Sebas&an Schornack University of Cambridge, Sainsbury Laboratory (SLCU)

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1 18 th Interna?onal Oil Palm Conference, Cartagena, Colombia Sebas&an Schornack University of Cambridge, Sainsbury Laboratory (SLCU) P. palmivora entering a leaf

2 Phytophthora sp. cause diseases in important crops Zoospore Host range narrow broader P. Infestans potato late blight P. ramorum sudden oak death University Davis P. sojae soybean root rot P. palmivora palm bud and cocoa fruit rot A. Dorrance (APSnet).

3 P. palmivora sporangia releasing zoospores Phytophthora palmivora is a cosmopolit In the tropics the most commonly encountered species is Phytophthora palmivora. P. palmivora diseases

4 Phytophthora palmivora causes oil palm bud rot Economically important hosts Oil palms Coconut Orchids Pineapple Rubber tree Durian Papaya Cocoa Citrus Picture: QuesadaLabNCSU

5 Phytophthora is a stealthy pathogen Biotrophy Necrotrophy

6 Phytophthora infec&on cycle Zoospore mycelia Sporangia and spores

7 P. palmivora forms haustoria in living plant cells Electron microscopy Light microscopy Fluorescence of a transgenic P. palmivora isolate

8 Microbes modify plant cell processes using secreted effector proteins SP RXLR effector domain effectors targets Alter plant cell processes fungus oomycete plant cell haustorium Help microbe colonize plant secreted by the pathogen into the host?ssue to bind to host proteins suppress host immunity, promote disease repertoire of effector- encoding genes varies significantly between Phytophthora isolates

9 Plant immunity: Plant resistance proteins recognise effectors effectors targets Alter plant cell processes fungus oomycete plant cell haustorium intracellular immune receptors effector- triggered immunity NB- LRR proteins recognise effectors or their ac?vi?es Are known NB- LRR proteins of other plants capable of detec?ng P. palmivora effectors?

10 Surveying effector resistance protein combina&ons Conserved Effector genes From pathogen MATCH! R gene candidates from oil palm genome Or Known R genes Expression in Nico:ana benthamiana using A. tumefaciens Wrong effector or wrong Resistance gene Effector + matching Resistance gene Limita&ons: R gene is func?onal in N. benthamiana, AVR and R gene are both cloned

11 P. palmivora belongs to a clade with no sequenced member species Phytophthora infestans (240 Mbp) - Solanum spp. P. palmivora (assumed < 90 Mbp) more than 200 hosts P. capsici (65 Mbp) - pepper, tomato, cucurbits P. sojae (95 Mbp) - soybean P. ramorum (65 Mbp) - various woody plants mul?locus phylogeny of Phytophthora from Blair et al Fungal Genet Biol

12 Genome sequencing of two P. palmivora isolates AJ LILI

13 Inden&fica&on of conserved effectors SP RXLR effector domain

14 It is beper to perceive early expressed effectors Early infec&on Late during infec&on

15 In the future: effector based resistance phenotyping AVRblb1 Avr3a AVR2 CRNs Blb (Rpi- blb1) Dms (R3a) Edn (Rpi- edn1) Snk (Rpi- snk1) Hjt (Rpi- hjt1) Sto (Rpi- sto1) Sto (Rpi- sto2)

16 R3a N. benthamiana is resistant to P. palmivora LILI (Colombia) Potato R3a transgenic tobacco Non- transgenic tobacco

17 R3a is effec&ve towards P. palmivora AJ (Indonesia) UV autofluorescence RFP merged WT R3a 3 dpi

18 Only one AVR3a variant is recognised by R3a AVR3a- EM Two alleles of AVR3a in Phytophthora infestans isolates SP RXLR Effector domain E M Potato R protein + R3a - R3a + INF1 (immunity triggering Phytophthora protein) AVR3a- KI K I

19 P. palmivora genome encodes AVR3a effectors P. Infestans (2) P. palmivora (9-15) P. Capsici (10) P. Sojae (2) P. Capsici (10)

20 RXLR effector domain R3a recognises some P. palmivora AVR3a variants Strong Weak None R3a HR Jus?ne Toulo,e

21 Some PpAVR3a variants suppress INF1 cell death Strong suppression weak suppression no suppression 35S-INF1 PpAVR3a Pi A VR3aKI PpAVR3a 35S-INF1 Pi A VR3aKI 35S-INF1 3B2 5A2 PpAVR3a Pi A VR3aKI 7C2 6B2 3C2 7B1 2A1 7A2 4A1 Strong Weak None R3a HR INF1 supp 4 3 2

22 Expressed AVR3a variants evade recogni&on by R3a SP RXLR effector domain

23 Extending R3a recogni&on spectrum Test improved resistance proteins with P. palmivora AVR3a variants

24 Effec&ve Classical resistance R genes fail by to removal deliver accessibility durable resistance factors Classical resistance Accessibility genes factor removal [not durable] [long las?ng resistance] Non- essen&al Microbe effector Plant Resistance protein acessibility factor no accessibility factor Resistant plant Disease Resistant plant barley mlo Medicago ram2 (Wang, Schornack et al, 2012, Curr. Biol.)

25 ram2 mutant inhibits P. palmivora appressorium forma&on WT % 100 % 80 % WT 100 ram2 mutant 60 % 40 % 20 % 0 % W T r a m 2 a pp r e ss o r i a N o app r e ss o r i a ram Wang, Schornack et al. (2012) Current Biol.

26 Cu&n monomers enhance appressorium forma&on and infec&on by P. palmivora 12 Control 10 Appressorium count With 16:0 OH- FA 0 EtOH 0.1% 16:0 OH- FA 10ug/ml Polypropylene surface Wang, Schornack et al. (2012) Current Biol.

27 Varia&on in monocot leaf colonisa&on Barley Wheat Suscep:ble Resistant Suscep:ble Resistant Barley and wheat accessions suscep&bility to P. palmivora 70 % spots infected Suscep:ble Resistant Ruth Le Fevre 0 Germplasm origin: M. Moscou (TSL) and RAGT Seeds Ltd.

28 Summary and Outlook P. palmivora uses effectors to cause disease Some effectors are present in all strains AVR3a effector family can be perceived by potato R3a Highly Expressed AVR3a variants evade recogni?on P. palmivora infects monocot grasses sources of gene?c varia?on Ø Transfer improved immune receptors into oil palms Ø Find gene?c basis for non- immunity resistance

29 Acknowledgements Collabora&ons/Material Sophien Kamoun (TSL, Norwich) Ma, Moscou (TSL Norwich) Giles Oldroyd (JIC Norwich) Joe Win (TSL, Norwich) Liliana Cano (NCSU, Raleigh) Diane Saunders (JIC, Norwich) Mike Coffey (UCR, Riverside) Mauricio Romero (Cenipalma) Carolin Alfs Abhishek Cha,erjee Edouard Evangelis& Stuart Fawke Aleksandr Gavrin Ruth Le Fevre Anna Gogleva Bridget O Boyle Clement Quan Jus&ne TouloPe Temur Yunusov

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