Transformation of Nicotiana benthamiana with the RNA-dependent RNA-Polymerase gene and the coat protein gene of Vicia cryptic

virus By Nadeem Yaqoob

Institute of Plant Diseases and Plant Protection

Supervisors:

Prof. Dr. Edgar Mai Prof. Dr. Gnther Scherer Prof. Dr. Hartmut Sttzel

Outline
Introduction Objectives Hypotheses Methodology Results Conclusions

Introduction
Plant viruses classification (based on type of genome) DNA RNA
dsRNA ssRNA

The International Committee on Taxonomy of Viruses httpwww.ncbi.nlm.nih.govICTVDiagramsplantrna.html

Introduction- dsRNA
Partitiviridae and Reoviridae Partitiviridae include three genera:
Partitivirus Alphacryptovirus Betacryptovirus

Examples of cryptic viruses:

Beet cryptic virus (BCV) Alfalfa cryptic virus (ACV 1) Vicia cryptic virus (VCV) Carnation cryptic virus (CarCV) White clover cryptic virus (WCCV)
Beet cryptic virus
Bar equals 100 nm (Tidona (Tidona & Darai, Darai, Springer, 2002)

Introduction- Vicia cryptic virus

Vicia cryptic virus (VCV) belongs to the genus Alphacryptovirus Has no impact on the yield of Vicia faba Isometric particles, about 30 nm in diameter No transmission by sap and grafting
(Kenten et al., 1978)

RdRp CP
VCV RNA1 & RNA 2

DsRNAs with molecular weights of 1.37 x 10 6 Da, 1.26 x 10 6 Da and 1.21 x 10 6 Da. (Abou-Elnasr et al., 1985) Genome consists of dsRNA, encoding RdRp and CP. Contains no movement protein

Introduction
Potato virus X (PVX)
Virions not enveloped 470-580 nm long; 11-13 nm in diameter Symmetry helical Distribution: through contact between the plants subjected to friction
Potato virus X x300,000
www.rothamsted.bbsrc.ac.uk/... www.rothamsted.bbsrc.ac.uk/...

Objectives
Generation of transgenic N. benthamiana expressing RdRp, CP or both Characterization of transgenic plants Evaluation of the susceptibility of transgenic plants to virus infection (PVX) Evaluation of RdRp and CP as silencing suppressors

Hypotheses Statement
Vicia cryptic virus RdRp and/or CP will affect the pathogenic behavior/ pathogenicity of the PVX in N. benthamiana Vicia cryptic virus will increase the symptoms / severity of the PVX RdRp and/or CP act as suppressors of gene silencing

Experiment 1 Transformation of Nicotiana benthamiana plants

Objective

Transform the Nicotiana benthamiana plants with CP and RdRp genes of

Vicia cryptic virus

Method- Cloning of VCV RdRp and CP

LB Kan pe35S RdRp tnos RB

LB

Kan

pe35S

CP

tnos

RB

Two cassettes having RdRp & CP

LB

Kan

pe35S

RdRp

tnos

pe35S

CP

tnos

RB

One cassettes having both CP & RdRp Not drawn to scale; Marker are omitted

Method- Transformation
Binary vector:pLX222 or pBIN19 Agrobacterium tumefaciens LBA4404 Explants in a petri plate: Size of each explant: 1 cm One transformation = 3 plates = 45 explants

Replications : VCV-RdRp : 4 VCV-CP: 8

http://www.agriculture.purdue.edu/agbiotech/images/leafdisk1.html http://www.agriculture.purdue.edu/agbiotech/images/leafdisk1.html

Results
Table : Number of calli and T0 plants achieved in 12 transformation experiments

Gene of VCV RdRp CP

Calli produced 143 62

Regenerated T0 plants 34 29

Plants PCR negative 14 9

Plants PCR positive 20 20

Fig: Different stages of plants growth

Results
Table: Segregation ratio for germinated seedlings from T0 plants in MS + Kanamycin medium. Chi square values (X) are significant when less than 3.38 at = 0.05.

Line CP 1 CP 5 RdRp 1 RdRp 7 RdRp 8

Green seedling s 80 78 95 31 92

Yellow seedling s 17 19 1 30 4

Total Segregatio seedling n s ratios 97 96 96 61 96 3:1 3:1 15:1 63:1 15:1

X(chi sq. values) 0.70 0.38 0.28 3.71 0.02

Fig: Germination of T0 seeds on Kanamycin 400gm/ MS medium

Experiment 2 Germination and growth comparison of selected transgenic Nicotiana benthamiana lines

Objective

Behaviour of selected transgenic lines during germination and growth

Method
Sowing of seeds 50 seeds in one pot 3 pots per line

Colection of data Counting of germinated seeds after 1st week Shifting seedlings in trays and counting :2nd week Measurement of height and counting of leaves: 3rd, 5th, 7th and 10th weeks

Results
Table: Germiantion of seeds in selected transgenic lines

Plant lines CP 1 CP 5 RdRp 1 RdRp 7 RdRp 8 Control

% germination after 7 days 71 68 88 86 83 96 b b b b b a

% germination after 14 days 77 83 85 82 83 97 b b b b b a

Results
2,5

1
A A A A A

12

Control CP RdRp Control = 96 CP1 n=115 CP5 n= 124 RdRP1 n=113 RdRp7 n= 84 RdRp8 n= 96

A B

2,0

10

Control CP RdRp Control n= 70 CP1 n= 70 CP5 n= 70 RdRp1 n= 70 RdRp7 n= 70 RdRp8 n= 70

Height (cm)

1,5

Height (cm)

1,0

0,5
2

0,0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

Plant lines

Plant lines

30

3
C BC D

60 A C AB Control CP RdRp Control n= 70 CP1 n= 70 CP5 n= 70 RdRp1 n= 70 RdRp7 n=70 RdRp8 n= 70

4
A BC C

A AB

AB

25

50

Control CP RdRp Control n= 25 CP1 n= 25 CP5 n= 25 RdRp1 n=25 RdRp7 n=25 RdRp8 n=25

20

40

Height (cm)

15

Height (cm)

30

10

20

10

0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

Plant lines

Plant lines

Fig : Comparison of plant heights between transgenic N. benthamiana (T1) coat protein lines (CP 1, CP 5) and RNA dependent RNA polymerase lines (RdRp 1; RdRp 7; RdRp 8) with non-transgenic N. benthamiana. 1 = at 3rd week of germination, 2 = at 5th week of gerrmination, 3 = at 7th week of germination and 4 = at 10th week of germination.

Results
Height comparison
50 CP1 CP5 RdRp1 RdRp7 RdRp8 Control

40

Height (cm)

30

20

10

0 3 5 7 10

Weeks
Fig: Comparison of leaf number between transgenic N. benthamiana, coat protein lines (CP 1, CP 5); RNA dependent RNA polymerase lines (RdRp 1, RdRp 7, RdRp 8) with non-transgenic N. benthamiana at 3rd, 5th, 7th and 10th week.

Results
7

1
A AB D CD BC A

20
Control CP RdRp Control n=96 CP1 n=115 CP5 n=124 RdRp1 n=113 RdRp7 n=84 RdRp8 n=96

A B

BC 15 D D

Control CP RdRp Control n= 70 CP1 n= 70 CP5 n= 70 RdRp1 n= 70 RdRp7 n= 70 RdRp8 n= 70

Number of leaves

Number of leaves

CD

10

0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

Plant lines

Plant lines

18 16 14

250

AB

A BC D CD BC

Control CP RdRp
200

4
A B B B

Control CP RdRp Control n= 25 CP1 n= 25 CP5 n= 25 RdRp1 n= 25 RdRp7 n= 25 RdRp8 n= 25

Number of leaves

12 10 8 6 4 2 0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

Number of leaves

Control n= 70 CP1 n= 70 CP5 n=70 RdRp1 n= 70 RdRp7 n= 70 RdRp8 n= 70

150 C

C 100

50

0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

Plant lines

Plant lines

Fig : Comparison of number of leaves between transgenic N. benthamiana (T1) coat protein lines (CP 1, CP 5) and RNA dependent RNA polymerase lines (RdRp 1; RdRp 7; RdRp 8) with non-transgenic N. benthamiana. 1 = at 3rd week of germination, 2 = at 5th week of gerrmination, 3 = at 7th week of germination and 4 = at 10th week of germination.

Results
Number of leaves comparison
250 CP1 CP5 RdRp1 RdRp7 RdRp8 Control

200

Number of leaves

150

100

50

0 2 4 6 8 10 12

Weeks
Fig: Comparison of leaf number between transgenic N. benthamiana, coat protein lines (CP 1, CP 5); RNA dependent RNA polymerase lines (RdRp 1, RdRp 7, RdRp 8) with non-transgenic N. benthamiana at 3rd, 5th and 10th week.

Results
Fresh weight comparison
60 A 50 BC 40 ABC BC AB C Control CP RdRp Control n= 25 CP1 n= 25 CP5 n= 25 RdRp1 n= 25 RdRp7 n= 25 RdRp8 n= 25

Weight (gm)

30

20

10

0 Control CP1 CP5 RdRp1 RdRp7 RdRp8

plant lines

Fig : Comparison of fresh weight between transgenic N. benthamiana, coat protein lines (CP 1, CP 5); RNA dependent RNA polymerase lines (RdRp 1, RdRp 7, RdRp 8) with non-transgenic N. benthamiana.

Experiment 3 Interaction of PVX with transgenic Nicotiana benthamiana plants

Objective

Evaluation of PVX interaction with transformed plants having CP and RdRp genes of

Vicia cryptic virus

Method
Mechanical inoculation
PVX strain: 0018

Five lines were tested by ELISA Observations: 4 and 7 days post inoculation

Results
3,0

2,5

A A

4 DPI 7 DPI

2
A A A

4 DPI 7 DPI CP: n = 16 RdRp: n = 24 Control 1: n =4 Control 2: n = 4

2,5

CP: n = 16 RdRp: n = 24
Absorbance (415 nm)

2,0

Absorbance (415 nm)

2,0 a 1,5 a 1,0 B 0,5 b 0,0 CP RdRp Control 1 Control 2 a

Control 1: n = 4 Control 2: n = 4

1,5

1,0 B 0,5 b 0,0 CP RdRp Control 1 Control 2

Plant lines

Plant lines

0,8

0,7
4 DPI 7 DPI

4
A A A

0,6
0,6

4DPI 7DPI

CP: n =16 RdRp: n =24

B

Absorbance (415 nm)

Absorbance (415 nm)

0,5 a 0,4 a 0,3 a B a

CP: n =16 RdRp: n =24 Control 1: n =4 Control 2: n =4

Control 1: n =4 Control 2: n =4

0,4 a 0,2 a a a

0,2

0,1
0,0 CP RdRp Control1 Control2

0,0 CP RdRp Control 1 Control 2

Plant lines

Plant lines

Fig : Quantification of PVX in transgenic N. benthamiana CP and RdRp lines at 4 and 7 d.p.i. 1 = experiment 1, 2 = experiment 2, 3 = experiment 3 and 4 = experiment 4.

Experiment 4 Suppression of gene silencing by CP and/or RdRp

Objective

Do CP and RdRp of VCV act as suppressors of gene silencing?

Mechanism of gene silencing

httpallserv.rug.ac.be~avierstrprinciplespcr.html

Peter M. Waterhouse NATURE | VOL 411 | 14 JUNE 2001 | www .nature. com

Adopted from Stram, 2006

Method- Cloning of VCV RdRp, CP

LB Kan pe35S CP tnos RB

LB

Kan

pe35S

RdRp

tnos

RB

LB

Kan

pe35S

GFP

tnos

RB

LB

Kan

pe35S

HC-Pro

tnos

RB

Fig:

Fig: Constructs used in gene silencing experiments, not drawn on scale

Method
Agroinfiltration:
N. benthamiana (16c line): 5 plants per treatment Agrobacterium tumefaciens: C58C1 Binary vector: pBIN19 Treatments applied:

GFP GFP+HC-Pro GFP+CP GFP+RdRp

green colour defuse/ red ring appears green colour persist/ no red ring appears

? ?

Results

GFP

HC-Pro+GFP

CP+GFP

RdRp+GFP

Fig: Effect of CP, RdRp,HC-Pro when applied with GFP and GFP alone in 16c plants.Top pannel 2d.p.i, middle 4 d.p.i and lower 7 d.p.i.

Results

GFP

HC-Pro+GFP

CP+GFP

RdRp+GFP

Fig: Effect of CP, RdRp,HC-Pro when applied with GFP and GFP alone in 16c plants.Top pannel 2d.p.i, middle 4 d.p.i and lower 7 d.p.i.

Conclusions
CP gene interfere while RdRp does not interfere significantly in the growth of N.

benthamiana
Transgenic plants having RdRp and CP gene of VCV are susceptible to PVX like nontransgenic plants

RdRp and CP gene does not act as suppressor for gene silencing

THE END