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Cochliobolus heterostrophus
Ophiobolus heterostrophus
(Drechsler, 1925)
Cochliobolus heterostrophus
(Drechsler, 1934)
Anamorphs:
Helminthosporium maydis
(Nisikado & Miyake, 1926)
Bipolaris maydis
(Shoemaker, 1959)
Drechslera maydis
(Subramanian & P.C. Jain,1966)
asexual reproduction
sexual reproduction
Journal Of Agricultural Research
Vol. XXXI. 1925 by Charles Drechsler
pathogen of corn
Southern Corn Leaf Blight
The Southern Corn Leaf Blight Epidemic
Canada
N. Dak.
Maine
Minn.
Vt.
Wis.
S. Dak.
N. Y.
Mich.
Iowa
Neb.
Pa.
Ill.Ill.
Ohio
Ind.
Md.
Mo.
Kan.
Va.
Ky.
N. C.
Tenn
Tenn.
Okla.
Ark.
S. C.
Miss Ala
Miss.
Ala.
Texas
Mexico
Ga.
Ga.
La
La.
La.
Fla.
Fla.
Fla.
March, 1970
July, 1970
Sept. 1970
1970’s -- the worst plant disease epidemic on record
• More than 30 states affected
• 15% corn crop destroyed • >$1 billion lost
Mass.
Con.
R.I.
Cochliobolus heterostrophus
two races
race O
race T
Texas male sterile (T)-cytoplasm corn
1970 Designation of race T (Smith et al; Hooker et al)
Race T produces a host selective toxin, T-toxin
OH OH O
O OH O
O OH O
O OH O
O
C41
OH OH O
O OH O
O OH O
O OH O
C39
OH OH O
O OH O
O OH O
O
C35
1971 Discovery of T-toxin in culture filtrates
(Lim & Hooker; Gracen et al)
The host: Corn
One of the world’s three
most important cereal
crops
40% of the world
corn crop is produced
in the United States
THE PRODUCTION OF HYBRID SEEDS
USING NORMAL PARENT
DETASSELED
FEMALE
INBRED
A
(MALE)
INBRED
B
(SEED PARENT)
Emasculation of Corn
T-cytoplasm corn, Cytoplasmic Male Sterility
and T-toxin Sensitivity
Cytoplasm
T-urf13
mitochondrion
nucleus
• T-corn: corn with Texas male
sterile cytoplasm
(T-cms, or T-cytoplasm)
• T-urf13- a hybrid gene consisting
of parts of 2 mitochondrial genes
and one chloroplast gene
URF13- a 13 kD protein
(mitochondrial inner membrane)
Male sterility
in T-corn
Sensitivity to
T- toxin
(modified from Wise et al, 1999)
race O
race T
race O
race T
(Tox -)
(Tox +)
(Tox -)
(Tox +)
Mitochondrial
inner membrane
N-cytoplasm corn
URF13
protein
T-cytoplasm corn
600 m.w.
Southern Corn
Leaf Blight
(1970)
T-cytoplasm corn
(genetic homogeneity)
T-URF-13, Expressed in E. coli,
Confers Sensitivity to T-toxin
race O
race T
Genetic Analysis of Tox1
Tox1: a single genetic locus
COCHLIOBOLUS
HETEROSTROPHUS
CORN
N
T
+
+
+
+++
race O (Tox-)
X
race T (Tox+)
1:1
Tox1
(Yoder and Gracen, 1975)
RFLP map of Cochliobolus heterostrophus
(Tzeng, et al, 1992, Genetics 130:81; Tasma and Bronson, 1998, Current Genetics 34:227).
6
12
Reciprocal
translocation
6;12 12;6
Race O
Race T
Chromosomes
Chromosomes
RFLP map of Cochliobolus heterostrophus
Tox1
(Tzeng, et al, 1992, Genetics 130:81; Tasma and Bronson, 1998, Current Genetics 34:227).
Generation of race T mutants deficient in
ability to produce T-toxin
Crosses
between
Naturally occurring
Single locus
race O X race T
Tox1
Tox1 mutants X race O or race T
(race T background)
Two loci,
Tox1A and Tox1B
(G. Yang; M. Rose)
Tox1 is two loci: physical evidence
race O
Tox1-
race T
Tox1+
12;6 12
T O
Tox1A
12;6
6;12
R.C4.186
R.C4.350
Tox1B
C4.PKS.13
T
6
1.2 Mb
‘extra’ DNA
in race T
12
6 + 12 < 12;6 + 6;12
(3.7 Mb)
(4.9 Mb)
(Kodama et al, 1999)
O
Tox1
O
T
O T
6 12;6
(Tzeng et al, 1992)
Tentative Model for Evolution of
Race T from Race O
1.2 Mb DNA
Tox1B
Insertion
6
12
Tox1A
Reciprocal
translocation
6
12
6;12 12;6
Race O
Hypothetical
Race T
Chromosomes
Intermediate
Chromosomes
Identification of Tox1 genes
Identification of Tox1 genes
Tox1A
 PKS1 = polyketide synthase
Tox1B
 DEC1 = decarboxylase
 RED1 = medium chain reductase/dehydrogenase
(G. Yang; M. Rose)
Assay of tox1- mutants on T-cms corn
race O
race T
pks1-
Tox1 genes are unique to race T
Probe

PKS1
9.4-
DEC1
23.1-
RED1
2.3-
Ge Yang
Mark Rose
Identification of Tox1 genes by
genome sequence analysis
“Shotgun” Approach to Sequencing the Cochliobolus
Genome
35 Mb
genomic DNA
shear and clone entire genome
sequence ends
contigs
meta contig/scaffold
assemble sequences
link contigs
Torrey Mesa Research Institute/Syngenta
Identification of Tox1 genes by Genome Sequence Analysis
• Query of the C. heterostrophus genome sequence database with
four known Tox1 genes (PKS1, PKS2, DEC1, and RED1)
• These genes mapped to three scaffolds:
•4FP (carrying PKS1)
•4LU (carrying PKS2)
•3PL (carrying DEC1 and RED1)
• These scaffolds were the smallest in the C. heterostrophus
genome assembly
• Genes embedded in highly repeated A+T rich DNA
C. heterostrophus Scaffold 4FP
23,099 kb
contig 1
contig 2
%AT 74
50
contig 3
78
contig 4
contig 5
64
71
23,099
1
PKS1
Scaffold 4FP is at Tox1A
PKS1 = polyketide synthase
Functional analysis of new Tox1 genes
Organization of Genes for T-toxin Biosynthesis at Tox1
C. heterostrophus
Tox1A
PKS1
PKS2
LAM1
A +T rich
DEC1
RED1 RED2
RED3
Tox1B
Didymella zeae
maydis
(Mycosphaerella zeaemaydis)
RED1
RED2
PKS1
Are there additional PKSs at Tox1 ?
• 25 PKSs in C. heterostrophus, including PKS1
and PKS2
• Screen for PKSs that are only in race T to
find PKSs at Tox1
Presence (+)/Absence (-) in race T and race O
raceT/raceO
PKS1
PKS2
PKS3
PKS4
PKS5
PKS6
PKS7
PKS8
PKS9
PKS10
PKS12
PKS13
+/+/+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
raceT/raceO
PKS14
PKS15
PKS16
PKS17
PKS18
PKS19
PKS20
PKS21
PKS22
PKS23
PKS24
PKS25
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+