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

To further explore the function of P0, a yeast two hybrid screen

Viral dsRNA RNA silencing is a mechanism used by plants as
Recognition of dsRNA an antiviral defense. Exogenous viral dsRNA is was performed to detect potential cellular interactors of P0 and
by DICER detected by DICER ribonucleases and cleaved to permitted to identify an interaction with sHSPs (Small Heat
produce siRNAs (20–25 bp). Shock proteins)

Processing by One strands of the siRNA is integrated into an Domains of Small Heat Shock Proteins
DICER active RISC (RNA induced silencing complex).
siRNA Then the RISC complex guides the cleavage of N-ter C-ter
homologous mRNAs, thereby preventing it from Variable
being used as a translation template. C-terminal
Degradation of Association with α-C extension
AGO1 AGO1 The RNA silencing suppressor protein P0 is
Variable N-terminal part Conserved C-terminal domain
encoded by the plant virus genus polerovirus.
This viral protein is able to suppress RNA
silencing by destabilizing ARGONAUTE 1 - α-C : Alpha Crystallin Domain
Blocking of the way
Recognition of (AGO1), the major component of RISC complex - Molecular mass : 14 – 20 kDa
Target viral RNA which carries the RNA slicer activity, thus - Act as molecular chaperones
blocking the RNA silencing mechanism. - Induced by stresses such as high temperature
Overview of RNA silencing Inhibition by P0 - 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
Digestion on recombinant plasmids
sHSP Nc
oI PCR from positive clones by BamHI.
ex + The sHSP is directly cloned into pGADT7
te
500 bp ns for the next two hybrid experiment.
pA

io
DH

n
GAL4 AD 8 kb
NcoI ligation
Am
Amp

T7
pr
r

LEU2 sHSP
500 bp 500 bp
es

E.Coli transformation
ni
lo
co
Amp

Digestion on recombinant plasmids

sHSP 17.6B class I clones verified by PCR
r

on

from positive clones by BglII.

6/10 clones appeared positive,11-20 not shown.
R
PC

Digestion by NcoI + CIP 8 kb

Extraction of recombinant plasmids then
digestion by restriction enzymes.
pA
DH

pGADT7 500 bp
GAL4 AD
8 kb
Amp

T7 LB+Amp
MCS
The sHSP gene in the vector from
r

U2
LE Only clone N.20 is positive
CIP : Alkaline Phosphatase, Calf Intestinal the clone N.20 has to be sequenced.

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 sHSP sHSP sHSP Vector ASK2

pGBKT7 17.4** 17.6C** 18.1** 17.7** -Control + Control

P0BW + + Weak + Nt Nt

P0CA + + - + - +
+ Vector control - - - - Nt -

Result from forward selection in yeast

Overview of the two-hybrid assay, checking for (SD-AHLW***) :
interactions between two proteins, called
here Bait and Prey. + : Interaction = 3-5
pA
DH clones/ 5 tested
TRP1 GAL4 DNA-BD
T7
appeared after 5 days,
pGBKT7 Recombinant plasmid contains
+ P0 and GAL4 DNA Binding site :
- : No growth (no interaction),
P0 P0 P0 acts as “Bait”. Weak : Weak interaction =
some yeast growth appeared
Ka
n
r

after 14 days,
Nt : Not tested.
pA
DH

pGADT7 Recombinant plasmid contains

+
GAL4 AD
sHSP and GAL4 Activation Domain : * P0BW and P0CA are the proteins encoded by Beet western yellows virus(BWYV)
Amp

sHSP T7 sHSP acts as “Prey”. and Cucurbit aphid-borne yellow virus(CABYV).

** 17.4CI, 17.6C CI, 18.1 CI, 17.7CII are different sHSPs proteins from Arabidopsis thaliana
r

LEU2
sHSP 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?