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)

Cochliobolus heterostrophus

Journal Of Agricultural ResearchVol. XXXI. 1925 by Charles Drechsler

asexual reproduction sexual reproduction

pathogen of cornSouthern Corn Leaf Blight

1970’s• More than 30 states affected• 15% corn crop destroyed • >$1 billion lost

-- the worst plant disease epidemic on record

July, 1970

Sept. 1970

N. Dak.

S. Dak.

Neb.

Kan.

Texas

Minn.

La.

Miss. Ala.

Fla.

S. C.

Tenn.

Ill.

Okla.

Ga.

Md.

Vt.

Mass.Con.

R.I.

Fla.

Ala

Mo.

Ark.

Md.

Mass.

Con.

Ill.

Ga.S. C.

La

Miss.

Iowa

Vt.

N. C.

Ky.

Wis.

Ind.Ohio

Mich.N. Y.

Pa.

W. Va.

Va.

N. J

.N

.H.

Maine

Tenn

Miss

Fla.

La.

Canada

Mexico March, 1970

The Southern Corn Leaf Blight Epidemic

1970 Designation of race T (Smith et al; Hooker et al)

two races

race T

Cochliobolus heterostrophus

race O

Texas male sterile (T)-cytoplasm corn

OHOH O OOH OH O OOH OO O O

C41OHOH O OOH OH O OOHO O O

C39

OHOH O OOH OH OO O O

C35

Race T produces a host selective toxin, T-toxin

1971 Discovery of T-toxin in culture filtrates (Lim & Hooker; Gracen et al)

The host: CornOne 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

URF13- a 13 kD protein (mitochondrial inner membrane)

Male sterility in T-corn

Sensitivity to T- toxin

(modified from Wise et al, 1999)

nucleus

Cytoplasm

mitochondrion

T-urf13

T-cytoplasm corn, Cytoplasmic Male Sterility and T-toxin Sensitivity

• 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

race O(Tox -)

race T(Tox +)

race O(Tox -)

race T(Tox +)

N-cytoplasm corn T-cytoplasm corn

Mitochondrialinner membrane

OH

O

O

O

URF13protein 600 m.w.

OH

O

O

O

ENVIR

ON

MEN

T

favorable weather

T-cytoplasm corn(genetic homogeneity)

Southern Corn Leaf Blight (1970)

C. h

eter

ostr

ophu

s

(the

rise

of ra

ce T

)

T-URF-13, Expressed in E. coli, Confers Sensitivity to T-toxin

race T

race O

Genetic Analysis of Tox1

Tox1: a single genetic locus

COCHLIOBOLUS HETEROSTROPHUS

race O (Tox-)

race T (Tox+)

CORNN T

+ +

+ +++

X

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).

Race O

Chromosomes

6 12

Race T

Chromosomes

6;12 12;6

Reciprocal translocation

RFLP map of Cochliobolus heterostrophus

(Tzeng, et al, 1992, Genetics 130:81; Tasma and Bronson, 1998, Current Genetics 34:227).

Tox1

Generation of race T mutants deficient in ability to produce T-toxin

Crosses

Naturally occurring race O X race T

between

Single locus Tox1

Two loci, Tox1A and Tox1B

Tox1 mutants X race O or race T(race T background)

(G. Yang; M. Rose)

Tox1 is two loci: physical evidence

race OTox1-

6

12

race TTox1+

< 6 + 12(3.7 Mb)

12;6 + 6;12 (4.9 Mb)

1.2 Mb ‘extra’ DNA in race T

12;6

6;12

T

OTox1

12;6 T

12 O

O

T

T12;6

O 6

(Tzeng et al, 1992)(Kodama et al, 1999)

R.C4.186R.C4.350

C4.PKS.13

Tox1A

Tox1B

Race O

Chromosomes

6 12

Tentative Model for Evolution of

Race T from Race O

Tox1B

Race T

Chromosomes

Tox1A

6;12 12;6

Reciprocal translocation

Insertion

1.2 Mb DNA

Hypothetical

Intermediate

6 12

Identification of Tox1 genes

Identification of Tox1 genes

PKS1 = polyketide synthase

DEC1 = decarboxylase

RED1 = medium chain reductase/dehydrogenase

(G. Yang; M. Rose)

Tox1A

Tox1B

race T race O pks1-

Assay of tox1- mutants on T-cms corn

Tox1 genes are unique to race T

PKS1

RED1

DEC1

Probe C. heterostrophus r

ace T

C. heterostrophus r

ace O

C. sativ

us

C. carbonum

C. victoriae

C. spicifer

2.3-

23.1-

9.4-

Ge YangMark Rose

Identification of Tox1 genes by genome sequence analysis

““Shotgun” Approach to Sequencing the Shotgun” Approach to Sequencing the CochliobolusCochliobolus Genome Genome

genomic DNA35 Mb

Torrey Mesa Research Institute/Syngenta

shear and clone entire genome

contigs assemble sequences

link contigsmeta contig/scaffold

sequence ends

• 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

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)

C. heterostrophus Scaffold 4FP

1 23,099

contig 1 contig 2 contig 5contig 4contig 3

%AT 74 50 78 64 71

23,099 kb

Scaffold 4FP is at Tox1A

PKS1

PKS1 = polyketide synthase

Functional analysis of new Tox1 genes

Organization of Genes for T-toxin Biosynthesis at Tox1

PKS2Tox1A

PKS1

LAM1

Tox1BDEC1 RED1 RED2 RED3

Didymella zeae maydis

(Mycosphaerella zeae- maydis)

RED1 RED2 PKS1

C. heterostrophus

A +T rich

• Screen for PKSs that are only in race T to find PKSs at Tox1

Are there additional PKSs at Tox1 ?

• 25 PKSs in C. heterostrophus, including PKS1 and PKS2

Presence (+)/Absence (-) in race T and race O

raceT/raceO

PKS14PKS15PKS16PKS17PKS18PKS19PKS20PKS21PKS22PKS23PKS24PKS25

PKS1PKS2PKS3PKS4PKS5PKS6PKS7PKS8PKS9PKS10PKS12PKS13

+/-+/-+/++/++/++/++/++/++/++/++/++/+

raceT/raceO

+/++/++/++/++/++/++/++/++/++/++/++/+