Exploiting the combination of natural and genetically engineered resistance to viruses impacting cassava production in Africa

Biocassava Plus 1st phase Objective: engineering virus-resistance trait using RNA interference to be combined with biofortification traits in cassava

Plant endogenous silencing machinery Systemic leaf Inoculated leaf Days after inoculation Inoculated leaf Fusaro and Waterhouse, 2006

Cassava silencing machinery against geminiviruses

Engineering virus resistance in crop plants

Targeting coding sequences (IR) Engineering geminivirus resistance in transgenic cassava Targeting coding sequences (IR) p35S 35S polyA sACMV intron asACMV asACMV sACMV

Immunity in transgenic lines Engineering geminivirus resistance in transgenic cassava Immunity in transgenic lines Biolistic delivery & agroinoculation CMD resistance screening in greenhouse (ETHz) Increasing virus load Agroinoculation

Engineering geminivirus resistance in transgenic cassava Resistance correlates with hairpin-derived small RNAs load in transgenic cassava lines

Engineering virus resistance in crop plants Development of reliable and sustainable CBSD resistance strategies in cassava 1. Reliable infection methods Tested scion High viral pressure 100% infection rate Infected rootstock Cleft grafting

Validation of the candidate genes in cassava Engineering virus resistance in crop plants Development of reliable and sustainable CBSD resistance strategies in cassava Reliable infection methods 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 Arabidopsis-potyvirus pathosystem Validation of the candidate genes in cassava

Engineering virus resistance in crop plants Development of reliable and sustainable CBSD resistance strategies in cassava Reliable infection methods Reliable virus quantitation methods 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

Engineering virus resistance in crop plants Development of reliable and sustainable CBSD resistance strategies in cassava 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, 8am

Target sequence selection Transgenic cassava production and selection Engineering CBSD resistance in transgenic cassava Workflow Target sequence selection Plasmid construction Transgenic cassava production and selection hairpins + others CBSV CP 3’end

Target sequence selection Transgenic cassava production and selection Engineering CBSD resistance in transgenic cassava Workflow Target sequence selection Plasmid construction Transgenic cassava production and selection hairpins + others CBSV CP 3’end

Target sequence selection Transgenic cassava production and selection Engineering CBSD resistance in transgenic cassava Workflow Target sequence selection Plasmid construction Transgenic cassava production and selection hairpins + others CBSV CP 3’end

Engineering CBSD resistance in transgenic cassava Workflow Symptom observation and virus load quantitation in rootstocks and scions by qPCR Transgenic cassava production and selection Material multiplication and grafting on CBSV-and UCBSV infected rootstocks Scion propagation and symptoms recording in leaves and roots

Engineering CBSD resistance in transgenic cassava Transgenic TMS60444 lines grafted on CBSV- and UCBSV-infected cassava rootstocks Complete immunity against CBSV Complete immunity against UCBSV CBSV UCBSV

Engineering CBSD resistance in transgenic cassava Transgenic TMS60444 lines grafted on CBSV- and UCBSV-infected cassava rootstocks CBSV immunity is maintained when grafted on CBSV hyper accumulator roostocks (AR34)

Engineering CBSD resistance in transgenic cassava Scions grafted for 4 months on rootstocks are multiplied and monitored for 7 months Wild-type Transgenics

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

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

ETH transformation platform for farmer-preferred cassava cultivars Engineering CBSD & CMD resistance in transgenic cassava ETH transformation platform for farmer-preferred cassava cultivars FEC induction and multiplication Cassava transformation Regeneration Rooting and molecular testing TME3 TME7 TME14 Zainuddin et al., Plant methods (2012)

Engineering CBSD & CMD resistance in transgenic cassava Production of transgenic TME7 and selection of single insertion, independent transgenic lines with high levels of hairpin-derived small RNAs.

Engineering CBSD & CMD resistance in transgenic cassava Transgenic TME7 lines grafted on CBSV- and UCBSV-infected cassava rootstocks All transgenic TME7 lines display complete immunity against CBSV All transgenic TME7 lines display complete immunity against UCBSV TME7 Wild-type TME7 transgenics CBSV UCBSV CBSV UCBSV

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 co-inoculated rootstocks Symptom observation and CBSV load quantitation in scions by qPCR by grafting & propagation by grafting

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 TMS60444 rootstocks EACMV + CBSV in wild-type TME7 scions Transgenic TME 7 maintain their CBSD resistance when exposed to co-infection CBSV + EACMV

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

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

Acknowledgements ETH Zurich Isabel Moreno Ravi Bodampalli Ima Zainuddin Wilhelm Gruissem Irene Zurkichen NRI Maruthi Gowda

http://www.pb.ethz.ch/research/cassava

http://www.pb.ethz.ch/research/cassava

Targeting non-coding sequences (IR) Engineering geminivirus resistance in cassava Targeting non-coding sequences (IR) p35S 35S polyA sACMV intron asACMV asACMV sACMV

Engineering geminivirus resistance in cassava

DNA methylation BCTV wt BCTV L2-1(mutant) BCTVL2-2 (mutant) Plants infected with mutant viruses show recovery phenotype Histone methylation CaLCuV in Arabidopsis ChIP with antibodies specific for the histone modifications Amplification of CaCuLV Intergenic Region