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

2. 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

3. 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

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

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

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

7. 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

8. Resistance gene cloning and analysis

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

10. Transgenic Lr34/Yr18 wheat lines confer rust resistance
Leaf rust
BW # # # # #190
seedling assay (100C) for Lr34

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

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

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

14. 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

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

16. 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

17. 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.

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

19. 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

20. 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 Aug 6;3(8):e2875)
Wheat rust Avr/effectors being identified

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

22. Hollaway et al field data from Horsham

23. 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

24. 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

25. Yr49 available in Avocet with tightly linked marker: contact Wolfgang

26. 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”