TRANSGENE SILENCING

Presented By,

POORANACHITHRA M

Ist M.Tech Biotechnology

Introduction:

Gene silencing (GS) is defined as a molecular process involved in

the down regulation of specific genes, and probably evolved as a

genetic defense system against viruses and invading nucleic acids.

In genetically modified plants, the introduced transgenes are

sometimes not expressed. They can be silenced.

Transgenes can also cause the silencing of endogenous plant genes

if they are sufficiently homologous, a phenomenon known as

transgene silencing or co-suppression.

Early Findings Of GS In Plants:

First discovered in plants by

R. Jorgensen in1990

When Jorgensen introduced a

re-engineered gene into petunia that

had a lot of homology with an

endogenous petunia gene, both

genes became suppressed!

Cont.…

Jorgensen tried to obtain transgenic petunias with greater amounts of anthocyanin pigments, by amplifying the gene activity of chalcone synthase.

Instead of obtaining deeper purples in the petals, white or chimeric flowers were produced.

The transgene was not expressed, and ended up silencing a homologue endogenous gene.

The phenomenon, named “co-suppression”

Wild-type petunia

producing purple

anthocyanin pigments

Chalcone synthase (CHS) is the enzyme at the start of the

biosynthetic pathway for anthocyanins

Anthocyanins

Chalcone synthase

(CHS)

Sense RNA

Antisense RNA

Sense construct:

PRO ORF

Endogenous genemRNA

Transgene

PRO ORF

mRNA

Protein translated

mRNA

mRNA

Extra protein translated

Antisense construct:

PRO

ORF

TransgeneSense-antisense duplex

forms and prohibits translation

Hypothesis: sense RNA production enhances pigmentation and antisense RNA production blocks pigmentation

Surprisingly, both antisense and sense gene constructs can inhibit pigment

production

Plants carrying CHS transgene

CaMV 35S pro : CHS CaMV 35S pro : CHS

Sense Antisense

OR

Silencing of transgenes:

Transgenes introduced into plants are frequently silenced by the

siRNA pathway

Silencing can be triggered by:

# Very high levels of gene expression

# dsRNA derived from transgenes

# Aberrant RNAs encoded by transgenes

Transgenes are silenced post-transcriptionally and transcriptionally

Routes Of GS In Plants:

Transcriptional gene silencing

siRNA mediated silencing

MicroRNA silencing

Virus induced gene silencing

Transcriptional gene silencing:

DNA methylation and chromatin remodeling play a major role in

transcriptional gene silencing (TGS), blocking gene expression.

In TGS, silenced transgenes coding regions and promoters are

densely methylated.

DNA methylation promotes protein binding that recognizes

methylated cytosine, leading to chromatin remodeling thus avoiding

the binding of transcription factors.

Co-suppression is a consequence of siRNA production

PRO ORF

Wild-typemRNA

mRNA

Protein translated

Endogenous gene

Sense RNA

Sense construct

Co-suppressed transgenic

PRO ORF

Co-suppression

PRO ORF

Endogenous genemRNA

siRNA produced

AGO

AGO AAAn

AGO AAAn

Most siRNAs are produced from transposons and repetitive DNA:

Most of the cellular siRNAs are derived from transposons and other repetitive sequences. In Arabidopsis, as shown above, there is a high density of these repeats in the pericentromeric regions of the chromosome.

Abundance of small

RNAsAbundance of transposon/

retrotransposons

Chromosome

Centromere

microRNAs – miRNAs:

miRNAs are thought to have evolved from siRNAs, and are

produced and processed somewhat similarly

Plants have a small number of highly conserved miRNAs, and a

large number of non-conserved miRNAs

miRNAs are encoded by specific MIR genes but act on other

genes – they are trans-acting regulatory factors

miRNAs in plants regulate developmental and physiological

events

MIR genes are transcribed into long RNAs that are processed to miRNAs

miRNAs are encoded by MIR genes.

The primary miRNA (pri-miRNA)

transcript folds back into a double-

stranded structure, which is

processed by DCL1.

The miRNA* strand is degraded

DCL

3'5' miRNA

miRNA*

3'5' pri-miRNA

miRNA

MIR gene

mRNA target

microRNAs - miRNAS

DCL

MIR gene

RNA Pol

AGO

RNA PolmRNA

AGO

AGOAAAn

AAAn

AAAnAAAn

microRNAs slice mRNAs or interfere with their translation

Model for RNAiBy “Dicer”

21-23 nt RNAs

Fig. 16.39, 3rd Ed.

ATP-dependentHelicase or Dicer

Active siRNA complexes = RISC

- contain Argonaute instead

of Dicer

Very efficient process because many small interfering RNAs (siRNAs) generated from a larger dsRNA.

Virus Induced Gene Silencing (VIGS):

Virus induced gene silencing is a technique designed to suppress

gene expression and study gene function in plants.

If viral transgenes are introduced and silenced, the

posttranscriptional process also prevents homologous RNA viruses

from accumulating; this is a means of generating virus-resistant

plants.

Applications:

Plant protection to multiple pathogens (viruses, bacteria) & Production of virus resistant plants through genetic transformation.

Food quality modification such as the reduction of caffeine levels in coffee beans and Increase the nutritional value of corn protein and tomatoes.

silencing of specific metabolic pathways (lignin synthesis, ethylene, allergens, caffeine and others)

Developmental and reproductive trait alteration in plants (induced male sterility and self-compatibility).

GS & Plant Disease Resistance:

Virus disease resistance in plants may be achieved by several approaches, including

# Coat protein mediated protection,

# Antisense RNA,

# Replicase mediated protection,

# PTGS / RNAi,

# Pathogen derived resistance (PDR)

Currently, RNAi is the major strategy in plant transformation for virus resistance.

Citrus tristeza virus (CTV) - resistant citrus plants, expressing the

coat protein of the virus has been obtained.

Resistant transgenic plums containing the silenced Plum pox virus

(PPV) coat protein gene.

Crown gall resistant apple tree roots were obtained by

transformation with transgenes designed to express double-stranded

RNA from the iaaM and ipt genes.

Thank you…!!