Exploiting the combination of natural and genetically engineered

Exploiting the combination of natural and genetically engineered resistance to viruses impacting cassava production in Africa 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 1

Biocassava Plus 1 st phase Objective: engineering virus-resistance trait using RNA interference to be combined with biofortification traits in cassava 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 2

Plant endogenous silencing machinery Systemic leaf Days after inoculation Inoculated leaf Fusaro and Waterhouse, 2006 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 3

Cassava silencing machinery against geminiviruses 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 4

Engineering virus resistance in crop plants 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 5

Engineering geminivirus resistance in transgenic cassava Targeting coding sequences (IR) as. ACMV 19. 06. 2012 s. ACMV intron Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch as. ACMV 35 S poly. A p 35 S 6

Engineering geminivirus resistance in transgenic cassava Immunity in transgenic lines Biolistic delivery & agroinoculation Increasing virus load 19. 06. 2012 Agroinoculation CMD resistance screening in greenhouse (ETHz) Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 7

Engineering geminivirus resistance in transgenic cassava Resistance correlates with hairpin-derived small RNAs load in transgenic cassava lines 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 8

Engineering virus resistance in crop plants Development of reliable and sustainable CBSD resistance strategies in cassava 1. Reliable infection methods Tested scion Infected rootstock High viral pressure 100% infection rate Cleft grafting 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 9

Engineering virus resistance in crop plants Development of reliable and sustainable CBSD resistance strategies in cassava 1. Reliable infection methods 2. Reliable virus quantitation methods Identification and evaluation of highly stable internal controls in the cassava-CBSV pathosystem Selection of the most stable candidates in the Arabidopsispotyvirus pathosystem Validation of the candidate genes in cassava 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 10

Engineering virus resistance in crop plants Development of reliable and sustainable CBSD resistance strategies in cassava 1. Reliable infection methods 2. Reliable virus quantitation methods 3. Appropriate target for broad spectrum resistance 2 species (UCBSV & CBSV) Some cultivars display resistance to UCBSV but not to CBSV Coat protein 3‘end sequence is conserved between species and isolates 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 11

Engineering virus resistance in crop plants Development of reliable and sustainable CBSD resistance strategies in cassava 1. 2. 3. 4. Reliable infection methods Reliable virus quantitation methods Appropriate target for broad spectrum resistance Robust cassava transformation and selection of best performing lines Thursday 21, Session 6, 8 am 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 12

Engineering CBSD resistance in transgenic cassava Workflow Target sequence selection Plasmid construction Transgenic cassava production and selection hairpins CBSV CP 3’end 19. 06. 2012 + others Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 13

Engineering CBSD resistance in transgenic cassava Workflow Target sequence selection Plasmid construction Transgenic cassava production and selection hairpins CBSV CP 3’end 19. 06. 2012 + others Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 14

Engineering CBSD resistance in transgenic cassava Workflow Target sequence selection Plasmid construction Transgenic cassava production and selection hairpins CBSV CP 3’end 19. 06. 2012 + others Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 15

Engineering CBSD resistance in transgenic cassava Workflow Transgenic cassava production and selection Material multiplication and grafting on CBSV-and UCBSV infected rootstocks Symptom observation and virus load quantitation in rootstocks and scions by q. PCR Scion propagation and symptoms recording in leaves and roots 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 16

Engineering CBSD resistance in transgenic cassava Transgenic TMS 60444 lines grafted on CBSV- and UCBSVinfected cassava rootstocks Complete immunity against CBSV Complete immunity against UCBSV 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch UCBSV 17

Engineering CBSD resistance in transgenic cassava Transgenic TMS 60444 lines grafted on CBSV- and UCBSVinfected cassava rootstocks CBSV immunity is maintained when grafted on CBSV hyper accumulator roostocks (AR 34) 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 18

Engineering CBSD resistance in transgenic cassava Scions grafted for 4 months on rootstocks are multiplied and monitored for 7 months Wild-type Transgenics 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 19

Engineering CBSD & CMD resistance in transgenic cassava Production of cassava resistant to CMD and CBSD • Fully transgenic: use of hairpins targeting conserved sequences of cassava geminiviruses and ipomovirus • Exploit and combine natural virus resistance to engineered resistance 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 20

Engineering CBSD & CMD resistance in transgenic cassava • Natural CBSD resistance: recently identified, not fully characterized • Exploit natural virus resistance and combine it to engineered resistance TME series 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 21

Engineering CBSD & CMD resistance in transgenic cassava ETH transformation platform for farmer-preferred cassava cultivars FEC induction and multiplication TME 3 TME 7 Cassava transformation Regeneration Rooting and molecular testing TME 14 Zainuddin et al. , Plant methods (2012) 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 22

Engineering CBSD & CMD resistance in transgenic cassava Production of transgenic TME 7 and selection of single insertion, independent transgenic lines with high levels of hairpin-derived small RNAs. 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 23

Engineering CBSD & CMD resistance in transgenic cassava Transgenic TME 7 lines grafted on CBSV- and UCBSV-infected cassava rootstocks All transgenic TME 7 lines display complete immunity against CBSV All transgenic TME 7 lines display complete immunity against UCBSV TME 7 Wild-type TME 7 transgenics CBSV 19. 06. 2012 UCBSV Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch UCBSV 24

Engineering CBSD & CMD resistance in transgenic cassava Is CBSD resistance maintained when transgenics are exposed to combined CBSD and CMD infections? Co-inoculation of CBSV and EACMV in cassava rootstocks Grafting of transgenic scions on coinoculated rootstocks by grafting & propagation by grafting 19. 06. 2012 Symptom observation and CBSV load quantitation in scions by q. PCR Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 25

Engineering CBSD & CMD resistance in transgenic cassava Is CBSD resistance maintained when transgenics are exposed to combined CBSD and CMD infections? EACMV + CBSV in wild-type TMS 60444 rootstocks EACMV + CBSV in wild-type TME 7 scions Transgenic TME 7 maintain their CBSD resistance when exposed to co-infection CBSV + EACMV 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 26

What’s next? Field trials (cassava community goal to have an impact for African farmers) Development of sustainable transgenic strategies for CMD resistance (ongoing) Further characterization of natural CBSD resistance and combination with CMD resistance Collaborative effort to address cassava viral diseases 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 27

What’s next? Field trials (cassava community goal to have an impact for African farmers) Development of sustainable transgenic strategies for CMD resistance (ongoing) Further characterization of natural CBSD resistance and combination with CMD resistance Collaborative effort to address cassava viral diseases 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 28

Acknowledgements ETH Zurich Isabel Moreno Ravi Bodampalli Ima Zainuddin Wilhelm Gruissem Irene Zurkichen NRI Maruthi Gowda 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 29

http: //www. pb. ethz. ch/research/cassava 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 30

http: //www. pb. ethz. ch/research/cassava 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 31

Engineering geminivirus resistance in cassava Targeting non-coding sequences (IR) as. ACMV 19. 06. 2012 s. ACMV intron Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch as. ACMV 35 S poly. A p 35 S 32

Engineering geminivirus resistance in cassava 19. 06. 2012 Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 33

DNA methylation BCTV wt BCTV L 2 -1(mutant) BCTVL 2 -2 (mutant) Plants infected with mutant viruses show recovery phenotype 19. 06. 2012 Histone methylation Ca. LCu. V in Arabidopsis Ch. IP with antibodies specific for the histone modifications Amplification of Ca. Cu. LV Intergenic Region Hervé Vanderschuren / ETHz – cassava research team / hvanderschuren@ethz. ch 34