NanoLuc as a tool to study infection with a plant virus

(1)

HAL Id: hal-01807560

https://hal.archives-ouvertes.fr/hal-01807560

Submitted on 5 Jun 2020

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Copyright

NanoLuc as a tool to study infection with a plant virus

Marie Ducousso, Sylvaine Buissinot, Véronique Brault, Martin Drucker

To cite this version:

Marie Ducousso, Sylvaine Buissinot, Véronique Brault, Martin Drucker. NanoLuc as a tool to study infection with a plant virus. 50. Jahrestreffen des Arbeitskreises ”Viruskrankheiten der Pflanzen”, Mar 2018, Bad Herrenalb, Germany. �hal-01807560�

(2)

NanoLuc as a Tool to Study Virus Infection

Marie Ducousso

1 , Sylvaine Boissinot

2 , Véronique Brault

2 , Martin Drucker

1,2

1 Virus Insect Vector Interactions, INRA UMR BGPI, Montpellier, France

2 ,Virology Vection, INRA UMR SVQV, Colmar, France

Firefly Luciferase

(61 kD)

Renilla Luciferase

(36 kD)

NanoLuc

(19 kD)

Split NanoLuc*

_{(7.2 + 11.8 kD)}

The smaller, the better...

Bioluminescence systems for tagging proteins

From: Hall et al. (2012) ACS Chem. Biol. 7, 1848-1857.

*Zhao et al. (2016) ACS Chem. Biol. 11, 132–138.

N65 5297 NanoLuc 4979

wt

N65

Luc

N65

+

C66 (Plant) NanoLuc

Experimental system

Turnip yellows virus

(Polerovirus)

+

Model: P. Bron, INSERM

Montpellier

5’VPg

0

2

4 3a

1

3

5 3’OH

RT cleavage site

Proline tail

CP

RTD

Montia perforata

Arabidopsis thaliana

Nicotiana benthamiana

TuYV-Luc and TuYV-N65 in plants

Agroinfiltration of N. benthamiana

pMDC43-66C-myc

LB

RB

Ka nR Hygro R

pBR322 ori

pVS1

2X35S promoter

C66 myc

Nos terminator

XbaI

SacI

9668 pb

pMDC43-HA-N65

LB

RB

Ka nR Hygro R

pBR322 ori

pVS1

2X35S promoter

N65 HA

Nos terminator

XbaI

SacI

9560 pb

Validation of split NanoLuc in plants

Agroinfiltration of N. benthamiana

TuMV constructs

Validation of TuYV-N65 and -Luc

kD

_w

t

95-

72-

34-

16-N

65 Lu

c

M

oc

k

55-RT

CP

Rub

Western blot: agroinoculated M. perforata leaves 15 dpi

ELISA and RT-PCR on systemic TuMV-Luc leaves

1

2

3

4

5

6

7

8 9 10 wt M

0

1

2

3 N65

Substrate only

Infiltrated zone

N65 + C66

C66

Systemic leaf

TuYV-Luc

5 dpi, inoculated leaf

Infiltrated zone

TuYV-Luc

N65 + C66

TuYV-N65 + C66

TuYV-Luc

5 dpi

~655 bp

M. perforata 15 dpi

1

2

3

4

5

6

7 wt

M

0

1

2

3 ELISA

RT-PCR

Plant

A

b

so

rb

an

ce

Plant

1

2

3

4

5

6

7 M

H

₂

O Ctl

+

Luc

_Luminescence

Plant

1

2

3

4

5

6

7

8 9 10

M H

₂

O Ctl

+

N. benthamiana 15 dpi

1

2

3

4

5

6

7

8 9 10 wt M

0

1

2

3 Plant

Plant

1

2

3

4 5 6

7

8 9 10

M H

₂

O Ctl

+

A. thaliana 15 dpi

A

b

so

rb

an

ce

A

b

so

rb

an

ce

Contributed equally

Bioluminescence

O

₂

_CO

2

Substrate

_red

+

Substrate

_ox

Conclusions

The auto-assembling split NanoLuc system is functional in plants → Construction of transgenic A. thaliana expressing N65 and C66 in progress

Insertion of N65 and NanoLuc to RTD yielded systemically infecting viruses, but RT accumulation decreased

NanoLuc insertion stable for at least one generation in M. perforata and N. benthamiana, but not in A. thaliana

TuYV-Luc and TuYV-N65 luminescent in plants

NanoLuc vs. GFP: + lower background, + greater observation depth, - substrate application, - limited observation time, - exposure time