T-DNA mediated insertional mutagenesis
T-DNA
LB RB
Wild type gene
Why T-DNA?
1.
Homologous Recombination in plants is inefficient.
2.
Active transposons do not exist in every species including Arabidopsis .
3.
Agrobacterium -mediated transformation is simpler: floral dip, leaf disk transformation.
4.
More likely to generate simpler full-length integration as compared to other methods.
5.
T-DNA insertion mainly occurs in transcriptionally active area of the genome.
6.
The mutated locus gets tagged.

Effect of tissue culture process on T-DNA tagging
Somaclonal variation
Root explant callus plant
Somaclonal variation
Root explant cocultivation callus plant
T-DNA insertion
Preliminary data
60 transformants: none of the visible mutation were linked to T-DNA.
101 regenerants (non-transformed) had 7 visible recessive mutants (identified by progeny analysis).
Suggestion by the authors: high number of mutations due to somaclonal variation rather than T-DNA insertion.

Isolation of a plant gene by T-DNA tagging:
The Plant Cell, Vol. 6, 1211-1225
Control of Arabidopsis Flower and Seed Development by the Homeotic
Gene APETALA2
K. Diane Jofuku, Bart G. W. den Boer, Marc Van Montagu and Jack K. Okamuroa

Tissue
Leaf
Stems
Seeds
Callus
Roots
1986
1986
1987
1987
1988
Cotyledons 1988
Inflorescence 1993
Inflorescence 1994 ref.
problem somaclonal variation ditto real mutants!!! Irreproducible.
somaclonal variation ditto ditto
Best procedure
Labor intensive

Seed Infection
1.
Germinating seeds were soaked in agro suspension.
2.
In 1985, a group showed that exogenous DNA is able to get into the cells of imbibing seeds of Arabidopsis and integrate into its genome.
3.
In 1987, Feldman and Marks followed it up by soaking arabidopsis seeds in
Agro suspension. They recovered several transformed seedlings.
4.
More than 14000 transformants have been generated by this method.
5.
T-DNA contained kanamycin resistant gene.
6.
57% contain single locus, 25% two linked or unlinked locus, 5% 3 locus,
5% 4 or more, 9% becoming KanS. Average number of insert/ locus= 1.5.
LB pBR322 neo pBR322 RB
Structure of inserts
10 lines were characterized: 7 were concatameric, each containing 2-5 T-DNA or parts thereof.
6/7 contained rearranged T-DNA. 6/10 were inverted repeats of RB region and 3/10 were inverted repeats of LB region. 70% contain LB-plant DNA junction or RB-plant DNA junction or both. 30% contain internal T-DNA/ plant DNA junction.

Mutants
Progeny analysis
Dominant phenotype will segregate as 3 mutant:1wild-type
Recessive phenotype will segregate as 1 mutant: 3 wild-type.
15-26% lines show visible phenotype (under normal growth condition)
Genetic redundancy in higher organisms
Majority of the mutants were recessive
Are mutants tagged?
Co-segregation analysis indicates that only 35-40% mutants are due to T-DNA insertion i.e. they are tagged.
Untagged lines
Untagged lines were used for cloning of the mutant gene by other techniques such as map based cloning technique. It has been found that all untagged lines contain short deletion in the mutant gene. This indicates abortive T-DNA integration resulting in deletion and phenotype.

Utility of T-DNA tagged population in Reverse Genetics
Have the gene but don’t know the phenotype? So design a
PCR primer in T-DNA border and another in the gene-ofinterest and identify your mutant by PCR screening. Need following:
1. Large number of T-DNA tagged mutants.
2. Random insertions
3. Pooled DNA.

Transposon tagging
Ac-Ds system: contain 11 bp terminal inverted repeat (TIR), create 8 bp target site duplication. The 4.5 kb Ac element codes for 3.5 kb mRNA for transposase.
En-Spm system: contain a 13 bp TIR and create a 3 bp target site duplication. En element is 8.3 kb long and contains 2 alternately spliced gene products, Tnp A and
Tnp D. Both are required for transposition.
Robertson’s mutator ( Mu )/ Mu DR: Very mobile, short (1.5 or 1.7 kb) in maize.
Cause high rate of mutation, somatic instability. MuDR encodes the MURA transposase required for Mu transposition and MURB, a helper protein implicated in insertion. All Mu elements share 215-bp terminal inverted repeat (TIR) sequences and the mobile Mu elements contain a highly conserved 32-bp MURA transposase binding site. Characteristic 9-bp host sequence duplications are generated during MuDR/Mu germinal insertion.

Maize Gene Discovery Project: RescueMu http://www.maizegdb.org/documentation/mgdp/education/

Tagging strategies
Random vs Targeted tagging.
Dilute somaclonal variation effect.
Use gametophytic promoters to drive transposase expression.
Proof of tagging
1. In case of excision system: phenotypic reversion.
2. Variegation in the presence of transposase
3. In case of non-excision system: co-segregation of mutant phenotype and specific element.

Classical examples:
Bz was isolated in 1984 by Ac tagging. Ac was present in very few copies.
A1 was isolated in 1985 by En and Mu tagging. Each of which were present in high copies, so a cross hybridization strategy was used to identify which En -tag or
Mu -tag contained A1 gene.
Once the tag is identified, it can be used to probe genomic library to isolate the wild-type gene.
Heterologous transposition
Ac-Ds in tobacco, rice, potato, tomato etc.
Ds or
Spm
X promoter Excision marker
Pro-transposase Pro-codA
[Pro: promoter; codA: negative selection marker]

Entrapment strategies: for the gene of unknown function
Genetrap.cshl.org/traps.htm
Enhancer Trap gene P
TIR MP marker TIR ancer enh
TIR: terminal inverted repeat
MP: minimal promoter
Promoter Trap
Native promoter
ATG marker Native ORF
Trn stop
Gene Trap
P ex1 ex2 ex3
TIR marker TIR ex4
Fusion protein Splice acceptor site
Transcription stop

Activation tagging
1.
Based on over-expression of native genes: gain-of-function.
2.
Addresses the problem of insertional mutagenesis: i) genetic redundancy ii) multiple inserts make gene isolation difficult.
iii) large number of T-DNA insertions are not linked with the mutant phenotype.
Bar selection gene
4X 35S RB Native ORF

Advantages of entrapment
1.
Can identify very low abundance transcripts. cDNA libraries do not well represent low abundance and very low abundance mRNA.
2.
Tissue specific transcripts can be identified: seedling cDNA libraries do have good representation of tissue specific transcripts.
3.
Temporally expressed transcripts can be identified: ditto problem with cDNA libraries.
4.
High frequency of promoter traps (30% in arabidopsis and similar frequencies in other species) suggests that T-DNA insertion is not random, that it is in transcriptional regions of genome.

Isolation of the tagged- or trapped gene
1. Plasmid rescue
2. Inverse PCR
3. TAIL-PCR
Gene function study of isolated unknown gene
1. Over-expression
2. Suppression
Stable transgenics i) Antisense ii) RNAi
Transient transgenics: VIGS

Applications of Gene Silencing
Antisense RNA approach: Antisense approach has been successfully used to down regulate or inhibit gene expression in E.coli
, C. elegans , D. discoideum , plants and vertebrates. Several mechanisms have been suggested based on studies:
1.
In C. elegans lin4 antisense RNA inhibits translation of the lin4 mRNA.
2.
Nuclear destabilization of the human eIF2a mRNA by an antisense transcript.
3.
Cytoplasmic destabilization of D. discoideum PSV-A mRNA by the antisense RNA
4.
Inhibition of the splicing of erbA gene transcript by antisense RNA in mammalian cells.
5.
In prokaryotes: inhibition of translational coupling by antisense RNA.
In principal, the loss of gene expression may or may not be accompanied by the loss of the respective mRNA.
Additional mechanism: Methylation of the respective gene copy in response to antisense expression.
It is unclear whether or not these mechanisms function simultaneously or function in different cell types.

Differential regulation of antisense machinery
Some genes are regulated by endogenous antisense transcript such as
D. discoideum prespore gene.
1. With the induction of antisense transcription, the half life of the mRNA is reduced from several hours to 15 min.
2. Inhibition of transcription (general) stabilizes the previously accumulated mRNA.
2. Antisense transcripts are accumulated only after the majority of the mRNA has been degraded.
These observations suggest: hybrid formation between sense and antisense and rapid degradation. It implies that sense and antisense transcripts can never accumulate simultaneously unless antisense machinery of hybrid formation and degradation is under differential control. Targets for this control would be the proteins that promote hybrid formation, proteins which resolve duplexes (helicase) and nucleases that degrade dsRNA.

Formation of dsRNA and degradation by dsRNases. In principle, an
RNA complementary to the target RNA should be able to form a duplex and if this duplex sequesters translation initiation signal or become substrate of dsRNases, inhibition of gene expression is expected. However, ineffectiveness of antisense approach has commonly been experienced. General factors that determine the effectiveness may be: physico-chemical principles that determine whether structural complementarity of folded RNAs is possible and rate at which pairing occurs and the binding pathway specificity.
Further mechanism of degradation may be a key factor in the success or failure of this technology.

PTGS for functional genomics
RNAi
Injection of dsRNA into nematodes can trigger specific (homologous) RNA degradation. RNAi has been utilized to study 4000 genes in C. elegans. In plants, dsRNA and sshairpin RNA have similar effect.
The predicted RNA structure and efficacy of gene-silencing constructs.
(Ref: Wesley et al., 2001, Plant J. 27: 581-590)
Constructs, controlled by Ubi1 promoter, silencing GUS in rice. Thick lines indicate a
560 nt GUS sequence; grey lines indicate non-GUS sequences; dashed grey lines indicate intron-junction sequences left after splicing; and short lines within the stem of hairpin structures indicate base pairing. Numbers in PTGS column indicate the percentage of plants showing GUS silencing; n = number of plants in each treatment.

From Ref: Wesley et al.
(Ref: Wesley et al., 2001, Plant J. 27: 581-590)
Using hpRNA constructs, we have obtained silenced plants for every gene that we targeted, irrespective of whether it was a viral gene, transgene or endogenous gene, and the silencing appears to be uniform within tissues in which the hpRNA is expressed. With ihpRNA constructs the efficiency averaged about 90%, and arms of 400 ±800 nt appear to be stable and effective. High levels of silencing were obtained with constructs having unmatched arm lengths, with arms as long as 853 nt or as little as 98 nt, and with arm sequences derived from coding, 3’ or 5’ untranslated regions of the target gene. These results suggest that ihpRNA constructs will be effective in a wide range of circumstances, and augur well for the generic use of the technology. The silencing was much more profound with ihpRNA constructs than either anti-sense or co-suppression constructs; some ihpRNA transformants were close to exhibiting a complete knockout of the target endogenous gene.
However, most of the ihpRNA plants showed dramatically reduced but detectable levels of target gene activity.

Virus induced gene silencing
RNA mediated defense mechanism is related to PTGS in which RNA are specifically degraded if they are similar in sequence to a transgene, especially if it is designed to produce dsRNA. This process is mechanistically similar between plants, animals and fungi. This approach can be applied to carry out Gene silencing of the native gene and study their function.
Procedure: Agro-infiltration
Vectors based on
TMV, PVX,
TGMV and
TRV.

Problems:
1. VIGS phenotype is superimposed on, and complicated by, chlorosis, leaf distortion, and necrosis.
2. Virus is not able to invade every cell, such that cells in which the target gene is not silenced may obscure VIGS phenotype.
3. Viruses commonly used in VIGS such as TMV, PVX and TGMV don't infect growing points of plant and therefore remain uninformative about genes effecting identity and development of plant tissue.
4. Variation between experiments in proportion of plants showing VIGS phenotype.
Better vector (Tobacco Rattle Virus): TRV is better because it induces mild symptoms, infects large area of adjacent cells and infects growing points.
TRV persists in ‘recovered’ plants. TRV is shown to be successful in
Nicotiana and Tomato. Its potential lies in potato and oats as well.

H
Flank
H
Plasmid Rescue
P1
E pUC19
H
P2
E
P1 pUC19
H
Restriction
E pUC19
Ligation
P1
P2
P2
Flank pUC19 pUC19
E
Flanking genomic seq.
P1 and P2 = vector sequences
Transform into E. coli

H
P1
Inverse PCR
P1
H
P2
H
HindIII cut followed by ligation
P2 PCR primer
H
H
Left flank
Right flank
Cloning and sequencing

AD
TAIL-PCR (Thermal Asymmetric Interlaced PCR)
P1 P2
LP1-AD or RP1-AD reaction
Target + non-target amplification
LP2-AD or RP2-AD reaction
LP3-AD or RP3-AD reaction
Target product
AD