2. 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

3. Outline
• Introduction
• Objectives
• Materials & Methods
• Results
• Conclusion

4. Introduction
TYLCD
Tomato total world production 153 million ton annually
Around 2 billion $ losses due to TYLCD annually (FAOSTAT, 2009)

5. TYLCV Symptoms
Shoots become distorted
with yellowing
Leaflets are reduced in
size and curled

6. TYLCV Vector
1–2 mm in length
Wide host range
Feed and lay eggs on
leaves undersurface
Whitefly Bemisia tabaci

7. TYLCV Structure
Electron
microscope image
of TYLCV

8. TYLCV Resistance
Breeding program to produce
TYLCV-resistance tomato lines
30 genes are preferentially over-expressed
in R line, of them is the
Elongation factor1-alpha gene.
Would the resistance collapse upon silencing EF1α?

9. Objectives
• Silencing the Elongation factor1-alpha gene in
TYLCV-resistant tomato plants
• Then, perceiving if the resistance will
collapse after silencing the gene

10. Materials & Methods
TYLCV infected susceptible (S) and resistant (R) tomato
plants in the field. Source: (Eybishtz et al., 2009)

11. Materials & Methods
SOL
Primers
DNA
PCR
PCR
products
TOPO II
E.coli

12. Materials & Methods Transformed
Bacteria

13. Virus-Induced Gene Silencing (VIGS)
TRV
Tobacco Rattle Virus

14. Silencing Signal

15. Monitoring Timeline

16. Results
M: 100bp ladder
SNP-PCR products of five R and five S individual plants
in hsp70 gene

17. PCR products of the designed
silencing insert
M: 100bp ladder NTC: non-template control
Amplified from five different R plants

18. 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)

19. Approximate histogram of EF1α gene
silencing
Averages of the first three weeks after TYLCV inoculation in
comparison with a non-silenced control plant

20. First week after TYLCV inoculation
qRT-PCR curves of representative sample replicates from EF1α-silenced R
plants and non-silenced control plant replicates

21. Second week after TYLCV inoculation
qRT-PCR curves of representative sample replicates from EF1α-silenced R
plants and non-silenced control plant replicates

22. Third week after TYLCV inoculation
qRT-PCR curves of representative sample replicates from EF1α-silenced R
plants and non-silenced control plant replicates

23. 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

24. Silenced and non-silenced R plants
At fifth week after
TYLCV-inoculation

25. Conclusion
The results display the collapse of TYLCV
resistance in R line tomato plants upon
EF1α gene silencing

26. Recommendations
Since 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