Identification of the PlantSystemic RNA Silencing Signal

2008 Summer HHMI Program

Simon JohnsonMentors: Dr. James C. Carrington – Professor and Director

Dr. Kristin Kasschau – Senior Scientific Coordinator Dr. Atsushi Takeda – Postdoctoral Researcher

Motivation:

-RNA silencing is important to almost all eukaryotic organisms

-RNA silencing is involved in gene regulation, development, and antiviral defense

-In plants, RNA silencing provides the primary defense against viral assault – first biologically significant role characterized

-We believe our experimental setup is applicable to multiple silencing pathways

Viral Infection in Host Plants

-Viral Entry

-Viral cell-to-cell movement through mesophyll cells

-Entry into and movement through phloem, resulting in systemic infection

In a plant with no defenses:

VIRUSVIRUS

SYSTEMIC SYSTEMIC INFECTIONINFECTION

PHLOEM = VasculaturePHLOEM = Vasculature

MESOPHYLL = Surrounding Leaf TissueMESOPHYLL = Surrounding Leaf Tissue

Plant Innate Immune Defense

-Molecular defense against viruses – Antiviral RNA Silencing

-Highly evolved defense strategy

-Halts the flow of genetic information and degrades viral genome

… plants are not defenseless:

RNA

Protein

Translation

BACKGROUND – RNA Silencing

RNA Silencing is:

-RNA mediated

-Homology dependent

-Potent and specific

-Short RNA molecules guide cleavage of their complementary sequence

RNA

Protein

Translation

ANTIVIRAL DEFENSE – RNA SILENCING

VIRUS

VIRUS FREEVIRUS FREE -Following viral entry, antiviral RNA silencing is initiated

-This response produces a signal transported from cell to cell and into vasculature

-The signal is thought to move faster than the virus, providing a systemic resistance against the pathogen

Suppression Suppression SignalSignal Suppression Suppression

SignalSignal

Suppression Suppression SignalSignal

ANTIVIRAL RNA SILENCING3-Phase Model Proposed by Dr. James Carrington et al.:

INITIAL PHASE:

•Structures in viral genome cleaved by Dicer-Like (DCL) enzymes into small interfering RNA (siRNA) duplexes

•The RNA silencing signal is amplified through a combination of target cleavage, siRNA primed polymerization, and subsequent cleavage by DCL

AMPLIFICATION PHASE:

The Systemic Phase

The molecular identity of the systemic signal is not known-We hypothesized that the signal is in the form of siRNA duplexes

-siRNA AGO complexes are thought to be an alternative possibility

The goal of my HHMI summer research is to determine the molecular identity of the systemic

signal as moves from phloem to mesophyll

SYSTEMIC:

A silencing signal is transported into neighboring cells and plant vasculature, resulting in a systemic resistance.

ANTIVIRAL RNA SILENCINGdsRNA or foldback

siRNA Duplexes

siRNA/AGOComplexes

Successful SilencingSuccessful Silencing

AGOAGO

AGOAGO

Further Clarification:

-Linear process within each cell. Each intermediate component is necessary

-At some point in the pathway, a component is also transported from cell to cell

-RNA silencing picks up from this point in the receiving cell

DICERDICER

CELL 1 CELL 2

Signal?Signal?

Signal?Signal?

Successful SilencingSuccessful Silencing

AGOAGO AGOAGO

Viral Suppressors of RNA Silencing

•Many viruses have evolved suppressors of antiviral RNAi

•Blocking RNA silencing, these suppressors restore infectivity

•These suppressors are diverse in structure and method of suppression

VIRUSVIRUS

RESTORED RESTORED INFECTIVITYINFECTIVITY

Suppression Suppression SignalSignal

Viral Suppressors Block RNAiViral Suppressors Block RNAi

Viral Suppressors of RNA Silencing

Viral Suppressorsof RNA SilencingViral Suppressorsof RNA Silencing

Two distinct suppressors are important here:

•siRNA duplex binding suppressor P19

•siRNA/AGO disrupting suppressor Fny2b

•We are using these suppressors to study the silencing signal

•Our system also uses a stable initiator of RNA silencing

•By interrupting RNA silencing at distinct steps, these suppressors allow us to examine the signal identity

Experimental SetupdsRNA or foldback

siRNA Duplexes

siRNA/AGOComplexes

P19P19

Fny2bFny2b

Successful SilencingSuccessful Silencing

AGOAGOAGOAGO

DICERDICER

Experimental Model – dsRNA Construct

•Our stable initiator of silencing is an engineered gene

•Following transcription, the intron is spliced out and the complementary fragments form a double stranded RNA structure

•This is cleaved by DCL4 and enters the antiviral silencing pathway

Experimental Model – dsRNA Construct

•According to our model, these siRNA enter directly into the systemic phase

Experimental Model – dsRNA Construct

•The target of these construct derived siRNA are mRNA of a subunit of an enzyme involved in chlorophyll production

•The result is chlorotic staining of affected areas

Above Left: Wildtype (Col-0) ArabidopsisAbove Right: SUL under a phloem specific promoter

The chlorotic staining provides for visual determination of RNA silencing functionality and

successful RNA silencing signal transport(Bleached Cell = RNA silencing successful)(Green Cell = no RNA silencing)

Right – GUS produced in PhloemLeft – Control (wildtype)

Experimental Model – Tissue Specific Expression

•Tissue specific promoters control the location of expression

•The initiator CH42 is expressed in phloem (see right)

•The suppressors are expressed in either phloem or mesophyll

•The resulting phenotypes provide visual evidence for signal identity

Experimental Model – Tissue Specific ExpressionFor comparison:

A protein expressed in phloem compared to SUL expressed in phloem

The RNA silencing signal is spread to neighboring cells

Experimental Model

Initiation of the RNAi Pathway

siRNA Duplexes

siRNA/AGOComplexes

PHLOEM MESOPHYLLPHLOEM MESOPHYLLEach possible combination has been produced, but 2 cases distinguish the proposed signal identities:

1)siRNA/AGO interfering proteins in phloem

2)Duplex binding proteins in mesophyll

This diagram give a represents our system

DUPLEX BINDING (P19)

DUPLEX BINDING (P19)

siRNA/AGO INTERFERING (Fny 2b)

siRNA/AGO INTERFERING (Fny 2b)

siRNA/AGO Interfering Protein Fny2b in Phloem•RNA suppression in the

phloem will be blocked regardless of signal identity; the phloem will remain green

•If siRNA/AGO complexes are the signal, the signal will be suppressed

•Mesophyll will also remain green

•If siRNA duplexes are the signal, transport will be successful

•Mesophyll will become photobleached

CASE 1:

Initiation of the RNAi Pathway

siRNA Duplexes

siRNA/AGOComplexes

Fny2b

PHLOEM MESOPHYLLPHLOEM MESOPHYLL

SuccessfulRNAi

siRNA Duplexes

siRNA/AGOComplexes

Successful RNAi

Duplex Binding Protein P19 in Mesophyll•RNA silencing in the phloem

will NOT be blocked, regardless of signal identity; phloem will be photobleached

•If siRNA/AGO complexes are the signal, the signal will NOT be suppressed

•Mesophyll will also become photobleached

•If siRNA duplexes are the signal, transport will be blocked

•Mesophyll will remain green

CASE 2:

Initiation of the RNAi Pathway

siRNA Duplexes

siRNA/AGOComplexes

PHLOEM MESOPHYLLPHLOEM MESOPHYLL

SuccessfulRNAi

siRNA Duplexes

siRNA/AGOComplexes

Successful RNAi

P19P19

Results:Our Preliminary Results Suggest that siRNA Duplexes DO

NOT Carry the Systemic Signal as it Exits Phloem

CASE 1: siRNA/AGO Suppressors in Phloem

-These suppressors were found to block the systemic signal

-This indicates that the signal is formed downstream of siRNA duplexes

-The function lf Fny2b must be verified to support this conclusion

SUC2:dsCH42 ♀ X SUC2:Fny2bHA ♂ SUC2:dsCH42 ♀ X SUC2:GUSHA ♂

GU

S C

on

tro

l

Co

l-0

(W

T)

SU

C2

:ds

CH

42

♀ X

S

UC

2:G

US

HA

♂

Fn

y2

bH

A

Co

ntr

ol

Co

l-0

(W

T)

SU

C2

:ds

CH

42

♀ X

S

UC

2:F

ny

2b

HA

♂

Results:Case 2: P19 in Mesophyll

-Mesophyll produced duplex binding suppressors appear to restrict photobleaching to phloem

-Our current model cannot account for this and the 2b in Phloem suppression simultaneously

-We must verify this phenotype

-One possible explanation is that the signal must be amplified in each receiving cell; Alternatively, Fny2b may have functions not yet characterized

SUC2:dsCH42 ♀ X CAB3:P19 ♂

P19

HA

Co

ntr

ol

Co

l-0

(WT

)

SU

C2:

dsC

H42

♀ X

CA

B3:

P19

HA

♂

SUC2:dsCH42 ♀ X SUC2:GUSHA ♂

GU

S C

on

tro

l

Co

l-0

(WT

)

SU

C2:

dsC

H42

♀ X

SU

C2:

GU

SH

A

♂

SUC2:dsCH42

Results:

-siRNA/AGO interfering suppressor 2b in mesophyll caused an apparent vein-restricted bleaching phenotype (left)

-siRNA duplex binding protein P19 in phloem caused complete suppression of photobleaching (right)

SUC2:dsCH42 ♀ X CAB3:Fny2bHA ♂

P19

HA

Co

ntr

ol

Co

l-0

(WT

)

SU

C2:

dsC

H42

♀ X

SU

C2:

P19

HA

♂

Fn

y2b

HA

Co

ntr

ol

Co

l-0

(WT

)

SU

C2:

dsC

H42

♀ X

CA

B3:

Fn

y2b

HA

♂

Control Crosses

SUC2:dsCH42 ♀ X SUC2:P19HA ♂

-These results were expected regardless of signal identity

SUC2:dsCH42 ♀ X SUC2:GUSHA ♂

GU

S

Co

ntr

ol

Co

l-0

(W

T)

SU

C2:

dsC

H42

♀

X

SU

C2:

GU

SH

A

♂

dsRNA or foldback

siRNA Duplexes

siRNA/AGOComplexes

Successful SilencingSuccessful Silencing

AGOAGO

DICERDICER

AGOAGO

P19

Fny2b

Discussion:

-These results are preliminary. We are currently selecting high expression homozygous single copy lines for our final results

-We are also:

>Testing the effects of growing conditions on the photobleaching phenotypes (to verify that the vein-restricted phenotype is not a result of stress)

>Verifying Fny2b function

>Checking the possibility that the construct itself is being silenced

>This information will allow us to analyze our findings

Conclusions:-If we can verify Fny2b function:

(a)siRNA duplexes do not carry the systemic signal from phloem to mesophyll

-If we can verify the vein restriction phenotype of mesophyll driven P19

(a)Downstream of signal movement into mesophyll cells, siRNA duplex production is needed for silencing to occur in these cells

(a) This may indicate that amplification of the signal is needed after the signal has been transported

dsRNA or foldback

siRNA Duplexes

siRNA/AGOComplexes

Successful SilencingSuccessful Silencing

AGOAGOAGOAGO

DICERDICER

Amplification of Signal?

siRNA/AGOComplexes AGOAGO

AGOAGO

siRNA Duplexes

Signal Movement

Thank You:

Howard Hughes Medical InstituteCollege of Science

Cripps Scholarship Fund

Dr. James C. Carrington

Dr. Kevin Ahern

Dr. Atsushi Takeda

Dr. Kristin Kasschau

The Carrington Lab