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)