126/09/2016

Ravi AnjanappaProf. Wilhelm Gruissem

Metabolic engineering of carbon pathways to enhance cassava

starch yields

CAssava Source-Sink

226/09/2016

Outline

Overview of the project

Transformation of farmer preferred cassava varieties

TME419

TME7

Evaluating tissue specific promoters

Promoters from potato and Arabidopsis

Cassava endogenous promoters Cassava cultivar production for FACE experiment Foliar double strand (ds) RNA spray Outlook

326/09/2016

RuBisCO Activase (RCase)

Tonoplast monosaccharide transporter (TMT)

Glycolate dehydrogenase (GlyDH)

ADP-glucose pyrophosphorylase (AGPase)

redirecting carbon flux towards sucrose

synthesis

redirecting carbon flux towards starch

synthesisGlucose-6-phosphate Translocator (GPT)

Plastidic nucleotide transporter (NTT)

Sucrose synthase 4 (SuSy4)

Glucan water dikinase (GWD)

Adenylate kinase (ADK)

RNAi-ACMV

Virus resistanceusing a hairpin-RNA construct to generate resistance to a common cassava pathogen, African Cassava Mosaic Virus

Inorganic carbon transporter B (ictB)

Regulating gene expression in photosynthetic and storage tissues to increase starch production

Source

Sink

Overview

OEP7 Hexokinase (OEP7-HK)

426/09/2016

1

2

3

4

5

6

7

8

9

Rcase -RuBisCO Activase TMT -Tonoplast monosaccharide transporterGlyDH -Glycolate dehydrogenase IctB -Inorganic carbon transporter B GPT -Glucose-6-phosphate Translocator NTT -Plastidic nucleotide transporter OEP7HK -Outer envelope protein 7 HK SuSy4 -Sucrose synthase 4 GDW -Glucan water dikinase ADK - Adenylate kinase

Above-ground

Root-specific

RNAi-ACMV: to generate resistancet to a common cassava pathogen, African Cassava Mosaic Virus

OverviewDifferent combinations of target genes in gene stacks

for generating transgenic cassava lines

Greenhouse grown cassava plants exude drops that are rich in sugar (data from Patrick Klemens)

526/09/2016

Cassava transformation overview

Adapted from:Bull et al. (2009)Nature Protocols

Cassava Transformation

626/09/2016

TME419 transformation

Friable embryogenic calli (FEC) obtained from TME419 (Herbicide-tolerant cassava project)

FEC transformed with pCambia1305.1 (GUS expressing)

Cassava Transformation

pro35S Reporter gene (GUS)

726/09/2016

TME419: Low transformation efficiency

FECs were transformed with pCAMBIA 1305.1 (35S::GUSPlus::NosT) and assessed for GUS expression.

Variety No. of FECs clumpstransformed

No of plantlets

60444 63 38

TME419 63 3

Cassava Transformation

826/09/2016

TME 7 is a farmer-preferred variety that is tolerant to CMD but susceptible to CBSD

Advantages:

CMD2 locus

Previously optimized transformation protocol

Transgenic TME 7 resistant to CBSD:A) & B) WT TME 7 inoculated with CBSV and UCBSVC) & D) Transgenic TME 7 inoculated with CBSV and UCBSV

Cassava Transformation

926/09/2016

TME7: CMD recovery phenotype

Rootstock: 60444-ACMV infected Rootstock: 60444-EACMV infected

Adrian Alder

Cassava Transformation

TME7 plants (scions) displaying recovery phenotype (cassava mosaic disease-free) when grafted on to 60444 cassava mosaic virus-infected rootstock.

1026/09/2016

TME7 friabale embryonic callus (FEC) has a tendency to become tetraploid

• Ploidy analysis:• Galbraith's buffer (Galbraith et al., 1983) for isolation of intact nuclei• Stained with Propidium iodide (nucleic acid binding fluorescent dye)• Attune NxT Flow Cytometer-ThermoFisher

Cassava Transformation

N2: 1572N4: 72

N2: 0N4: 883

1126/09/2016

TME7 Transformation 1

Cassava Transformation

• Tetraploid FEC (obtained from HT Cassava team) were transformed with all the 9 stacks + 35S::GUSPLUS control.

• GUS expression observed at 8 weeks of regeneration.

GUS expressing friable embryogenic calli (FEC)

1226/09/2016

TME7 Transformation 1

Cassava Transformation

• Regeneration failure.

A) TME 7 (tetraploid) FEC failed to develop cotyledons on regeneration media with hygromycin selection.

B) Cotyledons developed on regeneration media without hygromycin selection

A B

1326/09/2016

TME7 Transformation 2Cassava Transformation

• New TME7 Diploid FEC (from HT Cassava team)

• Transformed with 9 stacks + 35S::GUSPLUS (control)

• Currently: recovery stage (1 week post-transformation)

1426/09/2016

Summary

• TME419 FECs have low transformation efficiency and low regeneration rate.

• Tetraploid TME7 FECs have a better transformation efficiency but do not regenerate.

• Transforming new diploid TME7 FECs with all the 9 stacks and controls.

• Transforming the same stacks in diploid 60444 FECs as backups.

Cassava Transformation

1526/09/2016

Outline

Overview of the project

Transformation of farmer preferred cassava varieties

TME419

TME7

Evaluating tissue specific promoters

Promoters from potato and Arabidopsis

Cassava endogenous promoters Cassava cultivar production for FACE experiment Foliar double strand (ds) RNA spray Outlook

1626/09/2016

Analysis of Arabidopsis and potato promoter specificities

Sl. No. Promoter Source Tissue expression

Expression previously tested in cassava

1 StLS1 Potato leaf specific No2 CAB1 Arabidopsis leaf specific No3 RbcS3B Arabidopsis leaf specific No4 SSS Potato Root specific No5 STP Potato Root specific No6 GBSS1 Cassava Root specific Yes7 35S CaMV Constitutive Yes

Promoter Characterization

Variety: 60444

Constructs:

Candidate promoters

Reporter gene (GUS)

1726/09/2016

Analysis of Arabidopsis and potato promoter specificities

Sl. No. Promoter Source Tissue

expression No of transgenic lines1 StLS1 Potato leaf specific 082 CAB1 Arabidopsis leaf specific 083 RbcS3B Arabidopsis leaf specific 154 SSS Potato Root specific 135 STP Potato Root specific 186 GBSS1 Cassava Root specific 197 35S CaMV Constitutive 15

Promoter Characterization

1826/09/2016

Cassava cv. 60444 non-transgenic controlPromoter Characterization

1926/09/2016

Potato leaf-specific promoter (proStLS1)

Expected promoter activity: Leaf-specific expressionObserved promoter activity: Constitutive expression but stronger in leaves

Promoter Characterization

2026/09/2016

Arabidopsis chlorophyll a/b-binding promoter (proAtCAB1)

Expected promoter activity: Leaf-specific expressionObserved promoter activity: Leaf expression but also weak expression in roots

Promoter Characterization

2126/09/2016

Arabidopsis Rubisco small subunit 3B (proAtRbcS3B)

Expected promoter activity: Leaf-specific expressionObserved promoter activity: Leaf-specific expression

Promoter Characterization

2226/09/2016

Arabidopsis Rubisco small subunit 3B (proAtRbcS3B)

Expected promoter activity: Leaf-specific expressionObserved promoter activity: Leaf-specific expression

Promoter Characterization

2326/09/2016

Potato soluble starch synthase promoter (proStSSS)

Expected promoter activity: Root-specific expressionObserved promoter activity: Weak expression in leaves and roots

Promoter Characterization

2426/09/2016

Potato soluble starch synthase promoter (proStSSS)

Expected promoter activity: Root-specific expressionObserved promoter activity: Weak expression in roots and leaves

Promoter Characterization

2526/09/2016

Potato soluble starch synthase promoter (proStSSS)

Expected promoter activity: Root-specific expressionObserved promoter activity: Leaf and stem expression and low expression in roots

Promoter Characterization

2626/09/2016

Potato starch phosphorylase promoter (proStSTP)

Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression

Promoter Characterization

2726/09/2016

Potato starch phosphorylase promoter (proStSTP)

Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression

Promoter Characterization

2826/09/2016

Potato Starch phosphorylase promoter (proStSTP)

Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression

Promoter Characterization

2926/09/2016

Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression but weaker expression in roots

Cassava granule-bound starch synthase 1 promoter (proMeGBSS1)

Promoter Characterization

3026/09/2016

Cassava granule-bound starch synthase 1 promoter (proMeGBSS1)

Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression

Promoter Characterization

3126/09/2016

CaMV 35S promoter (pro35S)

Expected promoter activity: Constitutive expressionObserved promoter activity: Constitutive expression

Promoter Characterization

3226/09/2016

CaMV 35S promoter (pro35S)

Expected promoter activity: Constitutive expressionObserved promoter activity: Constitutive expression

Promoter Characterization

3326/09/2016

CaMV 35S promoter (pro35S) : positive control

Expected promoter activity: Constitutive expressionObserved promoter activity: Constitutive expression

Promoter Characterization

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2. Novel cassava endogenous promoters (cv. 60444)

Leaf : Top 3 leavesFR : Fibrous roots (<1.99mm)IR : Intermediate roots (2-3.99mm)SR : Storage roots (>6.00mm)

RNAseq performed on leaves and roots at various developmental stages

Miyako Keller, PhD thesis (2014)

Early Middle Late

Promoter Characterisation

35

RNAseq: Tissue specific cassava promoters (cv. 60444)

Constitutive

Leaf-specific

Root-specific

26/09/2016

Promoter Characterisation

3626/09/2016

Cloning tissue specific promoters for promoter::GUS fusionsS.no. Activity Phytozome Cassava Id Gene Function*

1 Constitutive Manes.16G030000 Unknown/no annotated domains

2 Manes.16G007300 Unknown/no annotated domains

3 Manes.14G071100 Unknown/no annotated domains

4

Leaf-specific

Manes.01G011500 Ribulose-1,5-bisphosphate carboxylase small subunit

5 Manes.10G027600 AAA FAMILY ATPASE

6 Manes.15G102500 Photosystem II 10 kDa polypeptide PsbR

7 Manes.17G066900 Chlorophyll A/B binding protein

8 Manes.01G175600 Chlorophyll A/B binding protein

9 Manes.12G097200 Manganese-stabilising protein / photosystem II polypeptide

10

Root-specific

Manes.09G108300 Unknown/no annotated domains

11 Manes.14G127100 Cytochrome P450

12 Manes.12G062400 Unknown/no annotated domains

13 Manes.07G039800 Unknown/no annotated domains

14 Manes.12G133500 Cytochrome P450

15 Manes.10G101200 Unknown/no annotated domains

* Annotations based on Phytozome AM560-2 reference genome v. 4

Promoter Characterisation

3726/09/2016

Transformation statusSl No. Novel tissue specific

promotersExpression Total No of

plantletsRooting test

positives

1 Manes.14G071100 constitutive 42 29

2 Manes.01G011500 Leaf-specific

31 06

3 Manes.15G102500 36 214 Manes.13G058600 24 175 Manes.12G062400 Root-specific 44 34

Promoter Characterization

Variety: 60444

Construct:

Candidate promoters

3826/09/2016

Cassava endogenous promoters

proManes.14G071100

Expected promoter activity: Constitutive expression Observed promoter activity: Constitutive expression

3926/09/2016

Cassava endogenous promoters

proManes.12G062400

Expected promoter activity: Root-specific expressionObserved promoter activity: Root-specific expression but also leaf expression in some lines

4026/09/2016

Cassava endogenous promoters

Expected promoter activity: Leaf-specific expressionObserved promoter activity: Constitutive expression

proManes.01G011500

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proManes.15G102500Cassava endogenous promoters

Expected promoter activity: Leaf-specific expressionObserved promoter activity: Strong expression in leaves but also weak expression in roots

4226/09/2016

proManes.13G058600Cassava endogenous promoters

Expected promoter activity: Leaf-specific expressionObserved promoter activity: Constitutive expression

4326/09/2016

Summary

• Promoters from Arabidopsis and potato:

• No root-specific promoter found in young plants• Could be due to sucrose media and early developmental stage.

• RbcS3B promoter is weak (compared to 35S), but is leaf specific

• Cassava promoters:• No tissue-specific promoter found but proManes.12G062400 is a

potential root-specific promoter and proManes.15G102500 potential leaf-specific promoter.

Cassava Transformation

4426/09/2016

Outlook• Transformation:

• First 9 stacks in TME7 diploid FECs and 60444 as backup

• Promoter characterization:• Insert copy number assay (Southern blotting)• Check promoter activity in glasshouse grown plants.• Quantify promoter activity (MUG assays) .• Additional 5 promoters (cytFBPase::GUS, cytFBPase::GFP, AtSUC2::GUS,

AtSUC2::GFP, B33::GUS) from other species being transformed

Sep-16 Oct-16 Nov-16 Dec-16 Jan-17 Feb-17 Mar-17 Apr-17 May-17 June-17

Transform gene stack- TME7

Transform gene stack- 60444

Gene stack- transgenic lines- TME7

Gene stack- transgenic lines- 60444

Timeline for generating transgenic plants

2016-2017

Transform promoter::GUS -60444

Promoter::GUS transgenic lines- 60444

4526/09/2016

Outline

Overview of the project

Transformation of farmer preferred cassava varieties

TME419

TME7

Evaluating tissue specific promoters

Promoters from potato and Arabidopsis

Cassava endogenous promoters Cassava cultivar production for FACE experiment Foliar double strand (ds) RNA spray Outlook

4626/09/2016

• All cultivars tested for CMD and CBSD resistance as well as cassava leaf blight (Xanthomonas axonopodis pv. manihotis (Xam))

1. TME693-IITA (17 jars, 51 plants) 2. TMS98/0002 (18 jars, 54 plants) 3. TMS98/0581-IITA (17 Jars, 51 plants) 4. TMS30572-IITA (17 jars, 51 plants) 5. TME7-IITA (18 jars, 54 plants) 6. TMS98/0505 (18 jars, 54 plants) 7. TMS01/1412 (20 jars, 60 plants) 8. TME 419 (20 jars, 60 plants)

• Received clearance from USDA in July 2016

• Shipped plants to Illinois in July 2016.

Production of cultivars for FACE experimentFace experiment

4726/09/2016

Outline

Overview of the project

Transformation of farmer preferred cassava varieties TME419

TME7

Evaluating tissue specific promoters Promoters from potato and Arabidopsis

Cassava endogenous promoters

Cassava cultivar production for FACE experiment Foliar double strand (ds) RNA spray Outlook

4826/09/2016

Spraying anti-viral dsRNA to control CMGs

• Rationale: Field control of CMD in susceptible cassava cultivars

• Direct foliar application of dsRNAs targeting CMGs.

• Milestones:• Large-scale production of dsRNAs• Testing formulations• Testing resistance against multiple

cassava mosaic geminivirus (CMG) isolates

Foliar dsRNA sprays

4926/09/2016

• Conserved sequences (across all 299 reported CMG isolates from Africa, based on Genbank) longer than 21nt

• Combined to form a single 100nt hairpin

• + hairpins against interesting endogenous cassava genes.

• Hairpins will be expressed in a RNAse- free E.coli strain for large-scale production.

Foliar dsRNA spraysExperimental Design

5026/09/2016

Foliar dsRNA sprays

Additional notes

• We will be testing some surfactants like Silwet-L77.

• Reports indicate we will need >50 µg of RNA per plant for silencing endogenous genes.

• Reports indicate silencing does spread from initial site of application, hence reducing the need for repetitive spraying.

Olivier Voinnet,  2005

5126/09/2016

Acknowledgements

Prof. Wilhelm GruissemMarius Rohner (CASS project, technical support)Emily McCallum (HTC project, technical advice)Devang Mehta (virus resistance)Matthias Hirsch-Hoffman (IT and database support)

BILL & MELINDA GATES Foundation