Amer MBT's Defense
-
Upload
amer-t-wazwaz -
Category
Documents
-
view
141 -
download
0
Transcript of Amer MBT's Defense
The Joint Biotechnology Master Program
Collapse of Resistance to Tomato Yellow Leaf Curl
Virus in Tomato upon Silencing the Elongation factor
1-alpha Gene
Amer Wazwaz
Supervisor: Dr. Omar Darissa
December 6th 2013
Outline
• Introduction
• Objectives
• Materials & Methods
• Results
• Conclusion
Introduction
TYLCD
Tomato total world production 153 million ton annually
Around 2 billion $ losses due to TYLCD annually (FAOSTAT, 2009)
TYLCV SymptomsShoots become distorted
with yellowingLeaflets are reduced in
size and curled
TYLCV Vector
Wide host range
1–2 mm in length
Feed and lay eggs on leaves undersurface
Whitefly Bemisia tabaci
TYLCV Structure Electron microscope image of TYLCV
TYLCV Resistance
30 genes are preferentially over-
expressed in R line, of them is the
Elongation factor1-alpha gene.
Would the resistance collapse upon silencing EF1α?
Breeding program to produce
TYLCV-resistance tomato lines
Objectives
• Silencing the Elongation factor1-alpha gene in TYLCV-resistant tomato plants
• Then, perceiving if the resistance will collapse after silencing the gene
Materials & Methods
TYLCV infected susceptible (S) and resistant (R) tomato plants in the field. Source: (Eybishtz et al., 2009)
Materials & Methods
SOL
Primers
DNA
PCR
PCR products
TOPO II
E.coli
Materials & Methods Transformed Bacteria
Virus-Induced Gene Silencing (VIGS)
TRV Tobacco Rattle Virus
Silencing Signal
Monitoring Timeline
Results
M: 100bp ladder
SNP-PCR products of five R and five S individual plants
in hsp70 gene
PCR products of the designed silencing insert
M: 100bp ladder NTC: non-template control
Amplified from five different R plants
BLAST @ NCBI
99% identity of the cloned fragment of EF1α gene with the
corresponding exon of the same gene of L. esculentum
(accession number X53043.1)
Approximate histogram of EF1α gene silencing
Averages of the first three weeks after TYLCV inoculation in comparison with a non-silenced control plant
First week after TYLCV inoculation
qRT-PCR curves of representative sample replicates from EF1α-silenced R plants and non-silenced control plant replicates
Second week after TYLCV inoculation
qRT-PCR curves of representative sample replicates from EF1α-silenced R
plants and non-silenced control plant replicates
Third week after TYLCV inoculation
qRT-PCR curves of representative sample replicates from EF1α-silenced R plants and non-
silenced control plant replicates
Semi-quantitative PCR
WF: non-silenced control plant with TYLCV inoculation
6 EF1α-silenced plants and a non-silenced control plant at the fifth week after
TYLCV inoculation, in reference to β-actin as a house keeping gene
Silenced and non-silenced R plants
At fifth week after TYLCV-inoculation
Conclusion
The results display the collapse of TYLCV
resistance in R line tomato plants upon EF1α
gene silencing
RecommendationsSince TYLCV resistance genes are generally
hierarchically organized in a network
Future work should aim at localizing the position of EF1α within a such network
This could be achieved by studying the effects of EF1α-silencing on other genes’ expression
within the network