Supplementary Material to:
Apple scar skin viroid naked RNA is actively transmitted by the whitefly Trialeurodes
vaporariorum
Yashika Walia, Sunny Dhir, Aijaz Asghar Zaidi and Vipin Hallan
1
Identification of cucumber phloem protein having ASSVd binding properties
Exudate sampling: 10-12 days old cucumber plants were cut below the cotyledons and the
exudate was collected and diluted 1/10 in a cooled reduction buffer (10mM Tris HCl [pH 8],
1mM EDTA, 10mM Dithiothreitol).1
Electro mobility shift assay (EMSA) of phloem proteins with ASSVd RNA transcripts:
Digoxigenin-labeled transcripts of ASSVd (5 ng) were incubated with serial dilutions of
cucumber phloem protein 2 (CsPP2) in binding buffer for 30 min on ice. Dilutions of the
cucumber phloem protein 2 made were 0, 100, 200, 400, 800, and 1600 ng and phloem
exudate dilutions prepared were 1/1600, 1/800, 1/400, 1/200, 1/100, 1/50 and 1/25. 5 µl of
diluted phloem exudate and CsPP2 were mixed with 5 ng (1µl) of RNA probe in Binding
buffer (10 mM Tris HCl [pH 8.0], 100 mM NaCl and 50% glycerol) to a total volume of 15
µl. The reaction mixture was incubated on ice for 30 minutes and then loaded on a 1%
agarose gel. The gel was blotted overnight onto nylon membrane and chemiluminescent
detection was performed using CDP star (Ambion, USA) as per manufacturer’s instructions.
Northwestern assays of phloem proteins with ASSVd RNA transcripts: The phloem exudate
was run on 12% SDS PAGE in duplicate. One gel was coomassie stained whereas the other
was electro-blotted onto nitrocellulose membrane. Northwestern assays were performed as
described previously.2 Membranes were incubated overnight in RN Buffer (10 mM Tris–HCl,
pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.05% Triton X-100, 1x Denhardt’s reagent) at 4ºC
temperature followed by a 3h incubation in RN buffer in the presence of the corresponding
digoxigenin-labeled RNA (25–50 ng/ml). The detection of RNA–protein complex was
performed as described before. A duplicate polyacrylamide gel was Coomassie blue-stained
and used as a control.
Cloning,
expression
and
purification
of
CsPP2:
5’ATGGTTTGCATAGAGACAGAA3’
The
and
primers
CsPP2f:
CsPP2r:
5’TTAGCATCCGCAACTGGATTGT3’ were used for amplifying the mRNA corresponding
to phloem protein 2 from cucumber plant.1 Primers were synthesized (PP2expcuch:
5’ACGGGATCCATGGTTTGCATAGAGACAGAA3’
and
PP2expcucc
5’GCGTGCGGCCGCGCGTGGATCCTTAGCAGC3’) for cloning the amplified PP2 gene
into the expression vector pET32a (Novagen).
2
The recombinant pET32a-PP2 construct was induced by addition of 1mM IPTG at
37°C for 4 hours and analysed by 12% SDS-PAGE. Two bands near the desired induced
protein were observed in the inclusion bodies. The induced protein was purified using the
protein purification kit (Novagen) as per manufacturer’s instructions and refolded to the
native structure using the protein refolding kit (Novagen). The purified protein was blotted
and detected using the anti-His antibody. Six consecutive injections of 500 μl (1 mg/ml, each)
purified phloem protein 2 were injected into a healthy white New Zealander male rabbits
(approximately four months old) weekly, with Freund’s complete/incomplete adjuvant
(Genei).
Immunoprecipitation and reverse transcription RT-PCR: Four weeks after inoculation with
ASSVd transcripts, phloem exudates (250 µl) were collected from excised petioles of healthy
and infected cucumber plants and diluted 1:5 in reduction buffer. Immunoprecipitation was
carried according to the MagnaRIP kit (Millipore). RNA was isolated using phenolchloroform extraction protocol and RT-PCR was performed using viroid-specific primers
ASSVdich (5’-GTCGACGAAGGCCGGTGAGAA-3’) residues 87-106 and ASSVdicc
(GTCGACGACGACAGGTGAGTT) complementary to ASSVd residues 87-67 (Walia et al;
2013). RT-PCR products were analyzed on 2% agarose gel.
Northwestern hybridization of purified PP2 with ASSVd RNA: The purified cucumber PP2
(100 ng) was run in triplicate on 12% SDS-PAGE. One of the gels was coomassie stained,
whereas the others were electroblotted on nitrocellulose membrane. One blot was hybridized
with DIG- labled ASSVd probe for Northwestern hybridization and another hybridized with
PP2 antisera for western hybridization. The northwestern hybridization and western
hybridizations were done using the protocol as described previously.
3
Supplementary figures
1
2
M
3
4
ASSVd
Ubiquitin
ligase
5
6
M
300 bp
200 bp
100bp
Figure S1. RT-PCR from ASSVd infected and healthy cucumber plants using viroid-specific
primers showing amplifications of ASSVd and ubiquitin ligase from infected cucumber (lane
1 and 2). Ubiquitin ligase was amplified from healthy cucumber (lane 3 and 4). Lane 5 and 6
shows ASSVd specific amplification from ASSVd cloned in a vector used as positive control.
Lane M is low range ladder (Thermo Scientific).
4
1
2
3
M
300bp
200bp
100bp
ASSVd
Ubiquitin
Ligase
Figure S2 Nucleic acids were recovered from the immunoprecipitate, subjected to RT-PCR with
ASSVd-specific primers, and analyzed on 2% agarose gel, Lane 1: RT-PCR from RNA isolated
from the ASSVd infected cucumber plant immunoprecipitate; Lane 2: RT-PCR of the RNA
isolated from healthy cucumber plant immunoprecipitate, Lane 3: PCR from total RNA isolated
Table E1. Primers used in this study
from ASSVd infected cucumber plant and Lane M: 100bp marker (Thermo Scientific)
5
1 2 3
4
5
6
1
2
3
4
5
1
6
2
3
4
5
6
Bound RNA
Bound RNA
Bound RNA
Free RNA
Free RNA
A
Free RNA
C
B
Figure S3 (a) Elector mobility shift assay (EMSA) of ASSVd RNA transcripts with the cucumber
phloem exudates at dilution 1/800, 1/400, 1/200, 1/100, 1/50 (lane 2- 6) and lane 1 free RNA
transcript, on 1% agarose gel ; (b) Autoradiograph of the gel showing EMSA of DIG-labelled
ASSVd RNA transcript with cucumber phloem exudates at dilution 1/800, 1/400, 1/200, 1/100,
1/50 and lane 1 free RNA transcript; (c) Autoradiograph showing binding of ASSVd RNA with
cucumber phloem exudates (1/50 dilutions) at 1, 0.9, 0.8, 0.7, 0.6, 0.5 M NaCl (lanes 1–6). The
complex was stable up to 0.7 M NaCl.
6
1
M
PP2
W
26 kda
PP2 on western blot
A
Figure S4: Hybridization of the ASSVd RNA to the phloem exudate protein of 26kda corresponding to
the PP2 protein. A: lane 1- Coomassie stained phloem exudate proteins; lane M- unstained protein
marker (Thermo Scientific). W: Autoradiograph of the Northwestern hybridization of the phloem
proteins with ASSVd RNA.
7
1
1
M
Purified
PP2
A
1
M
M
Purified PP2
B
Smaller protein binding
with ASSVd
C
Figure S5: Northwestern hybridization of the cucumber recombinant purified phloem protein 2 with
the ASSVd DIG-labelled transcripts. A: Coomassie stained purified CsPP2 in pET 32a vector; B:
Western hybridization of the purified protein CsPP2 antibody and C: Northwestern hybridization of the
purified CsPP2 with ASSVd transcripts (lane 1 - CsPP2 purified and M- Prestained protein ladder.
Note: The purified cucumber phloem protein 2 exists as two proteins of different molecular weights
and the protein with lower molecular weight has ASSVd RNA binding properties.
8
Coomassie stained
phloem exudate proteins
Western blot
CsPP2
A
CsPP2
B
Figure S6 Co-immonoprecipitation of CsPP2 from phloem proteins using CsPP2 antibody (A)
coomassie stained SDS-PAGE gel showing cucumber phloem proteins and (B) western blot of
immunoprecpitate with CsPP2 antibody showing the band corresponding to CsPP2.
9
1
2
3
M
ASSVd
Figure S7: RT-PCR performed from immunoprecipitate of in vitro bound ASSVd and TLCV
AV2 protein (lane 1), ASPV CP (lane 2) and CsPP2 (lane 3). Lane M: 100bp ladder (Thermo
Scientific). Viroid-specific amplification was obtained in case of CsPP2 only (lane 3) which
suggests specificity of the binding of CsPP2 to the viroid RNA. The methodology is same as
indicated on page 3 of this file.
10
S.No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Sample name
Cucumber
Cucumber
Cucumber
Cucumber
Cucumber
Bean
Bean
Bean
Bean
Bean
Tomato
Tomato
Tomato
Tomato
Tomato
Pea
Pea
Pea
Pea
Pea
Healthy bean
Healthy Cucumis
Healthy TV
Infected TV
Infected Cucumis
Infected bean
Infected tomato
Infected Pea 1
Infected pea 2
Plasmid with ASSVd insert
Plasmid with ASSVd insert
Plasmid with ASSVd insert
IDV* value
233
393
551
966
1025
658
560
829
890
800
800
800
460
630
530
580
310
498
284
284
409
403
389
961
453
1034
1204
1152
453
1077
1204
1800
Result
-ve
-ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
-ve
+ve
-ve
-ve
-ve Control value
-ve Control value
-ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
Table S1 Northern blot intensity data
*Integrated density value (IDV) = Σ (each pixel value − background)
IDV value for the negative control = 403 and 409
No. of plants infected through viroidiferous whitefly having IDV value above the threshold
value = 15
Total no. of plants which were fed by viroidiferous whitefly = 20
Percentage of plants infected = 15/20 (75%)
11
References
1. Gomez G, Pallas V. A long-distance translocatable phloem protein from cucumber forms a
ribonucleoprotein complex in vivo with Hop stunt viroid RNA. J. Virol. 2004; 78(18): 1010410110.
2. Marcos JF, Vilar M, Perez-Paya E, Pallas V. In vivo detection, RNA-binding properties
and characterization of the RNA-binding domain of the p7 putative movement protein
from Carnation mottle carmovirus (CarMV). Virology 1999; 255: 354–365.
12