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A vicilin-like seed storage protein, PAP85, is involved in Tobacco mosaic virus 1
replication 2
Cheng-En Chena, Kuo-Chen Yehc, Shu-Hsing Wud, Hsiang-Iu Wange, Hsin-Hung Yeha,b 3
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Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, 9
Taiwana; Research Center for Plant Medicine, National Taiwan University, Taipei, 10
Taiwanb; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 11
Taiwanc; Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwand; 12
Department of Computer Science, National Tsing Hua University, Hsinchu, Taiwane. 13
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Address correspondence to Hsin-Hung Yeh, [email protected]. 15
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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00268-13 JVI Accepts, published online ahead of print on 10 April 2013
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ABSTRACT 27
One striking feature of viruses with an RNA genome is the modification of the host 28
membrane structure during early infection. This process requires both virus- and 29
host-encoded proteins; however, the host factors involved and their role in this 30
process remain largely unknown. On infection with Tobacco mosaic virus (TMV), a 31
(+)RNA virus, the filamentous and tubular endoplasmic reticulum (ER) converts to 32
aggregations at the early stage and returns to filamentous at the late infectious stage, 33
termed the ER transition. As well, membrane- or vesicle-packaged viral replication 34
complexes (VRCs) are induced early during infection. We used microarray assay to 35
screen Arabidopsis gene(s) responding to infection with TMV in the initial infection 36
stage and identified an Arabidopsis gene, PAP85 (annotated as a vicilin-like seed 37
storage protein), with upregulated expression during 0.5- to 6-hr TMV infection. 38
TMV accumulation was reduced in pap85-RNAi Arabidopsis and restored to 39
wild-type levels when PAP85 was overexpressed in pap85-RNAi Arabidopsis. We did 40
not observe the ER transition in TMV-infected PAP85-knockdown Arabidopsis 41
protoplasts. In addition, TMV accumulation was reduced in PAP85-knockdown 42
protoplasts. VRC accumulation was reduced but not significantly (P=0.06) in 43
PAP85-knockdown protoplasts. Co-expression of PAP85 and the TMV main replicase 44
(P126) but not their expression alone in Arabidopsis protoplasts could induce ER 45
aggregations. 46
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INTRODUCTION 53
Most viruses causing important diseases in both animals and plants have a 54
single-stranded RNA genome of positive polarity [(+)RNA viruses]. One striking 55
feature of (+)RNA viruses is their modification of the host membrane structure for 56
replication. Once (+)RNA viruses enter host cells, they induce a change in membrane 57
structure and form membrane or vesicle packages of 50 to 400 nm in diameter (1); 58
these structures are free within the cytoplasm or are associated with surrounding 59
membrane (1, 2). The virus-induced organelles contain viral proteins, viral RNAs and 60
host factors usually termed viroplasm or viral replication complexes (VRCs) (3-6). 61
More viral genomic RNAs are then produced within the VRCs. The origins of 62
membranes forming VRCs are diverse in different types of invading viruses (1). 63
Some viral replicases, when expressed alone, can induce membrane 64
modifications. An example is the 1a protein of Brome mosaic virus (BMV). The 1a 65
protein is responsible for recruiting the 2a and viral RNA to the sites of replication in 66
yeast (7, 8). The protein 1a is localized on the ER, and expression of 1a alone can 67
induce the membrane to form spherule invaginations (9, 10). Modulating the relative 68
levels and interactions of la and 2a can change the membrane rearrangements from 69
small spherule invaginations to large multilayered double membranes (11). The main 70
replicases of viruses within the genus Tombusvirus target different membrane systems 71
and induce various changes in membranes. For example, the main replicase of Tomato 72
bushy stunt tombusvirus P33 targets peroxisomes and causes their progressive 73
aggregation (12); the main replicase P27 encoded by Red clover necrotic tombusvirus 74
induces perinuclear aggregation and many smaller aggregations derived from the ER 75
(13). As well, some viruses encoding proteins with no predicted enzyme activity 76
related to replication may also participate in modification of the membrane structure 77
for replication. The 6-kDa protein encoded by Tobacco etch virus (TEV) forms a 78
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membranous vesicle at ER exit sites similar to ER alterations in TEV-infected cells 79
(14, 15). 80
Besides virus-encoded proteins, some host factors, such as the chaperone 81
proteins heat shock protein 70 (Hsp70) and Hsp90 (for BMV, tombusviruses), 82
peroxisomal transport Pex19p (for Tomato bushy stunt virus), coat protein complex 83
I/II for retrograde and anterograde transport (for picornavirus and TEV) and 84
lipid-synthesis-associated proteins (for BMV, tombusviruses and Semliki Forest virus) 85
are also involved in this process (14, 16-25). More host factors may be involved in 86
this process, and their identification and understanding how they function may 87
provide opportunities for developing antiviral strategies. 88
The first-known and well-studied virus, Tobacco mosaic virus (TMV), belongs to 89
the (+)RNA viruses. The TMV replicases P126 and P183 (the read-through version of 90
P126) are the viral-encoded proteins required for virus replication. The main viral 91
replicase P126 can localize to the ER and modulate the formation of VRCs (26-28). 92
P126 does not contain transmembrane domains, and TMV may hijack Arabidopsis 93
Tobamovirus multiplication 1 (TOM1) genes for membrane anchoring. TOM1 protein 94
contains transmembrane domains that interact with TMV P126, which may serve as a 95
tethering factor for anchoring viral replicases to the ER membrane. Genetic screening 96
identified another gene, TOM2A, involved in accumulation of different strains of 97
TMV. TOM2, encoding a four-pass transmembrane protein, also interacts with TOM1 98
and was suggested to be associated with tobamovirus VRCs (29-31). 99
Besides the formation of VRCs, in early TMV infection, the filamentous and 100
tubular ER is converted into aggregations and returns to a filamentous and tubular ER 101
in the later infection stage, called the ER transition (32, 33). In TMV, the ER 102
transition was suggested to be the event for the conversion of smooth to rough ER by 103
recruiting the binding of ribosomes to smooth ER during TMV infection and is 104
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important for TMV to build up the infrastructure for protein synthesis and virus 105
replication (32). TMV P126 alone can induce VRC formation (26-28); however, 106
whether P126 can induce the ER transition remains to be resolved. 107
In this study, we focused on the early stage of virus replication to screen host 108
factors involved in TMV infection. TMV U1 strain was used to infect Arabidopsis 109
ecotype Col-0. Arabidopsis is a symptomless host of TMV U1 (34). This feature 110
suggests that this host has less complicated physiological responses as compared with 111
other symptom-developing hosts, making it simpler for our screening. We used 112
microarray analysis to identify 2 host genes, At3g28770 and At3g22640 (PAP85), 113
involved in TMV replication. At3g28770 encodes a functional unknown protein, and 114
At3g22640 encodes a vicilin-like seed-storage protein (PAP85) (35) (Table 1). 115
Because seed-storage protein is involved in membrane modification for transportation 116
(36-38) and TMV infection is also involved in membrane structure modification (32, 117
39), we initially focused on PAP85. TMV-induced ER transition was not observed in 118
PAP85-silenced cells. The expression of TMV P126 alone induced a VRC-like 119
structure but not the ER transition; however, co-expression of PAP85 and TMV P126 120
could induce an ER structure change similar to the ER transition during TMV 121
infection. 122
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MATERIALS AND METHODS 124
Constructs used in this study 125
Clones for cDNA microarray. The TMV wild-type infectious clone was previously 126
described (40). Two mutated clones, TMV*coat protein (CP) and TMV*movement 127
protein (MP), were first created by site-directed mutagenesis. TMV was used as the 128
template, and primer pairs U1-CP-F1/U1-CP-R1 and U1-CP-F2/U1-CP-R2 were used 129
in the PCR reaction to generate 2 overlapping fragments. The amplified fragments 130
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were gel-purified and mixed together in the molar ratio of 1:1 for 5 PCR reaction 131
cycles, then the primer pair U1-CP-F1/U1-CP-R2 was added for another 30 cycles. 132
The amplified products were digested with use of NcoI/BsiWI and separated on 1% 133
agarose gels to purify the fragments. The digested fragment was ligated to 134
NcoI/BsiWI-digested TMV by use of T4 DNA ligase (Promega; Madison, WI, USA) 135
to construct the TMV*CP. The construction of TMV*MP was essentially similar to 136
that for TMV*CP, except the primer pairs U1-MP-F1/U1-MP-R1 and 137
U1-MP-F2/U1-MP-R2 were used to amplify the first 2 overlapping fragments and 138
U1-MP-F1/U1-MP-R2 was used for the second amplification, and the amplified 139
fragment was digested with MfeI. TMV*CP and TMV*MP were both digested with 140
NcoI and BsiWI, and the 787- and 8294-kb fragments were gel-purified. The 2 141
purified fragments were ligated together with T4 DNA ligase (Promega) to create 142
TMV*CP.MP. Two stop codons were introduced in the CP (at the 14 amino acid 143
position) and MP (at the 24 amino acid position). TMV*rep was constructed by the 144
cut and fill-in method. TMV was digested by MluI and then treated with Klenow 145
enzyme (3’-5’ exonuclease, New England Biolabs, Beverly, MA) for 30 min to fill in 146
the overhangs. Then, the reaction was shifted to 65℃ for 20 min for Klenow enzyme 147
inactivation. The blunt end products were re-ligated by use of T4 DNA ligase 148
(Promega), which caused a frame shift (at the 249 amino acid position) and induced a 149
stop codon in the replicase open reading frame (ORF; at the 271 amino acid position). 150
Clone for RNA probe preparation. A fragment corresponding to 448 nt of the TMV 151
CP 3’ end was amplified by PCR with the template TMV and the primer pair 152
U1-CP-F2/U1 RE. The fragment was cloned into pGEM-T Easy vector (Promega) to 153
generate pGEMT-CP. 154
Clones for plant transformation. pB7GWIWG2(I)-PAP85 was constructed by use of 155
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Gateway technology (Invitrogen, Gaithersburg, MD, USA). A 130-bp DNA fragment 156
complementary to the PAP85 nucleotide positions 921-1050 was amplified by PCR 157
with the primer pair AttB1-PAP85F(921)/AttB2-PAP85R(1050). The PCR products 158
were inserted into the donor vector (provided by Invitrogen) by recombination to 159
generate an entry vector, then by another recombination with the destination vector 160
pB7GWIWG2(I) (VIB, Ghent, Belgium) (41) to generate pB7GWIWG2(I)-PAP85. 161
Clone for VRC observation. Red fluorescent protein (mCherry) was fused to the C 162
terminus of the MP of TMV to construct TMV-MP:mCherry. The construction of 163
TMV-MP:mCherry was essentially the same as for TMV*CP, except 2 overlapping 164
fragments were generated by PCR with the template TMV and the primer pair 165
U1-MP-F2/U1-MP-R and with the template pSAT6-mCherry-C1-B (a kind gift from 166
Dr. Lan-Ying Lee, Department of Biological Sciences, Purdue University) and the 167
primer pair MP:YFP F/MP:YFP R. The primer pairs U1-MP-F2/MP:YFP R were used 168
for secondary amplification, and the amplified products were digested with NcoI/PacI 169
and then ligated to NcoI/PacI-digested TMV to construct TMV-MP:mCherry. 170
Clone for PAP85 overexpression. The fragments of PAP85-GFP was amplified by 171
PCR with pCass2-PAP85-GFP as a template and the primer pair 172
PAP85F(CACC)/GFP R3. The PAP85-GFP fragment was cloned into the binary 173
vector pK2GW7 (VIB, Ghent, Belgium) by use of Gateway technology (Invitrogen). 174
PCR products were cloned into the pENTR/D-TOPO Gateway entry vector 175
(Invitrogen) following the manufacturer's recommendations. The pENTR/D-TOPO 176
recombinant construct was sequenced to confirm the accuracy of the cloned fragments, 177
then LR Clonase II enzyme (Invitrogen) was used to transfer the cloned fragments 178
into pK2GW7 to generate pK2GW7-PAP85-GFP. pK2GW7-*PAP85-GFP was 179
constructed by the cut and fill-in method. pK2GW7-PAP85-GFP was digested by use 180
of AseI and then treated with Klenow enzyme (3’-5’ exonuclease, New England 181
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Biolabs) for 30 min to fill in the overhangs. Then, the reaction was shifted to 65℃ 182
for 20 min for Klenow enzyme inactivation. The blunt end products were re-ligated 183
by use of T4 DNA ligase (Promega), which caused a frame shift (at the 7 amino acid 184
position) and induced a stop codon in the replicase open reading frame (ORF; at the 185
10 amino acid position). 186
Clones for subcellular localization analysis. The coding region of PAP85 and P126 187
was inserted in the vector pCass2 with a double 35S promoter (42) and fused with 188
GFP and red fluorescent protein, respectively. The coding region of PAP85 was 189
amplified by RT-PCR with template RNA extracted from TMV-infected plants. Total 190
RNA was treated with TURBO DNA-free kit to remove residual DNA (Ambion, 191
Austin, TX, USA), and total cDNA was synthesized by use of the High-Capacity 192
cDNA Reverse Transcription Kit (Applied Biosystems, Tokyo, Japan), then PAP85 193
was amplified by PCR with the primer pair PAP85 F(GFP)/PAP85 R(GFP). The 194
fragments were digested by use of EcoRI and ligated into EcoRI-digested pCass2 to 195
generate pCass2-PAP85. GFP was amplified by PCR with the template 30B-GFP (40) 196
and the primer pair GFP F2/GFP R2. NruI (blunt end) site and HindIII site were 197
incorporated by use of primers into the amplified GFP fragment. pCass2-PAP85 was 198
first digested by BclI, then treated with Mung Bean nuclease (NEB) to create a blunt 199
end, then digested with HindIII. The GFP fragments were digested by NruI and 200
HindIIII and ligated to the Mung Bean nuclease and HindIII-treated pCass2-PAP85 to 201
construct pCass2-PAP85-GFP. The construction of pCass2-mCherry-P126 was 202
essentially the same as for pCass2-PAP85-GFP, except TMV was used as a template 203
and the primer pair P126 F(mCherry)/P126 R(mCherry) was used to amplify P126. 204
The fragments were digested by KpnI and ligated into KpnI-digested pCass2 to 205
generate pCass2-P126. mCherry fragments were amplified by PCR with the plasmid 206
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pSAT6-mCherry-C1-B (a kind gift from Dr. Lan-Ying Lee) used as a template and 207
the primer pair mCherry F1/mCherry R1. The PCR fragments were digested with 208
SalI/PmlI and ligated to the SalI/PmlI-digested pCass2-P126 to construct 209
pCass2-mCherry-P126. All primer sequences are available upon request. 210
Plant materials and transgenic plants. Wild-type A. thaliana ecotype Col-0, 211
T-DNA insertion SALK lines, ER-YFP transgenic A. thaliana (YFP with an HDEL 212
ER retention signal peptide and the AtWAK2 signal peptide, which targets the 213
proteins to the ER) (43, 44) and pap85-RNAi transgenic lines were all grown at 22℃ 214
under long-day conditions (16-hr light/8-hr dark, 100-150 µE). The pap85-RNAi 215
transgenic lines were generated by infecting A. thaliana (Col-0) with Agrobacterium 216
tumefaciens GV3101 carrying pB7GWIWG2(I)-PAP85 (fragment 921-1050) by the 217
floral dip method (45). Progeny transformants were identified by germinating seeds 218
on Murashige and Skoog medium containing 50 µg/ml glufosinate ammonium (GA). 219
After 2 weeks, GA-resistant seedlings were transferred to soil, and seeds were 220
collected later. Individual homozygous lines in the T2 generation were obtained. 221
Three individual lines were chosen, and their T3 generations were used for the 222
following experiments. 223
In vitro transcription. Capped transcripts corresponding to the wild-type virus 224
and the constructed virus were synthesized by use of the mMESSAGE mMACHINE 225
T7 Kit (Ambion) as described (46-48), except that TMV and its derived plasmids 226
were linearized with KpnI. 227
Protoplast isolation and PEG transfection. Protoplasts were isolated from 6- 228
to 7-week-old A. thaliana expanded leaves as described (49) with some modification. 229
Leaves were cut into 0.5- to 1-mm strips with use of a clean razor. The leaves were 230
incubated in a Petri dish with enzyme solution containing 1% cellulose R10 (Yakult 231
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Honsha, Tokyo, Japan), 0.2% macerozyme R10 (Yakult Honsha), 0.4 M mannitol, 20 232
mM KCl, 20 mM MES, 10 mM CaCl2, and 0.1% BSA, pH 5.7, and incubated for 3 hr 233
in the dark. The protoplasts were harvested by spinning the enzyme solution at 100 × 234
g to pellet the protoplasts. Protoplasts were washed twice with W5 solution (154 mM 235
NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH adjusted to 5.7), then pelleted and 236
resuspended in MMg solution (0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7). 237
Protoplasts were transfected by the PEG method as described (49) with some 238
modification. Protoplasts (1×105 cells) were collected in a round-bottomed tube. 239
Nucleic acids (10 µg RNA transcripts for virus inoculation, 20 µg of each plasmid 240
DNA for subcellular localization analysis and the amount of dsRNA used for transient 241
RNAi induction), and 110 µl PEG/Ca solution (4 g PEG 4000, 3 ml H2O, 2.5 ml 0.8 242
M mannitol, 1 mM CaCl2) was added to the tube smoothly for incubation at 23℃ for 243
20 min, then the tube was diluted with 0.44 ml W5 solution. The solution was gently 244
mixed and centrifuged for 1 min to remove PEG. The protoplasts were resuspended in 245
10 ml W5 solution and incubated at 25℃ in dark. 246
RNA extraction. RNA used in northern blot analysis, quantitative RT-PCR 247
(qRT-PCR) and cDNA microarray analysis was extracted from protoplasts by the Pine 248
Tree Method (50) and dissolved in diethyl pyrocarbonate-treated water. For cDNA 249
microarray analysis, the quality of RNA was checked by use of 2100 Bioanalyzer 250
(Agilent Technologies, Palo Alto, CA). 251
Northern blot hybridization. T3 RNA polymerase and EcoRI-digested 252
pGEMT-CP plasmids were used to generate negative-sense digoxigenin (DIG)-labeled 253
minus-sense probes (Roche Applied Science, Indianapolis, IN, USA). Northern blot 254
hybridization was performed as described (48). Hybridization signals were detected 255
by use of the chemiluminescent substrate CDP STAR (Roche Applied Science) and 256
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blots were exposed to Fuji medical x-ray film (Fuji, Tokyo). 257
cDNA microarray fabrication and hybridization. The cDNA microarray glass 258
slides (including 11,500 Arabidopsis cDNA clones corresponding to 10,452 unique 259
genes) and techniques used in cDNA microarray screening were provided and 260
supported by the DNA Microarray Core Laboratory, Institute of Plant and Microbial 261
Biology, Academia Sinica (Taipei). Total RNA was extracted from TMV*CP.MP- or 262
TMV*rep-inoculated protoplasts, and RNA quality was analyzed by use of 2100 263
Bioanalyzer (Agilent Technologies). RNA derived from TMV*CP.MP- and 264
TMV*rep-inoculated samples was labeled with Cy5 and Cy3, respectively. Methods 265
for preparing the fluorescent probe and hybridization were as described 266
(http://ipmb.sinica.edu.tw/microarray/protocol.htm). The hybridization signals were 267
acquired with the use of Axon GenePix 4000B and analyzed by use of GenePix 4.0 268
(Axon Instruments, Foster City, CA, USA). 269
Real-time qRT-PCR. Extracted total RNA was treated with TURBO DNA-free 270
kit (Ambion) to remove residual DNA. An amount of 1 µg total RNA was used as a 271
template for synthesis of cDNA by use of the High-Capacity cDNA Reverse 272
Transcription Kit (Applied Biosystems). A one-fourth aliquot of the cDNA was used 273
as a template, and 2 × SYBR Green PCR master mix was added (Applied Biosystems). 274
qRT-PCR involved use of the ABI Prism 7000 sequence detection system (Applied 275
Biosystems). The primer sequences are available on request. Quantification of genes 276
involved RNA from 3 repeated individual experiments. For qRT-PCR reaction, each 277
sample was analyzed in triplicate. The relative quantification was calculated 278
according to the manufacturer’s instructions (Applied Biosystems). The Arabidopsis 279
UBQ10 gene was used as an internal quantification control. 280
Preparation of dsRNA and transient RNAi induction in Arabidopsis 281
protoplasts. dsRNA was designed and prepared as described (51) by use of the T7 282
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RiboMax Express RNAi system (Promega). The dsRNA of PAP85 was targeted to 283
922-1031 nt, that of At2g34700 to 110-219 nt, and that of At3g08670 to 942-1051 nt. 284
The dsRNA of GFP (689-818 nt) and At1g55940 (600-709 nt) were controls. The 285
primer sequences are available on request. 286
Agrobacterium-mediated transient complementation assays. Seven-week-old 287
Arabidopsis plants were used for agroinfiltration. Agrobacterium tumefaciens strain 288
GV3101 containing binary vector (pK2GW7 or pK2GW7-PAP85-GFP or 289
pK2GW7-*PAP85-GFP) was incubated with YEP (10 g/L yeast extract, 10 g/L 290
peptone, 5 g/L NaCl, pH 7.0) solid medium with kanamycin (50 µg/ml) and 291
rifampicin (50 µg/ml) and grown at 28℃ for 2 days. Before agroinfiltration, A. 292
tumefaciens was maintained at 28℃ overnight in YEP broth with kanamycin (50 293
µg/ml) and rifampicin (50 µg/ml). The preparation of A. tumefaciens suspension and 294
the agroinfiltration method were essentially as described (52). 295
Western blot hybridization. In total, 10 µg crude extracted protein was resolved 296
on 12.5% SDS-PAGE, then proteins were transferred to nitrocellulose membranes 297
(Whatman Protran, Dassel, Germany), which were probed with the primary antibody 298
monoclonal anti-GFP (1:2000 dilution; Sigma Aldrich, St. Louis, MO, USA), then 299
anti-mouse IgG-horseradish peroxidase-conjugated secondary antibody (1:20000 300
dilution). Signals were detected by use of enhanced chemiluminescence agents 301
(Amersham GE Healthcare, Little Chalfont, UK), and blots were exposed to Fuji 302
medical x-ray film (Fuji). 303
Confocal microscopy. Protoplasts used for observation of subcellular 304
localization were examined by confocal microscopy (Zeiss LSM 510 META NLO 305
DuoScan, Carl Zeiss, Jena, Germany). Protoplasts were harvested at 0.5 to 36 hr 306
post-inoculation by centrifugation at 100 × g for 5 min. For single fluorescent 307
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imaging of GFP or YFP, excitation wavelength was 488 nm, with detection 308
wavelength 500 to 587 nm. For imaging mCherry, excitation wavelength was 561 nm,, 309
with detection wavelength 575 to 630 nm. For simultaneous imaging of GFP and YFP, 310
we used the “emission fingerprinting method” and the software ZEN (Carl Zeiss). 311
Cells solely expressing GFP or YFP were first captured and stored in Lambda mode 312
(in ZEN) as a reference. Detection wavelength was 500 to 587 nm to capture both 313
GFP and YFP fluorescence, then META Unmixing (in ZEN) was used to distinguish 314
GFP and YFP signals. Images were assembled by use of ZEN. 315
316
RESULTS 317
Twelve genes upregulated during early TMV infection. Because we focused on the 318
early stage of virus replication and searched for gene(s) whose expression responded 319
to virus infection, we used the genome-wide screening tool, DNA microarray, to 320
identify host factors involved in virus replication. To help us more easily find the host 321
factor, we narrowed down our target gene(s) by experimental design before 322
microarray screening. First, we used the construct TMV*CP.MP with stop codons in 323
both the coat protein (CP) and movement protein (MP) (Fig. 1A,B). The CP and MP 324
are dispensable in virus replication. A replication-incompetent TMV, TMV*rep, with 325
truncated RdRp was constructed as a control (Fig. 1C). Genomic RNA (gRNA) and 326
subgenomic RNA (sgRNA) accumulated in TMV- and TMV*CP.MP- but not 327
TMV*rep-infected protoplasts at 12 and 24 hpi (Fig. 1D). 328
Next, we selected plant protoplasts and maximized virus transfection efficiency 329
to obtain a large amount of cells at the initial TMV replication stages for microarray 330
analysis. We optimized the TMV–Arabidopsis transfection efficiency by using the 331
TMV construct 30B-GFP to express GFP in cells (40). We achieved almost 70% TMV 332
transfection efficiency in repeated experiments (data not shown). 333
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Then, we determined the suitable timing for microarray analysis by conducting 334
time-course analyses of TMV*CP.MP accumulation. Viral gRNA and sgRNA were 335
visible at 8 hpi (data not shown). Therefore, the important virus–host interaction must 336
occur before 8 hpi in Arabidopsis protoplasts. 337
RNA was collected from TMV*CP.MP- and TMV*rep-infected protoplasts at 0.5, 338
4 and 6 hpi for microarray analysis. We used cDNA microarray (contains 10,452 339
genes) to screen for the relative transcriptome change of TMV*CP.MP- and 340
TMV*rep-inoculated samples at all 3 times, with 2 microarray analyses conducted at 341
each time (GEO accession no.: GSE45283). The microarray data were analyzed as 342
described (53), and the results for the 2 analyses at each time were highly correlated 343
(R2 = 0.90–0.96). 344
Initially, we looked for genes with consistent upregulation at all 3 times and 345
identified 12 genes (Table 1). The expression of the 12 genes was analyzed in at least 346
3 repeated experiments by qRT-PCR (data not shown). 347
TMV accumulation was reduced in At3g28770 mutant plants and 348
At3g22640-silenced protoplasts. From the SALK collection (http://signal.salk.edu/), 349
we found individual T-DNA insertion lines corresponding to 9 of the 12 genes. The 350
T-DNA insertion of each SALK line was confirmed by PCR (data not shown). We 351
obtained 8 homozygous mutant lines and 1 heterozygous mutant line (Table 1). We 352
inoculated TMV in the 9 Arabidopsis mutant lines, and examined TMV accumulation 353
by RT-PCR 20 days post-inoculation. TMV accumulation was greatly reduced in 354
SALK_022597 (At3g28770) mutant plants (Fig. 2A). 355
We were unable to find mutant lines corresponding to At2g34700, At3g22640 356
and At3g08670 (Table 1). Thus, we introduced in vitro-prepared dsRNAs (110 bp) 357
corresponding to the 3 genes into wild-type Arabidopsis protoplasts to induce RNA 358
interference (RNAi) before TMV infection. The selected dsRNA region (110 bp) was 359
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aligned with the TMV genome, and a search of the bl2seq database 360
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed no similar pairs. The expression of 361
each gene was analyzed (Fig. 2B). TMV accumulation was inhibited in protoplasts 362
pre-transfected with At3g22640-dsRNA treated protoplasts (Fig. 2C). 363
Detailed analysis of the role of At3g22640 in TMV accumulation. According 364
to the annotation, At3g28770 encodes a functional unknown protein, and At3g22640 365
encodes a vicilin-like seed-storage protein (PAP85) (35) (Table 1). Because previous 366
reports indicated that seed-storage protein is involved in membrane modification for 367
transportation (36-38), initially we focused on PAP85. The RNA level of PAP85 was 368
reduced to 38% of the buffer–pre-treated protoplast level in 369
PAP85-dsRNA–pre-treated protoplasts at 24 hpi (Fig. 3A), then TMV was inoculated 370
into these protoplasts. qRT-PCR revealed that PAP85-dsRNA–treated cells showed 371
knocked-down PAP85 level (Fig. 3B). TMV accumulation was reduced over time in 372
PAP85-dsRNA treated protoplasts but was detected in buffer- or GFP-dsRNA 373
pre-treated protoplasts as compared to buffer–pre-treated protoplasts (Fig. 3C). Cell 374
viability was similar among treatments as analyzed by fluorescein diacetate staining 375
(54) (data not shown). 376
To further validate the role of PAP85 in TMV replication in A. thaliana, we used 377
a 35S promoter to express PAP85 hairpin RNA and generate pap85-RNAi transgenic 378
lines (PAP85-knockout lines are not available in the SALK collection). We randomly 379
selected 24 homozygous T3 plants derived from 3 individual T1 plants (pap85-1, 380
pap85-2 and pap85-3). The mRNA level of PAP85 in these lines was maximally 381
reduced to 52% of the wild-type level (pap85-2), with no obvious phenotypes 382
observed (data not shown). 383
Because T-DNA insertion plants are not available for PAP85 and we could not 384
obtain strong PAP85-knockdown plants, adequate expression of PAP85 may be 385
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required for plant viability. However, we still used these plants for our inoculation test. 386
The mRNA level of PAP85 in plants was checked by qRT-PCR before TMV 387
inoculation, and the accumulation of TMV in inoculated and systemic leaves was 388
checked by qRT-PCR at 7 and 20 dpi, respectively (Fig. 4). Reduced TMV 389
accumulation was associated with PAP85 knockdown level in both TMV initially 390
inoculated and systemic leaves (Fig. 4). 391
Overexpression of PAP85 in pap85-RNAi Arabidopsis restored the 392
accumulation of TMV. To further validate the role of PAP85 in TMV accumulation 393
in Arabidopsis, we performed Agrobacterium-mediated transient overexpression 394
assays. We used the 35S promoter to transiently express PAP85::GFP 395
(pK2GW7-PAP85-GFP) or the same construct but introduced frameshift and stop 396
codons in PAP85 (pK2GW7-*PAP85-GFP) (Fig. 5A) in wild-type and pap85-RNAi 397
plants by Agrobacterium infiltration. The mRNA level of PAP85 in selected 398
pap85-RNAi plants was reduced to 64% of the wild-type level (pap85-2). 399
Agrobacterium carrying the pK2GW7 vector was used as a control. After 3 days, the 400
protein expression of PAP85::GFP was observed in 401
pK2GW7-PAP85-GFP–transfected plants (Fig. 5B). Similar mRNA levels of PAP85 402
were detected in pK2GW7-PAP85-GFP– and pK2GW7-*PAP85-GFP–transfected 403
wild-type or pap85-RNAi plants (Fig. 5B); however, PAP85::GFP was detected only 404
in pK2GW7-PAP85-GFP–transfected plants. 405
Next, we inoculated TMV transcripts in these leaves. The accumulation of TMV 406
was higher but not significantly in pK2GW7-PAP85-GFP– or 407
pK2GW7-*PAP85-GFP–transfected plants than in vector control 408
(pK2GW7)-transfected plants (Fig. 5C). TMV accumulation was lower in pK2GW7– 409
or pK2GW7-*PAP85-GFP–transfected pap85-RNAi lines than in vector 410
control-transfected wild-type Arabidopsis (p<0.05); however, in 411
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PAP85::GFP–overexpressed leaves, the accumulation of TMV recovered to a level 412
similar to that in vector control-transfected wild-type leaves (Fig. 5C). Thus, PAP85 413
protein but not mRNA is involved in TMV accumulation. 414
Expression pattern of PAP85 in Arabidopsis. We examined the expression 415
pattern of PAP85 in different tissues and developmental stages by use of 416
Genevestigator (https://www.genevestigator.com/gv/index.jsp) (Fig. 6A) and 417
qRT-PCR in Arabidopsis plants (Fig. 6B). PAP85 was mainly expressed in germinated 418
seeds and mature siliques. 419
ER morphology in TMV infected cells. To determine the role of PAP85 in 420
TMV infection, we used TMV-MP:mCherry to transfect the ER marker (yellow 421
fluorescent protein [YFP] fused with signal peptides targeting the ER derived from 422
the Arabidopsis gene AtWAK2 and the HDEL ER retention signal peptide) transgenic 423
Arabidopsis. Previous study revealed TMV VRCs easily observed in cells infected 424
with TMV when the TMV MP is fused with a green fluorescent protein (39). Thus, we 425
could observe the ER and TMV VRCs simultaneously. During the infection process in 426
Arabidopsis cells, the ER change in structure (Fig. 7A) was similar to that in 427
TMV-infected N. benthamiana (32): ER aggregations were formed initially (at 8-24 428
hpi) and returned to a filamentous structure after 36 hpi, and TMV VRCs were formed 429
and associated with ER aggregations at 8 to 24 hpi. 430
We delivered PAP85 dsRNA to ER-marker transgenic Arabidopsis cells, then 431
inoculated TMV-MP:mCherry to observe ER morphologic features and VRCs over 432
time. The ratio of cells showing VRCs was lower in PAP85-dsRNA–treated cells than 433
in untreated or At1g55940 dsRNA-treated cells (9% vs. 72% or 67%). As well, in 434
cells showing VRCs, the ER transition was observed in all examined untreated or 435
At1g55940 dsRNA-treated cells but not in 80% of PAP85-dsRNA–treated cells (Fig. 436
7A-C). Only PAP85-dsRNA–treated cells showed knocked-down PAP85 and reduced 437
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TMV levels (Fig. 7D,E). 438
ER structure with TMV P126 expressed alone or co-expressed with PAP85. 439
Previous reports indicated that TMV P126 alone can induce the VRC-like structure 440
(26-28); however, whether TMV P126 alone can induce the ER transition has not 441
been reported. To examine whether P126 alone can induce the ER transition, we 442
expressed P126 only in ER-marker transgenic Arabidopsis protoplasts and examined 443
the ER structure over time. We observed the VRC-like structure but not the ER 444
transition with P126 expression alone (Fig. 8A). 445
Our data indicated that PAP85 is involved in TMV-induced ER transition; 446
however, neither PAP85 nor TMV P126 alone could induce the ER transition (Fig. 447
8A-C). TMV P126 is the main protein expressed at the early stage of TMV infection 448
and PAP85 is the gene upregulated in this stage. We speculated that both TMV P126 449
and PAP85 are required for inducing the ER transition. Thus, we co-expressed 450
PAP85-GFP and TMV P126-mCherry (P126 fused with a red fluorescence protein) in 451
Arabidopsis protoplasts. In all examined cells, the ER filament/tubular-like structure 452
disappeared, and we observed ER aggregations similar to the ER transition at 12-24 453
hpi (Fig. 8D). 454
Induction of PAP85 expression in TMV-infected or TMV P126-expressed 455
cells. If PAP85 and TMV P126 are both required to induce ER aggregation similar to 456
the ER transition, TMV P126 expressed alone may not induce the ER transition 457
because its sole expression cannot induce the expression of PAP85. To test this 458
hypothesis, we examined the expression of PAP85 in mock, TMV-infected and TMV 459
P126-expressed protoplasts. We observed the induction of PAP85 only in 460
TMV-infected protoplasts (Fig. 9). 461
462
DISCUSSION 463
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Our knowledge of viruses helps in developing antiviral strategies and has led to 464
several landmark discoveries of cell biology (55-59). In this report, we identified a 465
host gene, PAP85, induced by TMV and involved in TMV accumulation. As well, our 466
data suggest that PAP85, together with TMV P126, is involved in the ER transition, 467
from a filamentous structure to aggregations and a return to a filamentous structure, a 468
phenomenon beginning early in TMV infection. Our studies help in understanding the 469
early event of TMV replication, provide an opportunity for developing antiviral 470
strategies. 471
Our data from knockdown and overexpression of PAP85 in TMV-infected cells 472
indicate that PAP85 is involved in TMV accumulation (Fig. 3-5). PAP85 encodes a 473
vicilin-like seed-storage protein (35). We searched for orthologous genes of PAP85 in 474
the database for Solanaceae (the primary host for TMV) (http://solgenomics.net/) (60). 475
The ortholog of PAP85 in Solanum spp. is SGN-U215172 and in Capsicum annuum 476
is SGN-U201542. PAP85 shares 34% and 39% protein sequence identity with 477
SGN-U215172 and SGN-U201542, respectively; however, the function of both genes 478
is currently unknown. The BLASTP best hit for PAP85 in GenBank, besides 479
Arabidopsis species, is another vicilin-like seed storage protein in Juglans nigra 480
(AAM54366.1). PAP85 and AAM54366.1 share 35% protein sequence identity. 481
However, the function of AAM54366.1 is also unknown. PAP85 contains 2 cupin 482
conserved domains; the BLASTP best hit for each cupin domain in GenBank, besides 483
Arabidopsis species, is the same protein in Prunus persica (EMJ26465.1). The 2 484
PAP85 cupin domains share 42% and 39% sequence identity with the 2 cupin 485
domains, respectively, of EMJ26465.1. The function of EMJ26465.1 has yet to be 486
identified. 487
Seed-storage proteins play roles as nutrient reservoirs. In Arabidopsis, the main 488
seed proteins are the 12S globulins and the 2S albumins, and PAP85 is similar to the 489
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12S globulins. Vicilin-like seed-storage proteins share striking similarities in 490
secondary and tertiary structure to the cupin protein superfamily of prokaryotic and 491
eukaryotic organisms (61). The cupin protein superfamily has many functions (62). 492
The characteristic cupin domain, the compact “jelly-roll” β-barrel, is thought to confer 493
a high degree of thermal stability and resistance to protease digestion. PAP85 has not 494
been extensively studied and is a silique development marker (35). PAP85 expresses 495
at a low level in most tissues and stages and abundantly in harvested seeds, embryos 496
and endosperm of developing seeds (63-65). Genome-wide analysis revealed the 497
upregulation of PAP85 in alpha-amanitin– and abscisic-acid–treated seeds and MINI 498
ZINC FINGER 1 protein-overexpressed seedlings and downregulation with knockout 499
of the abscisic-acid–upregulated receptor-like kinase RPK1 (63, 66-68). However, 500
current information about PAP85 does not help explain why PAP85 is involved in 501
TMV accumulation. 502
Seed storage proteins are reported to be translated in the ER, and the translated 503
protein is sorted to the Golgi body and then deposited in protein storage vacuoles or 504
results from protein accretion to form a protein body (PB) (36-38). Once a PB is 505
formed, seed proteins bypass the Golgi body and are transferred to the protein storage 506
vacuole or sometimes remain in cytoplasm (36-38). PBs originating from the tubular 507
ER and attaching to ribosomes were suggested to be ER bodies (36). The origin and 508
formation of the PB shares similarity to the ER changes observed in early TMV 509
infection (Fig. 7A). 510
In the tobamovirus genus, several host factors are involved in virus infection. 511
Tobacco eukaryotic elongation factor 1A; ARL8, a small GTP-binding protein; and 512
Hsp70 are components of a replication complex (69-72). Arabidopsis Rab GDP 513
dissociation inhibitor (GDI2) can interact with TMV P126 and may alter vesicle 514
trafficking to enhance the establishment of virus infection (73). In addition, 515
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Arabidopsis auxin/indole acetic acid proteins interact with TMV P126/183 replicase 516
and are involved in symptom development; the interaction may reprogram the auxin 517
response pathway to enhance virus infection (74-76). The Arabidopsis NAC-domain 518
transcription factor ATAF2 can interact with viral replicase and is involved in plant 519
defense responses against TMV (77, 78). Furthermore, a tobacco novel class II 520
KNOTTED1-like protein, NTH201, was found to assist viral cell-to-cell movement 521
and VRC formation in the early stage of TMV infection (79). TMV infection foci and 522
VRC (size and number) are altered in ATP-synthase gamma subunit (AtpC)- and 523
rubisco activase (RCA)-silenced leaves (80). However, all these host factors have not 524
been found to have direct effect on ER transition during tobamovirus replication. 525
Although the detailed mechanism of the role of PAP85 in TMV accumulation remains 526
elusive, our data show that 1) less ER transition in TMV-infected PAP85-knockdown 527
protoplasts (Fig. 7C) and 2) co-expression of PAP85 and TMV P126 but not either 528
protein alone can induce ER morphologic changes similar to the ER transition 529
induced by TMV infection, which supports PAP85 being involved in the ER 530
transition during TMV infection. Our data may also explain why TMV infection but 531
not TMV P126 overexpression induced the ER transition. We found increased levels 532
of PAP85 after Arabidopsis cells were infected with TMV but not with expression of 533
TMV P126 alone (Fig. 9). 534
Of note, our data seem to suggest that the VRC-like structure formation may be 535
independent of the ER transition because 1) in PAP85-knockdown cells, VRCs were 536
observed in cells without the ER transition and the VRC number was reduced but not 537
significantly (Fig. 7C); and 2) the expression of TMV P126 alone induced VRCs but 538
not the ER transition (Fig. 8A). However, small portions of ER aggregations (ER 539
transition) may be enough for TMV P126 to induce VRCs and we cannot rule out that 540
the ER transition is important for the formation of a VRC-like structure. 541
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A good plant host target for antiviral purposes should not have an essential 542
function in plants and likely remains dormant in most developmental stages. The 543
rationale is that if a gene is not necessary in most stages of plant growth but is 544
induced by virus infection, then designing strategies to prevent the gene induction by 545
viruses may prevent the virus from further infection without causing undue adverse 546
host effects. PAP85 may be needed for flower and seed development but is not 547
expressed in most growth stages (Fig. 6A,B). Thus, strategies such as using a 548
tissue-specific promoter to express dsRNA to prevent viruses inducing PAP85 may be 549
an effective antiviral strategy. 550
The formation of an ER body, including the PB, provides an alternative pathway 551
for protein transport (36). However, the biological and physiological processes 552
involved in this pathway remain largely unknown. Our data indicate that PAP85, 553
when expressed alone, is predominately localized to the ER and does not induce ER 554
aggregations. The expression of PAP85 modified the ER morphologic features only in 555
the presence of TMV P126. The induction of ER aggregations is protein specific 556
because abundant ER aggregations were not seen in all PAP85 and ER-marker 557
co-expressed cells (Fig. 8B). Thus, PAP85 has a role facilitating ER aggregations in a 558
protein-selective manner. However, its role in plant growth and development remains 559
to be further investigated. 560
561
ACKNOWLEDGEMENTS 562
We thank Shu-Jen Chou for technical support in microarray assay. This work was 563
supported in part by the DNA Microarray Core Laboratory, Institute of Plant and 564
Microbial Biology, Academia Sinica, Taiwan. Experiments and data analysis were 565
performed in part with use of the confocal microscope at the Scientific Instrument 566
Center of Academia Sinica and with the assistance of Shu-Chen Shen. We also thank 567
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Yi-Li Liu and I-Ching Huang for technical support in DNA sequencing. This work 568
was supported in part by the Department of Medical Research, National Taiwan 569
University Hospital. All authors have no conflicts of interest to declare. This research 570
was funded through grants from the National Science Council, Taiwan (no. 571
101-2321-B-002-048-). 572
573
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823
FIGURE LEGENDS: 824
FIG 1 Schematic representation of Tobacco mosaic virus (TMV) and derived 825
constructs and detection of virus accumulation. (A-C) Schematic representation of 826
TMV genomic RNA and specific mutants used in microarray analysis. Rectangles 827
represent open reading frames encoded by TMV genomic RNA. Wild-type TMV 828
encodes the 126-kDa and the read-through 183-kDa replicase proteins, the movement 829
protein (MP, 30 kDa) and coat protein (CP, 17.5 kDa). The mutated sequence in 830
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TMV*CP.MP and TMV*rep are indicated by red letters. (D) Detection of virus 831
accumulation by northern blot hybridization. Total RNA was purified from TMV- and 832
TMV-derived clone-infected protoplasts collected at 0.5, 12 and 24 hr 833
post-inoculation (hpi). Viral genomic RNA (gRNA) and CP subgenomic RNA 834
(sgRNA) are indicated. 835
836
FIG 2 Accumulation of TMV in Arabidopsis SALK mutant lines and double-stranded 837
RNA (dsRNA)-treated protoplasts. (A) TMV was inoculated in Arabidopsis mutant 838
lines. The Arabidopsis mutant lines from the SALK collection were characterized by 839
PCR to confirm their T-DNA insertion (data not shown). Total RNA was extracted 840
from systemic leaves at 20 days post-inoculation. TMV CP accumulation was 841
detected by RT-PCR. Ubiquitin 10 was used as a loading control. The lanes 1-9 842
represent SALK_149616C (1), SALK_131250 (2), SALK_136570C (3), 843
SALK_059297C (4), SALK_074253C (5), SALK_020627C (6), SALK_022597 (7), 844
SALK_115514C (8) and SALK_136572C (9) (corresponding to At5g18880, 845
At2g41550, At1g43910, At2g38970, At2g05250, At1g19900, At3g28770, At1g55940 846
and At5g12100, respectively). Wild-type (WT) Arabidopsis was used as a control. (B) 847
qRT-PCR analysis of mRNA levels of At2g34700, At3g22640 and At3g08670 848
relative to that in mock-inoculated protoplasts (set to 1). Protoplasts were treated with 849
buffer, GFP dsRNA and gene-specific dsRNA (designed from At2g34700, 850
At3g22640 and At3g08670). Total RNA was extracted at 24 hr post-inoculation (hpi). 851
Data are mean±SD from 3 experiments. (C) Arabidopsis thaliana protoplasts 852
pre-transfected with buffer (buf.) and dsRNA from GFP, At2g34700 (10), At3g22640 853
(11) and At3g08670 (12). After 24 hr, pre-treated protoplasts were transfected with 854
TMV, then cells were collected at 24 hpi. RT-PCR analysis of TMV CP level in cells. 855
Ubiquitin 10 was an internal control. 856
857
FIG 3 Accumulation of TMV in PAP85-dsRNA–treated Arabidopsis protoplasts. (A) 858
Arabidopsis thaliana protoplasts were transfected with buffer and dsRNA (from 859
PAP85 and GFP). Time course analysis of the quantity of PAP85 in Arabidopsis 860
protoplasts relative to that in mock-inoculated protoplasts at 0.5 hpi (set to 1) treated 861
with 10 µg dsRNA (derived from PAP85 and GFP). The protoplasts were collected at 862
0.5, 12 and 24 hpi. (B) After 24 hr, the pre-treated protoplasts were inoculated with 863
TMV, then collected at 0.5, 12 and 24 hpi. qRT-PCR analysis of PAP85 level in 864
TMV-infected protoplasts pre-treated with buffer, PAP85 and GFP dsRNA relative to 865
that in mock-inoculated protoplasts at 0.5 hpi (set to 1). (C) qRT-PCR analysis of 866
TMV level in A. thaliana protoplasts pre-transfected with buffer and dsRNA. The 867
RNA level of TMV in protoplasts pre-treated with buffer at 0.5 hpi was set to 1. Data 868
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are mean±SD from 3 individual experiments and were analyzed by Dunnett’s T test. *, 869
P < 0.05 compared with mock inoculation. 870
871
FIG 4 Accumulation of TMV in pap85-RNAi transgenic Arabidopsis. Eight T3 plants 872
(1-8) for each T1 plant (pap85-1, pap85-2 and pap85-3) were randomly selected for 873
TMV inoculation. qRT-PCR analysis of PAP85 and TMV accumulation relative to that 874
in WT plants (set to 1). Shows the expression of PAP85 before TMV inoculation 875
(PAP85) and TMV accumulation in inoculated (Ino.) and systemic leaves (Sys.) in 876
pap85-RNAi lines at 7 and 20 days post-inoculation (dpi), respectively. Data are 877
mean±SD from 3 repeated experiments. 878
879
FIG 5 Accumulation of TMV in PAP85-overexpressed pap85-RNAi transgenic 880
Arabidopsis. (A) Schematic representation of PAP85-GFP fusion protein. Rectangles 881
represent open reading frames encoding PAP85 and GFP. The mutated sequence in 882
PAP85 is indicated by red letters, and the created stop codon is indicated by italics. (B) 883
Western blot and RT-PCR analyses of PAP85 protein and mRNA levels in 884
PAP85-overexpressed leaves. Wild-type and pap85-RNAi (pap85) leaves were 885
transfected with Agrobacterium tumefaciens carrying pK2GW7, 886
pK2GW7-PAP85-GFP or pK2GW7-*PAP85-GFP. Total crude proteins and total RNA 887
were extracted from infiltrated leaves at 3 dpi. Monoclonal anti-GFP antibody was 888
used to detect GFP–tagged PAP85 protein. Loading control (Rubisco large subunit, 889
RbcL) for western blot and corresponding positions of marker proteins (size in kDa) 890
are indicated. UBQ10 was an internal control. (C) Binary vector-infiltrated 891
Arabidopsis leaves were inoculated with TMV. qRT-PCR analysis of TMV level in 892
inoculated leaves relative to that in wild-type leaves infiltrated with pK2GW7 (set to 893
1) at 7 dpi. Data are mean±SD from 3 individual experiments and were analyzed by 894
Dunnett’s T test. *, P < 0.05 compared with TMV-inoculated wild-type Arabidopsis 895
leaves pre-infiltrated with pK2GW7. 896
897
FIG 6 Expression analysis by in silico and qRT-PCR analysis at different plant 898
developmental stages. (A) In silico analysis of relative expression of PAP85 at 899
different developmental stages. Expression levels are shown as heat maps, with dark 900
red indicating maximal expression. Analysis involved use of Genevestigator. (B) 901
qRT-PCR analysis of the expression pattern of At3g22640 (PAP85) relative to that in 902
germinated seeds (set to 100%) at different developmental stages. Data are mean±SD 903
from 3 individual experiments. 904
905
FIG 7 Morphology of endoplasmic reticulum (ER) in PAP85-dsRNA pre-treated 906
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protoplasts infected with TMV-MP:mcherry. Analysis of protoplasts from ER-yellow 907
fluorescent protein (ER-YFP) transgenic Arabidopsis leaves pre-transfected with 908
buffer (A) or control dsRNA from At1g55940 (B) and PAP85 (C), then inoculated 909
with TMV-MP:mcherry and cells were collected at 0.5, 8-24 and 36 hpi. Scale bars 910
represent 10 µm. (D) qRT-PCR analysis of PAP85 level relative to that in 911
mock-inoculated protoplasts at 0.5 hpi (set to 1) and TMV-MP:mcherry-infected 912
protoplasts pre-treated with buffer, PAP85 or At1g55940 dsRNA at 0.5, 8-24 and 36 913
hpi. (E) qRT-PCR analysis of TMV-MP:mcherry level in A. thaliana protoplasts 914
pre-transfected with buffer and dsRNA. Total RNA was purified from protoplasts 915
(described in D). The mRNA level of TMV in buffer pre-treated protoplasts at 0.5 hpi 916
was set to 1. Data are expressed as mean±SD from 3 individual experiments. 917
918
FIG 8 ER morphology in P126-mcherry– and/or PAP85-GFP–expressed Arabidopsis 919
protoplasts. (A) Confocal microscopy of protoplasts from ER-YFP transgenic 920
Arabidopsis leaves inoculated with P126-mCherry (expressed by double 35S 921
promoter) at 0.5, 8-24 and 36 hpi. (B-D) Subcellular localization of PAP85 and TMV 922
P126 in Arabidopsis protoplasts. Transient expression (by double 35S promoter) of 923
GFP-tagged PAP85 (B) and mCherry-tagged P126 (C). (D) Transient co-expression of 924
GFP-tagged PAP85 and mCherry-tagged P126. Cells were examined by confocal 925
microscopy at 12-24 hpi. Scale bars represent 10 µm. 926
927
FIG 9 Relative expression of PAP85 in TMV P126-overexpressed or TMV-infected 928
Arabidopsis protoplasts. qRT-PCR analysis of expression of PAP85 in pCass2- vector, 929
pCass2-P126- and TMV-inoculated Arabidopsis protoplasts at 0.5 (A) and 24 hpi (B) 930
relative to that in mock-inoculated protoplasts (set to 1). Data are mean±SD from 3 931
individual experiments and were analyzed by Dunnett’s T test. *, P < 0.01 compared 932
with the mock. 933
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TABLE
Table 1. Genes induced in common by TMV*CP.MP at 0.5, 4 and 6 hours
post-infection
Locus no. Description Fold expression a
0.5 hr 4 hr 6 hr
(1) At5g18880 b
RNA-directed DNA polymerase-related family protein
(glucose transmembrane transporter activity)
3.45 3.89 2.93
(2) At2g34700 d
Pollen Ole e 1 allergen and extensin family protein 2.45 2.13 2.16
(3) At3g22640d PAP85 2.23 2.08 2.01
(4) At2g41550 b
Rho transcription termination factor 2.23 2.44 2.10
(5) At1g43910 b
P-loop containing nucleoside triphosphate
hydrolase-like protein, AAA-type ATPase
2.48 2.40 2.31
(6) At2g38970 b
Zinc finger (C3HC4-type RING finger) family protein
(ubiquitin-protein ligase activity)
2.36 2.30 2.24
(7) At2g05250 b
DNAJ heat shock N-terminal domain-containing
protein
2.12 2.05 2.33
(8) At1g19900 b
Glyoxal oxidase-related 2.54 2.05 2.23
(9) At3g28770c Unknown protein 2.53 2.35 2.10
(10) At1g55940b
Member of CYP708A, cytochrome P450 2.29 2.00 2.01
(11) At5g12100b
Pentatricopeptide (PPR) repeat-containing protein 2.11 2.04 2.09
(12) At3g08670d Hypothetical protein, related to oxidative stress 2.99 2.40 2.29
a Relative transcriptome ratio of TMV*CP.MP to TMV*rep in inoculated samples. b Arabidopsis homologous mutant is available. c Arabidopsis heterologous mutant is available. d Arabidopsis mutant is not available.
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419 2208 773 619 309 651 949 255 93
0% 100%
Percent of Expression Potential
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