Kolomiets

Current Lab Members :
Eli Borrego lox7, 8 and 9 mutants
Shawn Christensen lox10, 12 and opr2 mutants
Yuanxin Yan lox1, lox2 and opr7 and 8
mutants
Former Lab Members :
Xiquan Gao (now at TAMU, Dept. Biochemistry and
Biophysics) lox3 and other 9-
LOXs
Yong-Soon Park (now in Cornell) lox4 and lox5
mutants
Andriy Nemchenko (South-Western Medical Center)
lox10 and lox12 mutants
Jinglan Zhang (now Human Genetics Consulting
firm (OPR gene family )
LOX and OPR mutants harvest

University of Wisconsin-Madison:
Nancy Keller, Marion Brodhagen,
Sigal Brown Horowitz Aspergillus nidulans and A. flavus oxylipin mutants
Texas A&M University:
Seth Murray, Gerald de LaFuente Corn Genetics and Breeding
Won-Bo Shim, Tom Isakeit mycotoxin quantification
Göttingen University, Germany:
Ivo Feussner oxylipin profiling (42 diverse oxylipins)
USDA ARS, Peoria, IL:
Daren Brown microarray profiling of Fusarium verticillioides

1. Overview of my lab program:
Maize lipoxygenase (LOX) and oxo-phytodienoate
(OPR) gene families
2. Hypothesis for why oxydized fatty acids determine whether
corn will get contaminated with mycotoxins or be resistant.
3. Interactions of maize oxylipin mutants with Fusarium
verticillioides vs Aspergillus flavus
3. Initial evidence for oxylipin-mediated cross-talk using
oxylipin mutants of maize and A. flavus

Aspergillus flavus
colonizing maize kernel
Fungal Oxylipins:
blend A
Host Oxylipins:
blend B
Compatible:
Heavy colonization
Spore production high
Mycotoxin?
Incompatible:
Weak colonization
Spore production low
Mycotoxin?

Oxylipins are oxygenated lipids derived from
polyunsaturated fatty acids
a) linoleic acid (C18:2)
b) linolenic (C18:3)

Aldehyde
Green Leaf Volatiles
+
+
Epoxyalcohol
ω-oxo fatty acid
~400 plant oxylipins
identified to date
( I. Feussner, pers. comm. )
LA/LeA
HPO(D/T)E
Allenoxide
AOC
12-Oxophytodienoic acid
(12-OPDA)
Jasmonsäure
Plant “LOX Pathway”
H 2O
HO(D/T)E
Jasmonic acid
Divinylether
α-Ketol
+
γ-Ketol
KO(D/T)E

Aspergillus nidulans
Aspergillus flavus
(studies by N. Keller’s group)
Fusarium verticillioides?
Psi-factor producing
oxygenases (PPO),
LOX
psi-factors,
other oxylipins
Sporogenesis,
Mycotoxin synthesis
Quorum sensing
Pathogen
Reviewed in Brodhagen and Keller, 2006. MPP 7: 285

Hypothetical model of oxylipin-based cross-talk

Ongoing research towards testing hypothesis: Keller Lab

Ongoing research towards testing hypothesis: my lab
OPRs

13-LOXs:
Putative producers of
jasmonic acid (JA) and
green leaf volatiles (GLV)
Mostly 9-LOXs:
9-hydro(pero)xides
and other 9-oxylipins
ZmLOX12
ZmLOX7
ZmLOX1
ZmLOX2
0.05 changes
ZmLOX8
ZmLOX3
ZmLOX9
ZmLOX4
ZmLOX10
ZmLOX5
ZmLOX11
ZmLOX13
ZmLOX6
Nemchenko et al. 2006. J Exp Bot 14: 3767
Gao et al. 2008. Planta 227: 491
Park et al. 2010. Planta: in print
Kolomiets et al., unpublished

13-LOXs:
Putative producers of
jasmonic acid (JA) and
green leaf volatiles (GLV)
Mostly 9-LOXs:
9-hydro(pero)xides
and other 9-oxylipins
ZmLOX7
ZmLOX2
0.05 changes
ZmLOX8
ZmLOX9
ZmLOX4
ZmLOX10
ZmLOX5
ZmLOX11
ZmLOX13
C13 + C5
producing
HPL
Nemchenko et al. 2006. J Exp Bot 14: 3767
Gao et al. 2008. Planta 227: 491
Park et al. 2010. Planta: in print
Kolomiets et al., unpublished

ZmLOX10
ATG
I
lox10-1
Mutant alleles were genetically advanced into backgrounds of corn lines that are
either susceptible or resistant to fumonisin or aflatoxin contamination.
II
lox10-2
III
lox10-3
IV
TGA

1. Overview of the lab program:
Maize lipoxygenase (LOX) and oxo-phytodienoate
(OPR) gene families
2. Hypothesis for oxylipin-based cross-kingdom signal
communication
3. Interactions of maize oxylipin mutants with Fusarium
verticillioides vs Aspergillus flavus
3. Initial evidence for oxylipin-mediated cross-talk using
oxylipin mutants of maize and A. flavus

• Suspected carcinogen
• Inducer of Programmed Cell Death
Fusarium verticillioides
• inhibitor of sphingolipid biosynthesis
• leukoencephalomalacia in horses,
pulmonary edema in pigs
Fumonisin B1

nmol /g FW
8
7
6
5
4
3
2
1
0
WT 5-3
WT 5-1
KO 4-1
KO 4-2
9-HOD 9-KOD
Linoleic acid (18:2)-
derived oxylipins
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
WT 5-3
WT 5-1
KO 4-1
KO 4-2
9-HOT 9-KOT
Linolenic acid (18:3)derived
oxylipins

Secondary metabolism
Mycotoxins
Vegetative growth
+++
LOX3
+
Sporogenesis

Expression fold change in lox3 mutants vs WT
LOX10
LOX5
Microarray analysis of F. verticillioides infected lox3 mutant vs WT seed
20
16
12
8
4
WT
4 dpi 8 dpi
ZmLOX6 ZmLOX9 ZmLOX10 ZmLOX11 ZmOPR7 ZmOPR8
lox3
LOX3
LOX5
LOX10

WT lox5-3
*
WT lox5-3
FB1 (ng / kernel)
* P

Secondary metabolism
Mycotoxins
Vegetative growth
LOX5 - -
+++
LOX3
+
- -
LOX5
Sporogenesis

*
lox10-3 WT
FB1 (ng / kernel)
lox10-3 WT
lox10-3 WT
*
* P

Secondary metabolism
Mycotoxins
Vegetative growth
LOX5 - -
LOX10
LOX10
- -
- -
+++
LOX3
+
LOX5
- -
-
LOX10
Sporogenesis

Spores (106 / g of
kernel)
WT lox12
C
WT lox12
***
FB 1 ppm
***
WT lox12

Spores (10 6 / g of kernel)
WT lox3 lox12 lox3/12
b
a
c
b
FB 1 ppm
WT lox3 lox12 lox3/12 WT lox3 lox12 lox3/12
b
a
c
b

Secondary metabolism
Mycotoxins
Vegetative growth
LOX5 - -
LOX10
LOX10
- -
- -
+++
LOX3
+
LOX12 LOX3
LOX5
- -
-
LOX10
Sporogenesis

Aspergillus flavus
Aflatoxin
• potent hepatocarcinogen
• mutagen
• causes immunosuppression in animals

Surprisingly,
interaction of lox3, lox5 and lox10
mutants with Aspergillus flavus results
in completely opposite phenotypes
compared to Fusarium verticillioides

3.5 dpi
WT lox3-4

Silk-channel inoculation with Aspergillus flavus

Aflatoxin (ppb per ear)
1500
1000
500
0
2000
1500
1000
500
0
* *
* *
WT lox3-4
*
*
WT lox3-4
2000
1500
1000
500
0
1500
1000
500
0
*
*
WT lox3-4
WT lox3-4
** P

Initial tests of hypothesis of oxylipin-based cross-talk

Preliminary data:
LOX3 mutation of the host alters growth and development of
oxylipin mutants of Aspergillus flavus

Tx714 FR2128 Tx772
College Station field tests, 2006

College Station
2006
WT lox10-3
College Station
2007
WT lox10-3

An update:
Improving Maize Against Aflatoxin and Drought:
Translational Plant Breeding, Education, and
Extension

14 SNPs + 1 InDel
SNP-A/T
DS=T
7% - T
SNP-G/T
DS=T
6% - T
SNP-C/T
DS=C
62% - C
SNP-A/G
DS=A
2% - A
Alignment of ZmLOX5
SNP-A/G
DS=G
79% - G
SNP-A/G
DS=G
83% - G
DS = Derived State
Original State = Z. perennis
Located on Chromosome 5
SNP-C/T
DS=C
43% - C
SNP-C/G
DS=C
4% - C
SNP-C/G
DS=C
92% - C
28bp
INDEL
Va99
SNP-T/G
DS=T
45% - T
SNP-G/T
DS=T
6% - T
SNP-G/A
DS=G
16% - G
SNP-C/T
DS=C
77% - C
SNP-G/A
DS=G
20% - G

ZmLOX5 Duplica1on/Absence
Two Copies of ZmLOX5
Two Copies of ZmLOX5
ZmLOX5 Absent

Correlation between 100 Zmlox4 and Zmlox5 lines
3000
2500
2000
1500
1000
500
0
LSMEANS
Aflatoxin
y = 0.57x + 654
R² = 0.30
0 500 1000 1500 2000 2500 3000
Aflatoxin
Lowest five Hybrids
SC 212M zmlox5 128
Tzi 9 zmlox5 191
CML 91 zmlox5 195
Mp 04:96 zmlox5 215
NC370 zmlox5 220
Highest five Hybrids
NC330 zmlox5 2600
B73Htrhm zmlox5 2600
A680 zmlox5 2650
NC316 zmlox5 2700
A679 zmlox5 2850
Commercial Checks
BH 9014 zmlox5 86
26V21 zmlox5 113
28V81 zmlox5 116
DeKalb 68-05 zmlox5 105

A screenshot of the main page
http://aflatoxin.tamu.edu/

THANK YOU
Questions?????????