Poster Complet V2
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INTERACTION BETWEEN sHSP AND THE P0 PROTEIN, “A VIRAL SUPPRESSOR OF RNA SILENCING” P. Sanvittayagul, J. De Cillia, C. Sorin, V. Ziegler-Graff Phytovirus et Pathogenèse, Institut de Biologie Moléculaire des Plantes, 12 rue du Général Zimmer 67000, Strasbourg, France
Viral dsRNA
To further explore the function of P0, a yeast two hybrid screen was performed to detect potential cellular interactors of P0 and permitted to identify an interaction with sHSPs (Small Heat Shock proteins)
RNA silencing is a mechanism used by plants as Recognition of dsRNA an antiviral defense. Exogenous viral dsRNA is by DICER detected by DICER ribonucleases and cleaved to produce siRNAs (20–25 bp).
siRNA
Processing by DICER
Association with AGO1
Degradation of AGO1 Blocking of the way
Domains of Small Heat Shock Proteins
One strands of the siRNA is integrated into an active RISC (RNA induced silencing complex). Then the RISC complex guides the cleavage of homologous mRNAs, thereby preventing it from being used as a translation template.
N-ter
Overview of RNA silencing Inhibition by P0
Variable C-terminal extension
α-C
The RNA silencing suppressor protein P0 is encoded by the plant virus genus polerovirus. This viral protein is able to suppress RNA silencing by destabilizing ARGONAUTE 1 (AGO1), the major component of RISC complex which carries the RNA slicer activity, thus blocking the RNA silencing mechanism.
Recognition of Target viral RNA
C-ter
Variable N-terminal part
Conserved C-terminal domain
- α-C : Alpha Crystallin Domain -
Molecular mass : 14 – 20 kDa Act as molecular chaperones Induced by stresses such as high temperature Family of 17 genes in Arabidopsis thaliana
Cloning of the gene encoding Small Heat Shock Protein 17.6B Class I One part of my work consisted in cloning the Arabidopsis thaliana sHSP 17.6B CI genes sHSP
Nc
8 kb
GAL4 AD
pr Am Amp
NcoI ligation
r
T7 LEU2
sHSP 500 bp
lo co
PC
R
on
r
Amp
Digestion on recombinant plasmids from positive clones by BglII.
sHSP 17.6B class I clones verified by PCR 6/10 clones appeared positive,11-20 not shown.
Digestion by NcoI + CIP
8 kb
Extraction of recombinant plasmids then digestion by restriction enzymes.
DH pA
r
Amp
U2 LE
500 bp
ni
E.Coli transformation
pGADT7 8 kb
Digestion on recombinant plasmids from positive clones by BamHI.
The sHSP is directly cloned into pGADT7 for the next two hybrid experiment.
es
500 bp
DH pA
oI PCR ex + te ns io n
500 bp
GAL4 AD
LB+Amp
T7 MCS
CIP : Alkaline Phosphatase, Calf Intestinal
The sHSP gene in the vector from the clone N.20 has to be sequenced.
Only clone N.20 is positive
Yeast Two Hybrid System, Interaction Assay Between RNA Silencing Suppressor P0 And Small Heat Shock Proteins Two hybrid test between different Arabidopsis thaliana sHSPs and P0 from BWYV* and CABYV*. pGADT7
sHSP 17.4**
sHSP 17.6C**
sHSP 18.1**
sHSP 17.7**
Vector -Control
ASK2 + Control
P0BW
+
+
Weak
+
Nt
Nt
P0CA
+
+
-
+
-
+
Vector control
-
-
-
-
Nt
-
pGBKT7
+
Result from forward selection in yeast (SD-AHLW***) :
Overview of the two-hybrid assay, checking for interactions between two proteins, called here Bait and Prey. pA DH
TRP1 pGBKT7 + P0
+
: Interaction = 3-5 clones/ 5 tested appeared after 5 days, : No growth (no interaction), Weak : Weak interaction = some yeast growth appeared after 14 days, Nt : Not tested.
GAL4 DNA-BD T7
P0
Recombinant plasmid contains P0 and GAL4 DNA Binding site : P0 acts as “Bait”.
r
n Ka
DH pA
r
Amp
pGADT7 + sHSP LEU2
GAL4 AD T7
sHSP
Recombinant plasmid contains sHSP and GAL4 Activation Domain : sHSP acts as “Prey”.
*
P0BW and P0CA are the proteins encoded by Beet western yellows virus(BWYV) and Cucurbit aphid-borne yellow virus(CABYV). ** 17.4CI, 17.6C CI, 18.1 CI, 17.7CII are different sHSPs proteins from Arabidopsis thaliana belonging to class I or class II. *** SD medium without adenine, histidine, leucine and tryptophan.
We can conclude that there is an interaction between P0 and some sHSPs. However other sHSPs do not interact with P0 which indicates that there is a specificity of interaction between P0 and sHSPs. Perspectives : • To confirm the interaction between P0 proteins and sHSP another technique is needed such as Co-immunoprecipitation or FRET. • Is there a link between sHSP and RNA silencing ? What is the role of the interaction between P0 and sHSP in the inhibition of RNA silencing?
Viral dsRNA
To further explore the function of P0, a yeast two hybrid screen was performed to detect potential cellular interactors of P0 and permitted to identify an interaction with sHSPs (Small Heat Shock proteins)
RNA silencing is a mechanism used by plants as Recognition of dsRNA an antiviral defense. Exogenous viral dsRNA is by DICER detected by DICER ribonucleases and cleaved to produce siRNAs (20–25 bp).
siRNA
Processing by DICER
Association with AGO1
Degradation of AGO1 Blocking of the way
Domains of Small Heat Shock Proteins
One strands of the siRNA is integrated into an active RISC (RNA induced silencing complex). Then the RISC complex guides the cleavage of homologous mRNAs, thereby preventing it from being used as a translation template.
N-ter
Overview of RNA silencing Inhibition by P0
Variable C-terminal extension
α-C
The RNA silencing suppressor protein P0 is encoded by the plant virus genus polerovirus. This viral protein is able to suppress RNA silencing by destabilizing ARGONAUTE 1 (AGO1), the major component of RISC complex which carries the RNA slicer activity, thus blocking the RNA silencing mechanism.
Recognition of Target viral RNA
C-ter
Variable N-terminal part
Conserved C-terminal domain
- α-C : Alpha Crystallin Domain -
Molecular mass : 14 – 20 kDa Act as molecular chaperones Induced by stresses such as high temperature Family of 17 genes in Arabidopsis thaliana
Cloning of the gene encoding Small Heat Shock Protein 17.6B Class I One part of my work consisted in cloning the Arabidopsis thaliana sHSP 17.6B CI genes sHSP
Nc
8 kb
GAL4 AD
pr Am Amp
NcoI ligation
r
T7 LEU2
sHSP 500 bp
lo co
PC
R
on
r
Amp
Digestion on recombinant plasmids from positive clones by BglII.
sHSP 17.6B class I clones verified by PCR 6/10 clones appeared positive,11-20 not shown.
Digestion by NcoI + CIP
8 kb
Extraction of recombinant plasmids then digestion by restriction enzymes.
DH pA
r
Amp
U2 LE
500 bp
ni
E.Coli transformation
pGADT7 8 kb
Digestion on recombinant plasmids from positive clones by BamHI.
The sHSP is directly cloned into pGADT7 for the next two hybrid experiment.
es
500 bp
DH pA
oI PCR ex + te ns io n
500 bp
GAL4 AD
LB+Amp
T7 MCS
CIP : Alkaline Phosphatase, Calf Intestinal
The sHSP gene in the vector from the clone N.20 has to be sequenced.
Only clone N.20 is positive
Yeast Two Hybrid System, Interaction Assay Between RNA Silencing Suppressor P0 And Small Heat Shock Proteins Two hybrid test between different Arabidopsis thaliana sHSPs and P0 from BWYV* and CABYV*. pGADT7
sHSP 17.4**
sHSP 17.6C**
sHSP 18.1**
sHSP 17.7**
Vector -Control
ASK2 + Control
P0BW
+
+
Weak
+
Nt
Nt
P0CA
+
+
-
+
-
+
Vector control
-
-
-
-
Nt
-
pGBKT7
+
Result from forward selection in yeast (SD-AHLW***) :
Overview of the two-hybrid assay, checking for interactions between two proteins, called here Bait and Prey. pA DH
TRP1 pGBKT7 + P0
+
: Interaction = 3-5 clones/ 5 tested appeared after 5 days, : No growth (no interaction), Weak : Weak interaction = some yeast growth appeared after 14 days, Nt : Not tested.
GAL4 DNA-BD T7
P0
Recombinant plasmid contains P0 and GAL4 DNA Binding site : P0 acts as “Bait”.
r
n Ka
DH pA
r
Amp
pGADT7 + sHSP LEU2
GAL4 AD T7
sHSP
Recombinant plasmid contains sHSP and GAL4 Activation Domain : sHSP acts as “Prey”.
*
P0BW and P0CA are the proteins encoded by Beet western yellows virus(BWYV) and Cucurbit aphid-borne yellow virus(CABYV). ** 17.4CI, 17.6C CI, 18.1 CI, 17.7CII are different sHSPs proteins from Arabidopsis thaliana belonging to class I or class II. *** SD medium without adenine, histidine, leucine and tryptophan.
We can conclude that there is an interaction between P0 and some sHSPs. However other sHSPs do not interact with P0 which indicates that there is a specificity of interaction between P0 and sHSPs. Perspectives : • To confirm the interaction between P0 proteins and sHSP another technique is needed such as Co-immunoprecipitation or FRET. • Is there a link between sHSP and RNA silencing ? What is the role of the interaction between P0 and sHSP in the inhibition of RNA silencing?
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