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Identification of Tomato Yellow Leaf Curl Virus (TYLCV) Host Resistance Genes
Amer Wazwaz
Aug 30th 2012
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
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
INSERTIONAL MUTAGENESIS
CHEMICAL MUTAGENESIS FAST NEURON MUTAGENESIS
VIRUS-INDUCED GENE SILENCING
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
RNA-MEDIATED INTERFERENCE
similar to VIGS, but it is heritableDNA 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
INSERTIONAL MUTAGENESISDisruptions 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
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
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 gain-of-function mutations
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 large-scale microarray analyses and by protein-protein interaction studies, usually using the yeast two-hybrid system ~70 different genes preferentially expressed in R plants
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 singlenucleotide polymorphism (SNP) found in hsp70 gene
cDNA libraries from the S and R genotypeswere prepared and screened for genes preferentially
expressed in R tomato as described (Eybishtz et al. 2009)
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
Inoculation of tomato plants with different viruses
Five days after LeHT1 silencing20 silenced and 20 non-silenced plants were
inoculated 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)
PCR and semi-quantitative PCR
• Semi-quantitative PCR analyses of LeHT1 expression in not-silenced 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
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
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
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
Visualization of TYLCV in infected leaves by in situ hybridization
21 d.a.i, using a tetramethyl rhodamine labeled viral DNA probe
PCD-induced necrosis in TYLCV infected LeHT1-silenced 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
Detection of H2O2
Plants of the same age stained and not stained with DAB Release of H2O2 is visualized as a reddish brown precipitate
Oxidative burst-related damages detected with DAB
Necrosis in LeHT1-silenced R plants
(Ri:TRV-Hex) infected
with
Bean dwarf mosaic virus (BDMV),
Cucumber mosaic virus (CMV), and Tobacco mosaic
virus (TMV)
Ri:0 are virus-infected not
silencedplants
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
ConclusionMany 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 afterinoculation of additional DNA (Bean dwarf mosaic virus) and RNA viruses (Cucumber mosaic virus and Tobacco
mosaic virus)