1. Identification of Tomato Yellow Leaf Curl
Virus (TYLCV) Host Resistance Genes
Amer Wazwaz
Aug 30th 2012

2. Large numbers of plant genes with NO defined functions
Powerful in silico techniques have been developed for the
analysis of genome sequence information
But gene function must always be verified in vivo using
genetic analysis

3. Reverse Genetics
a powerful tool that establishes a direct link between the
biochemical function of a gene product and its role in vivo
RNA-MEDIATED
INTERFERENCE
CHEMICAL MUTAGENESIS
VIRUS-INDUCED GENE
SILENCING
INSERTIONAL
MUTAGENESIS
FAST NEURON
MUTAGENESIS

4. VIRUS-INDUCED GENE SILENCING
Cloning a 200–1300 bp cDNA fragment from a plant gene of
interest into a DNA copy of the genome of an RNA-virus
Transfecting the plant with this construct using Agrobacterium
Double-stranded RNA from the viral genome, including sequence
from the gene of interest, is formed during viral replication
The double-stranded RNA molecules are degraded into siRNA
molecules by the plant Dicer-like enzymes
Limited only by the host range of the virus used, TRV and ALSV
are the most common vectors

5. RNA-MEDIATED INTERFERENCE
similar to VIGS, but it is heritable
DNA construct that produces either s.s or d.s RNA
complementary to the gene of interest is introduced
into a cell
It activates the RNA silencing pathway and degrades
some or all of the transcripts from the gene of interest
Through amiRNA using promoters that are temporally or
spatially specific or inducible by exogenous factor
Partial loss of function can be achieved

6. INSERTIONAL MUTAGENESIS
Disruptions in target genes of interest through genome(wide insertions (insertional knockouts
Plants carrying an insertion in a gene of interest can be
identified by screening the population with PCR using one
gene-specific primer and one insertion based primer
Result in a total loss of function and the insertion can be
easily followed using PCR
Phenotypes may not be obvious if the gene function is
redundant
Insertions in essential genes will typically result in lethality

7. FAST NEURON MUTAGENESIS
Bombardment is used to generate deletions & chromosome
rearrangements of various sizes randomly in the genome
Seeds are mutagenised with fast neutron radiation and
deletions are identified by PCR using primer sequences
that flank the gene of interest
Laborious because of the number of plants that must be
screened
Has limitations in terms of the sizes of deletions that can
be recovered

8. CHEMICAL MUTAGENESIS
Chemical mutagenesis induced point mutations in DNA in all
species in which it has been tested
Mutations induced using these mutagens are distributed in
the genome randomly
As point mutations are less damaging, a high degree of
saturation can be achieved in a mutant population
facilitating examining of gene function on a genomic level
Unlike other reverse genetics techniques, chemical
mutagenesis can result in either loss-of-function or gainof-function mutations

9. Plant resistance to viruses is the outcome of interconnecting
gene networks and signaling pathways leading to inhibition of
virus replication and/or movement
These gene and protein networks have been revealed by largescale microarray analyses and by protein-protein interaction
studies, usually using the yeast two-hybrid system
~70 different genes preferentially expressed in R plants

10. Two inbred tomato lines
(line 902 is resistant to the virus (R
(line 906-4 is susceptible (S
R and S can be distinguished by a single
nucleotide polymorphism (SNP) found in hsp70 gene
cDNA libraries from the S and R genotypes
were prepared and screened for genes preferentially
(expressed in R tomato as described (Eybishtz et al. 2009

11. Silencing of the Hexose transporter
LeHT1 gene using a VIGS vector
cDNA encoding a fragment of the hexose transporter gene
(LeHT1 (518 to 1038
Fragment was T/A cloned into pDrive vector then excised and
ligated to TRVII vector using XbaI & KpnI
Plasmid was introduced into Agrobacterium by electroporation
Agrobacterium cells containing TRV-Hex and TRVI were
cultured in YEB medium for 48 h at 28C
Agroinoculated into 30 R and 30 S tomato seedlings at the
4-6 leaf stage

12. Inoculation of tomato plants with
different viruses
Five days after LeHT1 silencing
silenced and 20 non-silenced plants were inoculated 20
with TYLCV
Caging plants with viruliferous whiteflies for 3 days
(~ 30 insect /plant), kept at 24–27C
Plants were grown in a greenhouse at 18–24C, 16 h
light
Then, plants were mechanically inoculated with Bean
Dwarf Mosaic Virus (BDMV), Cucumber Mosaic Virus
((CMV) and with Tobacco Mosaic Virus (TMV

13. PCR and semi-quantitative PCR
• Semi-quantitative PCR analyses of LeHT1 expression in notsilenced susceptible (So:0) and resistant (Ro:0) plants, and
silenced resistant plants (Ro:TRV-Hex) 14 days after TRVHex
treatment
β-actin was used as an internal control in the same samples

14. Detection of small RNA related to silencing
the hexose transporter gene LeHT1
Northern blot-based detection of a 24-nucleotide-long RNA present
in LeHT1-silenced plants (Ro:TRV-Hex), but not in not silenced R
(Ro:0) plants
C 21-nucleotide-long primer used to label the LeHT1 probe, utilized
as size marker

15. TYLCV amounts and appearance of TYLCV
inoculated in virus-infected LeHT1-silenced
(Ri:TRV-LeHT1) and not-silenced (Ri:0) R plants
Comparison of TYLCV amounts estimated by semi-quantitative
PCR, 21 days after inoculation

16. TYLCV amounts and appearance of TYLCV
inoculated in virus-infected LeHT1-silenced
(Ri:TRV-LeHT1) and not-silenced (Ri:0) R plants
Growth inhibition and mild symptoms of Ri:TRV- LeHT1 plants
compared with infected not silenced R (Ri:0) and S (Si:0) plants

17. Visualization of TYLCV in infected leaves
by in situ hybridization
d.a.i, using a tetramethyl rhodamine labeled viral DNA probe 21

18. PCD-induced necrosis in TYLCV infected LeHT1silenced R plants
Necrosis on stem and petiole of TYLCV-infected LeHT1-silenced R
plants (Ri:TRV-Hex); no necrosis on not-silenced infected R (Ri:0)
and S (Si:0) plants, and on infected LeHT1-silenced S plants

19. Detection of H2O2
Oxidative
burstrelated
damages
detected
with DAB
Plants of the same age stained and not stained with DAB
Release of H2O2 is visualized as a reddish brown precipitate

20. Necrosis in LeHT1silenced R plants
(Ri:TRV-Hex)
infected
with
Bean dwarf mosaic
virus (BDMV),
Cucumber mosaic
virus (CMV), and
Tobacco mosaic
virus (TMV)
Ri:0 are virusinfected not
silenced
plants

21. Conclusion
The amounts of LeHT1 transcripts in R tomato was
about 120 times higher than in the S plants
The expression of the gene was reduced by
approximately 60-folds
The infected silenced plants contained
approximately 30,000 times more virus than
the untreated Ri plants

22. Conclusion
Many of the 70 genes were annotated as membranal,
suggesting that the mechanism of resistance might
involve: virus entry in the cell and/or cell-to-cell
trafficking
A second line of resistance was uncovered in the
silenced R plants in form of programmed cell death
((PCD
Not only upon inoculation of TYLCV, but also after
inoculation of additional DNA (Bean dwarf mosaic virus)
and RNA viruses (Cucumber mosaic virus and Tobacco
(mosaic virus