Use of Helper Component Proteinase Gene to Identify a New Egyptian Isolate of Watermelon Mosaic Potyvirus
In this study, an Egyptian isolate of potyvirus known
to be zucchini yellow mosaic potyvirus (ZYMV) was isolated from leaves
of the cultivar Giza 1 watermelon plants which cultivated in Agricultural
Experimental Research Station, Sids, Beni Suef, Egypt. The double antibody
sandwich (DAS)-enzyme-linked immunosorbent assay (ELISA) was carried out
using polyclonal antibodies specific to ZYMV. The helper component proteinase
gene (Hc-pro) of WMV was isolated by reverse transcriptase-polymerase
chain reaction (RT-PCR) using primers based on the DNA sequence of ZYMV
virus, strain KR-PA. The blast searches identify the Hc-pro sequence
as a part of Water Melon Mosaic Potyvirus (WMV). The sequence analysis
showed that the isolated Hc-pro gene belongs to a new isolate of
WMVs and its DNA sequence and deduced amino acid were compared with the
Hc-pro of the French, China and Pakistan isolates. This study showed
that the Egyptian isolate is closer to Pakistan isolate than French and
China isolates, however, the phylogenetic tree showed that using one gene
is not enough to relate these isolates to each other.
Plant viruses are a persistent threat toward production of both watermelon
and other cucurbits in Egypt as well as in the World. There are several
common viruses that can affect cucurbits, including zucchini mosaic potyvirus
(ZYMV), watermelon mosaic potyvirus (WMV), papaya ringspot potyvirus (PRSV)
(Verma, 1988) and cucumber mosaic cucumovirus (CMV). Depending on the
type of viral infection, the infected plants show stunted, mottled, or
crinkled light green colored leaves. Moreover, fruits may be irregular
in shape, mottled or warty. In Fact the superinfection is a frequent phenomena
of potyviruses which can be detected in infected cucurbit plants in particularly,
watermelon for example by PRSV and WMV. In Egypt, ZYMV is known to be
one of the most abundant potyviruses that infect cucurbits. However, the
common methods of detecting the infected cucurbit plants were based on
morphological and serological methods. Therefore, differentiation between
ZYMV infection and WMV infection is not convenient using the conventional
methods especially due to their morphological similarity although differentiation
among WMVs was done using monoclonal antibodies (Desbiez et al.,
2007). ZYMV was first reported in Cucurbita pepo, in Italy by Lisa
et al. (1981). Afterwards the presence of the virus was reported
in Algeria, Australia, Egypt, Germany, Israel, Japan, Jordan, Lebanon,
Morocco, Spain, Taiwan, England, Turkey and USA (Davis, 1986; Brunt
et al., 1996). WMV was not isolated or identified in Egypt and the
infected watermelons and cucurbits were known to be infected with ZYMV.
WMV is probably distributed worldwide (Purcifull et al., 1984)
mostly in temperate and Mediterranean regions and was first reported in
Citrullus lanatus by Webb and Scott (1965). They reported that
the WMV-infected watermelon showed yellowish (chlorosis), mosaic, mottled,
deformed or/and stunted leaves and stems. WMV is transmitted by at least
29 species of Aphids, such as Myzus persicae or Aphis craccivora
(Edwardson and Christie, 1986). WMV has been known to be transmitted by
mechanical inoculation and to infect more than 170 plant species belonging
to 27 families (Shukla et al., 1994). At the molecular level, WMV
is closely related to SMV in most of its genome and could consider as
a divergent strain of this virus, moreover, it also appears to share a
recombination with Bean common mosaic virus (BCMV) in the P1 coding region
(Desbiez and Lecoq, 2004). The first report of full-sequenced WMV was
from France (WMV-Fr) (Desbiez and Lecoq, 2004) followed by China (WMV-CHN)
(Wu et al., 2006) and Pakistan (WMV-Pk) (Ali et al., 2006).
Most of the genes that used to identify WMV were using the polyprotein
(P1) gene or the Coat Protein (cp) gene. Helper component proteinase (Hc-pro)
was not used before to relate WMVs to each other. Hc-pro known
to fulfill many functions in viral life cycle (Urcuqui-Inchima et al.,
2001) it was recognized as indispensable helper factor for virus host-to-host
transmission by Aphid vectors (Thornbury et al., 1985). It has
a protease activity to release itself from the precursor polyprotein (Oh
and Carrington, 1989). Not only Hc-pro has a general enhancer function
of infectivity and genome amplification but also cell-to-cell and systemic
movement in plant (Kasschau et al., 1997). Recently it has been
identified as a suppressor of Post Transcriptional Gene Silencing (PTGS)
(Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau
and Carrington, 1998, Pruss et al., 2004). Due to new molecular
methods have been emerged as a new trend to resist plant viruses such
as using RNA Interference (RNAi), it is important to identify and know
the genomic sequence of the viruses of interest. In this study we isolated
the Hc-pro from a viral isolate used to be known in Egypt as ZYMV
but post gene isolation and sequencing showed its similarity to WMVs.
This step was very important in order to resist WMV using molecular techniques
which would be impossible since it was known to be ZYMV. A comparative
study between the WMV-Eg and WMV-Fr, WMV-CHN and WMV-Pk was performed
to show the relationship of the Egyptian isolate to the other isolates.
MATERIALS AND METHODS
Virus Isolation and Propagation
Leaf samples of watermelon plants cv. Giza 1 showing virus-like symptoms
were collected from the open field at Agricultural Experimental Research
Station, Sids, Beni Suef Governorate, Egypt. These samples were transferred
to the lab in ice and kept at -20°C till used.
The infectious sap was prepared and then Eskandarani squash plants were
mechanically inoculated in the presence of an abrasive (Carborandom, 600
mesh) as described by Allam et al. (2000). The inoculated plants
plus the control were kept for symptoms developing under controlled greenhouse
at biocontainment rooms for 4 weeks.
The Double Antibody Sandwich (DAS)-enzyme-linked immunosorbent assay
(ELISA) was carried out using polyclonal antibodies specific to zucchini
yellow mosaic potyvirus (ZYMV) raised at AGERI, ARC, Giza, Egypt as described
by Clark and Adams (1977).
Total RNA was extracted from infected Eskandarani squash plants by
SV total RNA Isolation system (Promega) according to the manufacturer's
instructions. RT-PCR was performed according to the manufacturer's manual
of one step RT-PCR kit (QIAGEN) using Forward primer (ZvHpF) 5' ATG TCG
TCG CAA CCG GAA GTT CAG TTC TTC and Reverse primer (ZvHpR) 5' TTA CCA
ACT CTG TAA TGC TTC ATC TCG C designed according to the nucleotide sequence
of HC-pro of ZYMV strain KR-PA (AY278998, Kwon et al., 2005). The
reaction mixture (50 μL) was containing: 400 μM dNTPs, 0.6 μM
of each primer and 1.1 μg total RNA template. The thermal cycler
program was as the following: 50°C for 45 min; 95°C for 15 min
for one cycle followed by 40 cycles of 94°C for 45 sec, 50°C for
45 sec, 72°C for 2.5 min and 72°C for 10 min. The PCR product
was purified from the gel by the gel extraction kit (Qiagen) and cloned
into pGEM-T Easy vector system I (Promega) and transformed into Escherichia
coli JM109 strain.
Three different clones carrying Hc-pro were completely sequenced in
the genomic facility in AGERI, ARC, Egypt. Each clone was sequenced in
both directions by using M13F and M13R universal primers using the Big
Dye terminator reaction mix (Applied Biosystems). The sequences were run
on a 3700 ABI automated sequencer. Sequencher 4.1 (Gene Codes Corporation)
was used to assemble the contiguous sequences. BLAST searches (Altschul
et al., 1990) were performed on the resulted sequence to verify
authenticity before any phylogenetic analyses were undertaken. The DNA
and deduced amino acid (aa) sequences were also compared with the other
Hc-pro from closely related isolates of the same virus using GenDoc program.
The DNA sequence of the Hc-pro isolated from the WMV-Eg strain was
aligned using Lasergene Megaline program (DNAStar, Madison, WI) against
the three Hc-pro sequences from the completely sequenced WMVs (France
strain AY437609, Chinese strain DQ399708 and Pakistani strain AB218280).
The sequences used for alignment were trimmed to the size of the Hc-pro
gene without the primer sequences used for PCR. The alignments were performed
on the DNA and amino acid basis. The ancestral relationships among the
four WMVs are presented as a phylogenetic tree created by the lasergene
RESULTS AND DISCUSSION
In this investigation, an Egyptian isolate of WMV, used to be known as
ZYMV in Egypt, was obtained. This virus was isolated from watermelon leaves
naturally exhibited virus-like symptoms and propagated by mechanical inoculation
of the infectious sap obtained from infected squash plants Eskandarani
DAS-ELISA was used as a confirmatory test using polyclonal antibodies
specific to ZYMV which serologically related to WMV. ELISA showed that
no positive reaction was found in the healthy sample that used as a control
compared to the infected plants which showed values ranged from 0.853
to 0.918 at A405 nm (Table 1). The results
indicate that the virus under investigation is either ZYMV or related
||DAS-ELISA detection of the Egyptian isolate of WMV-Eg
used to be known as ZYMV in squash plants mechanically inoculated
with the infectious sap extracted from virus-infected watermelon samples
||RT-PCR analysis to confirm the WMV authenticity. M is
1 Kb ladder (Fermentas), W refers to the usage of WMV specific primers,
S refers to the usage of SMV specific primers and Z refers to the
usage of ZYMV specific primers
In Egypt, the virus under investigation was identified as ZYMV since
it usually gives positive results with DAS-ELISA against ZYMV antibodies
and since Watermelon is one of the variable hosts of ZYMV. During different
study to isolate Hc-pro from ZYMV, RT-PCR was used to isolate the Hc-pro
using primers designed based on the ZYMV-sequence alignment from the Basic
Local Alignment Search Tool (BLAST). The isolated ~ 1.3 Kb was cloned
in the pGEM-Teasy vector and sequenced. Results showed that the isolated
Hc-pro is similar to Hc-pro of WMVs.
In addition to perform RT-PCR and sequencing three times, another PCR
experiment was performed to eliminate the possibility of WMV superinfection
with other viruses such as SMV or ZYMV. The primers used were designed
to distinguish the WMV, SMV and ZYMV from each other by targeting unique
DNA sequence in each virus. All forward primers were designed based on
the 5 UTR of the coat protein DNA sequences and the reverse primers
were designed based on a unique region in the 3 UTR of each of the
viral genomes. The results showed expected band (~ 1kb) with the WMV-based
primers and no band with the SMV, or ZYMV-based primers (Fig.
From the previous results, the virus under investigation can identify
as a new Egyptian isolate of WMV (WMV-Eg). Due to the lack of information
provided for the WMV Hc-pro sequence, the alignment was performed using
the Hc-pro sequences from the complete genome of WMVs which were available
recently in 2004 for WMV-Fr (AY437609) isolated in France and 2006 for
WMV-CHN (DQ399708) and WMV-Pk (AB218280) isolated from China and Pakistan,
respectively (Desbiez and Lecoq, 2004; Ali et al., 2006; Wu et
al., 2006). For the sake of accuracy, the DNA sequence homologous
to ZYMV primers was excluded from the DNA alignment because their designs
were based on the ZYMV sequence. The DNA alignment of Hc-pro of WMV-Eg,
WMV-Fr, WMV-CHN and WMV-Pk showed 195 nucleotide differences out of 1318
bp, 145 of them were variable amongst all WMVs while 50 of them were exclusive
to WMV-Eg alone (Fig. 2). The nucleotide differences
in Hc-pro of WMV-Eg are the highest amongst all other WMVs, followed by
WMV-Fr, WMV-CHN and WMV-Pk which showed 30, 29, 18 exclusive nucleotide
differences, respectively. On the amino acid level, the alignment showed
21 amino acid (aa) differences amongst all 4 Hc-pro of WMVs, 13 a.a. of
them were variable among all 4 Hc-pro WMVs while 8 were exclusive to WMV-Eg
||Nucleotides alignment of Hc-pro among different WMVs
isolated from Egypt, France, China and Pakistan. Hc-pro of WMV-Eg
showed the highest exclusive variable nucleotides (50 nt) comparing
to other WMVs which showed 30, 29 and 18 nt differences in WMV-Fr,
WMV-CHN and WMV-Pk, respectively
The Hc-pro of WMV-Eg has the most exclusive a.a. differences followed
by WMV-Fr by 7 differences, however on the contrary to the DNA results,
WMV-Pk showed more a.a. differences (4) than WMV-CHN that has only one
a.a. exclusive difference (Fig. 3). Although the DNA
alignment showed nucleotide differences along the whole DNA sequence,
the a.a. differences were restricted to the first two third of the a.a.
sequence and the last third portion remained similar with only two a.a.
differences. This indicates that the 3 portion is the most conservative
domain of the Hc-pro protein. Hc-pro known to be consists of three regions:
An N-terminal region essential in aphid transmission, central region has
several functions such as RNA silencing suppression and a C-terminal region
has proteinase activity (Plisson et al., 2003).
||Amino acids alignment of Hc-pro. Hc-pro of the Egyptian
isolate (WMV-Eg) showed 8 exclusive a.a. which were the highest a.a.
differences comparing to other WMVs
The phylogenetic tree of Hc-pro on the basis of DNA and amino acids of
the 4 WMVs showed that the WMV-Eg is close to the WMV-Pk, while the WMV-Fr
is close to the WMV-CHN (Fig. 4 a and b).
In spite of that when the three WMVs (WMV-Pk, WMV-CHN and WMV-Fr) were
align together they showed confusing results. The alignment between the
three WMVs included the Hc-pro, coat protein, P1 and the whole genome
DNA on the basis of DNA and deduced a.a. sequences. WMV-Pk was distant
from the other 2 WMVs (WMV-CHN and WMV-Fr) when Hc-pro (Fig.
4c and d) and P1 (Fig. 4g and
h) were used for alignments on the basis of both DNA
and a.a. levels and also when whole genome was used on the basis of deduced
a.a. only (Fig. 4j).
||Phylogenetic tree of Hc-pro of the Egyptian, French,
Chinese and Pakistani WMV isolates based on DNA sequence (a) and deduced
amino acids (b). A comparison between the French, Chinese and Pakistani
WMV isolates were made on the DNA level for Hc-pro (c), CP (e), P1
(g) and the whole genome (I). While, the same was performed on the
deduced a.a. for Hc-pro (d), CP (f), P1 (h) and the whole genome (j).
The scale under the tree indicates the distance between sequences
in substitution event units
On the other hand, WMV-Pk was grouped with WMV-Fr and separated from
WMV-CHN on the basis of coat protein sequence (Fig. 4e,
f) alignments of both DNA and a.a. and on the basis
of whole genome sequence alignments of DNA only (Fig. 4i).
From the previous results we can include that the isolate under investigation
is a new Egyptian isolate of WMV (WMV-Eg) and it seems closely related
to the Pakistani isolate than the other 2 viruses, however, more sequences
need to be done to confirm that. Also we can conclude that the relation
between these viruses can not be confirmed by using one gene. Hence, the
CP which is the most common gene used to relate viruses in this group
is not legitimate since it showed the most controversy results amongst
the other genes.
We express our appreciation to Reda El-Sayed Salem from Dr. Salamas
Lab in AGERI, ARC, GIZA, Egypt for his help with ELISA test.
Ali, A., T. Natsuaki and S. Okuda, 2006. The complete nucleotide sequence of a Pakistani Isolate of Watermelon mosaic virus provides further insights into the taxonomic status in the Bean common mosaic virus subgroup. Virus Genes., 32: 307-311.
CrossRef | PubMed | Direct Link |
Allam, E.K., I. Sohair, El-Afifi and A.S. Sadik, 2000. Inclusion bodies as a rapid mean for detection of some plant viruses. Proceedings of the 9th Congress of the Egyptian Phytopathology Society, May 8-10, 2000, Giza, pp: 117-141.
Altschul, S.F., W. Gish, W. Miller, E.W. Myers and D.J. Lipman, 1990. Basic local alignment search tool. J. Mol. Biol., 215: 403-410.
CrossRef | PubMed | Direct Link |
Anandalakshmi, R., G.J. Pruss, X. Ge, R. Marathe, A.C. Mallory, T.H. Smith and V.B. Vance, 1998. A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. USA., 95: 13079-13084.
Direct Link |
Brigneti, G., O. Voinnet, W.X. Li, L.H. Ji, S.W. Ding and D.C. Baulcombe, 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J., 17: 6739-6746.
CrossRef | Direct Link |
Brunt, A.A., K. Craptree, M. Dallywitz, A. Gibs and L. Watson, 1996. Viruses of Plants Description and Lists from the Vide Database. University Press, Cambridge, UK.
Clark, M.F. and N.E. Adams, 1977. Characterization of the microtiter plate method of enzyme-linked immunoassay (ELISA), for the detection of plant viruses. J. Gen. Virol., 37: 475-483.
Davis, R.F., 1986. Partial characterization of zucchini yellow mosaic virus isolated from squash in Turkey. Plant Dis., 70: 735-738.
Desbiez, C. and H. Lecoq, 2004. The nucleotide sequence of Watermelon mosaic virus (WMV, Potyvirus) reveals interspecific recombination between two related potyviruses in the 5' part of the genome Arch. Virol., 149: 1619-1632.
CrossRef | Direct Link |
Desbiez, C., C. Costa, C. Wipf-Scheibel, M. Girard and H. Lecoq, 2007. Serological and molecular variability of watermelon mosaic virus (genus Potyvirus). Arch. Virol., 152: 775-781.
CrossRef | Direct Link |
Edwardson, J.R. and R.G. Christie, 1986. Viruses infecting forage legumes. Fla Agric. Exp. Stn Monog. No. 14, p: 454.
Kasschau, K.D. and J.C. Carrington, 1998. A counter defensive strategy of plant viruses: Suppression of posttranscriptional gene silencing. Cell, 95: 461-470.
Kasschau, K.D., S. Cronin and J.C. Carrington, 1997. Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase. Virology, 228: 251-262.
Kwon, S.W., M.S. Kim, H.S. Choi and K.H. Kim, 2005. Biological characteristics and nucleotide sequences of three Korean isolates of ZYMV. J. Gen. Plant Pathol., 71: 80-85.
Direct Link |
Lisa, V., G. Baccardo, G. D'Agostino, G. Dellavalle and M. d'Aquilio, 1981. Characterization of a potyvirus that causes Zucchini Yellow Mosaic. Phytopathology, 71: 667-672.
Direct Link |
Oh, C.S. and J.C. Carrington, 1989. Identification of essential residues in potyvirus proteinase HC-Pro by site-directed mutagenesis. Virology, 173: 692-699.
CrossRef | Direct Link |
Plisson, C., M. Drucker, S. Blanc, S. German-Retana, O. Le Gall, D. Thomas and P. Bron, 2003. Structural characterization of Hc-pro, a plant virus multifunctional protein. J. Biol. Chem., 278: 23753-23761.
Direct Link |
Pruss, G.J., C.B. Lawrence, T. Bass, Q.Q. Li, L.H. Bowman and V. Vance, 2004. The potyviral suppressor of RNA silencing confers enhanced resistance to multiple pathogens. Virology, 320: 107-120.
PubMed | Direct Link |
Purcifull, D., E. Hiebert and J.R. Edwardson, 1984. Watermelon mosaic virus 2. CMI/AAB Descr. Pl. Viruses No. 293, pp: 7.
Shukla, D.D., C.W. Ward and A.A. Brunt, 1994. Genome Structure, Variation and Function. In: The Potyviridae. CAB International, Cambridge, pp: 74-112.
Thornbury, D.W., G.M. Hellman, R.E. Rhoads and T.P. Pirone, 1985. Purification and characterization of potyvirus helper component. Virology, 144: 260-267.
Urcuqui-Inchima, S., A.L. Haenni and F. Bernardi, 2001. Potyvirus proteins: A wealth of functions. Virus Res., 74: 157-175.
CrossRef | PubMed |
Verma, A., 1988. The Plant Viruses The Filamentous Plant Viruses. Plenum Press, Vol. 4., New York, pp: 371.
Webb, R.E. and H.A. Scott, 1965. Isolation and identification of Watermelon mosaic virus 1 and 2. Phytopathology, 55: 895-900.