RESEARCH PROPOSAL UNDER PARB CGS SYSTEM
PROJECT ID NO. 127
1.
2.
3.
4.
Novel approach to generate wide spectrum
resistance to all cotton begomoviruses
infecting cotton and other cultivated crops
PARB THEME UNDER WHICH Theme -1: Enhancing Productivity on
Sustainable Basis of Major Farming
THIS PROJECT FALLS
Systems
Sub-theme 1.2: Cotton-wheat System
PARB SUB-THEME UNDER
WHICH THIS PROJECT
FALLS
Project group 1.2.1: Develop resistance to
PARB PROJECT GROUP FOR
gemini viruses (Leaf Curl
WHICH THIS PROJECT
Virus in cotton)
MATCH
PROJECT TITLE
To develop transgenic cotton plants with
RNAi-based resistance to cotton leaf curl
disease (CLCuD) caused by begomoviruses.
5.
OBJECTIVE OF THE
PROJECT
(Mission statement)
6.
ORGANIZATION SUBMITTING THE PROJECT
a. Name of the Host Organization:
University of the Punjab, Lahore.
b. Host Institute/Division/Section/Department: School of Biological Sciences
University of the Punjab, Lahore.
c. Administrative Contacts
i. Head of the Host Organization (VC/DG/DIRECTOR/etc
Name:
Prof. Dr. Mujahid Kamran
Title :
Vice Chancellor
Telephone: 042-923-1099, 042-923-1098
Email:
vc@pu.edu.pk
ii. Head of the Host Institution
Name : Prof. Dr. M. Akhtar, FRS
Title : Director General
Telephone: 042-9230960
Email: ma3@soton.ac.uk
Signature with date and seal
2
7.
COLLABORATING ORGANIZATION
a. Name of Organization:
b. Institute/Division/Section/
Biology
c. Administrative Contacts:
University of Toronto (Canada)
Department of Cell and Systems
Tamar Mamourian
i. Head of the Institution (Director/Chairman/Division Head etc.)
Name:
Title:
Telephone:
Email:
Dr. D. Goring
Professor and Chair
416 978 2378
d.goring@csb.utoronto.ca
Signature with date seal
8.
Pl. see separate sheet attached for signatures.
PROJECT MANAGER
Name:
Dr. Muhammad Saleem Haider
Title:
Assistant Professor
Organization/Institute: University of the Punjab
Qualification and Relevant Experience:
(CV Attached)
Telephone:
042-9231149
Mobile:
0333-4123220
Fax:
042-9231534
E mail:
haider65us@yahoo.com
Signature with date and seal
9.
COLABBORATING SCIENTIST
Name of the Team Leader: Dr. M.G. AbouHaidar
Institute:
University of Toronto, Canada
Qualification and Experience (Attach CV): (CV attached)
Telephone:
416 978 5615
Email:
mounir.abouhaidar@utoronto.ca
Fax: 416 978 5878
Signature with date and seal
April 3, 2008
3
10.
APPROVED BY
Name:
Dr. Mubarik Ali
Designation:
Chief Executive
Approval Date of PARB Board
of Governors:
14.4.2009
Signature with date and seal
11.
DURATION :
48 months
12.
DATE OF COMMENCEMENT
June, 1st 2009
13.
TOTAL PROJECT COST
28.139 million
14.
LOCATION OF THE
PROJECT
1. School of Biological Sciences, Punjab
University, Lahore.
2. Laboratory of Virology, Department of Cell
and Systems, University of Toronto, Canada
15.
BACKGROUND INFORMATION
i.
Problem to be addressed:
Begomoviruses are known to be a major problem in the cotton and other crops in
Pakistan particularly in the Punjab region. Crops infected with these viruses show a
notable decrease in the yield which results in major losses for farmers in particular
and for the nation’s economy in general.
ii. Relevance of the Project to the problem to be addressed:
The proposed research deals mainly in the development of transgenic cotton
plants which will be resistant to infection by cotton infecting begomoviruses. The
approach (described in this proposed project) for the development of transgenic plants
is not only novel but also the results are expected to exceed any of the previous
attempts to develop a wide spectrum resistance to an entire genus of viruses
(begomoviruses).
iii. Literature review preferably for the last 5 years.
Viruses of the genus Begomovirus (family Geminiviridae) are a major
constraint to the agricultural output of many tropical and sub-tropical countries
including Pakistan. The majority of the economically important geminiviruses fall
into the genus Begomovirus, which presently encompasses 682 isolates of approx.
200 species (Fauquet et al., 2008). These viruses are transmitted exclusively by the
whitefly Bemisia tabaci and affect almost all dicotyledonous crop species. The most
4
prominent example is cotton leaf curl disease (CLCuD), which is caused by a
complex of begomoviruses, and caused over US$ 5 billion losses to the Pakistan
economy during the mid to late 1990s. Occurrence of whitefly-transmitted diseases in
plants, particularly in vegetables, ornamental plants, agricultural and economic crops
presents a challenge for plant scientists concerned with the yield and quality in plant
production. In recent years, the role of whitefly and begomoviruses both in yield and
quality interest has been recognized.
In the present studies we will be using a beta satellite associated begomovirus
called Ageratum Enation Virus (AEV) as a model that we have recently isolated from
a weed plant Sonchus oleraceus , also found infecting a crop plant turnip (Brassica
rapa) under natural conditions of Punjab. AEV was first isolated in Nepal and it is
now present in Lahore, Pakistan (Tahir et al., unpublished). Similar to other
begomoviruses, AEV consists of a monopartite circular, single-stranded DNA
genome (DNA-A) enclosed in a characteristic twinned quasi-icosahedral particle
(Francki et al., 1980; Harrison, 1985). In addition, AEV is associated with a class of
single-stranded DNA satellites known as betasatellite (Briddon and Stanley, 2006).
Betasatellite is involved in host range determination and accumulation of both helper
virus and satellite molecules (Saunders et al., 2000; Briddon et al, Zhou et al., 2003;
and Qian and Zhou, 2005). On the other hand, DNA-A encodes all factors required
for virus replication, overcoming host defences, insect transmission and control of
gene expression (reviewed by Hanley-Bowdoin et al., 1999). AEV with its cognate
betasatellite causes characteristic symptoms such as yellowing of the leaves in
tabacum, downward leaf curl and necrosis in tomato, depression on the upper surface,
and enation-like structures on the undersides of the leaves in N. benthamiana and
tabacum. The clones are infectious to Nicotiana benthamiana, N. tabacum, tomato
and Ageratum conyzoides by agroinoculation (Tahir et al., unpublished work). AEV
is closely related to a number of viruses causing CLCuD which is a major constraint
to cotton production in Pakistan (Tahir et al., unpublished work). Its phylogenetic
analysis cluster it near cotton infecting begomoviruses. Therefore, the effort to
develop plant resistance against this virus is of economical benefit.
A successful method of developing plant resistant against viruses is by
targeting the RNA interference (RNAi) mechanism of the host cell. RNAi is an
evolutionary conserved mechanism in plants, animals, and fungi and is thought to
protect cells against invasive nucleic acids, such as viruses and transposons (Pooggin
et al., 2003). Post-transcriptional gene silencing (PTGS) is an example of RNAi
which is triggered by double stranded (ds) RNA. This dsRNA is processed via an
RNase III-like enzyme, called the Dicer, into short-interfering (si) RNAs. The
siRNAs guided by an endonuclease complex, called RISC, cleave a target RNA
homologous to the siRNAs themselves. As a result, PTGS plays a role in downregulating transcribed genes or terminating viral RNAs.
In experimental approaches, dsRNA expressed from a transgene in plants
illustrates the process of PTGS as well as de novo methylation of cognate DNA in the
genome (Sijen et al., 2001; Mette et al., 2000). Depending on the nature of the
transgene a difference in the silencing mechanism is observed. When dsRNA is
homologous to the coding region for the flower pigmentation genes in Petunia, PTGS
as well as methylation of the coding region was observed. On the other hand,
constructing the dsRNA homologous to the promoter sequences of the pigmentation
genes resulted in promoter methylation and transcriptional gene silencing (TGS)
(Sijen et al., 2001). Therefore, in both cases silencing was accompanied by the
methylation of DNA sequences that are homologous to the dsRNA. Sijen and
5
colleagues (2001) concluded that DNA methylation is an essential process for
regulating TGS and is important for reinforcing PTGS. Methylation of DNA in the
promoter region has also been implicated with reduced transcription levels (Fojtova
et al., 2003). Similarly, Mette et al. (2000) designed constructs to produce dsRNA
homologous to the promoter region of the gene for nopaline synthase in tobacco and
Arabidopsis. They observed that dsRNA is involved in promoter methylation which
in turn causes transcriptional gene silencing. Transcriptional gene silencing by
targeting promoter methylation proved to be an effective strategy for down-regulating
genes in plants.
This approach was adopted for developing plants resistant to begomoviruses.
Pooggin et al. (2003) showed that by using dsRNA to target a geminivirus promoter
one is able to develop plants with total virus resistant. Constructs were made by
inserting the bidirectional promoter of Vigna mungo yellow mosaic virus (VMYMV)
DNA A in forward and reverse directions, separated by a plant intron. Vigna mungo
plants formerly infected with the VMYMV showed complete recovery upon
bombardment of the leaves with the promoter construct. Involvement of PTGS was
also demonstrated as the interference signal spread throughout the entire plant.
Similarly, transgenic cassava plants expressing dsRNA cognate to the common
region-containing bidirectional promoter of African cassava mosaic virus (ACMV)
showed accelerated plant recovery after ACMV infection (Vanderschuren et al.,
2007). This finding correlated with the presence of transgene-derived siRNAs, 21-24
nt in length, which are presumably produced by the Dicer action. Vanderschuren et
al. (2007) concluded that the viral promoter and common region might undergo
siRNA-directed DNA methylation and histone modification that reduce both the
transcriptional activity of the promoter and/or impair the recruitment of DNA
polymerase necessary for replication via altered binding site properties. From the
above observations it is evident that transcriptional gene silencing by targeting viral
promoter sequences could render an effective method for plant resistance to
begomoviruses.
References
1. Briddon, R. W., Bull, S. E., Amin, I., Idris, A. M., Mansoor, S., Bedford, I.
D., Dhawan, P., Rishi, N., Siwatch, S. S., Abdel-Salam, A. M., Brown, J. K.,
Zafar, Y., and Markham, P. G. (2003). Diversity of DNA- β, a satellite
molecule associated with some monopartite begomoviruses. Virology, 312:
106–121.
2. Briddon, R. W., Stanley J. (2006). Subviral agents associated with plant
single-stranded DNA viruses. Virology, 344: 198-210.
3. Fauquet, C. M., Briddon, R. W., Brown, J. K., Moriones, E., Stanley, J.,
Zerbini, M., and Zhou, X. (2008). Geminivirus strain demarcation and
nomenclature. Archives of Virology 153: 783-821.
4. Fojtova M., Van Houdt H., Depicker A., Kovarik A. (2003) Epigenetic switch
from posttranscriptional to transcriptional silencing is correlated with
promoter hypermethylation. Plant Physiology, 133: 1240-50
5. Francki, R. I. B., Hatta, T., Boccardo, G. and Randles, J. W. (1980). The
composition of chloris striate mosaic virus, a geminivirus. Virology, 101:
233-241.
6
6. Hanley-Bowdoin, L., Settlage, S.B., Orozco, B.M, Nagar. S., and Robertson,
D. (1999). Geminiviruses: Models for plant DNA replication, transcription,
and cell cycle regulation. Crit. Rev. Plant Sci., 18:71-106.
7. Harrison, B.D. (1985). Advances in geminivirus research.
Phytopath, 23: 55-82.
Annu. Rev.
8. Mette M. F., Aufsatz W., Winden J., Matzke M. A., Matzke A. J. M. (2000)
Transcriptional silencing and promoter methylation triggered by doublestranded RNA. The EMBO Journal, 19: 5194-5201.
9. Pooggin M., Shivaprasad P. V., Veluthambi K., Hohn T. (2003) RNAi
targeting of DNA virus in plants. Nature, 21: 131-132.
10. Qian, Y., and Zhou, X. (2005). Pathogenicity and stability of a truncated
DNAâ associated with Tomato yellow leaf curl China virus. Virus Res. 109:
159–163.
11. Saunders, K., Bedford, I. D., Briddon, R. W., Markham, P. G., Wong, S. M.,
and Stanley, J. (2000). A unique virus complex causes Ageratum yellow vein
disease. Proc. Natl. Acad. Sci. USA, 97: 6890–6895.
12. Sijen T., Vijin I., Rebocho A., Blokland V. R., Roelofs D., Mol., N. M. J., and
Kooter M. J. (2001) Transcriptional and posttranscriptional gene silencing are
mechanistically related. Current Biology: 11: 436-440.
13. Tahir M., Haider S. M., Iqbal J., Akhtar M., Briddon W. R. (2008). Ageratum
enation virus causes yellow vein disease of Sonchus oleraceus. Unpublished
manuscript.
14. Vanderschuren H., Akbergenov R., Poogin M. M., Hohn T., Gruissem W.,
Zhang P. (2007) Transgenic cassava resistance to African cassava mosaic
virus is enhanced by viral DNA-A bidirectional promoter-derived siRNAs.
Plant Mol Biol, 64: 549-557.
16. PROJECT PLAN
a. Scientific/technical methodology (give details):
The present proposed study deals essentially with the determination of conserved
promoter regions of begomoviruses which are known to infect cotton plants. In addition, it
includes the designing of binary vector constructs to transform cotton plants and to generate
transgenic cotton plants. The main concept of this proposal is to use dsRNA sequences
complementary to the conserved sequences of the DNA polymerase promoter of presently
known begomoviruses which infect cotton plants. Large quantities of dsRNA sequences
targeting the promoter of cotton infecting begomoviruses will be continuously produced in
transgenic cotton plants. Large dsRNA sequences will be processed by the dicer enzyme to
generate short fragments of (21-22nucleotides) dsRNA. These stable dsRNA sequences will
trigger the methylation of viral promoters. Consequently, there will not be any transcription of
the DNA polymerase gene and the viral replication will be halted. This process is quite
different from the well established PTGS which degrades the mRNAs after transcription. This
process acts, unlike in the PTGS, prior to the transcription of the viral genes and in particular
the DNA polymerase gene. Consequently, neither there will be viral replication nor any disease
symptom expression and the plants will be totally immune to the target begomoviruses. Since
the betasatellite which determines the host range of the virus depends on the genomic DNA
7
(DNA A) for its replication and function, inhibiting the DNA polymerase will result in a wide
spectrum of resistance to begomoviruses.
We have carefully analyzed the intergenic region, where the promoter lies, of presently
known cotton infecting begomoviruses, in order to determine the degree of sequence identity
amongst these viruses. Sequence analysis revealed a high degree of DNA sequence identity in
the promoter region of these begomoviruses. The precautionary measure (avoided TATA box
region which is universal of viral and plant genes), we under took in designing dsRNA
construct. The dsRNA construct is designed to be transcribed from the strong 35S promoter in
the pCambia1200 plasmid and the nos-terminator. Single-stranded RNA transcripts will fold
back due to complementarity and will form a hairpin structure (See Fig.2B). It is possible that
the 5’end will be capped. However, the “dicer” will generate the typical 20-21 dsRNA
fragments which in theory should trigger the viral promoter region to be methylated.
Consequently, viral replication will be halted due to lack of transcripts and DNA polymerase
protein.
There are a lot of studies related to micro-RNAs where smaller (less than 20 nucleotides)
sequence matching was required to produce the required gene silencing. In other terms the
system seems to tolerate some mismatching. This becomes important in our case because some
promoter sequences of begomoviruses are not 100 % identical but probably they will be
methylated. The wide spectrum of resistance to all begomoviruses is a sine qua none condition
for the success of our proposed study.
b. Scientific/technical methodology (give details):
1. Year I & II:
Molecular Biology studies
1. Plasmid Construction

Preparation of two synthetic oligonucleotides. Insert 1 will be specific to the
intergenic region (IR) of AEV DNA-A. Insert 2 will be complementary to the
selected common sequence of IR of begomoviruses infecting cotton.

Selected target sequences will be inserted in forward and reverse direction,
separated by a synthetic intron (see Fig 1) between promoter and termination
sequences of CaMV. This arrangement will produce a dsRNA with a hairpin loop
(see Fig 2B).

Ecoli transformations with the produced constructs.

Maxi prep for sequencing and further subcloning.

Mobilization of the constructs into Agrobacterium tumefaciens by direct
transformation as described (Hoefgen and Willmitzer, 1988).
8
Figure 1. dsRNA construct: fragment of insert cloned between NcoI and NheI restriction
sites of pCambia1200. Hygromycin is the plant selection gene.
Insert 1: Entire intergenic region (IR) of AEV DNA-A from positions 2599-2750 and 1135 in forward and reverse direction separated by a synthetic intron (CTAGC).
Insert 2: AEV DNA A common region (CR) from the position 2726-2750 and 1-25.
 An alignment of intergenic region of AEV with cotton leaf curl virus isolates
showed high sequence identity (along 50 base pairs) with the promoter sequence
located in CR.
 This insert contains the nona-nucleotide motif conserved to all begomoviruses and
a GGTG intron characteristic of a promoter sequence (Fig 2A).
 BLAST analysis shows that Insert 2 has sequence identity with many
begomoviruses including cotton infecting begomoviruses.
9
A
NcoI
nona-nucleotides
GGTG motif
NheI
5’ –CATGGCTAAAGCGGCCATCCGTATAATATTACCGGATGGCCGCGATTTTTTTAAAGTGGCTAGCCCACTTTAAAAAAATCGCGGCCATCCGGTAATATTATACGGATGGCCGCTTTAGG
-3’
3’CGATTTCGCCGGTAGGCATATTATAATGGCCTACCGGCGCTAAAAAAATTTCACCGATCGGGTGAAATTTTTTTAGCGCCGGTAGGCCATTATAATATGCCTACCGGCGAAATCCGATC-5’
Synthetic Intron
B
CAUGG|
CUAAAGCGGCCAUCCGUAUAAUAUUACCGGAUGGCCGCGAUUUUUUUAAAGUGG C
U
A
G
GAUUUCGCCGGUAGGCAUAUUAUAAUGGCCUACCGGCGCUAAAAAAAUUUCACC C
G----^
Figure 2 A) Sequence of insert 2 illustrating the position of synthetic intron, nonanucleotides, GGTG motif and restriction enzymes.
B) Predicted hairpin secondary structure of the RNA transcript for insert 2.
2.


3.



Plant Transformation
Since AEV begomovirus can replicate in tobacco plants (Nicotiana benthamiana),
we are planning to first generate transgenic tobacco plants as a study model to
prove the concept for methylation of viral promoters leading to virus resistance.
N. benthamiana plants will be transformed with the two constructs (Insert 1 and 2)
by an agroinoculations, and hygromycin-resistant plants will be regenerated as
described (Radian-Sade et al., 2000).
o N. benthamiana leaves will be cut with a razor blade and inoculated with
the A. tumefaciens for 5 minutes.
o Leaves will be blotted on filter paper and placed for 2 days on MS medium
(2 mg/l kinetin, 1 mg/l indole acetic acid (IAA), and 100 μM
acetosyringone).
o Leaves will be then transferred to a regeneration medium composed of the
above MS medium plus 2 mg/l kinetin, 1 mg/l zeatin, 1mg/l IAA, 18 mg/l
hygromycin and 300 mg/l carbenicillin for 3 days.
o After 20-30 days post inoculation shoots and roots will be regenerated and
plantlets will be transferred and maintain in greenhouse.
Characterization of Transgenic Lines
N. benthamiana genomic DNA will be extracted from freeze-dried leaves
according to Soni and Murray (1994).
PCR and Southern (and Northern if required) analyses will be carried out
following standard protocols.
Hybridization probes will be specific for Inserts 1 and 2.
4. Biolistic Bombardment of AEV D NA A and betastatllite

Particle preparation and bombardment will be conducted as described by Knierim
and Maiss (2007).
o Full-length infectious DNA of AEV DNA A with its cognate betasatellite
have been previously cloned into pBS+ plasmid (Tahir et al., unpublished).
10
o DNA A and cognate betasatellite will be precipitated on gold microprojectiles and co-bombarded into tobacco plants with a particle inflow gun
using an air pressure of 3 bar and a vacuum of 200 mbar.
5. Virus Detection in Infected Plants



Total DNA will be extracted from bombarded plants following Soni and Murray
(1994) method.
Equal amounts of total DNA from each sample will be double-digested with NheI
and NcoI and analyzed using standard Southern blot protocol.
Accumulated viral DNA will be hybridized with a DIG-labeled probe specific to
AEV DNA A and its cognate betasatellite.
Year III: Production of transgenic cotton plants.






Transformation and regeneration of cotton plants (Gossypium hirsutum) through
infection by A. tumefaciens as previously reported by Rajasekaran and colleagues
(1996).
The same dsRNA constructs which will confer resistance to begomoviruses in
tobacco will be used to generate transgenic cotton plants.
o Five to 7-day old G. hirsutum seedlings will be used to prepare cotyledon
and hypocotyls explants.
o Explants will be co-cultivated with A. tumefaciens harboring dsRNA
constructs for Insert 2 specific for begomoviruses infecting cotton.
o Transfer explants on a solidified agar plate with callus inducing medium in
Petri dishes.
o Following co-cultivation, the explants will be washed thoroughly with MS
solution containing 200 mg/l cefotaxime and 200 mg/l carbenicillin, blotted
dry and placed on freshly prepared callus induction medium containing
10mg/l hygromycin.
o Leaves will be transferred to a regeneration medium and subsequently into
the Greenhouse for further development.
Characterization of transgenic cotton will be performed as described for tobacco
plants
Testing transgenic cotton plants for resistance to the available begomoviruses
under greenhouse conditions.
Methods of infection will include: 1) Biolistic bombardment of infectious clones 2)
Agrobacterium-mediated infection 3) Infection with Whiteflies (natural host).
Analysis of the resistance. Begomovirus replication and quantity of virus as well as
the severity of disease symptoms will be the landmark to determine the resistance
to virus infection. Virus quantities will be estimated by ELISA, or quantitative
PCR etc.
Year IV: Glass house tests for transgenic cotton plants.


Transgenic cotton plants tested in the lab will be exposed to virus inoculums under
glass house conditions to test virus resistance level.
Identical procedures will be used to assess the degree of resistance.
11
References
1. Hoefgen R and Willmitzer L. (1988) Storage of competent cells for Agrobacterium
transformation. Nucleic Acids Research 16: 9877.
2. Maiss E. (2007) Application of Phi29 DNA polymerase in identification and fulllength clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf
curl Thailand virus. Arch Virol, 152: 941-954.
3. Radian-Sade S., Perl A., Edelbaum O., Kuznetsova L., Gafny R., Sela I., Tanne E.
(2000) Transgenic Nicotiana benthamiana and Grapevine plants transformed with
Grapevine Virus A (GVA) sequences. Phytoparasitica, 28: 1-8.
4. Rajasekaran K., Grula J. W., Hudspeth R. L., Pofelis S., Anderson D. M. (1996)
Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding
mutant forms of acetohydroxyacid synthase. Molecular Breeding, 2: 307-319.
5. Soni R., Murray JAH (1994) Isolation of intact DNA and RNA from plant tissues.
Analyt Biochem, 218: 474-476.
12
a.
Item
Milestones:
Description
Achievement indicators
Risk involved
Scientists
Involved*
30.5. 2013
Saleem Haider*
AbouHaidar
30.11. 2009
AbouHaidar*
Saleem Haider
1.900
AbouHaidar*
Saleem Haider
AbouHaidar*
Saleem Haider
AbouHaidar*
Saleem Haider
0.600
rupees)
28.139
Objective
Development of transgenic cotton resistant
to all CLCuD strain/species
CLCuD resistant cotton plant
Output-1
Generation of proper binary plasmid AEV
construct
Restriction analysis/ Sequence
Designing of primer and amplification of
RNAi product
Ligation and cloning of the RNAi fragment
into pCambia 1300 vector
Verification of proper pCambia construct
PCR amplified product
-do-
30.7. 2009
Restriction analysis
-do-
30.9. 2009
DNA sequencing for the
proper insert.
-do-
30.11. 2009
Survey of field for CLCuD/ whitefly
sampling from cotton or other host plants
(exhibiting CLCuD like symptoms). Five
districts of Punjab i.e. Vehari, Multan,
Bahawalpur, Bahawalnagar and Rahim Yar
Khan and a neighbouring district of Sindh
from the cotton belt will be surveyed. On an
average twenty five samples will be collected
from each district.
Collection of CLCuD samples and whitefly
from all the above named districts during
cotton season
Collection of CLCuD samples of other host
plants from all the above named districts during
the season when cotton crop is not in the field
CLCuD like symptomatic plant
samples of cotton and other
hosts.
None
30.11. 2011
Saleem Haider*
0.450
Twenty samples of infected
cotton plants from each district at
different locations
Five suspected samples of
infected vegetables, ornamentals
or weeds from each district at
different locations
None
30.11. 2009
30.11. 2010
30.11. 2011
30.5. 2010
30.5. 2011
Saleem Haider
0.300
Saleem Haider
0.150
Activity 1
Activity 2
Activity 3
Output-2
Activity 1
Activity 2
Natural
recombination/
Spontaneous
emergence of a
new or
recombinant,
variant/strain/
species of
begomovirus
Cost (million
Completion
date
None
None
0.800
0.700
13
PCR amplified product/
Restriction analysis/ Sequence
analysis
Spectrophotometer reading
quantifying DNA
None
30.5. 2011
Saleem Haider
0.650
-do-
Saleem Haider
0.100
Detection of CLCuD through specific primers
Expected size PCR product on
agarose gel
-do-
Saleem Haider
0.100
Primer designing and production of full length
clones
Restriction analysis
-do-
30.12. 2009
30.6. 2010
30.12. 2010
30.1. 2010
30.7. 2010
30.1. 2011
30.4. 2010
30.10. 2010
30.4. 2011
Saleem Haider*
0.200
Output 3
Analysis of samples collected in output 2
Activity 1
Extraction of total DNA
Activity 2
Activity 3
Activity 4
Confirmation through sequencing of the clones
Sequence analysis
Output-4
Development of AEV Transgenic tobacco
PCR , Southern/Northern analysis
None
30.5. 2010
30.11. 2010
30.5. 2011
30.5. 2010
Activity 1
Agro-transformation of the proper pCambia
RNAi construct
Agrobacterium /biolistic inoculation of the
RNAi construct into tobacco
Colony growth on selective
antibiotics
Transgenic callus/plant showing
resistance against control
-do-
30.1. 2010
-do-
30.3. 2010
Activity 3
Verification for transgenic tobacco plants
PCR , Southern/Northern analysis
-do-
30.5. 2010
Output 5
Rearing of whiteflies and CLCuD
transmission studies
Whiteflies breeding in the cages
and evidence of transmission
through symptomatic and
control (symptom less) plants
-do-
Activity 1
To grow and maintain insect and disease free
cotton plants on regular basis
Collection of whitefly populations from the
field and rearing on healthy plants
Healthy cotton plants in the
glasshouse
Whiteflies breeding in the cages
-do-
Continuous
process
from 30.11.
2010 to end
of project
30.7. 2010
-do-
Maintenance of breeding populations and
identification of biotype through squash silver
leaf assay
Silver leaf effect on squash plant
will be produced, only if Bbiotype is present in field (only
nymphs of the B-biotype cause
-do-
Activity 2
Activity 2
Activity 3
-do
Rob Briddon
(NIBGE-Fsd)
Naeem Rashid
(SBS-PU, LHR)
Saleem Haider*
AbouHaidar
0.250
Saleem Haider*
AbouHaidar
Saleem Haider*
AbouHaidar
Saleem Haider*
AbouHaidar
Rob Briddon
Saleem Haider*
AbouHaidar
Saleem Haider*
2.000
Saleem Haider
0.150
30.9. 2010
Saleem Haider
0.200
Continuous
process from
30.11. 2010
to end of the
Saleem Haider
0.150
0.600
0.700
0.700
0.850
14
Activity 4
Activity 5
Output 6
Exposure of A-viruleferous whiteflies for
acquisition access period (diseased plants) and
transmission access period (healthy plants)
Shifting of whitefly inoculated plants from
insectary to glasshouse and their fumigation
over there
Testing for resistance of transgenic tobacco
plants
Activity 1
Agrobacterium and biolistic inoculation of
AEV infectious clones onto transgenic tobacco
Activity 2
Testing of resistance through whitefly
inoculation
Output-7
Generation of proper binary plasmid
CLCuD construct
Activity 1
silver leaf effect), that also
confirms the breeding of whitefly
population
Successful transfer of whitefly
population from step to step
Symptomatic and asymptomatic
behaviour of plants depending on
the nature of experiment
Transgenic tobacco plant showing
resistance to AEV
project
-do-
-do-
Saleem Haider
0.150
-do-
-do-
Saleem Haider
0.200
None
30.11. 2010
Saleem Haider*
AbouHaidar
1.200
-do-
30.9. 2010
Saleem Haider*
AbouHaidar
0.700
Appearance of no or mild
symptoms in transgenic tobacco
and exhibition of typical
symptoms of AEV in control
plants
Asymptomatic and symptomatic
response of transgene and control
plants respectively
Restriction analysis/Sequence
-do-
30.11. 2010
Saleem Haider*
0.500
None
30.11. 2010
AbouHaidar*
Saleem Haider
1.500
Alignment of IR sequences of all CLCuD
species/strains to develop a conserved sequence
RNAi insert/fragment
Synthesis of insert
-do-
30.1. 2010
AbouHaidar*
Saleem Haider
0.400
Restriction analysis
Activity 3
Ligation and cloning of the RNAi fragment
into pCambia 1300 vector
Verification of proper pCambia construct
Output-8
Production of CLCuD infectious clones
Activity 1
Restriction analysis for the infectious clones
Activity 2
Production and cloning of partial dimer
Activity 2
DNA sequencing for the proper
insert
Restriction analysis
-do-
30.5. 2010
-do-
30.11. 2010
None
30.11. 2011
Expected size restricted fragment
on the agarose gel
-do-
Expected size fragment on the
agarose gel
-do-
30.7. 2010
30.1. 2011
30.7. 2011
30.9. 2010
28.3. 2011
30.9. 2011
Naeem Rashid
Rob Briddon
AbouHaidar*
Saleem Haider
AbouHaidar*
Saleem Haider
Saleem Haider*
0.500
0.600
1.700
Saleem Haider*
AbouHaidar
0.300
Saleem Haider *
Rob Briddon
0.600
15
Activity 3
Confirmation of partial dimer through
restriction and sequence analysis
Activity 4
Agro-transformation of infectious clones
Output-9
Development of CLCuD resistant
Transgenic tobacco and cotton
(In addition to cotton cultivar Cocker-312,
two susceptible cotton cultivars such as S-12
& MNH-93 along with two other elite cotton
cultivars will be used)
Agro-transformation of the proper pCambia
RNAi construct
Agrobacterium /biolistic inoculation of the
RNAi construct into tobacco and cotton
Verification for transgenic tobacco and cotton
plants
Testing of CLCuD infectious clones
Activity 1
Activity 2
Activity-3
Output 10
Activity 1
To grow and maintain insect and disease free
tobacco and cotton
Activity 2
Agrobacterium mediated /biolistic inoculation
through infectious clones and maintenance of
infected plants
Testing of CLCuD resistant transgenic
tobacco and cotton
Output 11
Activity 1
Agrobacterium and biolistic inoculation of
CLCuD infectious clones onto transgenic
tobacco and cotton
Activity 2
Test of resistance through whitefly inoculation
Expected size restricted fragment
on the agarose gel and desired
sequence
Colony growth on the plates
under selective antibiotics
-do-
Saleem Haider*
0.500
Saleem Haider*
0.300
None
30.11. 2010
30.5. 2011
30.11. 2011
30.11. 2010
30.5. 2011
30.11. 2011
30.11. 2011
PCR , Southern/Northern analysis
AbouHaidar*
Saleem Haider
1.500
Colony growth on selective
antibiotics
Transgenic callus/plant showing
resistance against control
PCR , Southern/Northern analysis
-do-
30.4. 2011
0.500
-do-
30.7. 2011
-do-
30.11. 2011
Production of typical symptoms
of CLCuD
Symptomless tobacco and cotton
plants in plant growth chamber
None
30.5. 2012
-do-
28.2. 2012
Symptomatic tobacco and cotton
plants in plant growth chamber
-do-
30.3. 2012
AbouHaidar*
Saleem Haider
AbouHaidar*
Saleem Haider
AbouHaidar*
Saleem Haider
Saleem Haider*
AbouHaidar
Saleem Haider*
Arshad Javed
(SBS-PU, LHR)
Saleem Haider *
AbouHaidar
Appearance of no or mild
symptoms in transgenic tobacco
and cotton and exhibition of
typical symptoms of CLCuD in
control plants
Asymptomatic and symptomatic
response of transgenic and control
(non- transgenic) plants
respectively
Asymptomatic and symptomatic
response of transgenic and control
(non- transgenic) plants
respectively
None
30.5. 2012
AbouHaidar*
Saleem Haider
1.300
-do-
30.3. 2012
AbouHaidar*
Saleem Haider
0.800
-do-
30.5. 2012
Saleem Haider
0.500
-do-
0.600
0.400
1.400
0.500
0.900
16
Output 12
Testing of CLCuD resistant transgenic
tobacco and cotton T1 plants
Activity 1
Agrobacterium and biolistic inoculation of
CLCuD infectious clones onto transgenic
tobacco and cotton
Activity 2
Test of resistance through whitefly inoculation
Output 13
Testing of CLCuD resistant transgenic
tobacco and cotton T2 plants
Activity 1
Agrobacterium and biolistic inoculation of
CLCuD infectious clones onto transgenic
tobacco and cotton
Activity 2
Test of resistance through whitefly inoculation
Output 14
Take care of IPR and Bio-safety rules
Activity 1
To apply for patent
Activity 2
Bio-safety clearance and handing over the
product to PARB
Appearance of no or mild
symptoms in transgenic tobacco
and cotton and exhibition of
typical symptoms of CLCuD in
control plants
Appearance of no or mild
symptoms in transgenic tobacco
and cotton and exhibition of
typical symptoms of CLCuD in
control plants
Asymptomatic and symptomatic
response of transgenic and control
plants respectively
Appearance of no or mild
symptoms in transgenic tobacco
and cotton and exhibition of
typical symptoms of CLCuD in
control plants
Appearance of no or mild
symptoms in transgenic cotton
and exhibition of typical
symptoms of AEV in control
plants
Asymptomatic and symptomatic
response of transgene and control
plants respectively
To contact/approach the
concerned authorities
Patent with independent and
equal rights for PARB, Host
institute and collaborating
institute.
A certificate to PARB for
independent and equal IPR for
PARB along with Host institute
and collaborating institute.
None
30.11. 2012
AbouHaidar*
Saleem Haider
1.500
-do-
30.9. 2012
AbouHaidar*
Saleem Haider)
0.800
-do-
30.11. 2012
Saleem Haider
0.700
None
30.5. 2013
AbouHaidar*
Saleem Haider
1.400
-do-
31.11. 2012
AbouHaidar*
Saleem Haider
0.800
Saghir Ahmed
Arshad Javed
-do-
30.5. 2013
Saleem Haider
0.600
-do-
30.5. 2013
Saleem Haider*
0.500
-do-
28.1. 2013
AbouHaidar
Saleem Haider*
0.300
-do-
30.5. 2013
Saleem Haider*
AbouHaidar
0.200
Salaries
Equipment
Note: Management cost is inclusive in the estimated cost against each output.
Put * mark on the name of the activity in charge. Put % share of each scientist in ( ) for each activity, if not equal.
8.298
1.220
17
16.
PROJECT STAFF DESCRIPTION:
a. Host Institute
Additional staff requirements and their proposed qualifications
Name of
post
Research
Associate
No. of posts with Proposed
Experience Pay Package
justification
qualifications
1 (Molecular
Ph. D
Nil
Rs. 75,000 per month + 7% of
Virology work)
initial pay as annual increment +
one month pay as gratuity for
each completed year of service
in the project at the end of the
project
b. Collaborating Institute
Additional staff requirements and their proposed qualifications
Name of
post
Research
Associate
17.
No. of posts
Proposed
Experience Pay Package
with justification qualifications
1(Biotechnology MSc/M phil
Five years Rs. 60,000+ 7% of initial pay
work at Univ. of
experience as annual increment + one
Toronto,Canada)
month pay as gratuity for each
completed year of service in
the project at the end of the
project
SUMMARY OF THE BUDGET (Detail as Annex-
Combined Summary of the Project (Rs. Millions)
Item of Expenditure
Salaries
Operating
Equipment
Overseas Travel
Sub total
Management Cost (25% of the project
cost)
Incentives for Scientists (5% of the
project cost)
Incentive for PM (1% of the project
cost)
Sub-Total
G. Total
Research Phase
Demonstration
Year 1 Year 2 Year 3 Year 4 Total
1.719 1.832 2.045 2.702 8.298
3.561 2.942 2.513 0.950 9.966
1.170
0.500
6.950
1.738
0.000
0.300
5.074
1.269
0.000
0.600
5.158
1.290
0.000 1.170
0.400 1.800
4.052 21.234
1.013 5.309
0.434 0.317
0.322
0.253
0.088 0.063
2.260 1.649
9.210 6.723
0.073
1.685
6.843
0.045 0.269
1.311 6.905
5.363 28.139
1.327
18
Summary of Host Institution (Rs. Millions)
Item of Expenditure
Salaries
Operating
Equipment
Overseas Travel
Sub total
Management Cost (25% of the project
cost)
Incentives for Scientists (5% of the
project cost)
Incentive for PM (1% of the project
cost)
Sub-Total
G. Total
Research Phase
Demonstration
Year 1 Year 2 Year 3 Year 4 Total
1.719 1.832 2.045 2.702 8.298
1.906 1.557 1.328 0.625 5.416
0.695
0.500
4.820
1.205
0.000
0.000
3.389
0.847
0.000
0.600
3.973
0.993
0.000 0.695
0.000 1.100
3.327 15.509
0.832 3.877
0.301 0.212
0.248
0.208
0.088 0.063
1.594 1.122
6.414 4.511
0.073
1.314
5.287
0.045 0.269
1.085 5.115
4.412 20.624
0.969
Summary of Collaborating Institution (Rs. Millions)
Item of Expenditure
Operating
Equipment
Research Phase
Demonstration
Year 1 Year 2 Year 3 Year 4 Total
1.655 1.385 1.185
0.325 4.550
0.475 0.000 0.000
0.000 0.475
Overseas Travel
Sub total
Management Cost (25% of the project
cost)
Incentives for Scientists (5% of the
project cost)
Sub-Total
0.000
2.130
0.533
0.300
1.685
0.421
0.000
1.185
0.296
0.400
0.725
0.181
0.700
5.725
1.432
0.133
0.105
0.074
0.045
0.358
0.666
0.527
0.370
0.227
1.790
G. Total
2.796
2.212
1.555
0.952
7.515
19
18. BUDGET INSTALMENTS
Instalment
(Half
Yearly)
1st
2nd
3rd
4th
5th
6th
7th
8th
Total
Host
Institute(SBS)
Collaborating
Institute(U of T, Ca)
Total
4.000
2.400
3.000
1.500
3.500
1.800
2.800
1.624
20.624
1.800
1.000
1.300
0.900
1.000
0.550
0.500
0.465
7.515
5.800
3.400
4.300
2.400
4.500
2.350
3.300
2.089
28.139
19. INTERNATIONAL COLLABORATION
a)
b)
c)
Name of linking international institute(s) with justification
University of Toronto (To get guide lines for the high Tech Molecular Plant
Virology)
Type of collaboration: Molecular biology (construction of clones, constructs,
transformation) and some limited greenhouse testing
Scientist(s) involved. Graduate students, technical staff and postdoctoral fellows
from the labs of Professor AbouHaidar and Dr. M. Saleem Haider
20. INTRNATIONAL TRAVELS
i.
HOST INSTITUTE
a) Name of scientist(s):
Dr. Muhammad Saleem Haider,
b) Purpose of each visit:
To check the resistance of transgenic plants to CLCuD, Transfer of material and
training.
c) Name of institute(s) to be visited:
Department of Cell and Systems Biology, University of Toronto (Canada).
d) Number of visits
Two visit of two weeks each in alternate years
ii. COLLABORATING INSTITUTE
a) Name of scientist(s):
Prof. Dr. AbouHaidar
b) Purpose of each visit:
Transfer of material, reviewing experiments and training.
c) Name of institute(s) to be visited:
School of Biological Sciences, University of the Punjab, Lahore
20
d) Number of visits
Two visit of two weeks each in alternate years
21. IMPORT OF TECHNOLOGIES
a. Details of the expected imports of materials, chemicals, technologies, machines,
hybrids, germplasm, etc. to be imported. Some Begomovirus clones, cotton seeds
will be imported to the collaborating laboratory.
b. Item wise estimated cost and time schedule of import
22. COMMERCIALIZATION AND BENEFIT TO END USERS
i) Method of transferring results: Patent application for clones, constructs and
technology will be applied for and processed before the end of project.
ii) Agency/company/consultants involved in adaptation and adoption. Private and/or
public company will be responsible of marketing at the local or international level. In principle
Pakistani cotton farmers should obtain the transgenic cotton plants at no cost.
iii)
Expected benefits to end users.
Anticipated benef its of the present study
The present study will (at the end of the proposed project) produce transgenic cotton plants
which will have a wide spectrum of immunity to most (if not all) the begomoviruses which are
known for their deleterious and devastating effect on the cotton crop in Punjab and in Pakistan.
The usage of such resistant plants in the field will certainly reduce the virus load in the field and
in the wild plant species which they are used as a reservoir for the begomoviruses. Cotton farmer
will benefit enormously from those cotton plants with a wide spectrum of resistance. Other
agricultural plants will in the future be transformed in a similar fashion to produce other crops
resistant to begomoviruses.
Serious efforts will be made to obtain patent (s) for this technology. Pakistani company licensed
to market such a technology will result in financial gains and job creation. Other spin off will also
result from such technology and production.
23. FINAL REPORT SUBMISSION: May 30th, 2013
21
Annexure- I
DETAILED COSTS
(Million Rupees)
Host Institute (School of Biological Sciences, University of the Punjab, Lahore)
Budget Code Item of Expenditure
Research phase
Demonstration
Year 1 Year 2
Year 3
Year 4
Total
A. Salaries
Two Research Associates, one @ Rs.75000 PM &
1.620
1.733
1.847
1.960
7.160
one @ Rs. 60000 PM (7% increase on initial
pay/year) + Gratuity @ one month pay for each
year at the end of the project.
Gratuity
Daily wages labour (un-skilled) Rs 250/day
10 % bonus
Sub-Total (A)
B. Operational
Research Consumables
Glass wares
Plastic wares
Chemicals and Enzymes
Primers & sequencing
0.000
0.090
0.009
1.719
0.000
0.090
0.009
1.832
0.000
0.180
0.018
2.045
0.544
0.180
0.018
2.702
0.544
0.540
0.054
8.298
0.150
0.150
0.650
0.500
1.450
0.100
0.100
0.500
0.400
1.100
0.100
0.100
0.400
0.350
0.950
0.000
0.000
0.120
0.100
0.220
0.350
0.350
1.670
1.350
3.720
0.000
0.000
0.000
0.000
0.010
0.010
0.020
0.010
0.030
0.020
Selfing bags/tags/labels etc
Plant protection
Traveling Allowance
POL
Stationery
Communication costs (postage/phone/fax/
internet)
Advertisement costs
Printing costs
Total
Sub-Total (B)
C. Machinery and equipment
0.005
0.001
0.200
0.100
0.050
0.050
0.005
0.002
0.200
0.100
0.050
0.050
0.005
0.003
0.100
0.100
0.050
0.050
0.015
0.010
0.100
0.100
0.050
0.050
0.030
0.016
0.600
0.400
0.200
0.200
0.030
0.020
0.456
0.030
0.020
0.457
0.030
0.020
0.378
0.030
0.020
0.405
0.120
0.080
1.696
1.906
1.557
1.328
0.625
5.416
Freezer -80 small (1)
Freezer -20 (1)
Humidifier (1)
Manual pipettes (1)
Cool cabinet (1)
Air Conditioner for the insectary
Air Conditioner for the glass house
Digital camera (1)
Computer with accessories (1)
Sub-Total (C)
(D) Overseas Travel
Sub-Total (D)
TOTAL (E) = (A+B+C+D)
Management Cost (25% of the (E) cost)
TOTAL (F) = (E+MC)
Incentives for Scientists (5% of the (F) cost)
Incentive for PM (1% of the project cost)
Sub total (G)
TOTAL COST (F+G) Host Institute
0.300
0.030
0.040
0.075
0.050
0.040
0.040
0.020
0.100
0.695
0.500
0.500
4.820
1.205
6.025
0.301
0.088
0.390
6.415
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
3.389
0.847
4.236
0.212
0.063
0.275
4.511
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.600
0.600
3.973
0.993
4.966
0.248
0.073
0.321
5.287
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
3.327
0.832
4.159
0.208
0.045
0.253
4.412
0.300
0.030
0.040
0.075
0.050
0.040
0.040
0.020
0.100
0.695
1.100
1.100
15.509
3.877
19.386
0.969
0.269
1.238
20.624
Total
Research Material & Supplies
Fertilizer
Irrigation
22
Detailed Costs of the Collaborating Institution (U of T) (Rs. Millions)
Assignments to be accomplished
Generation of proper binary plasmid AEV construct (Output 1)
Generation of proper binary plasmid CLCuD construct (Output 7)
Generation
of proper
binary
plasmid CLCuD
construct
Development
of CLCuD
Transgenic
tobacco/cotton
(Output 9)
Testing of CLCuD transgenic tobacco/cotton (Output 11)
Testing of CLCuD transgenic tobacco/cotton T1 plants (Output 12)
Testing of CLCuV transgenic tobacco/cotton T2 plants and biosafety
(Output 13-14)
Total of the collaborating institute
Research Phase
Demonstration
Year 1 Year 2 Year 3 Year 4 Total
1.800 0.000 0.000 0.000 1.800
1.000
1.300
0.000
0.000
2.300
0.000
0.000
0.900
0.000
0.000
1.000
0.000
0.000
0.900
1.000
0.000
0.000
0.550
0.500
1.050
0.000
2.800
0.000
2.200
0.000
1.550
0.450
0.950
0.450
7.500