Outline
• Background
• Data we have
• Summary
• Future plan
Infection and haustorium
Hyphal plasma membrane
Hyphal Wall
Haustorial
Mother Cell
Intercellular Hypha
Neckband
Plant Cell
Haustorial Wall
Extrahaustorial
Membrane
Haustorium
Haustorial Plasma
Membrane
Plant Plasma
Membrane
Koch, 1983
Plant Cell Wall
Extrahaustorial
Matrix
Previous work
Concanavalin A Sepharose
2585 having records
Other no hits in database
454 sequencing for cDNA
BLASTX NR
4483 fungi contigs
RNA in haustoria
1568 having annotation
742 unknown function
275 under criteria
BestORF
SignalP
189 ORFs
TMHMM -30
TargetP -3
156 ORFs candidate effectors
40 Annotated
116 unknown or
under criteria
Purpose
• Which effector candidates could play
important roles in the infection on soybean
by P. pachyrhizi? (virulence or avirulence)
• How do they work?
• What are the soybean partner proteins?
• Is that possible to control ASR through those
mechanisms?
Screening using bacterial tools
JL Dangl, et al. (2006) The plant immune system. Nature 444: 323-329.
Pseudomonas fluorescens EtHAn
P. syringae pv tomato DC3000
P. syringae pv tomato DC3000 ∆CEL
Agrobacterium tumefaciens
Bacteria with
Effectors
Plants
Screening
expectation
Soybean
DC3000
Tobacco
HR suppression
Virulence factors
N. benthamiana
Arabidopsis
DC3000
Tomato
Soybean
Tobacco
EtHAn
N. benthamiana
HR induction
Avirulence factors
Arabidopsis
Tobacco
Agrobacterium
N. benthamiana
82 effector candidates were applied to 12 combinations of screening
PpEC23 has two conserved
cysteine-rich motifs
Large protein family; Special structure feature; Unique tandem repeat
PpEC23 is a ETI suppressor
DC3000
18 hpi
30 hpi
18 hpi
N. benthamiana
42 hpi
Transgenic N. benthamiana plants
confirmed the phenotype
OD 600 nm
DC3000
transgenic N. benthamiana
0.2
0.02
EV
FLAGPpEC23
18 hpi
Relative Expression Level
Expression level of
immune marker genes
PR1a
PR2
801
201
601
151
401
101
201
51
1
P = 0.0331
WRKY12
121
301
81
201
41
101
1
1
P = 0.0008
PI1
P = 0.0268
1
EV
P = 0.1064
FLAG23
16 hpi DC3000 @ OD 600 nm =0.02
Domain analysis of PpEC23
@ 0.02 18 hpi
@ 0.2
1
PpEC23CM2
PpEC23CM1
PpEC23C
PpEC23N
M
PpEC23F
2
DC/23F
DC/23CM2
DC/23N
DC/23CM3
2
DC/23C
DC/23CM4
2
PpEC23CM4
2
DC/23CM1
PpEC23CM3
1
DC3000
M
2
pEDV6
constructs
stands for AvrRps4 1-137 Aa.
PpEC23 is a PTI suppressor
DC3000
DC3000
253±82
ΔCEL
1824±202
ΔCEL/23
557±169
ΔCEL
EV
0
ΔCEL/23N
1861±221
ΔCEL/23C
587±185
ΔCEL/23CM1
1798±236
ΔCEL/23CM2
1768±198
ΔCEL/23CM3
1799±155
ΔCEL/23CM4
577±135
700
1400
2100
Expression level of
immune marker genes
Relative Expression Level
PR1a
7
PR2
WRKY12
33
33
25
25
5
81
17
17
9
9
3
1
PI1
121
41
P = 0.0033
1
P = 0.0038
1
1
P = 0.0119
EV
P = 0.2239
FLAG23
6 hpi EtHAn @ OD 600 nm =0.2
PpEC23 Dimerization
by Yeast Two Hybrid (Y2H)
Plates
SD/X/-LTH
pGBKT7
pGADT7
lam
PpEC23F
T
p53
SD/-LTHA
lam
PpEC23F
SD/-LT
lam
T
PpEC23F
T
Positive control
2 days
10-cysteine-motif 2 only is sufficient for dimerization,
C terminal low complexity motif is necessary for full dimerization
PpEC23 dimerization
by Bimolecular Fluorescence Complementation (BiFC)
YFP
DAPI
BF
Overlay
nYFP-PpEC23
cYFP-PpEC23
Bar = 20 µm
PpEC23 interaction proteins
by library screening using Y2H
Prey
AD
Prey
AD
Prey
AD
PpEC23
GmSPL
12like
AD
BD
LexA
operator
Promoter
lacZ/LEU2
Screening against a soybean library from heavily infected soybean plants
Interaction between PpEC23
and GmSPL12like by Y2H
SD/-LTHA
Plates
pGADT7
pGBKT7
SD/-LT
GmSPL12
SD/X/-LTH
SD/-LT
SPL-C
Empty vector
Full length clone
1017 Aa
Library clone
899 Aa
Truncated clone
SPL-C
517 Aa
Interaction between PpEC23
and GmSPL12like by BiFC
YFP / GFP
DAPI
BF
Overlay
nYFP-GmSPL12
cYFP-PpEC23
nYFP-PpEC23
cYFP-GmSPL12
GFP-GmSPL12
Bar = 20 µm
Interaction between PpEC23
and GmSPL12like by CoIP
FLAG-23
GFP
GFP-SPL
INPUT
+
+
+
+
CO-IP
+
-
+
+
@FLAG
@GFP
SPL proteins
Plant Innate
Immunity
Leaf
Development
AtSPL6
Toxin
Response
SPLs
Gibberelic
Acid Signaling
Vegetative
Phase Change
Flower & Fruit
Development
Plant
Architecture
Sporogenesis
Phenotypes after infection
of Downy mildew
Empty vector
GmSPL12likei
Third trifoliate
Second trifoliate
Bar = 1 cm
Future plan
 More phenotypes for PpEC23i and GmSPL12likei during
the infection by viral assays
 in situ immunolocalization for PpEC23 during infection
 GmSPL12like details
 More screenings are ongoing…
Thank you!
QUESTIONS?