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126/09/2016
Ravi AnjanappaProf. Wilhelm Gruissem
Metabolic engineering of carbon pathways to enhance cassava
starch yields
CAssava Source-Sink
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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
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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)
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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)
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526/09/2016
Cassava transformation overview
Adapted from:Bull et al. (2009)Nature Protocols
Cassava Transformation
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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)
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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
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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
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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.
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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
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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)
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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
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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)
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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
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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
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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)
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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
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1826/09/2016
Cassava cv. 60444 non-transgenic controlPromoter Characterization
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1926/09/2016
Potato leaf-specific promoter (proStLS1)
Expected promoter activity: Leaf-specific expressionObserved promoter activity: Constitutive expression but stronger in leaves
Promoter Characterization
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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
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2126/09/2016
Arabidopsis Rubisco small subunit 3B (proAtRbcS3B)
Expected promoter activity: Leaf-specific expressionObserved promoter activity: Leaf-specific expression
Promoter Characterization
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2226/09/2016
Arabidopsis Rubisco small subunit 3B (proAtRbcS3B)
Expected promoter activity: Leaf-specific expressionObserved promoter activity: Leaf-specific expression
Promoter Characterization
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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
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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
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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
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2626/09/2016
Potato starch phosphorylase promoter (proStSTP)
Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression
Promoter Characterization
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2726/09/2016
Potato starch phosphorylase promoter (proStSTP)
Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression
Promoter Characterization
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2826/09/2016
Potato Starch phosphorylase promoter (proStSTP)
Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression
Promoter Characterization
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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
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3026/09/2016
Cassava granule-bound starch synthase 1 promoter (proMeGBSS1)
Expected promoter activity: Root-specific expressionObserved promoter activity: Constitutive expression
Promoter Characterization
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3126/09/2016
CaMV 35S promoter (pro35S)
Expected promoter activity: Constitutive expressionObserved promoter activity: Constitutive expression
Promoter Characterization
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3226/09/2016
CaMV 35S promoter (pro35S)
Expected promoter activity: Constitutive expressionObserved promoter activity: Constitutive expression
Promoter Characterization
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3326/09/2016
CaMV 35S promoter (pro35S) : positive control
Expected promoter activity: Constitutive expressionObserved promoter activity: Constitutive expression
Promoter Characterization
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26/09/2016 34
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
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35
RNAseq: Tissue specific cassava promoters (cv. 60444)
Constitutive
Leaf-specific
Root-specific
26/09/2016
Promoter Characterisation
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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
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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
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3826/09/2016
Cassava endogenous promoters
proManes.14G071100
Expected promoter activity: Constitutive expression Observed promoter activity: Constitutive expression
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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
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4026/09/2016
Cassava endogenous promoters
Expected promoter activity: Leaf-specific expressionObserved promoter activity: Constitutive expression
proManes.01G011500
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4126/09/2016
proManes.15G102500Cassava endogenous promoters
Expected promoter activity: Leaf-specific expressionObserved promoter activity: Strong expression in leaves but also weak expression in roots
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4226/09/2016
proManes.13G058600Cassava endogenous promoters
Expected promoter activity: Leaf-specific expressionObserved promoter activity: Constitutive expression
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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
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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
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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
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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
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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
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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
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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
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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
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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