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