Identification of the Plant Systemic RNA Silencing Signal 2008 Summer HHMI Program 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 -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: VIRUS SYSTEMIC INFECTION PHLOEM = Vasculature MESOPHYLL = 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 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 Signal Signal Suppression Suppression Signal Signal SuppressionSignal

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 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 SILENCING dsRNA or foldback siRNA Duplexes siRNA/AGO Complexes Successful Silencing AGO AGO 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 DICER CELL 1 CELL 2 Signal? Successful Silencing AGO AGO

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 VIRUS RESTORED INFECTIVITY SuppressionSignal Viral Suppressors Block RNAi

Viral Suppressors of RNA Silencing Viral Suppressors of RNA Silencing Viral Suppressors of 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 Setup dsRNA or foldback siRNA Duplexes siRNA/AGO Complexes P19 Fny2b Successful Silencing AGO AGO DICER

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)

Right – GUS produced in Phloem Left – 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 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 Initiation of the RNAi Pathway siRNA Duplexes siRNA/AGO Complexes PHLOEM MESOPHYLL 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 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/AGO Complexes Fny2b PHLOEM MESOPHYLL PHLOEM MESOPHYLL Successful RNAi siRNA Duplexes siRNA/AGO Complexes 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/AGO Complexes PHLOEM MESOPHYLL PHLOEM MESOPHYLL Successful RNAi siRNA Duplexes siRNA/AGO Complexes Successful RNAi P19

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

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 SUC2:dsCH42 X SUC2:GUSHA 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 Control Crosses SUC2:dsCH42 X SUC2:P19HA -These results were expected regardless of signal identity SUC2:dsCH42 X SUC2:GUSHA dsRNA or foldback siRNA Duplexes siRNA/AGO Complexes Successful Silencing AGO DICER AGO 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/AGO Complexes Successful Silencing AGO AGO DICER Amplification of Signal? siRNA/AGO ComplexesAGO AGO siRNA Duplexes Signal Movement

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