PRESENTATION- 18.04.

23

SNEHA VERMA
UGC- JRF
NCCS
Identification of RNA
silencing suppressor encoded
by wheat blue dwarf (WBD)
phytoplasma
PRESENTATION- 18.04.23
• SYMPTOMS- dwarfism, witches’ broom, and yellow leaf tips in wheat
plants.
• 37 candidate effector proteins were transiently expressed in
Nicotiana benthamiana
• SWP1 induced witches broom in host.
• SWP11, induced both cell death and defence responses, including
H2O2 accumulation and callose deposition
• marker gene of the hypersensitive response, HIN1, and three
pathogenesis-related genes, PR1, PR2 and PR3, were significantly up-
regulated in leaves of N. benthamiana expressing SWP11
RNA silencing pathway
Known RSS in pathogen
• p19, encoded by tomato bushy stunt virus (TBSV), forms a homodimer
which specifically binds siRNA to interfere with the composition of
RISC to inhibit RNA silencing (Vargason et al. 2003).
• The protein 2b from cucumber mosaic virus (CMV) inhibits systemic
RNA silencing by binding both dsRNA/siRNA and inhibiting the
cleavage activity of AGO1.
• PSR1 encoded by Phytophthora can inhibit RNA silencing by targeting
PSR1-Interacting Protein 1 (PINP1), a novel component of the small
RNA pathway in plant.
Agrobacterium-mediated gene silencing model
to investigate RSS in WBD phytoplasma
• Agrobacterium-mediated gene silencing.
• existing experimental silencing system -Agrobacterium tumefaciens
was used to deliver the silencer GFP by agroinfiltration of GFP-
transgenic N. benthamiana line 16C (NbGFP). This treatment
abolished GFP expression, as evidenced by the absence of green
fluorescence at the site of infiltration upon UV illumination of NbGFP
plants. This initial effect was followed by a systemic fading of green
fluorescence by 3 weeks postagroinfiltration indicating that the GFP
transgene was systemically silenced (Voinnet and Baulcombe 1997;
Voinnet et al. 1998)
• The visible red fluorescence is emitted by chlorophyll upon UV
illumination. Potato virus X (PVX)-mediated delivery of SWP16.
• GFP-associated fluorescence was monitored frequently by UV
illumination during a 3-week period postinoculation.
• The results showed that green fluorescence gradually replaced red
fluorescence when the plants were infected with PVX/SWP16(+)
• By contrast, red fluorescence persisted on the silenced plants
inoculated with PVX/p19(–), similar to that observed for
noninoculated silenced plants. These observations confirmed that
SWP16 is a viral suppressor of Agrobacterium-mediated PTGS
SWP16 acts as RNA silencing suppressor
SWP16 supresses GFP silencing in
systemic leaves
Signal sequence of SWP16 was confirmed
using SIGNALP 3.0
YTK12 system & pSUC2
• The yeast YTK12 strain -invertase negative
• pSUC2 vector -invertase gene but lack Methionine (Met) and signal
peptide sequence – Negative control
• Avrb- SP – positive control
• Strains that are unable to secrete invertase can grow on CMD-W media
but not on YPRAA media
• The extracellular invertase enzyme activity was detected by the reduction
of 2,3,5-Triphenyltetrazolium Chloride (TTC) to insoluble red colored
1,3,5-Triphenylformazan (TPF)
pSUC2 Vector
Function of signal peptide was confirmed
using YTK12 system
pBI121- Binary Agrobacterium vector with a
GUS reporter gene for plant transformation.
• Vector used to transform GFP, P19, 37 effector proteins in N.
benthamia and to construct SWP16 transgenic plant A. thaliana.
• https://www.snapgene.com/plasmids/plant_vectors/pBI121
Generation of SWP16 transgenic A.
thaliana
• The SWP16 transgenic A. thaliana plants were generated using the floral-dip method
(Zhang et al. 2006a).
• The flowers of A. thaliana Col-0 were dipped in A. tumefaciens strain GV3101 with pBI121-
SWP16.
• Then seeds of dipped flowers were sterilized and sown in Murashige and Skoog medium
(MS) containing 50 ng l \hbox{-} 1 kanamycin and 50 ng l \hbox{-} 1 carbenicillin.
• Seeds (T1) were obtained from individual plants (T0) with antibiotic resistance and
separately sown in MS medium with 50 ng l \hbox{-} 1 kanamycin and 50 ng l \hbox{-} 1
carbenicillin.
• Seeds (≥50, T2) of plants (T1) with a 3:1 ratio of antibiotic resistance were selected as
single insertion lines and sown in the same medium.
• Seeds (T3) of plants (T2) with 100% antibiotic resistance were collected as homozygous
lines and used for further studies
Transgenic A. thaliana lines expressing SWP16 had no
significantly different phenotype to the wild-type Col-0
SWP16 interrupted accumulation of
miRNAs in SWP16 transgenic plants of A.
thaliana

miR159, miR164 and miR319 showed significant lower expression levels

 SWP16 is a pathogenic factor that enhance
entry and infection of pathogens into
plants

Potato virus X is a common virus in tobacco and causes leaf mottling and stunting SWP16 enhanced infection of PVX in N.
benthamiana