Breeding for resistance to necrotrophic fungal diseases of wheat. Kar-Chun

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1 Breeding for resistance to necrotrophic fungal diseases of wheat Kar-Chun

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3 The Australian Grain Belt

4 $$$ losses yield due to fungal disease in Australia Murray and Brennan (2009) AUD$1=GBP 0.62

5 Major necrotrophic fungal diseases of wheat in Australia NSDU Tan spot (TS) caused by Pyrenophora tritici-repentis (Ptr) Septoria nodorum blotch (SNB) caused by Parastagonospora nodorum

6 The disease cycle of Ptr The disease cycle of P. nodorum Production of secreted necrotrophic effectors Production of secreted necrotrophic effectors Disease is defined by a series of necrotrophic effector-plant receptor interactions Tan et al. (2010) *Simplified schematic illustration

7 Our approach Cheap and reliable markers for breeders SNB and TS Genetic resistance Marker-assisted breeding Effector-assisted breeding A simple and rapid screening approach for effector sensitivity

8 Three major effectors identified in P. nodorum Two major effectors identified in Ptr 1. SnToxA kda mature protein. Activity is light-dependent. 2. SnTox kda mature cysteine-rich protein with a chitin-like binding motif at the C-terminus. Activity is light-dependent. 3. SnTox kda mature protein with six cysteine residues. Activity is lightindependent. 1. PtrToxA Near-identical to SnToxA. Horizontally acquired possibly from P. nodorum. 2. PtrToxB 6.6 kda mature protein. Activity is light dependent. Fungal effector Wheat susceptibility quantitative train locus Gene ID SnToxA 5BL Tsn1 Protein kinase NB-LRR SnTox1 1BS Snn1 Receptor kinase SnTox3 5BS, 5DS Snn3 - Unknown Fungal effector Wheat susceptibility quantitative train locus Gene ID PtrToxA 5BL Tsn1 Protein kinase NB- LRR PtrToxB* 2BS Tsc2 - Unknown *Not in Australia yet

9 Inverse gene-for-gene interaction Wheat (Tsn1) Wheat (tsn1) WT (ToxA) Mutant 1 (toxa) Mutant 2 (toxa) Negative control Moffat et al. (2014) We can rapidly and accurately* assay for effector sensitivity in wheat *condition dependent

10 Using effectors as a predictor of disease susceptibility in Australian wheat cultivars Ptr - strong correlation between Tan spot and ToxA sensitivity Chlorosis/necrosis Mixed sensitivity Mild chlorosis Insensitive S-VS, susceptible-very-susceptible; S, susceptible; MS-S, moderately susceptible-susceptible; MS, moderately susceptible, MR-MS, moderately resistant-moderately susceptible; MR, moderately resistant. P. nodorum - poor correlation between SNB and effector sensitivity SnToxA, 1 and 3 may masked the effect of undiscovered effectors Tan et al. (2014) Crop Pasture Res

11 Constructed a P. nodorum deleted in ToxA, 1 and 3 P. nodorum SN15 toxa (Friesen et al Hygromycin R) P. nodorum SN15 toxa3 P. nodorum SN15 toxa13 Tan et al. (2015) Frontiers Plant Sci.

12 P. nodorum deleted in SnToxA, 1 and 3 (toxa13) is still infectious (delayed) P. nodorum toxa13 still produces necrosis-inducing factor(s) Australian wheat cultivars sensitivity to the toxa13 culture filtrate Chlorosis/necrosis Mixed sensitivity Mild chlorosis Insensitive S-VS, susceptible-very-susceptible; S, susceptible; MS-S, moderately susceptible-susceptible; MS, moderately susceptible, MR-MS, moderately resistant-moderately susceptible

13 We like choices Cheap and reliable markers for breeders SNB and TS Genetic resistance Marker-assisted breeding Effector-assisted breeding A simple and rapid screening approach for effector sensitivity

14 Effector sensitivity can be mapped to wheat chromosomes just like disease susceptibility/resistance Eg. SnTox3-Snn3 Chr 5B

15 Qualitative or quantitative? SnToxA SnTox1 SnTox3 Calingiri +++ Wyalkatchem Phan et al. (2016) Plant J. Commercial WA wheat varieties. Double haploid population (~200 lines) from Intergrain. SSR and SNP markers. We infiltrated the population with expressed SnTox1 and SnTox3 proteins, scored the symptom and performed QTL analyses. 0 = insensitive 1 = slight chlorosis 2 = chlorosis 3 = chlorosis with some necrosis 4 = necrosis

16 QTL analyses 1 Treatment Chromosome Treatment arm Chromosome QTL arm Locus/QTL Locus/QTL QTL LOD R 2 Effect LOD R 2 Effect flanking markers flanking markers SnTox1 SnTox1 1BS QSnb.fcu-1BS QSnb.fcu-1BS Snn1 151 Snn SnTox3 SnTox3 5BS QSnb.fcu-5BS QSnb.fcu-5BS Snn3 268 Snn BL Qsnb.cur-4BL wmc413 Qsnb.cur-4BL - wpt wmc wpt Seedling infection Seedling infection 1BS QSnb.fcu-1BS QSnb.fcu-1BS Snn1 Snn with SN15 with SN15 2AS1 Qsnb.cur-2AS1 Qsnb.cur-2AS1 gwm515 - gwm328 gwm gwm AL Qsnb.cur-3AL Qsnb.cur-3AL tpt wpt-4859 tpt wpt Adult plant Adult plant 1BS QSnb.fcu-1BS QSnb.fcu-1BS Snn1 7.3 Snn infection with SN15 infection with 6BS SN15 Qsnb.cur-6BS wpt-3168 Qsnb.cur-6BS - Xbarc146a wpt Xbarc146a Seedling infection Seedling infection 2AS1 Qsnb.cur-2AS1 Qsnb.cur-2AS1 gwm339 - gwm312 gwm gwm with tox1-6 with tox1-6 5BS QSnb.fcu-5BS QSnb.fcu-5BS Snn3 3.5 Snn BL Qsnb.cur-4BL Qsnb.cur-4BL barc163 - wpt-4243 barc wpt AL Qsnb.cur-3AL Qsnb.cur-3AL tpt wpt-4859 tpt wpt Seedling infection Seedling infection 2AS1 Qsnb.cur-2AS1 Qsnb.cur-2AS1 tpt-8937 gwm312 tpt gwm with toxa13 with toxa13 2DS 3AL Qsnb.cur-2DS Qsnb.cur-3AL Qsnb.cur-2DS cfd36 wpt cfd wpt Qsnb.cur-3AL tpt wpt-4859 tpt wpt Culture filtrate of Culture filtrate 2AS1 of Qsnb.cur-2AS1 Qsnb.cur-2AS1 wpt-9320 gwm328 wpt gwm toxa13 toxa13 3AL Qsnb.cur-3AL Qsnb.cur-3AL tpt wpt-4859 tpt wpt AS2 Qsnb.cur-2AS2 Qsnb.cur-2AS2 wmc382a - barc124a wmc382a barc124a BS Qsnb.cur-2BS wpt-6271 Qsnb.cur-2BS wpt-6311 wpt wpt Conclusion 1. Evidence of epistasis. 2. SnTox1-Snn1 interaction predominates over SnTox3-Snn3 (bench player). 3. SnTox1-Snn1 is important in seedling and adult SNB in key Australian wheat cultivars. 4. Australian cereal breeders are now enthusiastically adopting effectors and genetic markers in their breeding programme*. *Cockram et al. (2015) G3; J. Cockram and R. Downie

17 Adoption of tsn1 cultivars in Western Australia % tsn1 wheat 70% 2006 Present Effector-assisted breeding have been Successfully employed in the Australian cereal industry - saving growers an estimated AUD$50 millions per year in potential yield loss.

18 Acknowledgements Curtin University (Australia) Kasia Clarke Huyen Phan Eiko Furuki Caroline Moffat Richard Oliver (UK-based) Intergrain (Australia) Dan Mullan The Australian National University Peter Solomon The University of Western Australia Yit Heng Chooi USDA (USA) Timothy Friesen Justin Faris NIAB (UK) James Cockram Rowena Downie (Poster 29)