Nadeem MSc-Thesis Presentation.ppt

Transformation of Transformation of Nicotiana benthamianaNicotiana benthamiana with thewith theRNARNA--dependent RNAdependent RNA --Polymerase gene and the coat Polymerase gene and the coat

protein gene of protein gene of Vicia cryptic virusVicia cryptic virus

ByByNadeemNadeem YaqoobYaqoob

Institute of Plant Diseases and Plant ProtectionInstitute of Plant Diseases and Plant Protection

Supervisors: Supervisors: Prof. Dr. Edgar Prof. Dr. Edgar MaißMaißProf. Dr. Prof. Dr. GüntherGünther SchererSchererProf. Dr. Hartmut StützelProf. Dr. Hartmut Stützel

OutlineOutline

�� IntroductionIntroduction

�� ObjectivesObjectives

�� Hypotheses Hypotheses

�� MethodologyMethodology

�� ResultsResults

�� ConclusionsConclusions

�� Plant virusesPlant viruses cclassificationlassification(based on type of (based on type of genome)genome)

�� DNADNA

�� RNARNAdsRNAdsRNA

ssRNAssRNA

The International Committee on Taxonomy of VirusesThe International Committee on Taxonomy of Viruses

httpwww.ncbi.nlm.nih.govICTVDiagramsplantrna.htmlhttpwww.ncbi.nlm.nih.govICTVDiagramsplantrna.html

IntroductionIntroduction

��PartitiviridaePartitiviridae and and ReoviridaeReoviridae��PartitiviridaePartitiviridae include three genera: include three genera:

PartitivirusPartitivirusAlphacryptovirusAlphacryptovirusBetacryptovirusBetacryptovirus

��Examples of cryptic viruses:Examples of cryptic viruses:Beet cryptic virusBeet cryptic virus (BCV) (BCV) Alfalfa cryptic virusAlfalfa cryptic virus (ACV 1) (ACV 1) Vicia cryptic virusVicia cryptic virus (VCV) (VCV) Carnation cryptic virus (Carnation cryptic virus (CarCVCarCV))White clover cryptic virusWhite clover cryptic virus (WCCV)(WCCV)

Beet cryptic virusBeet cryptic virusBar equals 100 nm (Bar equals 100 nm (TidonaTidona & &

DaraiDarai, Springer, 2002), Springer, 2002)

IntroductionIntroduction-- dsRNAdsRNA

�� Vicia cryptic virusVicia cryptic virus (VCV) belongs to (VCV) belongs to the genus the genus AlphacryptovirusAlphacryptovirus

�� Has no impact on the yield of Has no impact on the yield of Vicia Vicia fabafaba

�� Isometric particles, about 30 nm in Isometric particles, about 30 nm in diameter diameter

�� No transmission by sap and graftingNo transmission by sap and grafting((KentenKenten et al.,et al., 1978) 1978)

�� DsRNAsDsRNAs with molecular weights of with molecular weights of 1.37 x 10 1.37 x 10 66 DaDa, 1.26 x 10 , 1.26 x 10 66 DaDa and and 1.21 x 10 1.21 x 10 66 DaDa. . ((AbouAbou--ElnasrElnasr et al., et al., 1985)1985)

�� Genome consists of Genome consists of dsRNAdsRNA, , encoding RdRp and CP. Contains encoding RdRp and CP. Contains no movement proteinno movement protein

RdRpRdRp

CPCP

IntroductionIntroduction-- Vicia Vicia crypticcryptic virusvirus

VCV RNA1 & RNA 2VCV RNA1 & RNA 2

IntroductionIntroduction

Potato virus X Potato virus X (PVX)(PVX)

�� VirionsVirions not enveloped not enveloped

�� 470470--580 nm long; 11580 nm long; 11--13 nm in diameter13 nm in diameter

�� Symmetry helicalSymmetry helical

�� Distribution: through contact between the Distribution: through contact between the

plants subjected to frictionplants subjected to friction

Potato virus XPotato virus X x300,000x300,000www.rothamsted.bbsrc.ac.uk/www.rothamsted.bbsrc.ac.uk/......

��Generation of transgenic Generation of transgenic N.N. benthamianabenthamiana expressingexpressingRdRpRdRp, CP or both, CP or both

��Characterization of transgenic plantsCharacterization of transgenic plants

��Evaluation of the susceptibility of transgenic plants to Evaluation of the susceptibility of transgenic plants to virus infection (PVX)virus infection (PVX)

�� Evaluation ofEvaluation of RdRpRdRp and CP as silencing suppressorsand CP as silencing suppressors

ObjectivesObjectives

�� VVicia cryptic virusicia cryptic virus RdRp and/or CP will affect the RdRp and/or CP will affect the pathogenic behavior/ pathogenic behavior/ pathogenicitypathogenicity of the PVX in of the PVX in N. N. benthamiana benthamiana

�� Vicia cryptic virusVicia cryptic virus will increase the symptoms / severity will increase the symptoms / severity of the PVXof the PVX

�� RdRp and/or CP act as suppressors of gene silencingRdRp and/or CP act as suppressors of gene silencing

HypothesesHypotheses StatementStatement

Experiment 1Experiment 1

Transformation ofTransformation ofNicotiana benthamianaNicotiana benthamiana plantsplants

ObjectiveObjective

TransformTransform thethe NicotianaNicotiana benthamianabenthamianaplantsplants withwith CP andCP and RdRpRdRp genes of genes of

Vicia Vicia crypticcryptic virusvirus

MethodMethod-- CloningCloning of VCVof VCV RdRpRdRp and CPand CP

Two cassettes havingTwo cassettes having RdRpRdRp & CP& CP

One cassettes having both CP &One cassettes having both CP & RdRpRdRp

Not drawn to scale; Marker are omittedNot drawn to scale; Marker are omitted

RdRpRdRp tnostnospe35Spe35S RBRBLBLB KanKan

CPCP tnostnospe35Spe35S RBRBLBLB KanKan

RdRpRdRp tnostnospe35Spe35SLBLB KanKan CPCP tnostnospe35Spe35S RBRB

MethodMethod-- TransformationTransformation

http://www.agriculture.purdue.edu/agbiotech/images/leafdisk1.htmhttp://www.agriculture.purdue.edu/agbiotech/images/leafdisk1.htmll

ExplantsExplants in ain a petri platepetri plate::

SizeSize of of eacheach explantexplant: 1 cm² : 1 cm² One One transformationtransformation = 3 = 3 platesplates = 45 = 45 explantsexplants

ReplicationsReplications ::

VCVVCV--RdRpRdRp : 4: 4VCVVCV--CP: 8CP: 8

Binary vector:pLX222 or pBIN19Binary vector:pLX222 or pBIN19AgrobacteriumAgrobacterium tumefacienstumefaciens LBA4404LBA4404

ResultsResults

Fig: Different stages of plants growthFig: Different stages of plants growth

Table : Number of Table : Number of callicalli and T0 plants achieved in 12 transformation experimentsand T0 plants achieved in 12 transformation experiments

2092962CP

201434143RdRp

Plants PCR positive

Plants PCR negative

Regenerated T0 plants

Calliproduced

Gene of VCV

ResultsResults

Fig: Germination of T0 seeds on Fig: Germination of T0 seeds on KanamycinKanamycin 400gm/ MS medium400gm/ MS medium

0.0215:196492RdRp 8

3.7163:1613031RdRp 7

0.2815:196195RdRp 1

0.383:1961978CP 5

0.703:1971780CP 1

X²(chi sq. values)

Segregation

ratios

Total seedling

s

Yellow seedling

s

Green seedling

sLine

Table: Segregation ratio for germinated seedlings from T0 plants in MS + Kanamycinmedium. Chi square values (X²) are significant when less than 3.38 at α = 0.05.

GerminationGermination and growth and growth comparisoncomparison

of of selectedselected transgenic transgenic

Nicotiana benthamianaNicotiana benthamiana lineslines


ObjectiveObjective

BehaviourBehaviour of of selectedselected transgenic transgenic

lineslines duringduring germinationgermination and growthand growth

MethodMethod

SowingSowing of of seedsseeds

50 50 seedsseeds in in oneone potpot3 3 potspots per lineper line

ColectionColection of of datadata

CountingCounting of of germinatedgerminated seedsseeds afterafter 11stst weekweek

ShiftingShifting seedlingsseedlings in in traystrays and and countingcounting :2:2ndnd weekweek

MeasurementMeasurement of of heightheight and and countingcounting of of leavesleaves: 3: 3rdrd, 5, 5thth, 7, 7thth and and 1010thth weeksweeks

ResultsResults

Table: Germiantion of seeds in selected transgenic lines

97 a96 aControl

83 b83 bRdRp 8

82 b 86 bRdRp 7

85 b88 bRdRp 1

83 b68 bCP 5

77 b71 bCP 1

% germination after 14 days

% germination after 7 days

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. = at 3rd week of germination, = at 5th week of gerrmination, = at 7th week of germination and = at 10th week of germination.

Plant lines

Control CP1 CP5 RdRp1 RdRp7 RdRp8

Hei

ght (

cm)

0

5

10

15

20

25

30ControlCPRdRp

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

C BC

D

A

CAB

Plant lines


Hei

ght (

cm)

0

10

20

30

40

50

60ControlCPRdRp

Control n= 25CP1 n= 25CP5 n= 25RdRp1 n=25RdRp7 n=25

RdRp8 n=25

A

BC C

AAB

AB

Plant lines


Hei

ght (

cm)

0,0

0,5

1,0

1,5

2,0

2,5ControlCPRdRpA

AA

AA

B

Control =

CP1 n=CP5 n=RdRP1 n=RdRp7 n=

RdRp8 n=

115

96

12411384

96

Plant lines


Hei

ght (

cm)

0

2

4

6

8

10

12ControlCPRdRp

A A A

B B B

Control n= 70CP1 n= 70

CP5 n= 70RdRp1 n= 70 RdRp7 n= 70 RdRp8 n= 70

1 2

3 4

1 23 4

ResultsResults

Weeks

3 5 7 10

Hei

ght (

cm)

0

10

20

30

40

50CP1CP5RdRp1RdRp7RdRp8Control

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. benthamianaat 3rd, 5th, 7th and 10th week.

Height comparison

ResultsResults

ResultsResults

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. = at 3rd week of germination, = at 5th week of gerrmination, = at 7th week of germination and = at 10th week of germination.

1 23 4

Plant lines


Num

ber

of le

aves

0

1

2

3

4

5

6

7ControlCPRdRpAB

DCD

BC

A A

Control n=96CP1 n=115CP5 n=124RdRp1 n=113RdRp7 n=84

RdRp8 n=96

Plant lines


Num

ber

of le

aves

0

5

10

15

20ControlCPRdRp

A

BC

DD

CD

B

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

Plant lines


Num

ber

of le

aves

0

2

4

6

8

10

12

14

16

18ControlCPRdRp

ABBC

DCD BC

A

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

1 2

3

Plant lines


Num

ber

of le

aves

0

50

100

150

200

250ControlCPRdRp

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

A

CC

B

BB

4

Weeks

2 4 6 8 10 12

Num

ber

of le

aves

0

50

100

150

200

250CP1CP5RdRp1RdRp7RdRp8Control

Number of leaves comparison

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. benthamianaat 3rd, 5th and 10th week.

ResultsResults

ResultsResults

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.

Fresh weight comparison

plant lines


Wei

ght (

gm)

0

10

20

30

40

50

60ControlCPRdRp

CP1 n= 25CP5 n= 25RdRp1 n= 25RdRp7 n= 25

BC BC

ABCAB

A

C

RdRp8 n= 25

Control n= 25


Interaction of PVXInteraction of PVX with transgenic with transgenic Nicotiana benthamianaNicotiana benthamiana plantsplants

ObjectiveObjective

Evaluation of PVX Evaluation of PVX interactioninteraction withwith transformedtransformed

plantsplants havinghaving CP andCP and RdRpRdRp genes of genes of

Vicia Vicia crypticcryptic virusvirus

MethodMethod

Mechanical inoculationMechanical inoculationPVX PVX strainstrain: 0018: 0018

Five Five lineslines werewere testedtested byby ELISAELISA

ObservationsObservations: 4 and 7 : 4 and 7 daysdays postpost inoculationinoculation

ResultsResults

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

CP RdRp Control 1 Control 2

Abs

orba

nce

(415

nm

)

0,0

0,5

1,0

1,5

2,0

2,5

3,04 DPI7 DPI

a

a a

b

A A

A

B

Plant lines

CP: n = 16

RdRp: n = 24

Control 1: n = 4

Control 2: n = 4

Plant lines


Abs

orba

nce

(415

nm

)

0,0

0,5

1,0

1,5

2,0

2,54 DPI7 DPI

CP: n = 16

RdRp: n = 24

Control 1: n =4

Control 2: n = 4a a

a

b

A

A

A

B

Plant lines

CP RdRp Control1 Control2

Abs

orba

nce

(415

nm

)

0,0

0,2

0,4

0,6

0,84 DPI7 DPI

CP: n =16

RdRp: n =24

Control 1: n =4

Control 2: n =4

a

A A

B

aa

a

A

Plant lines


Abs

orba

nce

(415

nm

)

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

4DPI7DPI

CP: n =16

RdRp: n =24

Control 1: n =4

Control 2: n =4

AA A

B

a

a

aa

1 2

3 4

1 2 3 4


Suppression ofSuppression of gene silencing by gene silencing by

CP and/CP and/oror RdRpRdRp

ObjectiveObjective

Do CP andDo CP and RdRpRdRp of VCV of VCV actact as as

suppressorssuppressors ofof gene silencinggene silencing??

httpallserv.rug.ac.be~avierstrprinciplespcr.html

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

Mechanism of gene silencingMechanism of gene silencing

Adopted from Stram, 2006

MethodMethod-- CloningCloning of VCVof VCV RdRpRdRp, CP, CP

CPCP tnostnospe35Spe35S RBRBLBLB KanKan

GFPGFP tnostnospe35Spe35S RBRBLBLB KanKan

HCHC--ProPro tnostnospe35Spe35S RBRBLBLB KanKan

RdRpRdRp tnostnospe35Spe35S RBRBLBLB KanKan

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

MethodMethod

AgroinfiltrationAgroinfiltration::N. N. benthamianabenthamiana (16c line): 5 plants per treatment (16c line): 5 plants per treatment

AgrobacteriumAgrobacterium tumefacienstumefaciens: : C58C1C58C1

Binary vectorBinary vector: pBIN19: pBIN19

Treatments applied:Treatments applied:

GFPGFP green green colourcolour defuse/ red ring appearsdefuse/ red ring appears

GFP+HCGFP+HC--ProPro green green colourcolour persist/ no red ring appearspersist/ no red ring appears

GFP+CPGFP+CP ??

GFP+GFP+RdRpRdRp ??

ResultsResults

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.

RdRp+GFPHC-Pro+GFPGFP CP+GFP

ResultsResults

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.

ConclusionsConclusions

�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 non-transgenic plants

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

THE ENDTHE END

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Nadeem MSc-Thesis Presentation.ppt