A Novel approach to study relationship between Cotton leaf curl virus (CLCuV)
and Beta satellites
Ahmad Ali Shahid*, Aleena Khalid, Syed Muhammad Nauman Gillani, Wajeeha Batool Chaudhary, Muhammad Azmat Ullah Khan
Centre of Excellence in Molecular Biology, Thoker Niaz Baig, University of the Punjab, Lahore, Pakistan.
Cotton is very important cash crop of Pakistan and due to non-motile nature of the plants it is susceptible to different kinds of biotic and
abiotic stresses. Lethal threat to cotton is cotton leaf curl virus (CLCuV) which belongs to family Geminiviridae and genus Begomovirus.
Geminiviridae is often associated with sub viral agents called DNA satellites. These small pathogenic viruses which are transmitted by
insects are responsible for various diseases in cotton crop. These defective interfering particles are associated with the infection like
Begomovirus which is having monopartite and bipartite genomes. Our study is concerned with the DNA ß or betasatellite component
of cotton leaf curl virus which is playing a tender role in cotton crop damage. Plants positive for betasatellites were confirmed for this
study first by using specific primers. Then viral titre of full length virus or original virus was identified by using molecular techniques
like southern blotting and real time PCR. After the detection of defective virus by southern blotting correlation study has been done
between defective betasatellites and cotton leaf curl virus effected plants. Plants containing different symptoms were used in this study.
Viral titers for different plants was compared with the positive plants for betasatellites. This correlation study between betasatellites and
CLCuV depicts the role of betasatellites in the symptom advancements and their number is higher in highly affected plants. If infection
is acute then number of betasatellites is higher. Our study helps to focus on the need to investigate another class of defective virus
(alphasatellites) and their role in the infection.
Cotton, CLCuV, Begomovirus, DNA, betasatellites, Suthern blotting, Real time PCR.
Agriculture industry forges ahead to the development of the
economy of Pakistan since independence. Agriculture is providing
crops like wheat, rice, barley, sugarcane, cotton etc that account
for more than 75% of total crop output. Agriculture subsidizes 21.5
% of GDP and utilizes 45 percent of nation’s work resources of
Pakistan(Tariq, Afzal, Hussain, & Sultana, 2007).
Cotton leaf curl virus is the most threatening disease amidst
defective DNA diseases having a dysfunctional mosaic pattern,
upward and downward curling of the leaf. In 1979, cotton leaf curl
virus disease was first reported in cotton crop in Multan, Pakistan
which is a tropical hot region. Cotton leaf curl virus disease
emerged lately in 1991 as the epidemic outbreak and in next year’s
it reduced the production drastically from 12.80 million bales in
1991 to 8.4 million bales in 1994(M. Hussain, Azhar, Khan, & Ali,
2012). This virus has single stranded DNA as defective particle.
Defective interfering particles contain both RNA and DNA in
genome are quite persistent in producing viral infections (A. S.
Huang, 1973). Most of the cases, interfering ability of defective
particles is not affirmed to significant level therefore called
defective DNA or sub genomic DNA (Patil & Dasgupta, 2006).
The genus Begomovirus find to consists of geminiviruses
which are transmitted through whitefly as vector and in which
preponderances have two types of genome or bipartite genome
comprising two ssDNA components, known as DNA A and
DNA-ß (Amudha et al., 2010). Some begomoviruses possess a
single genomic component that has all machinery mandatory for
multiplication of normal virus and locomotion within the plant.
Defective interfering particles are half in size from the genomic
DNA present in plants infected with viruses (Paximadis & Rey,
Southern blotting is a molecular technique in which DNA
fragments are transferred from gel to nitrocellulose membrane
and hybridized by probe. Southern blotting could be performed
by using restriction endonucleases digested DNA most frequently
(T. Brown, 1993) so defective particles and be studied through this
2. Materials and method :
Selection of plant materials
Leaf samples of the cotton variety MNH-786 were taken
from a cotton experimental field located in CEMB (Centre for
Excellence in Molecular biology). Virus resistant cotton plants
were categorized in (1) highly symptomatic that have high level
of leaf curling either upward or downward direction. The veins of
the leaves are thicker on the underside of the leaf. (2) Moderately
symptomatic that have viral attack up to a certain limit. They have
less amount of symptoms appeared on them. They were being
chooses and (3) resistant that are virus free control plants have
also chosen for virus correlation study.
2.1 Designing of primer and probe for betasatellites:
Primer were constructed for the C1 portion of betasatellite.
The sequence was accessed from NCBI with accession number FR873751.1 and following primers were designed i.e., BetaC1-F 5’TTCCTATTCGCATACAACGG’3, BetaC1-R 5’ATGCATTGCTGGTTTGTGTT’3.
2.2 Amplification and cloning of CLCuV betasatellite:
CTAB method with same adaptations as (Khan et al., 2015) was
used to extract genomic DNA of transgenic cotton plants. To check
the concentration and quantity of extracting DNA, it was running
on 0.8% gel. 0.8% agarose. After isolation PCR was performed.
Reaction mix comprises 2 μL of rolling circle amplification product
template having the concentration of 10ng/ μL 2.5ul of 10X PCR
buffer (Fermentas cat#B34), 2.5 μL of 2mM dNTPs, 1.5ul of
MgCl2( (Fermentas cat# R0971), 1ul of each forward primer (C1-
F) and reverse primer (C1-R) having the concentration of 5 μL Taq
polymerase enzyme (Fermentas cat# EP0071) were used. 14 μL
of water was added to makeup final volume of reaction. The PCR
product was viewed at 1.5 % agarose gel in 1X TAE buffer. PCR
conditions are given below. DNA product was ligated in TA vector
with the aid of Ptzr/T and positive colonies were confirmed by
PCR after plasmid isolation.
PCR conditions for betasatellites image shows the denaturation,
annealing, extension temperatures alongside their respective time
2.3 Southern blotting :
Southern blotting is the technique used to detect DNA sequences
in DNA samples. It’s the transfer of electrophoretic separated
fragments on a nitrocellulose membrane resulting in immobilization
of DNA. After immobilization DNA is subjected to hybridization
by using specific probe (M. S. Clark, 2013). It comprises of
following steps i.e., (1) DNA purification (2) Gel electrophoresis
(3) Denaturing and blotting (4) Hybridization (5) Washing and
autoradiography. At first a gel was run with 50 bp ladder and
DNA template along with positive cotton betasatellite DNA. Gel
was run for 3 hours without any break at 100 V. After the gel was
properly run it was placed under UV. After it was observed under
UV gel was depurinated by adding depurination solution (0.25 N
HCL) in tray. Tray was washed with autoclaved water twice for 15
min. After this denature the gel by adding denaturation solution
(0.5N NaOH, 0.5M Nacl, H2O) and put on shaker for about 45
minutes. Gel was washed with autoclaved water twice. Gel was
neutralized with neutralization solution (1M Tris PH: 8, 1.5M
Nacl,) for 30 minutes. The southern blot was performed according
to the protocol of Southern, 1973. (Bajwa et al. 2013)
nitrocellulose membrane placed on UV to cross
link the DNA.
2.4. Real time PCR :
Real time PCR is an advancement in polymerase chain reaction
in which quantity of nucleic acids from a mixture can be done by
fluorescent detection of PCR products. Specific and non specific
probes are used (Arya et al., 2014) with the same condition
3. Results :
3.1 Sampling groups based on symptoms
Plants (Gossyypium hirsutum) were being selected on the basic
of the severity of the infection and no infection at all. They were
categorized in three groups. Sampling was done from the fields in
center of excellence in molecular biology Lahore.
Characterization of sampling plants of Gossypium
hirsutum (a) mild symptomatic plant with less severe symptoms of
leaf curling (b) resistant plant (c) highly symptomatic plant.
3.2 Confirmation of DNA in CLCuV infected plant :
DNA from a cotton variety grown in the fields of Center of
excellence in molecular biology was extracted by CTAB method.
Expected DNA concentration in the gel is shown in the figure.
Extracted DNA from plants. Here M represents 1
kb marker, DNA of resistant plant is in lane 1-2, DNA of mild
symptomatic plant is in lane 3-4, DNA of highly symptomatic
plant is in lane 5-6.
Plasmid Isolation of betasatellites (extraction, cloning,
After the transformation the LB plates having ampicilin resistance
spread with the product and keep on incubation for whole night at
37°C (figure 4).
Plasmid isolation for selected white colonies. M is the l kb
ladder, lane 1-13 shows the betasatellites obtained after Miniprep.
3.3 Southern blotting :
The gel was then placed in assembly to transfer the DNA from
gel to nitrocellulose membrane. DNA of cotton plants Gossypium
hisutum was blotted on nitrocellulose membrane and hybridized
with probe. Defective DNA ß virus has been detected in plants
affecting with cotton leaf curl virus through southern blot analysis.
The picture 5 shows the 1 as the DNA from plant which was
mild symptomatic, 2 represents resistant plant, 3 represents
the plant having highly symptomatic of cotton leaf curl virus
while 4 represents plant positive for betasatellites. So defective
betasatellites have been detected by southern blotting from
different plants having different status of symptoms and infection
Agarose gel 1.5%, run for three hours at 80 V, M1 is
the 50 kb ladder; lane 1-3 is DNA, lane 4 is positive betasatellite
Hybridization pattern on nitrocellulose membrane in which the M is ladder, lane1-3 are DNA samples from three plants. Lane 4 represents the positive plant.
Comparison of both figures shows the successful transformation of
DNA from the agarose gel to nitrocellulose membrane. Scale helps
to know the exact location of the DNA present on gel and then
membrane after hybridization. So defective particles have been
identified through southern blotting.
3.2 Real time PCR :
Defective DNA or betasatellites are evident from the real time
PCR results. This graph shows the quantitative values of the viral
particles inside each plant having different level of symptoms.
In figure 7, plant 1 is showing high number of betasatellites as
compared to the resistant plant which is none. So defective DNA
particles tend to hamper the infection and they may associate with
the infection depending upon the replication machinery from their
Real time PCR analysis
Plant1 is plant which is highly symptomatic, Plant 2 is moderately
symptomatic, Plant 3 is resistant to cotton leaf curl virus, Positive
4. Discussion :
Cotton is being attacked by plenty of diseases. Among all CLCuV
considered to be threatening in case of loss bringing maladies.
Defective DNA particles are more common in infections. Aside
from the lengthy viral genomes, some DNA viruses that affect
plants are usually smaller in size (subviral molecules). In several
cases, their interfering nature is not affirmed to significant level
therefore called defective DNA or sub genomic DNA (Marcus &
Sekellick, 1977). Full virus or helper virus present in plants, often
contain small versions of the DNA they rendered to be defective
and have an interfering nature (A. S. Huang, 1973). In our study
prime focus is on the role of betasatellites or defective interfering
viral DNA of cotton leaf curl virus in the pathogenicity of the
disease. As defective DNA are found to involve in the symptom
amelioration of a disease. Their presence is burden on the helper
virus because they hinder the work of helper virus. In this study
positive virus affected plants for betasatellites have been identified.
In order to compute the betasatellites probes and primers were
designed as shown in figure. After the transformation, platting
white colonies were picked and Miniprep was performed. DNA
from the samples was confirmed through southern blotting
(Figure 1). Hybridization of the betasatellites probe with the DNA
fragments on the nitrocellulose membrane indicates the DNA
fragments contain sequences complementary to the betasatellites
evident from the figure 6. So defective virus (betasatellites) have
been detected through southern blotting in virus affected plants
which are burden on the full length and helper viruses.
Real time PCR was performed to quantify the viral titre of the
plants. It clearly depicts the level of viral components in the plants
and the viral titres vary in different plants which are at different
level of infection (vary from highly symptomatic to moderately
symptomatic and totally resistant plants). Number of betasatellites
tends to be higher in plants with high level of infection (Figure 7).
Real time analysis shows the numbers of betasatellites tend to be
higher in plants which were highly symptomatic as represented in
graph. Betasatellites are present in fewer amounts in plans having
moderate symptoms of diseases. And it’s almost absent in resistant
This correlation study between betasatellites and CLCuV depicts
the role of betasatellites in the symptom advancements and their
number is higher in highly affected plants as explained earlier in
real time figure 7. If infection is acute then number of betasatellites
is higher. They also compete with helper virus when co inoculated.
Our study helps to focus on the need to investigate about another
class of defective virus (alphasatellites) and their role in the
infection. There is an urgent need to introduce or breed crops with
broad-spectrum resistance to cope with possible emergence of new
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