with 40-day-old WT

and satBaMV-transgenic N.

benthamiana (sat). SatBaMV

RNA and P20 protein

accumulation were examined by

RNA gel blot and immunoblot

analysis, respectively. rRNA and

Coomassie blue staining were

used for loading controls. Four

independent biological samples, each involving four plants, generated similar results. (B) Leaf 7 (L7) and leaf 8 (L8) were

detached before grafting. L6, L9, and L10 near the graft union were harvested at 12 days after grafting (DAG) and L11 at

15 DAG. (C) Leaves L7 to L11 were detached immediately after grafting; L6 and L12 were harvested at 15 DAG.

(D) RT-PCR and tissue blot detection of satBaMV RNA in grafting scions after dark treatment. The dark treatment of

scions started at 12 DAG for 3 days. RNA and protein extracted from L6, L12-L14, and shoot apex (SA) were sampled at

15 DAG and examined by RT-PCR and immunoblot analysis, respectively. Plants without dark treatment (w/o dark

treatment) were used as controls. rRNA and Coomassie blue staining were used for loading controls. The tissue blots

from left to right were prepared from grafting stem tissues between L6 to L12, followed by hybridization with

satBaMV-specific probe. Four independent biological samples, each involving four plants, generated similar results.

(E) Detection of transgene RNA in transgenic stocks and WT scions after grafting. WT plants were grafted onto

transgenic N. benthamiana expressing GFP (GFP) or BaMV capsid protein (CP) or transgenic N. tabacum expressing

Cucumber mosaic virus satRNA (satCMV). RT-PCR analysis of mRNA level at 15 DAG. Four independent biological

samples, each involving four plants, generated similar results.

Figure 3. In situ RT-PCR Detection of SatBaMV. The grafting experiment was illustrated as in Fig. 2B. Stems and petioles

between L6 and L9 were harvested at 12 DAG for in situ RT-PCR detection. (A-F) Presence of satBaMV RNA in the stem (A, B), petiole (C, D) and leaf

midrib (E, F) of satBaMV transgenic N. benthamiana stock. (G-L) Presence of satBaMV RNA in the stem (G, H), petiole (I, J) and leaf

midrib (K, L) of WT scion. (M, N) Detection of SIEVE ELEMENT OCCLUSION 1 (SEO1) mRNA in the

WT scion stem. SEO1 mRNA was restricted to the sieve element (Ernst et al., 2012). Arrows in (M) indicate sieve element.

(O, P) Detection of TOBACCO POLYPHENOL OXIDASE 1 mRNA in the WT scion stem, which was exclusively present in flower organs (Goldman et al., 1998), as a negative control.

(Q, R) Detection of satBaMV RNA without reverse transcriptase in the reaction in the WT scion stem as control for non-specific amplification during RT-PCR.

The results of in situ RT-PCR were observed by confocal laser scanning microscopy. Green signal represents incorporation of Alexa Fluor 488-labeled nucleotides during specific amplification of target genes indicated beside images and the respective merged image, with bright-field image in the right panel. Four independent biological samples, each involving four plants, of in situ RT-PCR detection generated similar results. Scale bars: 100 μm. Pi, pith; Xy, xylem; Co, cortex; Ep, epidermis; Ph, phloem; IP, internal phloem; EP, external phloem; Pa, parenchyma; Me, mesophyll.

Figure 4. Identification of P20-interacting Protein Complex from Grafting N.

benthamiana Leaves with HV-dependent or -independent SatBaMV Infection by Co-immunoprecipitation (co-IP).

(A) Coomassie blue staining of total protein extracted from leaves of healthy or BaMV (pKB) + satBaMV (pKF4) (Liou et al., 2013) co-infected N. benthamiana at 7 DPI.

(B) Co-IP protein complexes by pre-immune IgG (PIS) or anti-P20 IgG (P20) were separated by SDS-PAGE. Protein bands were visualized by silver staining; frames indicate protein bands excised for LC-MS/MS protein identification. The gel is representative of 3 independent experiments.

(C-D) Detection of P20 and fibrillarin in co-IP complex from anti-P20 IgG or PIS antibody. Input (-) and complex were separated by SDS-PAGE followed by immunoblot analyses with anti-P20 (C) or anti-FIB IgG (D). (E) HV-independent grafting experiments are illustrated in the left as in Fig. 2B. Total protein was extracted from WT scion leaves after grafting at 15 DAG. Co-IP and protein analysis were performed as in (B).

Figure 5. Fibrillarin Silencing Suppresses SatBaMV Trafficking. (A) Accumulation of satBaMV RNA and fibrillarin mRNA and protein in N. benthamiana leaves from

satBaMV transgenic stock and grafted WT and TRV-induced fibrillarin silenced (Fib-s) scions. Plants were first infiltrated with A. tumefaciens strain LBA4404 carrying binary vectors expressing pTRV1 and pTRV2-fibrillarin (Fib-s); 7 days later, a fib-s scion was grafted onto satBaMV transgenic line 2-6 (sat). At 9 DAG, stock and scion leaves were harvested for RNA gel blot and immunoblot analyses. CBB: Coomassie Brilliant Blue staining. Four independent biological samples, each involving four plants generated similar results. Actin and CBB were used as loading controls.

(B) Accumulation of satBaMV RNA, coilin and fibrillarin mRNA and fibrillarin protein in N. benthamiana leaves from satBaMV transgenic stock and grafted WT, fibrillarin and coilin RNAi (Shaw et al., 2014) transgenic scions. RNA and protein analysis were performed at 9 DAG as described in (A). Four independent biological samples, each involving four plants generated similar results.

(C) Statistical analysis of satBaMV accumulation in grafted coilin or fibrillarin RNAi transgenic scions. Values are normalized against the WT sample. Data are mean ± SD from four independent biological samples, each involving four plants and were analyzed by Student t test. The asterisk represents significant difference between WT and fibrillarin RNAi lines (* = P < 0.001, ns = not significant).

(D, E) Accumulation of BaMV and satBaMV in WT, coilin, and fibrillarin RNAi transgenic plants. RNA gel blot analyses of BaMV and satBaMV RNAs in WT, coilin and fibrillarin RNAi transgenic plants agroinfected with pKB (B) or pKB + pKF4 (B+S) in inoculated leaves (IL) harvested at 5 DPI (D) and uninoculated upper leaves (UL) at 15 DPI (E). Four independent biological samples, each involving four plants, generated similar results. “−“: uninoculated (healthy) plant control. rRNA: loading control.

Figure 6. Mobile SatBaMV–P20 Complexes Contain Fibrillarin (FIB), TGBp1, TGBp2, TGBp3, CP, BaMV, and SatBaMV RNAs in N. benthamiana Agroinfected with pKB and pKF4.

(A, B) WT N. benthamiana plants were agroinfected with pKB (B) or pKB + pKF4 (B+S). Healthy (H) leaves were used as a control. Total proteins were extracted from uninoculated upper leaves of healthy plants or agroinfected plants at 15 DPI, and co-IP was performed with anti-P20

(A) or anti-fibrillarin (FIB) (B) IgG followed by protein A agarose immunoprecipitation. Input (−) and eluted (+) proteins were separated by SDS-PAGE followed by immunoblot analyses with anti-P20, anti-FIB, anti-TGBp1, anti-TGBp2 or anti-CP IgG. Lane 6 was loaded in 20-fold dilution. CBB indicates the input protein before co-IP. Four independent biological samples generated similar results.

(C) Genomic map of BaMV infectious clone pCB-P3HA (Chou et al., 2013). N. benthamiana plants were inoculated with WT pCB (B), pCB + pCBSF4 (B+S) or pCB-P3HA (B-P3HA), or pCB-P3HA + pCBSF4 (B-P3HA+S). Co-IP and immunoblot analyses were as described in (A, B), except that the HA antibody was used for immunoprecipitation. Input proteins before co-IP were detected by immunoblot against actin and CBB staining. Four independent biological samples generated similar results.

(D, E) Immunoblot analysis of TGBp3HA in co-IP complex. Co-IP with anti-P20 (D) or anti-FIB (E) IgG followed by protein A agarose. Input (−) and eluted (+) proteins were separated by SDS-PAGE followed by immunoblot analyses with anti-HA antibody. Lane 6 was loaded in 20-fold dilution. Four independent biological samples were examined.

(F, G) RT-PCR detection of BaMV (left panels) and satBaMV RNA (right panels) in co-IP fractions. RNA was extracted from anti-P20 (F) or anti-FIB (G) IgG co-IP fractions from healthy (H) or agroinfected with pKB (B) or pKB + pKF4 (B+S) N. benthamiana leaves. RT-PCR was used to detect the presence of BaMV and satBaMV RNAs. Amplified products were separated with agarose gel and the expected sizes of the BaMV (0.8 kb) and satBaMV (0.8 kb) fragments were shown. Four independent biological samples generated similar results.

(H) RT-PCR detection of satBaMV RNA in co-IP fractions from non-grafted or grafted plants. The grafting experiment was illustrated in Fig. 2B. Total protein from non-grafted WT (lane 1) or grafted L6 (lane 2) and L9 (lane 3) leaves was extracted and used for co-IP at 15 DAG. RNA was extracted and detected by RT-PCR from anti-P20 or anti-FIB IgG co-IP fractions. Four independent biological samples were examined.

Figure 7. Subcellular Localization of satBaMV-Encoded Protein P20 and Fibrillarin in N. benthamiana Epidermal Cells. Proteins were fused to eGFP or DsRed/mCherry and expressed in N. benthamiana leaves by agro-infiltration. Two days after agro-infiltration, leaf sections were examined under a confocal laser scanning microscope.

(A) P20-eGFP localizes to the nucleus and cell periphery as punctate structures (a). The cell periphery is shown through the merging of the fluorescent signal with the bright field (b). (B) Localization of P20-eGFP and mCherry-NbFIB2 in nucleus and nucleolus. Epidermal cells expressing P20-eGFP (a-c) or mCherry-NbFIB2 (d-f), or co-expressing both proteins (g-i) were examined. The nuclear area is indicated by the dashed circle.

(C) Localization of P20-eGFP peripheral punctate adjacent to

plasmodesmata. P20-eGFP co-expressed with TMVMP-DsRed (a-f) or callose deposition (as stained with aniline blue) (g-l). The images in the dashed square areas of (a-c) and (g-i) are shown magnified in (d-f) and (j-l), respectively.

(D) Localization of P20-eGFP after plasmolysis. The leaf tissues were plasmolyzed with 1 M NaCl before confocal microscopy. Images show localization of P20-eGFP (a) and TMVMP-DsRed (b) and merged images (c and d) in the plasmolyzed cell. The plasmolyzed region is labeled (Plasmolysis). Green, red and blue represent eGFP, DsRed/mCherry and aniline blue signals, respectively. Arrows and arrowheads indicate nucleus and nucleolus, respectively. Open triangles indicate the plasmodesmata. Scale bars: 40 μm in (A), 5 μm in (B) and (D), 10 μm in (C, a-c and g-i) and 2 μm in (C, d-f and j-l). Individual and merged images were edited using Photoshop CS5.

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DOI 10.1105/tpc.16.00071; originally published online October 4, 2016;Plant Cell

Taliansky, Ban-Yang Chang, Yau-Heiu Hsu and Na-Sheng LinChih-Hao Chang, Fu-Chen Hsu, Shu-Chuan Lee, Yih-Shan Lo, Jiun-Da Wang, Jane Shaw, Michael

Trafficking of a Subviral Satellite RNA in PlantsThe Nucleolar Fibrillarin Protein is Required for Helper Virus-Independent Long-Distance

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