Triple Rust Initiative Project GRDC-CSIRO co-investment in ACRCP

TRI leverage : partnering for greater impact: ANU, Prof Adrienne Hardham: how rusts cause disease : flax rust effectors $ to ANU (ARC) 2. UQ, Prof Bostjan Kobe: molecular structure of R proteins and rust effectors $ to UQ (ARC) 3. CSIRO – 2 Blades - Cobbitty: molecular basis of wheat stem rust pathogenicity (stem rust genomics) ($ to CSIRO) 4. ANU – CSIRO – Cobbitty : Molecular basis of stripe rust pathogenicity (Dr John Rathjen, ARC Future Fellow) to $ to ANU 5. ACIAR and Gates – stem rust markers and non-host R Rust being seen as a ‘national priority’ for research

Triple Rust Initiative research objectives Breeders’ markers for industry impact Cloning resistance genes from wheat for molecular analysis and transformation breeding Principles of synthetic resistance genes for breeding

Recognition and Resistance R genes encode recognition components of plant immune system Recognition Signalling TIR / CC NBS LRR Lr21 Sr35 Sr33 resistant susceptible

Model of R protein activation INACTIVE ADP bound Avr Nucleotide exchange ATP hydrolysis ACTIVE ATP bound Defense signalling

Resistance protein function: direct versus indirect recognition Avr Direct recognition Wheat rust R genes? Avr HP HP* R Indirect Recognition

Direct protein interaction = rust resistance ppppp Synthetic R proteins for rust resistance resistance protein Rust avirulence proteins Direct protein interaction = rust resistance No interaction = rust susceptibility Engineer cloned Resistance gene Direct protein interaction = rust resistance

Resistance gene cloning and analysis

Lr34/Yr18 : a new class of resistance gene ABC Transporter protein +Lr34/Yr18

Transgenic Lr34/Yr18 wheat lines confer rust resistance Leaf rust BW #5 #98 #171 #175 #190 seedling assay (100C) for Lr34

Lr34 transgenic barley is resistant at ambient temperatures Puccinia hordei Transgenic barley –ve Lr34 Transgenic barley +ve Lr34 Transgenics are also stem rust resistant

Lr34 How does it work, what does it transport???

More than one gene for Lr34 resistance ? Av H45 Het Lr34 transporter enzyme X Y outside

Chinese landraces: A useful genetic resource to dissect Lr34/Yr18 transporter function 129 (from 152) Chinese landraces carry Lr34 gene specific marker Field rust evaluation 25 accessions-completely susceptible Lr34-R Lr34-R Wu Ling

Sr2 and Lr46/Yr29 cloning Fine mapped and BAC contigs constructed Not NB-LRR or ABC transporters

Rpg1 studies in transgenic wheat Stem rust APR gene from barley for North America (but not Australia !?) Durable in NA except for 1 race that “disappeared” Not effective against Ug99 stem rust Cloned by Kleinhofs Had not been tested in transgenic wheat ************** 2 constructs tested in transgenic wheat ‘native’ with its own promoter engineered with strong promoter for over-expression NO RESISTANCE TO AN AUSTRALIAN STEM RUST SEED SENT TO US FOR TESTING

Resistance gene cassettes for GM wheat Lr34/Yr18 Yr36 Lr21 Sr33 Unit segregation- simpler breeding Our vision for future rust resistance breeding is to deliver marker packages that lead to highly effective and durable gene combinations, And to use our knowledge of genes involved in race-specific and race non-specific responses to identify new specificities or partial resistance with the ultimate goal of rust proofing the Australian wheat industry.

Efficient transformation a requirement for cassette delivery Agrobacterium transformation of wheat in CSIRO is now highly efficient 50% of embryos produce a transgenic

Rust resistance diversity: We access the entire wheat gene pool outside NHR from rice? Engineered R Triticeae Aegilops Sr32 Sr39 Transgenes Sr33, 45, 46 Aegilops SrR Sr31 Secale Bread wheat Lr34/Yr18 Lr46/Yr29 Sr25 Sr26 Lr19 Agropyron 10 10 20 Sr2 Sr13 Landraces APR Sr22 30 Triticum Hordeum Rpg1 Efficient gene cloning from ‘aliens’ needed

How can we tell if cloned R genes have different specificities? Cassettes (and conventional gene stacks) must be A + B +C and not A +A +A Important issue with R genes that provide resistance against all races Sr31 is not identical to SrR shown by Ug99 Potato blight provides an example: cloned Avr (effector) genes can be used to differentiate R gene specificities (Vleeshouwers VG et alPLoS One. 2008 Aug 6;3(8):e2875) Wheat rust Avr/effectors being identified

APR genetics and stacks “near immunity can be achieved by 3-5 APR genes” Which ones and which combinations?

Hollaway et al field data from Horsham

Quantification of stripe rust fungal biomass on zero, single and double APR gene combinations in Avocet background 6755 5616 4475 3335 ug chitin/gm tissue 2195 1056 Avocet Lr46/Yr29 Lr46/Yr29 Lr34/Yr18 Lr34/Yr18 + Ayliffe et al; field site at PBI Cobbitty in 2011

Dissecting components of near immunity stripe rust APR genes from Parula F3 derivatives from Avocet x Parula Genotyped with markers 1 – Parula 2 - +Lr34/Yr18, +Lr46/Yr29 3 - +Lr34/Yr18 4 - +Lr46/Yr29 5 - -veLr34/Yr18, -veLr46/Yr29 6 – Avocet Stripe rust-natural infection, Black Mountain, Canberra 2010 1 2 3 4 5 6

Yr49 available in Avocet with tightly linked marker: contact Wolfgang

Enrich breeding germplasm for Sr2, Lr34, Yr29, Yr49 etc “the rust resistance backbone for wheat” Evans Lagudah “in the fight against rust you use everything you have” Bob McIntosh ie R and APR genes “you can never have enough resistance genes”