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Natália T. Marques, Ângela A. Costa, Tatiana R. Martins, Gustavo Nolasco

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Natália T. Marques, Ângela A. Costa, Tatiana R. Martins,

Gustavo Nolasco

Images from Florida Division of Plant Industry Archive

Citrus tristeza virus and Tristeza symptoms

c) Quick decline

d) Stem pitting

a) Mild

b) seedling yellows

Introduction

Photo from Lee RF (by Roistacher,

C.N.)

Photo from Roistacher,

C.N.http://ecoport.org/ep?SearchType=slideshowView&slideshowId=97&checkRequired=YHttp://

Citrus tristeza virus (CTV) is a filamentous virus with 20 Kb

Closteroviridae family

Coat protein is formed by p25 (CP) and p27 (CPm)

CTV is transmitted by man and by aphids:

- Toxoptera citricida (Kirk.) is the most efficient

- Aphis gossypii (Glover).

- Aphis spiraecola

Introduction

Gp3a-201(8)

Gp3b-Q32

Gp4-206

GpM-201(25)

Gp2-440

Gp1-Q3

Gp5-199

Mild stem pitting

Mild

Mild

Severe stem pitting and

quick decline

Quick decline

Mild stem pitting

in some varieties

Quick decline

Citrus tristeza virus

Seven phylogenetic

distinct groups based on:

a) nucleotide variability of

the CP (p25) gene

b) the same phylogenetic

structure is maintained

throughout the 3´region

(p20, p23)

c) distinct CTV groups are

also related with different

plant syndromes

Introduction

RNA silencing mechanism

a) Induced by dsRNA

b) RNA is processed to small RNA fragments of

21-24 nt - – siRNAs –

c) siRNA incorporates RISC

complex.

d) Complementary RNA

sequences are

degraded/ translation

is blocked.

Introduction

CTV genes with a special function: suppressors of the RNA silencing

mechanism

CTV encodes at least three distinct suppressors of the RNA silencing

mechanism : p25 (CP), p20 and p23 proteins

Protein CP (p25) – suppresses at the intercellular levelLu et al., 2004, PNAS, vol.101, nº44, 15742–15747)

Protein p23 and p20 – suppresses at the intracellular level

Introduction

The success of a viral infection results from the

competition between the plant RNA silencing

mechanism and the ability of the virus to suppress it.

Although different p20 and p23 suppressor abilities were found

between CTV groups, it was not possible to draw a relationship

between the severity of the group and the ability of the suppressorprotein.

Objective

To assay the suppressor activity when both p20 and

p23 proteins act together

Introduction

Agroinfiltration of Nicotiana benthamina line 16C

Induces post-

transcriptional gene

silencing of GFP

Co-infiltration

with p23 or p20 proteins

+

Methodology for transient expression

+

Co-infiltration with GFP+p20+p23

CTV Group 3a M

p23 clone 11.5 25.4

p20 clone 11.4 25.11

Differences on the suppressor activity were monitorized by:

UV observation

Northern blot

Real time RT-PCR

Methodology

RNA was extracted from infiltrated leaf patches

5 days post-infiltration (dpi) / 9 days post-infiltration

WT

16C

N. benthamiana plants

under UV light

Results

At 5-6 dpi a red ring forms around the agroinfiltrated area

At 6-8 dpi is observed the mobile silencing signal moving on

vasculature.

Transient expression of GFP is maintained only for 3 dpi.

A mobile silencing signal (short-range)

was formed and moves 10-15 cells around the infiltrated area.

3 dpi 5 dpi 7 dpi

GFP

A mobile silencing signal (long-range)

enters the vascular system and moves to new leaves

Results

• In plants agro-inoculated with CTV suppressors is expected that

transient expression of GFP is maintained longer.

• 7 dpi - local silencing was observed in all leaves co-inoculated with

p20 (+GFP) or p23 (+GFP).

Results

GFP

GFP+p23 (Gp 3a)

TAV 2b

3 dpi 5 dpi 7 dpi 9 dpi

5S RNA

GFP siRNAs

Real-time RT-PCR

Results

Northern blot5dpi

24 nt marker

• Non-inoculated 16C plants shows the normal level of GFP mRNA in

plants; values obtained were used as a reference to determine the

relative level of GFP mRNA in samples.

• Reference gene: ubiquitin.

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

18,0

20,0

16C NI GFP p20 3a p23 3a p20+p23

3a

p20 M p23 M p20+p23

M

Tav-2b

Re

lati

ve

GF

P m

RN

A a

bu

nd

an

ce

5 Dpi

9 Dpi

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

18,0

20,0

16C NI GFP p20 3a p23 3a p20+p23

3a

p20 M p23 M p20+p23

M

Tav-2b

Re

lati

ve

GF

P m

RN

A a

bu

nd

an

ce

5 Dpi

9 Dpi

Results

• Observation of co-inoculation of GFP with p23 + p20 at 5 dpi and 9

dpi under UV light

Northern blot5dpi

24 nt marker GFP siRNAs

5S RNA

16C NI GFP p20_3a p23 3a p20+p23_3a p20M p23M p20+p23M

Results point to a synergetic action when both p23 andp20 suppressors from CTV act together.

Conclusions

Team members

Anahi Dandlen

Ângela Costa

Débora Lopes

Gonçalo Silva

Gustavo Nolasco

Natália Marques

Paulo Gouveia

Tatiana Martins

Acknowledgements