Overview of VIRCA Project

Douglas Miano

Biotech Communication Stakeholders Workshop

Hilton Hotel, Nairobi, September 14, 2012

VIRCA PARTNERS

Donald Danforth Plant Science Center St. Louis, Missouri

National Crops Resources Research Institute Namulonge, Uganda

Kenyan Agricultural Research Institute Nairobi, Kenya

International Institute for Tropical Agriculture BecA, Nairobi, Kenya

VIRCA – Virus Resistant Cassava for Africa

Cassava production in the world

Continent Area (ha) Production (‘000 MT)

Yield (t/ha)

% Global Production

Africa 11,662,941 103,423 8.87 52.9

Asia 3,508,103 58,373 16.64 29.8

Americas 2,683,268 33,601 12.52 17.2

Oceania 16,314 177 10.87 0.1

World 17,870,626 195,574 10.94 100

Source: FAO (2005)

Basic food for > 600 million people in the world

1. Cassava flour – Bakery and Confectionery

2. Cassava chips/pellets & leaves – Livestock feeds

3. Sweeteners – Food industry

4. Starch – paper, wood, batteries, pharmaceuticals and textile industry

5. Ethanol – Biofuel, distilleries

Food and Beverage

Starch

Ethanol

Animal Feed

Paper

Textile

CASSAVA Deg. Plastics Wood

Industrial uses of Cassava

Cassava – Strategic Priority for East & Central Africa

Omamo et al. 2006. IFPRI Report 150

Cassava: 2nd most important subsector for growth-inducing development

Challenges to cassava production

• Diseases and pests

- CMD, CBSD, CBB, whiteflies, mites, etc

• Nutritional quality

- protein, micronutrients (iron, Zinc & vitamin A) contents, trace minerals

• Post-harvest physiological deterioration

• Cyanogenic potential

• Lengthy growing cycle and recalcitrant nature of cassava

- results in slow breeding

• Cassava brown streak

disease (CBSV) also

becoming a major threat

Disease constraints

Combined annual economic loss of

US$ 180 million in East Africa

Necrotic rot of roots

• Cassava mosaic disease (CMD) has been the main disease constrain

-1st reported 1894 in present day Tanzania

-Caused an epidemic in the 1990s

- Africa losses an average 30-40% (15-24 million

tonnes; $6-25 billion/year)

Cassava susceptible to CMD

CMD resistant TME204

Challenge to management of the two diseases

• Continuous presence of the virus and the vector throughout the year

• Attachment of farmers to particular cultivars which are susceptible to the disease

• Lack of resistant / tolerant genotypes preferred by farmers

VIRCA project was initiated to combat these virus problems

Way forward?

Virus resistant cassava for Africa (VIRCA) Project

Goals

Deliver farmer-preferred transgenic cassava

resistant to CMD and CBSD to farmers in East

Africa

Capacity building (human and institutional)

African cassava mosaic virus (ACMV)

East African cassava mosaic virus (EACMV)

East African cassava mosaic Malawi virus (EACMMV)

East African cassava mosaic Cameroon virus (EACMCV)

East African cassava mosaic Zanzibar virus (EACMZV)

East African cassava mosaic Kenya virus (EACMKV)

South African cassava mosaic virus (SACMV)

Indian cassava mosaic virus (ICMV)

Sri Lankan cassava mosaic virus (SLCMV)

Africa

India

East African cassava

mosaic virus cluster

Cassava-infecting begomoviruses

• 9 distinct species, 7 in Africa

• ACMV and EACMV types most prevalent

Mal71 Mal65 Mal63 Mal57 Mal77 Ug46 Mal9 Mal25 Mal58 Ug45 Ug53 Ug52 Mal72 PANG ZANZ

Nampula1-1 Zanzibar7-1

Zanzibar11-1 Zanzibar6-2

CHAKE WETE KOR9 KOR1

Naliendele3-1 Mal17 Mal27

Tan70 Zanzibar8-2 HAND Kibaha10-2

Mo83 Zanzibar13-1

TanZ MLB9

MLB3 Mal18

Kikonde11-5 Kikonde10-1 Shirazi7-1

Nyumbasita5-4 Chumani Mbandar12-1

BSA2 Ug BSA4

Kilifi20-1 Kilifi20-3 Mwalumba16-2 Namulonge

Ke54 Diani3-1

Denyenye1-2 Kilifi18-2 LWR2

U23 HMA9

UgNamEBW Kabanyoro4-3 IGA8

NTG10 Ug16 UG42 Mal10

100

100

5475

100

99

86

100

5761

99100

100

99

99

8699

7694

89

76

67

100

5957

99

5170

78

71

656877

96

96

95

88

76

0.02

Coat protein

CBSV

‘UCBSV’

UgNamEBW NTG10 IGA8 HMA9 LWR2

Ke54 Ke125

BSA2 BSA4

MLB9 TanT8

MLB3 TanC9

ZANZ PANG

KOR9 KOR1

WETE CHAKE

Tan70 Mo83 HAND KenE

TanAB30 TanZ

100

98

8896

100

9955

99

75

100

83

53

100

8796

5097

58

76

62

0.05

HAM1h

Two Distinct Viruses

Main target was CMD

• Coat protein based

• Replication mediated

• G5 protein based

• Defective DNA based

• RNAi (gene silencing)

Phase I: Gene discovery and proof of concept

Xba I

Kpn I

BamH I BstB I

Sense

Anti Sense

All the constructs were initially made in pILTAB-0588 vector carrying the Cassava Vein Mosaic Virus & 35S promoters and NOS poly A sequence using Xba I & Kpn I and BamH I & BstB I restriction enzymes

Cloning in sense & anti-sense into pILTAB 0588

Non-transgenic cassava variety 60444 infected with K201

Transgenic cassava variety 60444 infected with K201

• Transgenic cassava lines showing resistance to EACMV; the effect of RNA-silencing constructs targeting the virus rep gene

• Clone K201 92% homology to EACMV-UG, 80-100% infectivity, faster progression of symptoms

Entries being evaluated in the CFT

Entry Line Vector Gene of interest Comments

1 P670-007 pILTAB670 AC2 AC2 from EACMV-UG

2 P670-010 pILTAB670 AC2 AC2 from EACMV-UG

3 P560-001 pILTAB560 Full Length AC1 AC1 from EACMV-UG

4 P560-008 pILTAB560 Full Length AC1 AC1 from EACMV-UG

5 P560-011 pILTAB560 Full Length AC1 AC1 from EACMV-UG

6 P561-009 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG

7 P561-013 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG

8 P561-021 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG

9 P506-027 pILTAB506 Cry-G5 Nuclear targeted

10 P506-036 pILTAB506 Cry-G5 Nuclear targeted

11 P505-027 pILTAB505 Cry-G5 Nuclear targeted

12 TME14 Nontransgenic None CMD resistant

13 TMS 60444 Nontransgenic None Susceptible control

14 TMS 30572 Nontransgenic None Resistant control

Transgenic trials for CMD resistance

NBA Permit No.: BA/KARI/G22/6(2)

CMDK-1

Transgenic Virus-Resistant Cassava

Confined Field Trial Plot Sketch

KARI-Alupe Substation

Transgenic and Control Cassava Plants

CMD Infected Cassava Plants

Guard Row Cassava Plants

Incineration Pit

Gate

Guard House

Store

Chain

linked fence

topped with

barbed wire

Foot

Bath

Width = 60m

Length

= 7

5m

• Planting of CMDK-1 at KARI Alupe: 11th May, 2011

CMD incidence

CMD Resistance to Transgenic Events

0%

20%

40%

60%

80%

100%

120%

1 3 5 7 9 11 13 15 17 19 22 24 26 28 30 32 34

WAP

CM

D In

cid

en

ce

(%

)

p505-027

p506-027

p506-036

p560-001

p560-008

p560-011

p561-009

p561-013

p561-021

p670-007

p670-010

TME14

TMS 30572

TMS 60444

Disease Severity

CMD Resistance to transgenic events

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

1 5 9 13 17 22 26 30 34

WAP

CM

D s

ev

eri

ty (

1-5

)

p505-027

p506-027

p506-036

p560-001

p560-008

p560-011

p561-009

p561-013

p561-021

p670-007

p670-010

TME14

TMS 30572

TMS 60444

CMD severity (C-terminal)

CMD Resistance to transgenic events

1.00

1.50

2.00

2.50

3.00

3.50

4.00

1 5 9 13 17 22 26 30 34

WAP

CM

D s

ev

eri

ty (

1-5

)

p561-009

p561-013

p561-021

TME14

TMS 30572

TMS 60444

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14% p

osi

tive

pla

nts

in

fect

ed

wit

h A

CM

V a

nd

EA

CM

V-

UG

Lines

ACMV Positive EACMV-UG Posotive

CMD1a: Virus Infection 7 MAP

Gene Silencing Lines n = 621

4 lines have been selected and are being tested for stability in the field

CMD1a: Storage Root Yield (Tons/Ha)

7.5

4.6

1.2

4.4

0.6 0.7

18.5 16.9

12.8

31

20.5

26.9

4

23.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14

7X

Lines

Cassava brown streak disease (CBSD) CFT

• To be conducted in KARI-Mtwapa, Coast region

• Application approved by NBA

Transgenic Virus-Resistant Cassava

Confined Field Trial Plot Sketch

KARI-Mtwapa Station

Incineration

Pit

Gate

Guard

House

Store

Chain linked

fence topped with

barbed wire

Foot Bath

Width = 80m

Length

= 8

7m

Root severity of CBSD in selected lines

Phase II: Product development

Initial target of two products

– TME204 – Natural resistant to CMD but

highly susceptible to CBSD

– Ebwanatareka – Susceptible to both CMD

and CBSD

• Regeneration and transformation of other

local popular varieties going on

Product 1 –TME204 modified for resistance to CBSD

Target pathogens - cassava brown streak virus & Uganda cassava brown streak virus

against

CBSV

against

CBSUV

pILTAB5001

sense strand

800 bp

intron

CsVMV

promoter antisense strand

5.2 kb

ΔFL CP 900 kb ΔFL CP 900 kb ΔFL CP

Lead construct

T-nos

ΔFL CP 900 kb

Proven efficacious as pILTAB718 in GH grafts and in CFT

Score Foliar Symptom Description

1 no symptoms on leaves or stems

2

mild/slight vein yellowing or chlorotic

blotches on leaves no brown

streaks/lesions on green stem

portions

3

mild/slight vein yellowing or chlorotic

blotches on leaves mild brown

streaks/lesions on green stem

portions

4

severe/extensive vein yellowing

or chlorotic blotches on leaves severe

brown streaks/lesions on green stem

portions no defoliation, stem dieback

or stunting

5

severe/extensive vein yellowing

or chlorotic blotches on leaves severe

brown streaks/lesions on green stem

portions defoliation, stem dieback or

stunting

Score Root Symptom Description

1 no symptoms on storage roots

2 less than 5% of storage root tissue

is necrotic

3 5-10% of storage root tissue is

necrotic

4 10-50% of storage root tissue is

necrotic

5 More than 50% of storage root

tissue is necrotic

CBSD symptom scoring scale

• Background - Farmer preferred cultivar Ebwanateraka:

• Ebwanateraka – previously most widely grown in Uganda – also good fit

in Kenya, perhaps throughout Lake Victoria region

• Outstanding public pull-through expected due to variety popularity

• High yield (potential ~32t/ha when disease-free), sweet, mealy, soft, early

maturing (12 months), long-lasting in the field >24 months, desirable upright

architecture

• Next step in complexity for technical and regulatory development

• Broad spectrum control of virus disease complex (CMD & CBSD)

• EACMV-UG

• ACMV

• CBSV

• CBSUV

• siRNA based resistance

2. Broad spectrum VR in Ebwanateraka

CMD and CBSD Resistant Ebwanateraka Resistance to CMD & CBSD – Engineered resistance to CMD & CBSD based

on siRNA technologies

Genetic insert – Single copy insert with no vector backbone

Disease resistance – Less than 20% CMD incidence, maximum root CBSD

severity score 2

Yield – At least 50% yield improvement over non-transgenic control, usable

storage root yield of al least 20t/ha

Duration of effect – resistance to both CMD and CBSD maintained over at

least 3 vegetative cycles

Response to other pathogens – efficacy against CMD & CBSD not

compromised by co-infection with CBB

Response to other stresses – virus resistance not adversely affected by

drought, temperature extremes, plant nutrition or insect attack

Agronomic characteristics – no negative pleiotropic effects on plant vigour,

morphology, quantity and quality of storage roots & vegetative

propagation

Mammalian toxicity - no increased toxicity or allergenicity to humans or

animals

Product #2 Description

Implementation

Executive

VIRCA Management and Oversight Structure

Communication

Monitoring

Anton

Bua

Claude

Fauquet

Paul

Anderson

PI and Director of Scientific and Technical Affairs

Director of Biosafety and Regulatory Affairs

Director of Product Development and

Communication Affairs in East Africa

VIRCA Executive Council

VIRCA Product Development Team

VIRCA Stake Holders Meeting

VIRCA Steering Committee

KARI

SCIFODE

(Champions &

working groups)

NARO/

NACRRI

ISAAA- AfriCentre

(Champions &

working groups)

VIRCA Communication Team

VIRCA DONORS