Molecular Variation of Potato virus Y Isolated from Egypt
Potato Virus Y (PVY) is one of the most important viruses affecting cultivated potatoes in Egypt. Different potato plants were collected from an experimental station in Giza Governorate, Egypt and were tested using RT-PCR. PVY was amplified using primers represented portion of the Coat Protein (CP) gene and 3' Untranslated Regions (UTR). Phylogenetic tree showed two main strain groups: Group I regroups PVYN and PVYNTN stains while Group II includes PVYO, PVYW and PVYN:O strains. The Egyptian PVY isolate was clearly classified within group I and was more closely related to PVYNTN strains. Ten nucleotide substitutions resulted in 3 conserved amino acid substitutions (V1→I, G7→E, M or V and S8→G) and were able to differentiate between both groups. The partial coat protein region was more diverse than that of the 3'UTR (92.6-100% and 97.7-100% identity, respectively). The 3'UTR of the Egyptian isolate showed RNA secondary structures different from those of the 5 PVY strains.
June 20, 2011; Accepted: July 27, 2011;
Published: October 25, 2011
PVY belongs to the potyvirus genus, Potato virus Y is an important pathogen
in Solanaceous crops. The level of damage to crop is determined by the strain
of PVY infecting the plants, the viral load, the time at which infection occurs
as well as the tolerance the host possesses toward the virus (Warren
et al., 2005). PVY is naturally transmitted by aphids in a non-persistent
manner with great efficiency, causing epidemics in potato, tomato, pepper, tobacco
and other solanaceous plants (De Bokx and Huttinga, 1981).
PVY has a single positive-sense genomic RNA~10 kb long and forms flexuous virions.
The genomic RNA contains a unique ORF encoding a polyprotein which is processed
into functional viral proteins by virus-encoded proteases (P1, HC-Pro and NIa
(Riechmann et al., 1992)). The CP gene is the
gene most frequently used for studies of genetic diversity in Potyviruses
(Shukla et al., 1994).
Based on symptomatology in tobacco plants, PVY isolates were divided into two
major pathology groups: the common or ordinary strain, PVYO that
induces mosaic or vein clearing symptoms in tobacco; and the necrotic strain,
PVYN that induces systemic Vein Necrosis (VN) in tobacco (De
Bokx and Huttinga, 1981), Subsequently it has been recognized that each
of these two strains contain subgroups and that other distinct strains do exist
(Singh et al., 2008). In the necrotic, PVYN
group, two main recombinant types were identified initially: PVYNTN
and PVYN-Wi (Nie and Singh, 2003; Piche
et al., 2004). PVYNTN was first reported from Hungary
in 1984 as a distinct subset of isolates within the PVYN strain that
were capable of inducing tuber necrosis in potato tubers, often referred to
as Potato Tuber Necrotic Ringspot Disease (PTNRD) (Beczner
et al., 1984).
Several studies based on the molecular and phylogenetic characteristics of
the PVY strains were previously performed by studying the different coding and
non-coding regions of the genome (Boonham et al.,
1999; Fanigliulo et al., 2004). The 3' Untranslated
Regions (UTR) and Coat Protein (CP) coding sequences have been used for the
identification and classification of many plant viruses (Yun
et al., 2002). In this study, RT-PCR was used for the specific detection
of PVY in potato samples. We analyzed and compared the nucleotide and the deduced
amino acid sequence of C-terminal portion of the CP gene and entire 3' UTR of
an Egyptian PVY isolate with those of previously reported. Phylogenic and secondary
structure analysis was conducted to differentiate between PVY groups.
MATERIALS AND METHOD
Virus source: Eight potato samples (leaves and tubers) showing viral infection were collected from an experimental station in Giza Governorate. PVY isolate exist in our laboratory was used as a reference in RT-PCR.
RNA extraction: Total RNA extraction was done using extraction kit (RNeasy Mini Kit IAGEN# 74903).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR): PVY was amplified using primers represented C-terminal portion of the CP gene and entire 3' UTR (Table 1). Reverse transcription and PCR were carried out according to the manufacturer's recommendations using ONE STEP RT-PCR kit (QIAGEN). The RT-PCR conditions were 30 min at 50°C, 2 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 45°C, 1 min at 72°C and 10 at 72°C. The amplified product was resolved by electrophoresis in 1% agarose gel.
Sequence analysis: The amplified PCR product of sample n° 7 corresponds
to portion of CP gene and 3' UTR was purified using QIAquick PCR purification
kit (Qiagne Inc., Mississauga, ON, Canada). DNA sequencing was carried out with
the Taq dye terminator cycle sequencing kit (Applied Biosystems) and an Applied
Biosystems 373A sequencer. DNA sequencing was carried out in one direction using
the PCR product specific primer PVYv. The sequence was edited using Chromas
Pro Version 1.34 software. The Egyptian isolate of PVY was compared with PVY
sequences in the NCBI database with the program BLAST. Sequences were manipulated
using BIOEDIT, Nucleotide and amino acid sequences were aligned using the multiple
sequence alignment program CLUSTALW (Thompson et al.,
1994) implemented in MEGA software (Kumar et al.,
2001). The phylogenetic relationships of sequences were inferred and compared
using the neighbor-joining algorithm (Saitou and Nei, 1987)
included in MEGA software (Kumar et al., 2001).
Bootstrapping of 1000 replicates was carried out (Felsenstein,
||Primers for RT-PCR-amplification
Potato virus Y detection using
RT-PCR: PVY was detected in 4 out of 8 samples using RT-PCR (Table
2). PCR fragment of about 650 bp corresponding to the C-terminal region
of CP and 3' UTR portion was produced for samples No. 1, 5, 7 and 8, however,
no PCR product was detected with the negative control (healthy tobacco leaves)
(Fig. 1a, b).
Comparative sequence analysis: One out of 4 PCR-positive samples (sample
No. 7) was sequenced. The sequence was edited using Chromas Pro Version 1.34
software, resulted in 593 nucleotides. The Egyptian isolate was compared with
PVY sequences in the NCBI database with the program BLAST. Nucleotide sequence
obtained in this study is deposited into GenBank (accession number JF698682).
||RT-PCR results of the PVY-field collected infected samples
|Fig. 1 (a-b):
||Agarose gel electrophoresis analysis of RT-PCR products of
PVY-collected samples from field. (a) M: 100 bp DNA ladder. L1, 2, 3, 5
and 6 correspond to samples 1-5, respectively. L4 and 7: Negative control
(healthy tobacco leaves). (b) L1: Positive control (PVY-infected tobacco
leaves collected from our greenhouse); L2-L4 correspond to samples 6, 7
and 8, respectively
The isolate named "PVY-Egypt-Medhat". Thirty six sequences correspond to the
5 PVY strains: PVYN, PVYNTN, PVYO, PVYW
and PVYN:O were retrieved from the GenBank database (accession
numbers are shown in Fig. 2-4. Three more
sequences (only coat protein sequence) correspond to 3 Egyptian isolates were
retrieved from GenBank (AF522296.1, GU550076.2 and GU980964.1) were used in
the comparison (El-Mohsen et al., 2003).
Ten nucleotide substitutions located at positions 1-39 at the CP portion were
able to differentiate between the necrotic (PVYN and PVYNTN)
and the ordinary (PVYN:O, PVYO and PVYW) groups
(Fig. 2-4). Our Egyptian isolate was located
within the necrotic group, however, the 3 other Egyptian isolates retrieved
from GenBank were classified within the ordinary group (Fig. 3,
4). Only 3 out of the 10 positions resulted in amino acid
substitutions: (V1→I), (G7→E, M, or V) and (S8→G)
||Multiple alignments of the nucleotide sequence of partial
CP region and the 3'UTR region of PVY isolates. Sequences related to group
II, in comparison with group I, are parenthesized, however, the 3 positions
characterize PVYN:O subgroup are arrowed. The single nucleotide
substitution (T436→C) located in the 3'UTR region and differentiate
between the Egyptian and the other PVY isolates is boxed in bold. Numbers
on top represent the partial CP and the 3'UTR region position. Only the
differences are shown
||Multiple alignments of the C-terminal region of the CP amino
acid of PVY isolates. The consensus AFDF and QMKAAAL sequence motifs are
boxed. Sequences related to group II, in comparison with group I, are arrowed.
Numbers on top represent the deduced CP amino acid position. Only the differences
The CP region was more diverse than the 3'UTR nucleotide sequences (92.6-100%
and (97.7-100% identity, respectively). However, the identity within groups
was less diverse (96.3-97.5% and 97.7- 98.7% for CP and 3'UTR, respectively).
PVYN:O subgroup was characterized by the presence of 2 nucleotide
substitutions did not result in amino acid changes in the CP portion (T42→C
and C234→T) and one nucleotide change in the 3'UTR region (T513→C).
Sequence motifs AFDF and QMKAAAL were found within the CP region (position 13
and 33, respectively) (Fig. 3). The Egyptian isolate of PVY
was characterized by the presence of nucleotide substitution (C436→T)
located in the 3'UTR region and differentiate between our isolate and the other
PVY isolates (Fig. 2). Interestingly, a substitution from
S44→P was detected in 14 out of 23 sequences of group I, however,
this substitution was conserved in all sequences of group II (Fig.
||Phylogenetic tree of the PVY Egyptian isolate and selected
PVY isolates based on the analysis of the nucleotide sequence of partial
CP region, The evolutionary history was inferred using the Neighbor-Joining
method. The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) is shown next to the branches
Phylogenetic analysis: We investigated in more details the genetic relationships
of PVY isolates. The phylogenetic relationships of sequences were inferred and
compared using the neighbor-joining algorithm (Saitou and
Nei, 1987) included in MEGA software (Kumar et al.,
2001). Bootstrapping of 1000 replicates was carried out (Felsenstein,
1985). The obtained phylogenetic tree using the nucleotide sequences of
the CP divided the sequences into two main groups: Group I regroups PVYN
and PVYNTN strains while Group II include PVYN:O,
PVYO and PVYW strains (Fig. 4). The
phylogenetic profile was similar to the nucleotide and the predicted amino acid
substitutions described above (Fig. 2, 3).
|Fig. 5 (a-b):
||Comparison of the RNA secondary structures of the 3' UTR of
the Egyptian PVY isolate and the consensus sequence of PVYNTN subgroup,
predicted by the use of the mFOLD version 3.2 program (Zucker,
1989). The RNA secondary structures of the Egyptian isolate differed
from that of PVYNTN consensus sequence by the presence of a multi-loop
with 3 hairpins (boxed) instead of forming interior loop. RNA secondary
structures obtained with the other strains was similar to that obtained
The Egyptian isolate was clustered with group I and was more closely related
to isolates: NTN-UK, NTN-Germany NTN-USA, NTN-Hungery, K- Koria and N-Greece
(99.6-100% identity). However, the 3 other Egyptian isolates retrieved from
GenBank were clustered with group II.
Secondary structure prediction: To investigate the possible changes
in the predicted secondary structure of the consensus sequence of the 5 PVY
strains in comparison with the Egyptian isolate, mFOLD version 3.2 program (Zucker,
was used. The RNA secondary structures of the Egyptian isolate differed from
that of PVYNTN consensus sequence by the presence of a multi-loop
with 3 hairpins (boxed) instead of forming interior loop (Fig.
5). RNA secondary structures obtained with the other strains was similar
to that obtained with PVYNTN. The Egyptian isolate was characterized
by the presence of nucleotide substitution (C436→T) located
in the 3'UTR region which differentiated between our isolate and the other PVY
isolates. This substitution might be the reason of changes resulted in the secondary
structures. No clear secondary structure changes were found using CP or 3'UTR
between the 5 strains.
As expected in previously studies, PVY sequences were clustered in 2 groups
(necrotic and ordinary groups) according to sequence alignment and phylogenetic
analysis. These 2 groups did not correlate to the geographical distribution.
They dont possess a spatial identity and can evolve and adapt from country
to another, abstraction to the environmental conditions (Feki
and Bouslama, 2008).
Specific differences were observed between the necrotic (group I) and the ordinary
(group II) groups at the first 10 amino acids of the CP. No notable difference
was observed between the PVYN and the PVYNTN strains.
Group II was characterized by the presence of amino acid substitution S44→P.
Also, 3 nucleotide substitutions at positions 42, 234 and 513 distinguished
PVYN:O subgroup. This differences my play a role in necrotic symptoms
for group I and veinal necrosis in group II. This data was in agreement with
those reported by Feki and Bouslama (2008). Interaction
between the PVY CP and the plant proteins expression might play probably an
important role in the virus symptoms (Feki et al.,
2005; Hofius et al., 2007). It was described
for numerous plant viruses, any sequences of the viral genome corresponding
to open reading frames, regulatory elements, non-coding sequences or silent
mutations could be involved in virus symptom induction (Van
Der Vossen et al., 1996; Cecchini et al.,
1997; Hirata et al., 2003). Contrarily, Yun
et al. (2002) suggested that the C- terminal portion of CP and 3'
UTR sequences does not always explain symptom variations; and symptom determinants
for necrosis in tobacco leaves and potato tubers are located in the HC-Pro gene
and in the NIa, NIb and N-terminal portion of CP, respectively.
In the 3' UTR of the Egyptian isolate, there was a substitution from cytosine
to thymine at position 436. This substitution might be the reason of secondary
structure change from interior loop exists in the 5 subgroups to a multi-loop
with 3 hairpins in our isolate. Moury (2010) reported
that 3 Chilean isolates were predicted to possess two stem-loop structures instead
of one in groups N, O or C.
In this study, we showed that our Egyptian PVY isolate is clearly classified within group I, however, the 3 other previously reported Egyptian isolates were located within group II. The Egyptian isolate is more closely related to subgroup PVYNTN. More biological studies will support this classification. Also, one nucleotide change in the RNA secondary structures characterized the Egyptian isolate; however, we need to study the effect of this substitution. It will be necessary to analyze genes or sequences involved in host selection and symptom developments.
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