P. palmivora entering a leaf
Sebas&an Schornack University of Cambridge, Sainsbury Laboratory (SLCU)
h,p://www.schornacklab.net; @dromius
18th Interna?onal Oil Palm Conference, Cartagena, Colombia
Zoospore
Phytophthora sp. cause diseases in important crops
University Davis
A. Dorrance (APSnet).
P. Infestans – potato late blight P. ramorum – sudden oak death
P. sojae – soybean root rot P. palmivora – palm bud and cocoa fruit rot
narrow broader Host range
Phytophthora palmivora is a cosmopolit
P. palmivora diseases
In the tropics the most commonly encountered species is Phytophthora palmivora.
P. palmivora sporangia releasing zoospores
Picture: QuesadaLabNCSU
Phytophthora palmivora causes oil palm bud rot
Economically important hosts
Oil palms Coconut Orchids Pineapple
Rubber tree
Durian Papaya Cocoa Citrus
Biotrophy Necrotrophy
Phytophthora is a stealthy pathogen
mycelia
Sporangia and spores
Zoospore
Phytophthora infec&on cycle
Electron microscopy Light microscopy
Fluorescence of a transgenic P.
palmivora isolate
P. palmivora forms haustoria in living plant cells
effectors
fungus
oomycete
haustorium
plant cell
Alter plant cell processes
targets
Help microbe colonize plant
Microbes modify plant cell processes using secreted effector proteins
• 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
RXLR SP effector domain
effectors
haustorium
Alter plant cell processes
targets
effector-‐ triggered immunity
Plant immunity: Plant resistance proteins recognise effectors
fungus
oomycete
plant cell intracellular immune receptors
• 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?
Surveying effector – resistance protein combina&ons
Conserved Effector genes From pathogen
R gene candidates from oil palm genome
Or Known R genes
MATCH!
Expression in Nico:ana benthamiana using A. tumefaciens
Limita&ons: R gene is func?onal in N. benthamiana, AVR and R gene are both cloned
Effector + matching Resistance gene
Wrong effector or wrong Resistance gene
mul?locus phylogeny of Phytophthora from Blair et al. 2008 Fungal Genet Biol
Phytophthora infestans (240 Mbp) - Solanum spp.
P. palmivora belongs to a clade with no sequenced member species
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
LILI
AJ
Sequencing @Kamounlab
Genome sequencing of two P. palmivora isolates
Inden&fica&on of conserved effectors
Edouard Evangelis?
RXLR SP effector domain
It is bePer to perceive early expressed effectors
Edouard Evangelis?
Early infec&on
Late during infec&on
AVR2
Edn (Rpi-‐edn1)
Hjt (Rpi-‐hjt1) Snk (Rpi-‐snk1)
AVRb
lb1
Blb (Rpi-‐blb1)
Sto (Rpi-‐sto1)
Avr3a
Sto (Rpi-‐sto2)
Dms (R3a)
CRNs
In the future: effector based resistance phenotyping
Vivianne Vleeshouwers et al
Non-‐transgenic
tobacco
Potato R3a transgenic
tobacco
R3a N. benthamiana is resistant to P. palmivora LILI (Colombia)
WT
R3a
3 dpi
UV autofluorescence RFP merged
R3a is effec&ve towards P. palmivora AJ (Indonesia)
+ INF1 (immunity triggering
Phytophthora protein)
AVR3a-‐EM
AVR3a-‐KI
E M
K I
SP RXLR
Two alleles of AVR3a in Phytophthora infestans isolates
+ R3a - R3a Potato R protein
Only one AVR3a variant is recognised by R3a
Effector domain
P. palmivora genome encodes AVR3a effectors
P. Capsici (10)
P. Infestans (2)
P. Sojae (2) P. Capsici (10)
P. palmivora (9-‐15)
+++
-‐-‐
-‐-‐
-‐-‐
-‐-‐
+
+++
+
+
-‐-‐
R3a recognises some P. palmivora AVR3a variants
Jus?ne Toulo,e
Strong Weak None
R3a HR 1 3 5
RXLR effector domain
3C2
Pi A VR3aKI
3B2
2A1
4A1
7A2
5A2
6B2
7C2
7B1
Pi A VR3aKI
Pi A VR3aKI
PpAVR3a PpAVR3a
PpAVR3a
Some PpAVR3a variants suppress INF1 cell death
35S-INF1 35S-INF1
35S-INF1
Strong suppression weak suppression no suppression
Strong Weak None
R3a HR 1 3 5
INF1 supp 4 3 2
Expressed AVR3a variants evade recogni&on by R3a
RXLR SP effector domain
Extending R3a recogni&on spectrum
Test improved resistance proteins with P. palmivora AVR3a variants
Classical R genes fail to deliver durable resistance
Resistant plant
Non-‐essen&al Microbe effector
Plant Resistance protein
Classical resistance genes [not durable]
Disease
Resistant plant
no accessibility
factor
acessibility factor
Accessibility factor removal [long las?ng resistance]
• barley mlo • Medicago ram2 (Wang, Schornack et al, 2012, Curr. Biol.)
Effec&ve resistance by removal accessibility factors
WT
ram2
0 %
20 %
40 %
60 %
80 %
100 %
120 %
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
100
10
100 10
WT
ram2 mutant
ram2 mutant inhibits P. palmivora appressorium forma&on
Wang, Schornack et al. (2012) Current Biol.
Cu&n monomers enhance appressorium forma&on and infec&on by P. palmivora
Control
With 16:0 OH-‐FA
Polypropylene surface
0
2
4
6
8
10
12
EtOH 0.1%
16:0 OH-‐FA 10ug/ml
Appressoriu
m cou
nt
Wang, Schornack et al. (2012) Current Biol.
Varia&on in monocot leaf colonisa&on Barley
Suscep:ble Resistant
Wheat Suscep:ble Resistant
0
10
20
30
40
50
60
70
80
90
% sp
ots infected
Barley and wheat accessions suscep&bility to P. palmivora
Suscep:ble Resistant
Germplasm origin: M. Moscou (TSL) and RAGT Seeds Ltd. Ruth Le Fevre
• 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
Summary and Outlook
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)
Acknowledgements
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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