Nuevos Mecanismos Moleculares De Resistencia De Arabidopsis Thaliana
(D15)
´
γ
β β
3
β
β
β
5
7
Chorismate ASA1 ASA2 Anthranilate
TSA1 Indole TSB1, TSB2
COOH
NH2
N H Tryptophan CYP79B2 YUCCA ATR1 CYP79B3 AtDof COOH
Myb51 N OH N H N H Indole-3-acetic acid (IAA) Indole-3-acetaldoxime (IAO X) CYP71A13 CYP83B1 Indole-3-acetonitrile (IAN) C-S lyase dIhydrocamalexic acid UGT74B1 CYP71B15 (PAD3)
N S Desulfoglucosinolate N ST5a H Camalexin - O O S O NO HO O OH S OH OH N H Indol-3-ylmethyl glucosinolate (I3G)
CYP81F2
- O O S O CH3 NO O HO O OH S OH OH N H 4-methoxy-Indol-3-ylmethyl glucosinolate (4MI3G)
β γ
9
Tryptophan
CYP79B2 CYP79B3
CYP71A13 N PAD3 S
IAO X Camalexin N H GSH
PAD2
GSH
NH2 OH
S PEN2 O N C N H I3G H + S I3A RA GSH CYP81F2 CH3 O OH NH2 S O PEN2 C N + H 4MI3G N S H 4MI3A RA
β
11
13
β
15
Carotene
Zeaxanthin
ABA1/ZEP VDE
Antheraxanthin
ABA1/ZEP VDE Violaxanthin
ABA4/NSY Isomerase?
Trans-neoxanthin Isomerase
9-cis-violaxanthin
9-cis-neoxanthin
NCEDs
CHLOROPLAST
Xanthoxin
ABA2 ABA3 Abscisic aldehyde
AAO3 MoCo
Abscisic acid
CYTOPLASM
17
β β
19
β
21
A B 0 1 2
3 4 5
23
µ
μ
µ
β
µ µ µ
µ µ
´ ´
´ ´ ´ ´
´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ β ´ ´ ´ ´
µ
5 12 dpi 4 d d d c,d 3 c b 2
Disease Rating a 1 a 0 -1 -1 WT 8 3 rx i irx9-1 rx1 27435 irx1-6 i g agb1-1 t4 a at3g16920
A B 5 10 dpi WT sid2NahG ein2 coi1 4
3 * * * * * 2
Disease Rating 1
0 irx1 irx1 irx1 irx1 irx1 WT irx1 coi1 sid2 ein2 sid2 NahG ein2 coi1 NahG NahG irx1 coi1 irx1 irx1 sid2 irx1 irx1 ein2 irx1
C WT irx1 NahG irx1 NahG ein2 irx1 ein2 coi1 irx1 coi1 M Pc M Pc M Pc M Pc M Pc M Pc M Pc M Pc dpi 0 1 3 1 3 013130131301313 0 1 3 1 3 013130131301313 PDF1.2
PR-1
PcITS
βββ−β−−−TUB
D 5 E NahG 7 dpi WT NahG ein2 ein2 4 * *
3 * * 2 * Disease Rating * * 1
0 irx1 irx1 irx1 WT
irx1 irx1 ein2 ein2 ein2 agb1 NahG ein2 NahG NahG NahG
irx1 ein2 ein2 irx1 NahG NahG
irx1 µ
6 WT * 5 irx1-6 ixr1-2 4 * * 3 * * 2 ±
n-fold WT expression 1 0 20 50 80 50 20 74 48 60 99 93 1 2 6680 2 3 5 G G 4 G 4G 5G T1 T2 G T4 T T A A T2 A A A A
70
60
50
40
30 up-regulated genes 20 irx5 /
irx1 10 induced by hormonesor stress % 0 T s BA MJ E ns ae s SA cae ta e A erea si s tr n r e ring stress e f lt p in sy Drought B. ci . Sa Wounding tumefaciens M. P P. A. cichoracearum Osmotic s E. ≥
response to stress response to abiotic or biotic stimulus
cytosol
cell wall signal transduction
transporter activity other enzyme activity
hydrolase activity transport
other cytoplasmic components
developmental processes transferase activity
0 1 2 3 4 5 6 Normalized frequency ≥
60
50
40
30
down-regulated genes 20 irx5 /
irx1 10 repressed orhormones by stress % 0 T e e s s g A a a ht s n BA MJ E S rea ens es A e i ic ng tr tre in c s stans oug s s ndi c fa er e yri u . e p Dr tic o B m Salt W M. P. inf P. s cichoracearum A. tu . Osmo E ≥
cell wall response to abiotic or biotic stimulus plasma membrane response to stress receptor binding or activity extracellular kinase activity signal transduction other membranes transporter activity hydrolase activity transcription factor activity transferase activity transport transcription protein binding
0 1 2 3 4 5 8 10 12 Normalized frequency
≥
A 5 6 dpi 4 * 3 2
* * *
Disease Rating 1 0 1 1 1 6 2 WT WT
abi1- µ agb1- aba1- aba1- aba3-
Ler Col-0 B WT(L er ) aba3-2 abi1-1 MP MPMP 0 dpi 0 1 3 1 3 0 1 3 1 3 0 1 3 1 3 PcITS
A
WT (Col-0) irx1-6 aba1-6 aba1-6 irx1-6
B C WT (Ler ) irx3-1 aba3-2
irx1-6 aba1-6 aba3-2 irx3-1
α β µ µ
A B
LTP M TH
LTP M TH LTP M + TH 100 + -
80
60 100 92.4 40 31.7 13.7
20 12.9 % growth inhibition 3.5 0 P. cucumerina R. solanacearum C
Col-0 irx1-6 Col-0 (TH + LTP) α γ
±
µ
A B * 500 MOCK MOCK Pc BMM 300 Pc BMM *
350 200
200 * 125 100 * 100
n-fold expression 75 5 50 * * * 25 * 0 0 PR-1 PDF1.2 PR-4 LTP3 CYP79B2 CYP79B3 CYP81F2 PAD3
A B 1400 160 WT Mock 1200 WT P. cucumerina 140 irx1 Mock 120 1000 irx1 P. cucumerina 100 800 80 600 Peak Area 60
400 40 200 20 nd nd 0 0 I3G 4MI3G 1MI3G Camalexin I3A RA
- O - O - O O O O N S S S S NH2 O O O OH NO CH3 NO NO HO O HO HO O O S OH O OH OH O S OH S OH S OH N H N CN OH OH OH H H N N N S H H O H3C
β
A 5 10 dpi * 4 ** 3 2 Disease Rating 1 B 0 210 3 dpi
200 10 8 β β 6
expression (n-fold WT) (n-fold expression 4
tub 2 β nd nd 0 Pc WT
pad3 cyp79B2 cyp79B3
cyp79B2 cyp79B3
β
A c 350 3 dpi 300 16 14 12 10 b 8 b 6 expression (n-fold WT) (n-fold expression 4 a a tub 2 a β
Pc 0 B 5 6 dpi d β 4 d β c 3 c
2
Disease Rating b 1 a 0 T x1 n3 3 3 3 W ir e n B B p pe 9 9 p7 p7 x1 y y ir 2c 2c 9B pB 7 cy yp 1 c irx
γ
AB Adapted P. cucumerina isolate 1.2 3 dpi Pc BMM BMM) 1.0 Pc Pc
0.8
0.6
20 µm 0.4 * tub expression (n-fold expression (n-fold tub
Non-adapted P. cucumerina isolates β
Pc- 0.2 Pc 1187 * 0
C 5 7 dpi
4
3 Pc 2127
2 Disease Rating 1 * * 0
Pc BMM Pc 1187 Pc 2127
µ β β
Pc BMM 7 dpi 5 Pc 1187 * * Pc 2127 4 * * 3 2
Disease Rating * 1 0 Col-0 irx1 sid2 ein2 jar1 agb1
A C E 210 35 40 3 dpi 3 dpi 3 dpi 30 35 200 25 30 10 25 20 8 20 15 6 15 4 10 10
tub expression (n-fold Col0) (n-fold expression tub 2 5
β 5
Pc 0 0 0 B D F 7 dpi 7 dpi c 6 dpi b 5 e e 5 5 e 4 4 4 d b,c,d c,d c,d b a a a 3 3 b 3 a,b,c b a,b 2 a 2 a 2 Disease Rating 1 1 1
0 0 0 3 0 3 3 3 2 B3 0 2 2 2 l - d 1 l1 -1 -3 -1 2 3 9 l - n F F B B g 2 l -0 2 3 d o e 1 1 9 o a A 9 n a C p 8 8 7 C p 1 7 ol en en p pad 2pad p p p 7 p C p Co pe p n cyp7 y y y p y en2pad32 c c c y c pe p B 2 2 c 2 n B B e 9 9 p 7 7 yp79 p p c y y c c
β β
A Pc BMM 7 dpi Pc 1187 5 * ** * Pc 2127 * 4 * * 3 * * 2 *
Disease Rating * 1
0 Col-0 irx1 pen2 cyp81F2 pen3 pad2 pad3 pen2 cyp79B2 pad3 cyp79B3 B Pc BMM Pc 1187 Pc 2127
Col-0
irx1
pad2
cyp79B2 cyp79B3 20 µm
A B
MOCK 1 dpi 400 MOCK 1 dpi 600 * Pc BMM Pc BMM * 500 Pc 1187 250 Pc 1187 Pc 2127 Pc 2127 400
100 * 140 120 * 100 * 40
n-fold expression 80 60 40 20 20 * * *** 0 0 PR1 PDF1.2 LTP3 CYP79B2 CYP79B3 PAD3
µ
NH 2 Tryptamine N H
Indole N H CH3 N
CH3 Gramine N H OH
S O RA C N S H
CH3 O NH2 4MI3A N H
NH2 I3A N H N S Camalexin
N H Thionin
0 100 200 300 400 500 600
EC50 (µM)
A B
Pc BMM 7 dpi *** * Pc BMM 5 Pc 1187 Pc 2127 * * 4 ** 3 Pc 1187
2 *
Disease Rating *
1 Pc 2127
20 µm 0 Col-0 irx1pen3 irx1 pen3 cyp79B2 irx1 cyp79B2 irx1 cyp79B3 cyp79B2 cyp79B3 cyp79B2 cyp79B3 cyp79B3
100 d 80 c,d c rate 60 b 40
penetration a 20 a 0 Col-0 irx1 pen3 irx1 cyp79B2 irx1 pen3 cyp79B3 cyp79B2 cyp79B3
γ
A B 7 dpi 3 dpi 5 * 4 * 4 * 3 3 2 * 2 * * Disease Rating * 1
1 -tub expression (n-fold wt)
β * Pc 0 2
0 6 6 2 1 6 6 2 1 1 3 er L Col-0 irx1- Col-0 irx1- aba3- aba1- aao3- agb1- aba1- aao3- agb1- aba2- aba2- C
Col-0 aba1-6 aba2-1 aao3-2 Ler aba3-2
β
40000 350 Col-0 * Col-0 aba1 -6 aba1 -6 35000 300 *
30000 250 25000 200 * 20000
n-fold Col-0 * 150 * 15000 * 100 10000
5000 50 * 0 0 0 0 0 0 0 0 0 1 2 7 2 0 2 2 6 4 4 7 5 2 3 4 4 8 4 1 7 9 1 4 3 0 3 5 5 G G G G G G G 2 5 2 3 4 5 5 T T T T T T T A A A A A A A
±
A B -20 2
Pc -6 t=0 -6 -6 -6 M -6 -6 Pc aba1 WT M WT WT WT t=0 aba1 aba1
C aba1 _wt t0 (213)
85
0 66 62 10 425 921 aba1 Pc _M (497)
wt Pc _M (1474)
-30 3
_M _M _WT t0 _WT Pc aba1 Pc aba1 WT
AB C
UP aba1 _wt 12 80 t0 (134) 10 60 8 44 6 40 0 4 38 52 4 20 2 0
Normalized frequency 416 0 ET MJ SA % of UP-regulated genes 510 ABA UP aba1 cell wall E. orontii
Pc _M (472) nematode B. cinerea B.
UP wt Pc _M extracellular P. syringae other binding M. persicae M. infestans P. (1016) Golgi apparatus A. tumefaciens A. other membranes transporter transporter activity response to stress plasma membrane transferase activity cichoracearum E. other enzyme activity
electron transport/energy pathways response to abiotic or biotic stimulus
≥ ≥ ≥
ABC DOWN 40 aba1 _wt t0 (79) 70 30 60 50 20 46 40 0 10 30 26 7 6 20 4 10 3 12 0 2 Normalized Frequency 1 ET MJ %Down-regulated genes 413 SA
DOWN aba1 ABA 0 Pc _M (25) E. E. orontii nematode DOWN wt B.cinerea P.syringae M. persicae M. cell wall P. infestans Pc _M (458) A.tumefaciens
extracellular E. E. cichoracearum
hydrolase activity other membranes
cell organization and biogenesis
≥ ≥ ≥
AB Cluster IV
-30 3 40
30
_M 20 _WT t0 Pc 10 ACC SA aba1 WT MJ
Normalized Frequency 0 cell wall extracellular and biogenesis cell organization
I hydrolase activity Cluster other membranes IV 8 6
4
2
Normalized Frequency 0
I activity
W-BOX other enzyme or biotic stimulus energy pathways transporter activity
TTGACT response to abiotic nucleic acid binding 4.3 x 10 -4 electron transport or Cluster III
10 8 III 6 GCC-BOX 4 GCCGCC 2 1.01 x 10 -7 W-BOX Normalized Frequency 0 TTGACT 4.3 x 10 -4 cell wall biotic stimulus response to stress transporter activity energy pathways electron transport or Cluster II response to abiotic or 25
20
15
II 10 5
Normalized Frequency 0 cell wall extracellular or biotic stimulus energy pathways other membranes response to abiotic electron transport or
N N N N ≥
A B C
5 5 30 8 dpi 8 dpi 3 dpi d d 25 4 c 4 c 20 3 a,b 3 a,b b 15 2 2 a 10 Disease Rating 1 1 a c
-tub expression (n-fold wt) 5 β a,b a
0 0 Pc 0 Col-0 aba1 coi1 coi1 aba1 Col-0 aba1 aos aos aba1 Col-0 aba1 aos aos aba1
β
≥
response to abiotic or biotic stimulus cell wall plasma membrane response to stress extracellular kinase activity plastid signal transduction receptor binding or activity transporter activity ER other enzyme activity other membranes cytosol electron transport or energy pathways transport transferase activity chloroplast protein binding 0 1 2 3 4 Normalized Frequency
≥
-30 3 _M _M
_WT t0 Pc WT WT aba1 aba1 Pc aba1 ACC SA MJ
ABA
≥
A B
response to stress response to abioticor -20 2 biotic stimulus other enzyme activity
_M transporter activity _M WT t0
Pc chloroplast _WT aba1_ irx aba1 Pc WT transport Cluster I 01 23 4 5678 910 I Normalized Frequency
cell wall
extracellular Cluster II 0 2 4 6 8 10 12 14 16 18 20 Normalized Frequency
II N N
N N ≥
-30 3 _Mk WT t0
Pc _WT ACC WT WT SA aba1_ irx MJ ABA
ż
?
?
Resistance to necrotrophs
ż
β
≤
≤
≤
≤