-
This article was downloaded by: [41.34.179.10]On: 10 March 2015,
At: 00:58Publisher: Taylor & FrancisInforma Ltd Registered in
England and Wales Registered Number: 1072954 Registered office:
MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK
Biotechnology & Biotechnological EquipmentPublication
details, including instructions for authors and subscription
information:http://www.tandfonline.com/loi/tbeq20
Altered Gene Expression: Induction/Suppressionin Leek Elicited
by Iris Yellow Spot Virus Infection(IYSV) Egyptian IsolateElsayed
Elsayed Hafeza, Ahmed A. Abdelkhaleka, Abeer Salah El-Deen Abd
El-Wahabb &Fatma Hassan Galalca City of Scientific Research and
Technology Applications, Arid Lands CultivationResearch Institute,
Alexandria, Egyptb Cairo University, Faculty of Agriculture, Giza,
Egyptc Cairo University, Faculty of Science, Giza, EgyptPublished
online: 16 Apr 2014.
To cite this article: Elsayed Elsayed Hafez, Ahmed A.
Abdelkhalek, Abeer Salah El-Deen Abd El-Wahab & Fatma
HassanGalal (2013) Altered Gene Expression: Induction/Suppression
in Leek Elicited by Iris Yellow Spot Virus Infection (IYSV)Egyptian
Isolate, Biotechnology & Biotechnological Equipment, 27:5,
4061-4068, DOI: 10.5504/BBEQ.2013.0068
To link to this article:
http://dx.doi.org/10.5504/BBEQ.2013.0068
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy
of all the information (the “Content”) containedin the publications
on our platform. However, Taylor & Francis, our agents, and our
licensors make norepresentations or warranties whatsoever as to the
accuracy, completeness, or suitability for any purpose ofthe
Content. Any opinions and views expressed in this publication are
the opinions and views of the authors,and are not the views of or
endorsed by Taylor & Francis. The accuracy of the Content
should not be reliedupon and should be independently verified with
primary sources of information. Taylor and Francis shallnot be
liable for any losses, actions, claims, proceedings, demands,
costs, expenses, damages, and otherliabilities whatsoever or
howsoever caused arising directly or indirectly in connection with,
in relation to orarising out of the use of the Content.
This article may be used for research, teaching, and private
study purposes. Any substantial or systematicreproduction,
redistribution, reselling, loan, sub-licensing, systematic supply,
or distribution in anyform to anyone is expressly forbidden. Terms
& Conditions of access and use can be found at
http://www.tandfonline.com/page/terms-and-conditions
http://www.tandfonline.com/loi/tbeq20http://www.tandfonline.com/action/showCitFormats?doi=10.5504/BBEQ.2013.0068http://dx.doi.org/10.5504/BBEQ.2013.0068http://www.tandfonline.com/page/terms-and-conditionshttp://www.tandfonline.com/page/terms-and-conditions
-
4061
Article HttP://DX.DOi.OrG/10.5504/BBeQ.2013.0068 A&eB
AGricUltUre AND eNVirONMeNtAl BiOtecHNOlOGY
© Biotechnol. & Biotechnol. eq. 27/2013/5
Biotechnol. & Biotechnol. eq. 2013, 27(5),
4061-4068Keywords: Allium porrum, iris Yellow Spot Virus, iYSV,
thrips, defense system, up-regulated and down-regulated genes
IntroductionBy the end of 2006 at least 47 plant species have
been reported to be infected naturally by iris Yellow Spot Virus
(iYSV) under field conditions (39). The natural hosts of IYSV
include: iris, onion, leek, chive, shallot (A. cepa var.
ascalonicum), garlic, some wild Allium species, prairie
gentian/lisianthus (Eustoma russellianum, E. grandiflorum),
Alstroemeria sp., amaryllis (Hippeastrum hybridum), Amaranthus
retroflexus, and Portulaca oleracea (21). the genus Allium includes
a number of economically important cultivated species, including
the bulb onion, chive (A. schoenoprasum), garlic (A. sativum), and
leek (A. porrum) (10). At least 18 other Allium species are
consumed as fresh vegetables, pickled, or used as flavoring. leek
is one of the most valuable species, with production concentrated
in europe and the orient (10). other studies
report that onion, leek, garlic, and their relatives are used in
traditional medicine especially in the treatment of common cold and
influenza and as antimicrobial agents (40).
iYSV is a member of the genus Tospovirus, family Bunyaviridae,
and this virus has a segmented, single-stranded RnA genome (large,
medium and small RnAs) (27). it is well known that iYSV is
biologically transferred by onion thrips, Thrips tabaci (30), and
there is no evidence of virus transmission through seeds.
it is well known that the plant immune system can develop
resistance to pathogens but this phenomenon has still not been well
characterized. Leisner et al. (26) described a form of
developmental resistance in Arabidopsis to Cauliflower mosaic virus
(caMV). A lot of evidence suggests that symptom development in
plants might involve programmed changes in expression of host genes
and symptom patterns are highly dependent on the genetic background
of both the host and the virus (12). Moreover, it was observed that
the viral infection affects or alters the pattern of host gene
expression (4, 47) and
ALTERED GENE EXPRESSION: INDUCTION/SUPPRESSION IN LEEK ELICITED
BY IRIS YELLOW SPOT VIRUS INFECTION (IYSV) EGYPTIAN ISOLATE
Elsayed Elsayed Hafez1, Ahmed A. Abdelkhalek1, Abeer Salah
el-Deen Abd el-Wahab2, Fatma hassan Galal3 1City of Scientific
Research and Technology Applications, Arid Lands Cultivation
Research Institute, Alexandria, Egypt2Cairo University, Faculty of
Agriculture, Giza, Egypt3Cairo University, Faculty of Science,
Giza, EgyptCorrespondence to: Elsayed Elsayed HafezE-mail:
[email protected]
ABSTRACTLeek (Allium porrum) has become one of the major leafy
vegetable crops in Egypt and all over the world. Iris yellow spot
virus (IYSV) was observed on leek plants in eight different
governorates in Egypt, in fields near to onion fields. The symptoms
in infected leek plants included yellowish or tan diamond-shaped or
irregularly shaped lesions on leaves and flower stalks. Necrotic
areas developed on leaves, and some plants had elongated brown
lesions or brown flecks resembling thrips injury. Moreover, thrips
larvae and adults feed with a punch-and-suck behavior that removes
leaf chlorophyll, causing white to silver patches and streaks,
which were observed on all infected plants. The plant samples were
collected and subjected to ELISA test using IYSV polyantiserum. The
results revealed that about 90 % of the collected samples with
symptoms were positive. For further confirmation, the ELISA
positive samples were subjected to PCR amplification using nuclear
coat protein specific primers. The PCR results were in agreement
with the results obtained by ELISA. Thrip tabaci adults were reared
on the infected plant and biological transfer was performed onto
new healthy plants. After 15 days to 4 weeks post inoculation,
symptoms were observed on the plant. Biologically infected plant
samples were collected at different times after thrips-inoculation
and the extracted RNA was subjected to Real Time PCR using the coat
protein gene primers. The results showed that the expression of the
coat protein fluctuated but reached its peak on day five (264 %).
Differential display technique was performed on the newly infected
plant tissues to identify changes in gene expression in leek
elicited by IYSV that causes a symptomatic phenotype. Both up- and
down-regulated genes were observed in infected plants conjugated
with the healthy ones. Sequence analysis of the up-regulated genes
was performed and the encoding sequence analysis showed that the
obtained genes include: MFS family protein, Pathogenesis-related
protein, Mitogen-activated protein kinase, N-acetylserotonin
O-methyltransferase-like protein, Serine/threonine-protein kinase,
and Putative Retrosat2 Ty3-Gypsy_retroelement. On the other hand,
only one down-regulated gene was identified, alpha-tubulin
suppressor-like protein. Most of the identified genes are suppress
defensin genes (innate/adaptive). It can be concluded that viral
infection is capable of inducing a huge number of genes which are
important in the plant immune system.
Dow
nloa
ded
by [
41.3
4.17
9.10
] at
00:
58 1
0 M
arch
201
5
-
4062 © Biotechnol. & Biotechnol. eq. 27/2013/5
responses such as gene silencing may be specifically connected
to antiviral pathways (1, 17, 38). By the end of the 19th century,
confirmed evidence was accumulated that virus spread is correlated
with dramatic changes in cellular metabolism and gene expression
(5, 47). Aranda et al. (4) added that a number of genes have been
identified whose level of expression is regulated by infection with
compatible viruses, and cole et al. (16) reported that the temporal
induction of plant defenses in ‘columbia’ may provide a new tool
for studying plant defenses in Nicotiana.
to study the genes regulated (gene expression alteration) when
the virus successfully makes complete infection followed by symptom
appearance, differential display-PcR (DD-PcR) is commonly
preferred. DD-PcR provides a powerful method for the rapid
identification of differentially expressed genes, and has been used
successfully to identify such genes in both the host and the
pathogen during infections (35). Benito et al. (7) noted that it
was difficult to prove to what extent the viral symptoms cause or
are a consequence of such changes. For that reason they used DD-PcR
to screen the expression changes for some genes due to the viral
infection. they also added that DD-PcR helped to understand the
virus replication, signal transduction during plant responses to
virus infection, or the mechanisms by which the symptom response is
enacted.
in the present study we used DD-PcR and real-time PcR to prove
that iYSV infection in leek plants induces many changes in gene
regulation and expression. these changes included the up- or
down-regulation and changes in the level of defensin gene
expression. Moreover, studying the structure and function of these
genes is a good indicator for the symptoms severity and the viral
propagation.
Materials and MethodsSource of inoculums and biological
transmission the source of the viral inoculums used in this study
was previously characterized and identified (23). Both larvae and
adults of T. tabaci (kindly provided from the Department of
economic entomology, Faculty of Agriculture, cairo University) were
reared on infected leek plants (the infection was previously
demonstrated). leek seedlings grown from seeds and testing negative
for iYSV by eliSA were used (as test plant) for thrips transmission
experiments.
A single adult thrips was placed on each healthy seedling leaf
for 2 days. Virus presence was later determined by eliSA. Adult T.
tabaci was confined for up to 4 days on bean pods to lay eggs. once
the eggs had hatched, the larvae were collected for up to 12 h and
used for virus acquisition. Alternatively, first instar larvae were
reared on infected leeks for 2 h and then transferred to healthy
leek seedlings in another cage. healthy plants exposed to thrips
for inoculation access feeding were kept under greenhouse
conditions and observed for symptom development for 4 weeks.
Uninfected, healthy onion plants grown in an insect-proof
greenhouse were used as negative controls in PcR.
Mechanical transmission and biological transmissionMechanical
transmission tests were made by homogenized samples of
iYSV-infected plants separately in 0.01 mol/l sodium phosphate
buffer (ph 7.0) containing 0.1 % sodium sulfite. The sap was used
to inoculate leek with carborundum 600 mesh. Plants were kept under
greenhouse conditions. Symptoms were monitored during the 4-week
period and the symptom-less plant were examined by PCR, using
specific primers for the viral coat protein gene (to confirm that
the symptom-less plants were healthy).
Biological transmission. Virus-free first instar thrips larvae
reared on bean pods were removed from the rearing plants and placed
on the virus source plant for acquisition access time (AAt) before
being used as adults to inoculate leek test plants. After the
acquisition period, these larvae were reared to emergence of adults
and were transferred onto healthy leek seedlings for a 24 h
inoculation access time (iAt). then, the thrips were removed by
spraying the plants with Malathion (0.01 %) and the plants were
monitored for appearance of symptoms under greenhouse conditions.
All transmission experiments were compared with control
treatment.
Extraction of total RNA from plant tissuesAbout 100 mg of leek
plant leaves (healthy and infected) were subjected to RnA
extraction using Rneasy Mini Kit according to the manufacturer’s
instructions (qiAGen, Germany). the extracted RnA was dissolved in
DePc-treated water, quantitated spectrophotometrically and analyzed
in a 1.2 % agarose gel.
Reverse transcription-polymerase chain reaction (RT-PCR)Reverse
transcription reactions were performed in a total volume of 25 μL.
The reaction mixture containing 2.5 μL of 5x buffer contained Mgcl2
(25 mmol/L), 2.5 μL of 2.5 mmol/L dNTPs, 4 μL of oligo (dT) primer
(20 pmol/μL), 2 μg of the extracted RNA and 200 U reverse
transcriptase enzyme (MLV, Fermentas, USA). RT-PCR amplification
was performed in a thermal cycler (eppendorf, Germany) programmed
at 42 °c for 1 h, followed by 72 °c for 10 min; and the resultant
cDnA was then stored at -20 °c until used.
Detection of IYSV in the leek inoculated cultivars the iYSV
nucleoprotein (NP) gene was amplified using specific PCR according
to Pappu et al. (28). In the PCR reaction mix 2 μL of the amplified
cDNA were added to 2.5 μL of taq polymerase buffer 10x (Promega,
Madison, USA), 2.5 μL of 25 mmol/L MgCl2, 2 μL of 2.5 mmol/L dNTPs,
2 μL of 20 pmol/μL of each primer (Forward: TAA AAC AAA CAT TCA AAC
AA, and Reverse: CTC TTA AAC ACA TTT AAC AAG CAC) and 0.2 μL Taq
polymerase (5 U/μL), and sterile h2O was added up to 25 μL. The PcR
reaction conditions were: Initial denaturation at 95 °C for 5 min,
followed by 34 cycles at 95 °C for 1 min, at 54 °c for 1 min, and
at 72 °c for 1 min. Final extension at 72 °c was done for 10 min.
the amplification products were separated by 1.5 % agarose gel
electrophoresis in 0.5x tBe buffer.
Dow
nloa
ded
by [
41.3
4.17
9.10
] at
00:
58 1
0 M
arch
201
5
-
4063© Biotechnol. & Biotechnol. eq. 27/2013/5
Real -time PCR analys of NP expression in the biologically
inoculated plant tissuesthe cDnA of the plant samples was subjected
to semi-quantitive PcR using the nP primers (mentioned above). the
25 μL real-time PCR reaction mix contained: 12.5 μL of 2x
Quantitech SYBR® Green RT Mix (Fermentase.com), 1 μL of 25 pmol/μL
forward primer, 1 μL of 25 pmol/μL reverse primer, 1 μL of cDNA (50
ng), 9.25 μL of RNase-free water. Samples were spun before loading
in the rotor wells. the real-time PCR program was as follows:
initial denaturation at 95 °C for 10 min; 40 cycles at 95 °C for 15
s; annealing at 47 °C for 30 s and extension at 72 °c for 30 s.
Data acquisition was performed during the extension step. this
reaction was performed using a Rotor-Gene-6000-system (qiagen,
USA). the18S rRnA gene was used as a housekeeping gene (reference
gene) in this test.
Data analysisComparative quantification analysis was done using
the Rotor-Gene-6000 Series Software based on the following equation
(37).
Ratio target gene expressionexperimental/control) =
= Fold change in target gene expression (expt/control)Fold
change in reference gene expression (expt/control)
Fold change in target gene expression=
= copy number experimental (expt/control)copy number control
Fold change in reference gene expression =
= copy number reference (expt/control)copy number control
Differential display for detection of up–down regulated defense
genesSix different arbitrary primers (Table 1) were used to scan
the mRnA genes in infected plants at intervals and in healthy
plants as well. total RnA extracts were subjected to cDnA
synthesis, using one oligo (dt) primer as previously described. The
amplified cDNA was used as a template for the differential display
reaction. the PcR reaction was performed in a 25 μL reaction
mixture containing: 2.5 μL of 10× TaqDnA polymerase buffer, 2.5 μL
of 50 mmol/L MgCl2, 2 μL of each primer (40 pmol/μL) (Table 1), and
0.25 μL of Taq polymerase (AmpliTaq, Perkin–Elmer, 5 U/μL), 2.5 μL
of cDNA, 2.5 μL of 4 mmol/L dNTPase, and 12.75 μL of dH2o. the PcR
reaction was performed in a 9700 thermal cycler (Perkin–Elmer) and
the PCR conditions were as follows: initial denaturation at 95 °C
for 5 min, followed by 40 cycles (94 °C for 1 min; 53 °C for 1 min;
and 72 °C for 2 min) and a final extension at 72 °C for 10 min. the
reaction was stored at 4 °c until used. the PcR products were
separated in a 1.5 % agarose gel.
Cloning of the up-regulated DNA bandsThe resultant PCR product
was excised from the gel and purified using a qiA quick gel
extraction kit (qiagen inc., Germany). Purified DNAs were ligated
into the pGEM-T vector (Promega co., USA). Recombinant DnA plasmids
were then sequenced
using an automated sequencer (Macrogene company, Korea), with
forward universal primer. DnA homology searches were carried out
with the ncBi data bases, using BlASt (3).
TABLE 1Sequence of the primers used in this study
Primer Sequence 5`-`3chi15 GGYGGY tGGAAtGARGGeZ46 tGG AGG AAG
GtG GGP3n Att AGA tAc cct DGt AGt ccRapd1 tGc cGA Gct GRapd2 AtG
ccc ctG tRapd3 AGc cAc cGA A
Sequence analysisAnalysis of nucleotide and deduced amino acid
sequences was carried out using editSeq-DnAstar inc., expert
Sequence Analysis software, Windows 32 Edit Seq 4.00 (1989~1999)
and exPasy database online. Blast search for alignment of the
obtained sequence with the published ones was done using the
database of the national centre for Biotechnology information
(ncBi). Phylogentic analysis was carried out using the MeGA4
program (43).
Results and DiscussionBiological transmission and symptoms
appearanceIYSV was first reported on leek seed crops in 2006 in the
Willamette Valley (36). this virus is known to infect other Allium
spp., including onion and garlic (36). it has been reported that
iYSV is transmitted by the onion thrips (Thrips tabaci) but has not
been shown to be transmitted by other thrips species (20). the
results from our study revealed that viral symptoms appeared in the
biologically inoculated plants (using Thrips tabaci) 10 days post
inoculation. the inoculated leek plants showed spindle-shaped,
straw-colored, irregular lesions with occasional green islands. the
severity of the symptoms increased with the incubation time (data
not shown). on the other hand, after mechanical inoculation, no
symptoms were observed on the inoculated plants. Widana Gamage (48)
worked on iYSV on leek (Allium porrum) in Sri lanka and reported
that straw-colored spots (2 mm to 3 mm in diameter), surrounded by
a greenish halo and a necrotic area were observed on the plant
leaves.
Detection of IYSVIYSV was detected using ELISA and a specific
primer of the tospovirus nP gene. the biologically inoculated plant
tissues gave positive results with the iYSV antiserum (data not
shown). the positive eliSA samples were tested by PcR using the NP
gene specific primers. The results shown in Fig. 1 revealed that
the NP primers successfully amplified a 1.1 kbp amplicon of the
viral nP gene, while there was no amplicon with the healthy plants
(negative controls). these results were in agreement with our
previous observations (23).
Dow
nloa
ded
by [
41.3
4.17
9.10
] at
00:
58 1
0 M
arch
201
5
-
4064 © Biotechnol. & Biotechnol. eq. 27/2013/5
Fig. 1. Rt-PcR for the nP gene in the infected leek plants after
10 days post thrips-transmission. Lane M: 1 kb Ladder DNA Marker;
Lane H: genetic pool for a healthy plant; Lanes 1–10: inoculated
plants after 1, 2, 3, 5, 7, 9 and 10 days, respectively.
Level of NP gene expression in infected plant tissues in this
study, real-time PcR results revealed that the expression of the nP
gene in the inoculated plants increased gradually as compared with
control (healthy) plants. the expression of the NP gene reached its
peak at day five (264 %), whereas the lowest expression was
observed at day two post inoculation (152 %) (Table 2). Similar
results were obtained by Pappu et al. (29). They reported that real
time PCR and ELISA could be considered rapid and sensitive
detection methods for iYSV and would facilitate accurate virus
diagnosis in onion plants.
TABLE 2Fold change and the ratio of nP gene expression, using
the real-time PcR
Sample Amplification Fold change in reference gene
expressionFold change in target
gene expressionRatio target gene
expression %
h (control) 0.01
34
1 0.021 0.61 61 1.79 1792 0.52 52 1.52 1523 0.71 71 2.08 2085
0.90 90 2.64 2647 0.79 79 2.32 2329 0.59 59 1.73 17310 0.78 78 2.29
22918S (house) 0.34 34 1 100
TABLE 3Isolated genes and their molecular sizes and functions in
the plant defense.
Gene name Length (bp)Up- or down- regulated Function
MFS family transporter 180 Up-regulated • Secondary carriers
transporting small solutes in response to chemiosmotic ion
gradients.Mitogen-activated protein kinase 256 Up-regulated • Plays
a role in resistance to certain pathogens.
Pathogenesis-related protein 320 Up-regulated
• Function as signals that spread “news” of the infection to
nearby cells.
• infections also stimulate the cross-linking of molecules in
the cell wall and the deposition of lignin, responses that set up a
local barricade that slows spread of the pathogen to other parts of
the plant.
• inactivate the proteins secreted by the parasites in the
invaded plant tissues
Serine/threonine-protein kinase 341 Up-regulated • Plays a role
in the regulation of cell proliferation, programmed cell death,
cell differentiation, and embryonic development.n-acetylserotonin
o-methyltransferase like protein 334 Up-regulated
• Part of the melatonin pathway, which is the first line of
defense against internal and environmental oxidative stressors.
Alpha-tubulin suppressor-like protein 343 Down-regulated
• Associates with beta-tubulin to form tubulin dimmer, which
polymerizes to form microtubules; relative distribution to nuclear
foci increases upon DnA replication stress.
Putative retroelement 294 Up-regulated • Plays an important role
in plant evolution.
Dow
nloa
ded
by [
41.3
4.17
9.10
] at
00:
58 1
0 M
arch
201
5
-
4065© Biotechnol. & Biotechnol. eq. 27/2013/5
Fig. 2. Differential display PcR for the infected plant tissues
collected at different time intervals. Six arbitrary primers
(chi15, eZ46, P3n, Rapd1, Rapd3 and Rapd2) were used. Lane M: DNA
marker; lane H: healthy plant: Lanes 1–10: plant tissues collected
1, 2, 3, 5, 7, 9 and 10 days after thrips transmission,
respectively.
Dow
nloa
ded
by [
41.3
4.17
9.10
] at
00:
58 1
0 M
arch
201
5
-
4066 © Biotechnol. & Biotechnol. eq. 27/2013/5
they also suggested that the two methods (eliSA and real-time
PcR) could aid the better understanding of the disease epidemiology
and the development of management strategies. our study suggested
that real-time PcR could be a good method for determination of the
viral propagation profile inside infected tissues. Boonham et al.
(9) used real-time PCR for virus detection in thrips, which is
considered the biological vector for tomato spotted wilt virus
(tSWV).
Differential display PCR (DD-PCR)Six different arbitrary primers
corresponding to well-known defense genes were used in the DD-PcR
reactions to study the induced/suppressed genes as a result for the
viral infection. the six primers yielded more than 160 different
bands (Fig. 2). there were 11 up-regulated bands and two
down-regulated ones. Seven bands were selected and sequenced; their
names and functions are summarized in Table 3. our results
confirmed the importance of differential display for discovering
and identifying new genes. Several studies (2, 41, 42) compared RnA
microarray and the differential display technique and concluded
that DD-PcR is a method that allows systematic comparison of
expressed mRnA in the cells. They added that DD-PCR is becoming
more efficient today in isolating and characterizing genes
differentially expressed among cells, tissues or individuals.
Sequence analysis of the up–down regulated genesPlants are known
to defend themselves against attacks from pathogens, such as
viruses, bacteria, fungi, invertebrates, and sometimes other
plants, by altering the host gene expression (14). The seven
sequenced genes coding for: Major Facilitator Superfamily (MFS)
family transporter, mitogen-activated protein kinase,
pathogenesis-related (PR) protein, n-acetylserotonin
o-methyltransferase-like protein, Serine/threonine-protein kinase
and Putative Retrosat2 ty3-Gypsy_retroelement (up-regulated genes);
and only one down-
regulated gene, encoding for the alpha-tubulin suppressor-like
protein.
Paulsen and Skurray (31) reported that MFS represent nearly a
half of the solute transporters encoded within the genomes of
microorganisms and they are also widespread in higher organisms.
Pearson and lipman (32) postulated that MFS was originally believed
to function primarily in the uptake of sugars but it was later
found that drug efflux systems and citric acid cycle metabolites
belong to this family (22). concerning the plant mitogen-activated
protein kinase (MAPK), Zhang and Klessig (49) reported that the
plant MAPK cascade is one of the points for observation of
different pathogens and pathogen-derived elicitors and they are
highly conserved modules in all eukaryotes. in plants, MAPK
pathways are involved in many aspects of the cell development
regulation, growth, and programmed cell death and in responses to
abitotic and biotic stresses including pathogen attack (15).
Pitzschke and Hirt (33) reported that the MAPK genes in the
Arabidopsis genome are activated by a diversity of stimuli
including abiotic stresses, pathogens and oxidative stress
(34).
one of the isolated genes was the PR gene and it is already
known that PR genes play an important role in the plant defense
against pathogens. Van loon et al. (46) showed that there is an
induction of plant defense proteins which commonly evolved as
pathogenesis-related (PR) proteins. Durrant and Dong (18) confirmed
that the accumulation of PR proteins is very often associated with
systemic acquired resistance (SAR) against a wide range of
pathogens. the detection of the PR gene in the infected tissue and
not in the non-infected tissues suggested that this gene is related
to defense genes and they are underlying the gene–gene type
interaction.
Accordingly, melatonin also plays a vital role in plant defense.
tan et al. (44) reported that n-acetylserotonin
o-methyltransferase-like protein is considered part of the
Fig. 3. Phylogenic tree for all the obtained genes based on the
deduced amino acid sequences (Mega 4 program).
Dow
nloa
ded
by [
41.3
4.17
9.10
] at
00:
58 1
0 M
arch
201
5
-
4067© Biotechnol. & Biotechnol. eq. 27/2013/5
melatonin pathway, which is the first line of defense against
internal and environmental oxidative stressors. they found the
n-acetylserotonin o-methyltransferase-like protein in different
concentrations in corn, rice, wheat, barley, and oats (44).
Additional effects of melatonin on the cytoskeleton in plants were
previously reported by Jackson (25) in the endosperm of the
amaryllidacean Scadoxus multiflorus (syn. Haemanthus katherinae)
and by Banerjee and Margulis (6) in the epidermal cells of Allium
cepa.
Retrotransposons play an important role in the evolution and
function of the plant genome, through gene copying or gene transfer
inside the plant genome. the results from our study that the
isolated Putative Retrosat2 ty3-Gypsy_retroelement was induced in
the infected plants but completely shutdown in the healthy plants,
are in agreement with previous findings (for review see [45]) that
most retrotransposons are activated by stress and environmental
factors. cheng et al. (13) isolated 90 differentially expressed
genes when Nicotiana benthamiana was induced with Bamboo mosaic
virus infection and suggested that these genes played a role in the
resistance to viral infection. our results are also in accordance
with those of Boller and Felix (8) that viruses are a serious
threat, due to their high mutation rate, which makes them better
able to evade host defense systems. We assume that the transposon
element may be directed by the virus shut down some plant defensin
genes or modify their structure.
on the other hand, the alpha tubuline suppressor gene was
down-regulated. this gene family is known to be differentially
expressed during plant development in a tissue-specific manner
and/or in response to environmental conditions (11). heinlein et
al. (24) reported that, during pathogen infection, microtubules
have a role in the spread of tobacco mosaic virus from cell to
cell. Furthermore, it has been described that fungal infection can
lead to local microtubule depolymerisation (19). In line with these
findings, it could be speculated that the viral infection triggers
the plant cells to suppress the tubulin genes in an attempt to
prevent the viral movement from cell to cell.
the obtained results indicated that in the course of the viral
infection some of the plant defense genes were induced and/or
suppressed. these genes, in a normal state, work in harmony to
resist the plant pathogens. A pathogen will be able to infect a
plant cell, when it has the tools to suppress these genes or to
shut down the majority of these genes. this assumption was
confirmed by the phylogenetic analysis which grouped the up- and
down-regulated genes into two main clusters, A and B (the
down-regulated gene did not cluster as a separate group). Cluster A
included two sub-clusters: sub-cluster 1 including MAPK and PR
protein gene, and sub-cluster 2 including Serine/threonine-protein
kinase and Alpha-tubulin suppressor-like protein. Cluster B also
included two sub-clusters: sub-cluster 1 including Putative
retroelement, and sub-cluster 2 including MFS family transporter
and n-acetylserotonin o-methyltransferase-like protein.
ConclusionsEgyptian leek fields showed symptoms of Iris yellow
spot virus infection. the infection by this virus affects the
defense system of the leek plants. the plant response against the
viral infection was projected when different up-regulated genes
were observed. on the other hand, some of the genes could be shut
down by the viral silencing genes.
Conflict of Interestthe author(s) declare that they have no
competing interests.
REFERENCES1. Al-Kaff N.S., Covey S.N., Kreike M.M., Page A.M.,
et al.
(1998) Science, 279, 2113-2115.2. Al-Taweel K., Fernando W.G.D.
(2011) Americas Journal of
Plant Science and Biotechnology, 5(S1), 1-10.3. Altschul S.F.,
Gish W., Miller W., Myers E.W., Lipman D.J.
(1990) J. Mol. Biol., 215, 403-410.4. Aranda M.A., Escaler M.,
Wang D., Maule A.J. (1996) P. Natl.
Acad. Sci. USA, 93, 15289-15293.5. Balachandran S., Xiang Y.,
Schobert C.,Thompson G.A.,
Lucas W.J. (1997) P. Natl. Acad. Sci. USA, 94, 14150-14155. 6.
Banerjee S., Margulis L. (1973) Exp. Cell Res., 78, 314-318.7.
Benito E.P., Prins T., Kan A.L. (1996) Plant Mol. Biol., 32,
947-957.8. Boller T., Felix G. (2009) Annu. Rev. Plant Biol.,
60, 379-406.9. Boonham N., Smith P., Walsh K., Tame J., et al. (
2002) J.
Virol. Methods, 101(1-2), 37-48.10. Brewster J.L. (1994) Onions
and Other Vegetable Alliums,
cAB international, oxon, UK, 1-22.11. Carpenter J.L., Ploense
S.E., Snustad D. P., Silflow, C.D.
(1992) Plant Cell, 4, 557-571.12. Cecchini E., Al Kaff N.S.,
Bannister A., Giannakou M.E., et
al. (1998) J. Exp. Bot., 49, 731-737.13. Cheng S.F., Huang
Y.-P., Wu Z.-R., Hu C.-C., Hsu Y.-H.,
Tsai C.-H. (2010) BMc Plant Biol., 10, 286.14. Cohn J., Sessa
G., Martin G.B. (2001) curr. opin. immunol.,
13(1), 55-62.15. Colcombet J., Hirt H. (2008) Biochem. J., 413,
217-226.16. Cole A.B., Király L., Lane L.C., Wiggin B.E., et al.
(2004)
Mol. Plant Microbe in., 17(9), 976-985.17. Covey S.N., Al-Kaff
N.S., Langara A., Turner D.S. (1997)
nature, 385, 781-782.18. Durrant W.E., Dong X. (2004) Annu. Rev.
Phytopathol., 42,
185-209.19. Garavaglia B.S., Thomas L., Zimaro T., Gottig N., et
al.
(2010) BMc Plant Biol., 10, 51.20. Gent D.H., du Toit L.J.,
Fichtner S.F., Mohan S.K., et al.
(2006) Plant Dis., 90, 1468-1480.21. Ghotbi T., Shahraeen N.,
Winter S. (2005) Plant Dis., 89, 425-
429.22. Griffith J.K., Baker M.E., Rouch D.A., Page M.G.P., et
al.
(1992) Curr. Opin. Cell Biol., 4, 684-695.
Dow
nloa
ded
by [
41.3
4.17
9.10
] at
00:
58 1
0 M
arch
201
5
-
4068 © Biotechnol. & Biotechnol. eq. 27/2013/5
23. Hafez E.E., Abdelkhalek A.A., El-Morsi A.A., El-Sbahaby O.A.
(2012) Plant Dis., 96, 594.2.
24. Heinlein M., Epel B.L., Padgett H.S., Beachy R.N. (1995)
Science, 270, 1983-1985.
25. Jackson W.T. (1969) J. Cell Sci., 5, 745-755.26. Leisner S.,
Turgeon R., Howell S.H. (1993) Plant Cell, 5, 191-
202.27. Moyer J.W. (1999) In: Encyclopedia of Virology (A.
Granoff,
R.G. Webster, eds.), Academic Press, San Diego, cA,
1803-1807.
28. Pappu H.R., du Toit L.J., Schwartz H.F., Mohan S.K. (2006)
Arch. Virol., 151, 1015-1025.
29. Pappu H.R., Rosales M., Druffel K.L. (2008) Plant Dis.,
4(92), 588-594.
30. Pappu H.R., Jones R.A.C., Jain R.K. ( 2009) Virus Res., 141,
219-236.
31. Paulsen I.T., Skurray R.A. (1994) Trends Biochem. Sci., 18,
404.
32. Pearson W. R., Lipman D. J. (1988) P. Natl. Acad. Sci. USA.,
85, 2444-2448.
33. Pitzschke A., Hirt H. (2006) Plant Physiol., 14,
1351-356.34. Pitzschke A., Schikora A., Hirt, H. (2009) Curr. Opin.
Plant
Biol., 12, 1-6.35. Poirier G.M., Pyati J., Wan J.S., Erlander
M.G. (1997)
nucleic Acids Res., 25, 913.36. Pscheidt J.W., Ocamb C.M. (2012)
Pacific Northwest Disease
Management Handbook, oregon State extension Service, p. 715.
37. Rasmussen R. (2001) In: Rapid Cycle Real-time PCR, Methods
and Applications (S. Meuer, c. Wittwer, K. nakagawara, eds.),
Springer Press, heidelberg, 21-34.
38. Ratcliff F., Harrison B.D., Baulcombe D.C. (1997) Science,
276, 1558-1560.
39. Robène-Soustrade I., Hostachy B., Roux-Cuvelier M., Minatchy
J., et al. (2006) Plant Pathol., 55, 288.
40. Schwartz H.F., Mohan S.K., Havey M.J., Crowe F.J. (2006) In:
Compendium of Onion and Garlic Diseases and Pests, 2nd ed. (H.F.
Schwartz, S.K. Mohan, Eds.), APS Press, St. Paul, MN, i-xii.
41. Seufi A.M., Hafez E.E., Galal F.H. (2011) BMc Mol. Biol.,
12, 47.
42. Seufi A.M., Galal F.H., Hafez E.E. (2012) PloS one., 7(8),
e42795.
43. Tamura K., Dudley J., Nei M., Kumar S. (2007) Mol. Biol.
evol., 24, 1596-1599.
44. Tan D.X., Hardeland R., Manchester L.C., Korkmaz A., et al.
(2011) J. exp. Bot., 63, 577-597.
45. Todorovska E. (2007) Biotechnol. Biotech. eq., 21(3),
249-305.46. Van Loon L.C., Rep M., Pietersen C.M.J. (2006) Annu.
Rev.
Phytopathol., 44, 135-162.47. Wang D., Maule A.J. (1995)
Science., 267, 229-231.48. Widana Gamage S.M.K. (2010) Plant Dis.,
94(8), 1070-1072.49. Zhang S., Klessig D.F. (1998) P. Natl. Acad.
Sci. USA., 95,
7433-7438.
Dow
nloa
ded
by [
41.3
4.17
9.10
] at
00:
58 1
0 M
arch
201
5
