siRNA Silencing of PVX Coat Protein Gene Affects Accumulation of Viral RNA in Potato and Tobacco Plants
This study, aims at determination of efficiency of micro interfering
RNA (miRNA) to develop ability of virus resistance against Egyptian PVX
isolate (PVX-Eg2) in both potato (Solanum tuberosum L. cv. Spunta)
and tobacco (Nicotiana benthamiana). RNA constructs of Sense (PVX-Eg2cpVs),
antisense (PVX-Eg2cpCs) and sense/antisense were designed, cloned and
sub- cloned for gene transfection using Agrobacterium inoculation
technique. Two to 3 leaf-stage seedlings of potato (Solanum tuberosum
L. cv. Spunta) and tobacco (Nicotiana benthamiana) were
inoculated with the three previous constructs. The construct-treated plants
were mechanically inoculated with the PVX-Eg2 isolate. Bioassay and PCR
amplification have been able to evaluate transfected-plant resistance
against PVX-Eg2 that is caused by siRNA of PVX-Eg2cp. PCR amplification
has been able to detect PVX viral genome in all challenged plants those
infiltrated with either pFGC5491 vector without insert, or with sense
construct and also with antisense construct. Bioassay has confirmed same
previous statement. Nine out of 10 sense/antisense-transfected potato
plants were negatively reacted with both bioassay and PCR amplification.
Same negative reaction has been viewed using both bioassay and PCR for
sense/antisense transfected-tobacco plants. Seven out of 10 proved they
are PVX-Eg2 resistant.
Potato (Solanum tuberosum L.) is one of the most important
economic crops in Egypt concerning the production, farm income and exportation.
It is used for human consumption, animal feed and as a source of starch
and alcohol for bio fuels (Horton, 1992). It is affected by more than
20 different viral diseases (Smith, 1931). Potato X potxvirus (PVX)
was first recognized on Solanum tuberosum in UK (Smith, 1931).
The most important viruses affecting potato crop in Egypt are Potato
X potxvirus (PVX), Potato Y potyvirus (PVY) and Potato leaf
roll luteovirus (PLRV). These viruses have been shown to cause great
economic losses and considered to be the major limiting factors of potato
production (El-Amrety, 1970). Potato virus X can be found wherever potatoes
are grown in Egypt (El-Amrety, 1970). The yield reduction, caused by this
virus, is usually less severe unless PVX occurs together with other viruses
(Salazar, 1996). PVX, which has long history in Egypt, was isolated and
studied by El-Amrety (1970), Fahmy and Mohamed (1984), Yousef (1992),
Abdel-Salam et al. (1994) and Soliman et al. (2000).
PVX-Eg2 isolate was isolated from diseased potato plants in 2006. PVX-like
symptoms i.e. mild mosaic, mottling and stunting were shown. Isolated
virus was maintained on Nicotiana tabaccum (L.
cv. White Burley) in Department of Plant Virus and Phytoplasma Research,
Plant Pathology Research Institute, ARC (Soliman et al., 2006).
Nucleotide sequence of a selected fragment of the coat protein gene of
the previous isolate was submitted in the GeneBank under the name of PVX
(PVX-Eg2) and Accession No. DQ412730 (Soliman et al., 2006).
PVX has a positive-sense genomic RNA consists of five open reading frames
(ORFs) numbered 1 to 5. Twenty nucleotides (59-to-39 direction encode
polypeptides of 166 kDa (viral replicase), 24 kDa (p24), 12 kDa (p12),
8 kDa (p8) and 25 kDa viral coat protein) (Orman et al., 1990).
Post-transcriptional gene silencing (PTGS) is a universal mechanism by
which plants are able to switch off the expression of target genes via
the reduction of steady state levels of specific RNAs. Double-stranded
RNA (dsRNA) can silence genes by developing degradation of homologous
RNA in the cytoplasm and by directing methylation of homologous nuclear
DNA sequences (Bass, 2000; Hammond et al., 2001; Voinnet, 2001;
Waterhouse et al., 2001).
MATERIALS AND METHODS
Design and Synthesis of Oligonucleotide Primers Total RNA was isolated from the PVX-Eg2-infected potato plants,
maintained at Dept. of Plant Virus and Phytoplasma Research, Plant Pathology
Research Institute, ARC, Egypt in 2002, using RNeasyÂ® Plant
Mini Kit obtained from QIAGEN according to manufacturer`s instructions.
Pair of primers specific for a part of the coat protein gene of PVX (selected
fragment) was designed according to available sequences from the GenBank.
The forward primer (PVXcpVs) with XbaI and AscI restriction
enzyme sites (underlined) is as follow: 5` TCTAGA GGCGCGCC AGTGGTATGGAACTGGATGC
3`. The complementary primer (PVXcpCs) with BamHI and SwaI
restriction enzyme sites (underlined) is as follow: 5`GGATCC ATTTAAAT
TCGTTGGATTGTGCCCTGGC 3`. All the restriction sites were added at 5` of
the designed primers to facilitate the cloning of the selected domains
in the binary vector. The expected size of the RT-PCR product is 259 bp.
Total RNA extracted from PVX-Eg2-infected potato plants was used as template
for each RT-PCR amplification reactions using one-step RT-PCR reaction
kit as instructed from QIAGENTM. PCR-amplified fragments were
separated by gel electrophoresis using 1.5% agarose gels in 0.5X TBE buffer,
stained with ethidium bromide and visualized with UV illumination using
Gel Documentation System.
Cloning and Sequencing of RT-PCR Product (Selected Fragment) RT-PCR product was ligated into PinPointâ„¢ Xa-1 T-Vector (PinPointâ„¢
Xa-1 T-Vector System I, PROMEGA). The recombinant plasmids were introduced
into E. coli strain DH5Î± as described by manufacturer`s instructions.
DNA was prepared from selected white colonies, digested with BamHI
and fractionated on agarose gels using 1 kb DNA ladder (Promega). The
nucleotide sequence of clones having a 259 bp insert were selected for
dideoxy sequencing [ABI PRISMTM Dye Terminal Cycle Sequencing Ready Reaction
Kit with AmpliTaq DNA Polymerase, FS (Biotechnology Department University
of Wisconsin, Madison, USA) and performed on 377 XL automated DNA Sequencer
(Applied Biosystem Co.). The nucleotide sequence was compared and analyzed
using DNAMAN Sequence Analysis Software (Lynnon BioSoft. Quebec, Canada)
with those of PVX isolates available in GenBank.
Sub-Cloning of the Selected Fragment into Binary Vector The selected fragment was cloned into the binary vector pFGC5941
(Fig. 1) (supplied from the University of Arizona, USA)
in both orientations once by the SwaI and AscI to give the
orientation and another time with BamHI and XbaI to give
the antisense orientation leaving the Chalcon Synthase (ChaS)
intron in the middle.
Preparation of Sense, Antisense and Sense/Anti-Sense Constructs Prepare the sense construct, 40 Î¼L of DNA plasmid (pPVXcpGS)
were digested using 50 U of SwaI (FERMENTAS), 10 Î¼L of 10X
buffer O and dH2O was added to final volume of 100 Î¼L.
The reaction tube was incubated at 30Â°C in water bath for 4 h then
50 U of AscI (FERMENTAS) and 10 Î¼L of 10 X buffer Tangoâ„¢
were added to the reaction tube and incubated at 37Â°C for 4 h. Prepare
the antisense construct, 40 Î¼L of DNA plasmid (pPVXcpGS) were digested
using 50 U of BamHI (PROMEGA), 10 Î¼L of 10 X buffer E, 5 Î¼L
of BSA (100 Î¼g Î¼L-1), 50 U of XbaI, 10 Î¼L
of 10 X buffer D and dH2O was added to final volume of 100
Î¼L. The reaction tube was incubated at 37Â°C in water bath for
4 h. Prepare the sense/antisense construct, 50 Î¼L of DNA plasmid
(pPVXcpGS) were digested using 50 U of each of BamHI and
XbaI enzymes, 10 Î¼L of multicore buffer (PROMEGA), 5 Î¼L
of BSA (100 Î¼g Î¼L-1) were added and dH2O
was added to final volume of 100 Î¼L. The reaction tube was incubated
at 37Â°C in water bath for 4 h. Prepared DNA was electrophoresed in
2% agarose gel prepared in 0.5% TBE buffer, stained with ethidium bromide
and visualized by using UV transilluminator. The digested band was cut
out with a clean sharp scalpel for gene cleaning. The binary vector pFGC5941
was digested with same two restriction enzymes in each case. One hundred
nanogram of digested DNA vector were added to a clean tube and then 17
ng of the digested DNA of selected fragment were added (binary vector
and selected fragment were digested with the same restriction enzymes).
One microliter of 10 X ligase buffer and 1 U of T4 DNA ligase
were added to the ligation mixture. Nuclease-free water was added to the
ligation mixture to final volume of 10 Î¼L, the mixture was incubated
at 4Â°C for overnight.
Transformation of the Constructs into E. coli Competent Cells Competent cells E. coli (strain DH5Î±) were thawed on
ice for 30 min and then 100 Î¼L transferred to a pre-chilled 5 mL
tube and 5 Î¼L of ligated DNA added and incubated on ice for another
30 min. Cells were heat-shocked for 2 min at 42Â°C by immersing the
tube into a water bath for 2 min and then incubated on ice for 2 min.
One milliliter of LB medium was added and cells grown for 1 h at 37Â°C
in shaking water bath. Transformed cells were plated on the surface of
prepared plates (LB solid medium with 50 Î¼g mL-1 kanamycin)
for overnight at 37Â°C and bacterial colonies containing recombinant
plasmids were selected for further characterization. The enzyme digestion
for the DNA minipreps of the selected colonies of constructs (psPVXcpGS
[sense], pasPVXcpGS [anti-sense], ps/asPVXcpGS [sense/anti-sense]) were
carried out using the restriction enzymes SwaI and AscI
(sense), BamHI and XbaI (anti-sense) and AscI and
Transformation into Agrobacterium tumefaciensLBA4404 One microgram of plasmid DNA was added to the cells. Freezing the
cells in liquid nitrogen was preformed. The cells were thawed by incubating
the tubes in a 37Â°C water bath for 5 min. One milliliter of YEP medium
was added to the tube and incubated at 28Â°C for 2-4 h with gentle
shaking. This period allowed the bacteria to express the antibiotic resistance
genes. The tubes were centrifuged for 30 sec in a table centrifuge. The
supernatant solution was discarded and the cells were resuspended in 0.1
mL YEP medium. The cells were spread on a YEP agar plate containing 50
Î¼g mL-1 kanamycin and 50 Î¼g mL-1 streptomycin.
The plate was incubated at 28Â°C. Transformed colonies should appear
in 2-3 days.
Gene Delivery Using the Syringe-Spotting Technique After Agrobacterium tumefaciens LBA4404 has been transformed
with vectors harboring different constructs using chemical transformation,
5 mL culture was grown overnight at 28Â°C in the appropriate antibiotic
selection medium. The next day, the culture was inoculated into a 50 mL
LB medium containing the selective antibiotics. The culture was grown
overnight in a 28Â°C shaker. Agrobacterium cells were harvested
and resuspended in infiltration media [10 mM MgCl2, 10 mM MES
(2-N-morpholino ethane sulfonic acid) and 20 Î¼M acetosyringone (4`-Hydroxy-3,5-dimethoxyacetophenone)],
adjusted to OD600 of 2.0 and left at room temperature for 3
h. Potato (Solanum tuberosum L. cv. Spunta) and tobacco
(Nicotiana benthamiana) plants (10 plants in each treatment) in
2-3 leaf-stage seedlings were infiltrated with Agrobacterium with
the constructs psPVXcpGS, pasPVXcpGS, ps/asPVXcpGS separately and pFGC5941
(without construct as a control) using syringe without needle 5 mL syringe
(Syringe-Spotting Technique (SST) developed by Johansen and Carrington
Constructs Used for Plant Transformation The following vectors and constructs were used in this study (Fig.
||Construct A (binary vector pFGC5941 without any insert).
This construct was used as negative control.
||Construct B (binary vector pFGC5941 with the sense orientation of
the silencing trigger sequence).
||Construct C (binary vector pFGC5941 with the antisense orientation
of the silencing trigger sequence).
||Construct D (binary vector pFGC5941 with sense/antisense orientation
of the silencing trigger sequence separated by the ChaS intron
to create the dsRNA inside the host cells).
Inoculation with PVX-Eg2 Potato and N. benthamiana plants previously transformed with
different constructs were infected with Egyptian isolate of PVX-Eg2 using
mechanical transmission as follows: sap from infected plants was extracted
from young potato leaves with neutral or weakly basic 0.05 M phosphate
Constructs used to transform potato
and N. tabaccum plants. 1: a plant binary vector, 2: 35S
with sense orientation of silencing construct, 3: 35S with antisense
orientation of silencing construct and 4: 35S with sense/antisense
orientations of silencing construct
(pH 7.2). The sap was clarified through two layers of cheesecloth and
the plants were dusted with carborundum. The crude sap was used as an
inoculum into 2-3 leaf-stage healthy seedlings. All inoculated plants
were kept under controlled conditions in the growth chamber with 16 h
of light and 8 h of darkness and at 25Â°C.
Evaluation of the Resistance Developed Against PVX-Eg2 Screening
Using Viral Symptoms
Resistance developed against PVX-Eg2, in both potato and N. benthamiana
plants those were transformed with different constructs and inoculated
with the Egyptian isolate of PVX-Eg2, was evaluated by daily observation
for the development of virus symptoms.
Screening with RT-PCR Total RNA was used as template for each RT-PCR amplification reactions
using QIAGENTM One-Step RT-PCR Kit. PVX-Eg2cp gene was detected
by PCR using primers for CP gene (Shalaby et al., 2002). Forward
primer (PVXCPvEcoRI) sequence was: 5`-GATAGAATTCAGAT GACTACACCAGCCAACACC-3',
with EcoRI restriction enzyme site (underlined) at the 5` end and
the complementary primer (PVXCPcNcoI) sequence was: 5`-TACGCGTCGGTTCCATGGACGT
AGTTATGGTGG-3`, with NcoI restriction enzyme site (underlined)
at the 5` end. PCR-amplified fragments were separated by gel electrophoresis
using 1% agarose gels in 0.5 X TBE buffer, stained with ethidium bromide
and visualized with UV illumination using Gel Documentation System. The
expected size of the PCR product was 750 bp.
Amplification of the Selected Fragment RT-PCR amplification of viral RNA was carried out on the total RNA
isolated from PVY-Eg2-infected plants using specific primers designed
to amplify the selected fragment of the coat protein gene of PVX-Eg2cp.
Electrophoresis analysis of RT-PCR product of the selected fragment showed
a single amplified fragment of 259 bp while no fragment was amplified
from potato healthy plant (Fig. 3).
Enzyme Digestion, Cleaning and Cloning of the Selected Fragment into
PinPointâ„¢ Xa-1 T-Vector Several white colonies resistant to kanamycin were selected to test
for recombinant plasmids containing the selected fragments. Enzyme digestion
for PinPointâ„¢ Xa-1 T-Vector carrying the DNA
||Agarose gel electrophoresis analysis of RT-PCR amplified
products M: 100 bp DNA ladder (Gibco BRL); L1: total RNA extract from
potato leaf sample infected with PVX-Eg2, L2: healthy potato sample
||Enzyme digestion of the recombinant plasmids from the
cloning of the selected fragment. M: 1 Kb DNA ladder (PROMEGA); L1
and L2: DNA minipreps digested with BamHI
minipreps of the selected colonies of the three constructs i.e., (psPVXcpGS)
for sense and (pasPVXcpGS) for anti-sense and (ps/asPVXcpGS) for sense/anti-sense
was done using restriction enzymes SwaI and AscI for having
sense orientation and BamHI and XbaI for having anti-sense
orientation as well as AscI and XbaI for having sense/anti-sense
orientation. Cleaning was done, for the cut band using sharp scalpel,
using QIAEX II Agarose Gel Extraction Protocol. It is shown in Fig.
4 (lower bands with size 259 bp).
Nucleotide Sequence Analysis Nucleotide sequencing of the RT-PCR product in the recombinant plasmid
for the selected fragment of PVX-Eg2cp was completed to confirm PCR-produced
fragment is belonged to Potexvirus
Nucleotide sequence of the selected
fragment of the coat protein gene of the Egyptian isolate of PVX
(PVX-Eg2) and the predicted amino acid
PVX symptoms appeared on tobacco
(a) and potato (c) plants treated with sense or antisense constructs
after challenging with PVX-Eg2 compared with no symptoms on tobacco
and potato plants those were treated with sense/antisense construct
(b and d, respectively)
group sequence available at GeneBank. Nucleotide sequence is shown in
Fig. 5. Sequence of PVX-Eg2cp fragment was submitted
in the GenBank under Accession No. DQ412730.
Sub-Cloning of the Selected Fragment into Binary Vector The selected fragment was sub-cloned into the binary vector pFGC5941
in both orientations (sense and antisense) once by the SwaI and
AscI to direct the sense orientation and another time with BamHI
and XbaI to direct the antisense orientation leaving the Chalcon
Synthase (ChaS) Intron. The Intron (ChasS) was left in the
middle of the third construct (sense/antisense).
Evaluation of the Resistance Developed Against PVX-Eg2 Screening Using
Bioassay Typical PVX symptoms were appeared in all positive control (non-infiltrated)
plants three weeks after PVX-Eg2 inoculation. Symptoms were also recorded
on all of infiltrated plants with the following: a) pFGC5491 vector without
any insert, b) sense construct, c) antisense construct. Agro-infiltrated
plants with sense/antisense construct did not produce viral symptoms (Fig
PCR amplification for the potato
(a) and tobacco (b) plants agro-infiltrated with sense construct.
M: 1 Kb DNA ladder (PROMEGA). Sense: 10 extracts of potato plants
agro-infiltrated with sense construct, pFGC: pFGC5491 vector without
insert, I: PVX-Eg2-infected plant, C+: positive control, H: healthy
plant (negative control)
Screening Using RT-PCR PCR was carried out, as more sensitive test, to confirm previous
bioassay results. Figure 7a and 8a show
positive amplification of the expected size (750 bp) fragment that was
obtained with the following: a) non-infiltrated potato plants, b) infiltrated
potato plants with (b1) pFGC5491 vector without any insert, (b2) sense
construct indicating the presence of viral genome in challenged plants.
Figure 7b and 8b show same positive
amplification of the expressed size (750 bp) fragment that was obtained
with the following: a) non-infiltrated tobacco plants, b) infiltered tobacco
plants with (b1) pFGC5491 vector without any insert, (b2) antisense construct;
indicating the presence of viral genome.
Infiltrated tobacco and potato plants with sense/antisense construct
gave negative amplification indicating the absence of the viral genome
in 9 out of 10 potato plants and in 7 out of 10 tobacco plants (Fig.
Twenty one to twenty three nt RNA known as siRNA is a key component
of gene silencing (Voinnet, 2002; Llave et al., 2002). This study
has used 21 nt as siRNA. One conserved, un-translated sequence from the
coat protein gene of PVX-Eg2 (PVX-Eg2cp) was selected to develop resistance
against PVX-Eg2. Successful application of gene silencing technique to
develop resistance against potato virus X (PVX-Eg2) is shown in Fig
6 and 9. Previous studies on virus resistance by
post-transcriptional gene silencing depended highly on parts of one gene
to switch off or eliminate the viral replication in the host plants (Beachy,
1997). PTGS with the subsequent production of virus-derived siRNAs in
the case of potato virus X-infected plants was published by Hamilton and
Baulcombe (1999). This induced defense is characterized by sequence specific
resistance against virus infection
PCR amplification for the potato
(a) and tobacco (b) plants agro-infiltrated with antisense construct.
M: 1 Kb DNA ladder (PROMEGA), Antisense: 10 potato plants agro-infiltrated
with antisense construct, pFGC: pFGC5491 vector without any insert,
I: PVX infected plant, C+: positive control, H: healthy plant (negative
(Ratcliff et al., 1999). PTGS using anti-sense or co-suppression
constructs usually results in only a modest proportion of silenced individuals
(Wesley et al., 2001). This study showed the potential of sense/antisense
construct encoding self complementary hairpin RNA (hpRNA) to efficiently
silence PVX-Eg2 genes. Inclusion of an intron in these constructs (Fig.
3) has a consistently enhanced effect. This technique is supported
by Wesley et al. (2001) who explained that Intron-containing constructs
(ihpRNA) generally gives 90-100% of independent transgenic plants showing
silencing. Agrobacterium tumefaciens-mediated transient expression
system, which was used in this study, is a versatile tool to rapidly introduce
genes into plant tissue. This system has also been used effectively by
Johansen and Carrington (2001) as a mean to deliver RNA silencing inducers
and suppressors into transgenic plants that express a silencing reporter
gene. Moreover, results of this study has been approved by Feng et
al. (2003) who explained that modified PVX-CP gene and wild-type CP
gene, those were inserted into binary vector under the control of a promoter
and these constructs were transferred into tobacco genomes via Agrobacterium
mediated method, have been analyzed using Northern blot of RNA isolated
from these plants showed that the silencing ratio of the modified gene
in transgenic tobaccos was higher than that of the wild gene. Syringe-Spotting
Technique that was used in this study has been efficient to deliver the
different constructs might be due to the fact that small leaf tissues
of newly developed seedlings posses a large amount of meristematic cells,
which provide the best host-cells for both the bacterium to attached and
the DNA to express this was supposed by Eckardt (2002). Moreover, amplicon-mediated
gene silencing provides an important new strategy for the consistent activation
of gene silencing in transgenic plants (Angell and Baulcombe, 1997). Transfection
of siRNA is commonly transported through the phloem (Ruiz-Medrano et
al., 2001; Lucas et al., 2001) and that regulation of RNA trafficking
plays an important role in resistance development and to its role in
PCR amplification for the potato
(a) and tobacco (b) plants agro-infiltrated with sense/antisense
construct. M: 1 Kb DNA ladder (PROMEGA), Sense/Antisense: 10 potato
plants agro-infiltrated with sense/antisense construct, pFGC: pFGC5491
vector without any insert, I: PVX infected plant, C+: positive control,
H: healthy plant (negative control)
PTGS (Vance and Vaucheret, 2001; Haywood et al., 2002). Potato
and N. benthamiana plants transformed with the empty vector and
later inoculated with PVX developed diseases symptoms 2-3 weeks after
inoculation (Fig. 6-8) indicating that the binary vector
alone was unable to develop resistance against PVX. Similarly, all potato
and N. benthamiana plants transformed with sense construct (Fig.
7) were susceptible to PVX-Eg2 infection indicating that mRNA resulted
from the transcription of the sense silencing did not code for any protein
and did not anneal to the viral mRNA during the viral infection cycle.
Also, both potato and tobacco plants those were transformed with antisense
construct, had mild symptoms with PVX-Eg2 infection suggesting that the
rate of disease development in these plants was lower (Fig.
8). High level of resistance was induced in potato and N. benthamiana
plants against PVX-Eg2 (Fig. 6, 9)
when they were transformed with sense/antisense construct. These results
demonstrate that the sense/antisense orientation could produce dsRNA folding
by the complementarities between the sense and the antisense orientations
of the silencing genes after the splicing of the ChaS intron post-transcriptionally.
siRNA technology could be used as significant virus-control measure.
As indicated high rate (nine potato plants out of 10) were resistant to
PVX-Eg2 when they were transformed with sense/antisense construct. Also,
seven tobacco plants out of 10 ones were resistant to PVX-Eg2 when they
were transformed with the previous construct.
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