2008 Summer HHMI Program
Identification of the Plant
Systemic RNA Silencing
Signal
Simon Johnson
Mentors: 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
SYSTEMIC
INFECTION
In a plant with no defenses:
-Viral Entry
VIRUS
-Viral cell-to-cell movement
through mesophyll cells
-Entry into and movement
through phloem, resulting in
systemic infection
PHLOEM = Vasculature
MESOPHYLL = Surrounding Leaf Tissue
Plant Innate Immune Defense
… plants are not defenseless:
-Highly evolved defense
strategy
-Halts the flow of genetic
information and degrades
viral genome
RNA
Translation
-Molecular defense against
viruses – Antiviral RNA
Silencing
Protein
RNA
Silencing by
RNA
Cleavage or
Translational
Repression
BACKGROUND – RNA Silencing
RNA Silencing is:
-RNA mediated
Translation
-Homology dependent
RNA
-Potent and specific
-Short RNA molecules guide
cleavage of their
complementary sequence
Protein
RNA
Silencing by
RNA
Cleavage or
Translational
Repression
ANTIVIRAL DEFENSE – RNA SILENCING
VIRUS FREE
Suppression
Signal
Suppression
Signal
VIRUS
-Following viral entry, antiviral
RNA silencing is initiated
-This response produces a
signal transported from cell to
cell and into vasculature
Suppression
Signal
-The signal is thought to
move faster than the virus,
providing a systemic
resistance against the
pathogen
ANTIVIRAL RNA SILENCING
3-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
AMPLIFICATION PHASE:
•The RNA silencing signal is amplified through a combination of
target cleavage, siRNA primed polymerization, and subsequent
cleavage by DCL
The Systemic Phase
SYSTEMIC:
A silencing signal is transported
into neighboring cells and plant
vasculature, resulting in a
systemic resistance.
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
ANTIVIRAL RNA SILENCING
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
dsRNA or
foldback
DICER
Signal?
siRNA
Duplexes
Signal?
siRNA/AGO
Complexes
AGO
AGO
AGO
Successful Silencing
CELL 1
AGO
Successful Silencing
CELL 2
Viral Suppressors of RNA Silencing
•Many viruses have evolved
suppressors of antiviral
RNAi
•Blocking RNA silencing,
these suppressors restore
infectivity
RESTORED
INFECTIVITY
VIRUS
Suppression
Signal
•These suppressors are
diverse in structure and
method of suppression
Viral Suppressors
Block RNAi
Viral Suppressors of RNA Silencing
Two distinct suppressors are
important here:
Viral Suppressors
of RNA Silencing
•siRNA duplex binding
suppressor P19
•siRNA/AGO
disrupting
suppressor Fny2b
Experimental Setup
•We are using these
suppressors to study the
silencing signal
dsRNA or
foldback
•Our system also uses a stable
initiator of RNA silencing
siRNA
Duplexes
P19
DICER
Fny2b
•By interrupting RNA silencing
at distinct steps, these
suppressors allow us to
examine the signal identity
siRNA/AGO
Complexes
AGO
AGO
Successful Silencing
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) Arabidopsis
Above 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)
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
Right – GUS produced in Phloem
Left – Control (wildtype)
•The resulting phenotypes provide visual
evidence for signal identity
Experimental Model –
Tissue Specific Expression
For comparison:
A protein expressed in phloem compared to SUL expressed
in phloem
The RNA silencing signal is spread to neighboring cells
Experimental Model
Each 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
PHLOEM
MESOPHYLL
Initiation of the
RNAi Pathway
DUPLEX
BINDING
(P19)
siRNA
Duplexes
siRNA/AGO
INTERFERING
(Fny 2b)
siRNA/AGO
Complexes
CASE 1:
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
PHLOEM
MESOPHYLL
Initiation of the
RNAi Pathway
siRNA
Duplexes
siRNA
Duplexes
Fny2b
siRNA/AGO
Complexes
Successful
RNAi
siRNA/AGO
Complexes
Successful
RNAi
CASE 2:
Duplex Binding Protein P19 in Mesophyll
•RNA silencing in the phloem
will NOT be blocked,
PHLOEM
regardless of signal identity;
Initiation of the
phloem will be photobleached RNAi Pathway
•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
siRNA
Duplexes
siRNA/AGO
Complexes
Successful
RNAi
MESOPHYLL
P19
siRNA
Duplexes
siRNA/AGO
Complexes
Successful
RNAi
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
SUC2:dsCH42 ♀
X
SUC2:GUSHA ♂
Col-0 (WT)
SUC2:dsCH42 ♀ X SUC2:GUSHA ♂
GUS Control
SUC2:dsCH42 ♀
X
SUC2:Fny2bHA ♂
Col-0 (WT)
Fny2bHA Control
-The function lf Fny2b must be
verified to support this
conclusion
SUC2:dsCH42 ♀ X SUC2:Fny2bHA ♂
Results:
Case 2: P19 in Mesophyll
-Mesophyll produced duplex
binding suppressors appear to
restrict photobleaching to
phloem
-One possible explanation is
that the signal must be
amplified in each receiving cell;
Alternatively, Fny2b may have
functions not yet characterized
SUC2:dsCH42
SUC2:dsCH42 ♀
X
SUC2:GUSHA ♂
Col-0 (WT)
SUC2:dsCH42 ♀ X SUC2:GUSHA ♂
GUS Control
SUC2:dsCH42 ♀
X
CAB3:P19HA ♂
Col-0 (WT)
-We must verify this phenotype
SUC2:dsCH42 ♀ X CAB3:P19 ♂
P19HA Control
-Our current model cannot
account for this and the 2b in
Phloem suppression
simultaneously
Results:
Control Crosses
-siRNA duplex binding
protein P19 in phloem
caused complete
suppression of
photobleaching (right)
SUC2:dsCH42 ♀
X
SUC2:P19HA ♂
SUC2:dsCH42 ♀ X SUC2:P19HA ♂
Col-0 (WT)
SUC2:dsCH42 ♀
X
CAB3:Fny2bHA ♂
Col-0 (WT)
Fny2bHA Control
SUC2:dsCH42 ♀ X CAB3:Fny2bHA ♂
P19HA Control
-siRNA/AGO interfering
suppressor 2b in
mesophyll caused an
apparent vein-restricted
bleaching phenotype
(left)
dsRNA or
foldback
P19
DICER
siRNA
Duplexes
Fny2b
Successful Silencing
AGO
SUC2:dsCH42 ♀ X SUC2:GUSHA ♂
SUC2:dsCH42 ♀
X
SUC2:GUSHA ♂
AGO
Col-0
(WT)
siRNA/AGO
Complexes
GUS
Control
-These results were
expected regardless of
signal identity
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
dsRNA or foldback
DICER
siRNA
Duplexes
siRNA/AGO
Complexes
AGO
AGO
Signal Movement
Amplification of Signal?
(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
siRNA
Duplexes
siRNA/AGO
Complexes
AGO
AGO
Successful Silencing
Thank You:
Howard Hughes Medical Institute
College of Science
Cripps Scholarship Fund
Dr. James C. Carrington
Dr. Kevin Ahern
Dr. Atsushi Takeda
Dr. Kristin Kasschau
The Carrington Lab