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: [email protected] 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 ([email protected]) 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
6
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
7
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
8
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
790
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792
793
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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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