a genomic approach to mycotoxin control - cnrmycoglobe.ispa.cnr.it/brussel/usa-kendra.pdf · a...

A Genomic Approach toA Genomic Approach to

Mycotoxin ControlMycotoxin Control

Deepak Deepak BhatnagarBhatnagar

David KendraDavid Kendra

United States Department of AgricultureAgricultural Research Service

Fusarium Fusarium verticillioidesverticillioidesCornCornFumonisinsFumonisins

Fusarium graminearumFusarium graminearumCorn, wheat, barleyCorn, wheat, barleyTrichothecenesTrichothecenes

Aspergillus Aspergillus flavusflavus

Aspergillus Aspergillus parasiticusparasiticusCorn, cottonseed,Corn, cottonseed,peanuts, peanuts, treenutstreenuts

AflatoxinsAflatoxins

Producing FungiProducing FungiCommoditiesCommodities

AffectedAffectedToxinToxin

In U.S.A. mycotoxins of In U.S.A. mycotoxins of majormajor concern include:concern include:

MYCOTOXINS OF U.S. INTEREST MYCOTOXINS OF U.S. INTEREST

PenicilliumPenicillium cyclopiumcyclopium

Aspergillus Aspergillus flavusflavusCorn, Corn, kodokodo milletmilletCyclopiazonicCyclopiazonic acidacid

Fusarium graminearumFusarium graminearumCorn, hayCorn, hayZearalenoneZearalenone

AspergillusAspergillus sppspp..

PenicilliumPenicillium sppspp..Apples, wheat straw Apples, wheat straw

residueresiduePatulinPatulin

Aspergillus Aspergillus ochraceusochraceus

PenicilliumPenicillium verrucosiumverrucosiumWheatWheatBarley, oats, corn, Barley, oats, corn,

othersothers

OchratoxinOchratoxin AA

Producing FungiProducing FungiAffected Affected

CommoditiesCommoditiesToxinToxin

The following mycotoxins are also receiving The following mycotoxins are also receiving significantsignificant attention:attention:

MYCOTOXINS OF U.S. INTERESTMYCOTOXINS OF U.S. INTEREST

The U.S. Food and Drug Administration has issued regulatory limits for aflatoxins.

REGULATIONREGULATION

Food and Drug Administration (1996)Food and Drug Administration (1996)

Action levelsAction levels

((µgµg/kg, ppb)/kg, ppb)Food and FeedFood and Feed

2020

0.50.5

2020

300300

100100

200200

300300

2020

Human foods (except milk)Human foods (except milk)

MilkMilk

Animal feeds (except as listed below)Animal feeds (except as listed below)

Cottonseed meal (as a feed ingredient)Cottonseed meal (as a feed ingredient)

Corn and peanut products for breeding beef cattle, Corn and peanut products for breeding beef cattle,

swine and mature poultryswine and mature poultry

Corn and peanut meal for finishing swineCorn and peanut meal for finishing swine

Corn and peanut meal for feedlot beef cattleCorn and peanut meal for feedlot beef cattle

Corn for immature animals and dairy cattleCorn for immature animals and dairy cattle

Current aflatoxin action levelsCurrent aflatoxin action levels

FDA has issued guidelines or advisories for other mycotoxins:

REGULATIONREGULATION

**PatulinPatulin Technical Symposium (FDA), Feb.18Technical Symposium (FDA), Feb.18--19, 2003, Orlando, Florida19, 2003, Orlando, Florida

(none), recommendation: good agricultural(none), recommendation: good agricultural

and manufacturing practicesand manufacturing practices

OchratoxinOchratoxin AA

50 50 µgµg /kg (ppb) in apple juice/kg (ppb) in apple juicePatulinPatulin**

22--4 4 ppmppm for human foodsfor human foods

55--100 100 ppmsppms for animal feedsfor animal feeds

Fumonisins Fumonisins

(B1, B2, B3) (B1, B2, B3)

1 mg/kg (1 mg/kg (ppmppm) for finished wheat products ) for finished wheat products for human usefor human use

55--10 10 ppmppm for other food and feedfor other food and feed

DeoxynivalenolDeoxynivalenol

ECONOMIC IMPACT OF MYCOTOXIN

CONTAMINATION

ECONOMIC IMPACT OF MYCOTOXIN ECONOMIC IMPACT OF MYCOTOXIN

CONTAMINATION CONTAMINATION

U.S. Food and Drug AdministrationU.S. Food and Drug Administration

(based on sum of the value of food and feed losses)(based on sum of the value of food and feed losses)

estimated annual crop losses of: estimated annual crop losses of:

$932 million per year (direct costs)$932 million per year (direct costs)

$466 million per year (mitigating costs)$466 million per year (mitigating costs)

$6 million per year (livestock costs)$6 million per year (livestock costs)

from contamination with 3 mycotoxins:from contamination with 3 mycotoxins:

AflatoxinsAflatoxins, Fumonisins, , Fumonisins, DeoxynionlenolDeoxynionlenol

________________________________________________________________________________________

**Reviewed in U.S. Council for Agricultural Science Reviewed in U.S. Council for Agricultural Science

and Technology Task Force Report, 2003.and Technology Task Force Report, 2003.

MYCOTOXIN CONTAMINATIONMYCOTOXIN CONTAMINATION

1.1. PostPost--HarvestHarvest

2.2. PrePre--HarvestHarvest

Post-Harvest Contamination

can be managed by

Pre-Harvest Control

PostPost--HarvestHarvest Contamination Contamination

can be managed bycan be managed by

PrePre--HarvestHarvest ControlControl

Strategies to Control PreStrategies to Control Pre--Harvest Harvest

Mycotoxin Contamination:Mycotoxin Contamination:

1.1. Cultural PracticesCultural Practices

2.2. Enhancing host resistanceEnhancing host resistance

-- Plant breeding / Genetic EngineeringPlant breeding / Genetic Engineering

3.3. Biological ControlBiological Control

4.4. Understanding basic biology / toxin Understanding basic biology / toxin

producing ability of the fungusproducing ability of the fungus

1.1. How are toxins synthesized by the How are toxins synthesized by the

producing organismproducing organism

2.2. Where are they produced in the cells of the Where are they produced in the cells of the

producing organismproducing organism

3.3. What environmental factors affect the What environmental factors affect the

synthesis of the toxin and the mechanism synthesis of the toxin and the mechanism

of the effectof the effect

For Control of toxin formation it isFor Control of toxin formation it is

important to know:important to know:

Mycotoxin Biosynthesis:Mycotoxin Biosynthesis:

•• Chemistry of toxin formationChemistry of toxin formation-- Precursor IdentificationPrecursor Identification

•• Biochemical StudiesBiochemical Studies-- Enzyme StudiesEnzyme Studies

•• GeneticsGenetics-- Gene CloningGene Cloning

USDA/ARSUSDA/ARS

Southern Regional Research CenterSouthern Regional Research Center

New Orleans, LANew Orleans, LA

Aflatoxin Biosynthesis GroupAflatoxin Biosynthesis Group

Deepak Deepak BhatnagarBhatnagar

Jeffrey CaryJeffrey Cary

PerngPerng--KuangKuang ChangChang

Thomas E. ClevelandThomas E. Cleveland

Kenneth EhrlichKenneth Ehrlich

JiujiangJiujiang YuYu

Collaborative EffortCollaborative Effort

Gary Payne

N. C. State Univ.John Linz / Frances Trail

Michigan State Univ.

Nancy Keller

Univ. Wisconsin-MadisonCharles Woloshuk

Purdue Univ.

Fun Sun Chu

Univ. Wisconsin-Madison

Deepak Bhatnagar / Thomas E. Cleveland

USDA-ARS-SRRC

Joan Bennett

Tulane University

USDA/ ARS/National Center for USDA/ ARS/National Center for

Agriculture Utilization Research, Peoria, ILAgriculture Utilization Research, Peoria, IL

Trichothecene Biosynthesis GroupTrichothecene Biosynthesis Group

Nancy AlexanderNancy Alexander

Daren BrownDaren Brown

Anne DesjardinsAnne Desjardins


Susan McCormickSusan McCormick

Ron PlattnerRon Plattner

Robert ProctorRobert Proctor

(Rex Dyer (Rex Dyer -- PostPost--doc)doc)

known roleunknown roleno role and examinednot examined

0 kb 4 8 12 16 20 24 28 32 36 40 44 48 52 56

Core Fusarium trichothecene gene cluster

orfF methytransferaseorfE unknown orfD unknownorfC beta-1,3-glucanosyltransferase orfB acetylesterase

orfA tyrosinase

TRI8 Similar to extracellular lipase from Candida albicansTRI7 AcetylesteraseTRI3 15-decalonectrin 15-0-acetyltransferaseTRI4 Cytochrome P450TRI6 DNA Binding Positive Acting Transcription FactorTRI5 Trichodiene Synthase (sequiterpene cyclase)TRI10 Regulation TRI9 Unknown TRI11 P450 or isotrichodermin C-15 hydroxylase (CYP 65A1TRI12 Trichothecene Efflux Pump

orfG deacetylase orfH unknown orfI dehydrogenase orfJ NADH-cytochrome b5 reductaseorfK cytochrome P450orfL unknown

TrichothecenesTrichothecenes biosynthesisbiosynthesis

USDA/ ARS/National Center for USDA/ ARS/National Center for

Agriculture Utilization Research, Peoria, ILAgriculture Utilization Research, Peoria, IL

Fumonisin Biosynthesis GroupFumonisin Biosynthesis Group


Robert ButchkoRobert Butchko

Anne DesjardinsAnne Desjardins


Ronald PlattnerRonald Plattner


Gene Predicted Function Based on

Name Sequence Similarity

FUM1 Polyketide synthase

FUM6 Cytochrome P450 Monooxygenase

FUM7 Dehydrogenase

FUM8 Aminotransferase

FUM3 Dioxygenase

FUM10 Fatty Acid-CoA Synthase

FUM11 Tricarboxylate Transporter


FUM13 Short-chain Dehydrogenase/Reductase

FUM14 Peptide Synthetase Condensation Domain


FUM16 Fatty Acid-CoA Synthetase

FUM17 Longevity Assurance Factor

FUM18 Longevity Assurance Factor

FUM19 ABC Transporter

Fumonisin Biosynthetic Gene Cluster

10 20 30 40 500kb scale

FUM

13FUM

14FUM

15FUM

16FUM

17FUM

18FUM

19

FUM

2

FUM

3FUM

10FUM

11

FUM

8

FUM

7

FUM

6

FUM

1

Proposed Fumonisin Biosynthetic Pathway

Proposed Fumonisin Proposed Fumonisin

Biosynthetic Biosynthetic

Pathway Pathway

HFB3 HFB4

FB4

FB2

FB3

FB1

FUM11

HO OH

O OOHO

S OH

O OOHO

FUM7

CoA

FUM10

Mitochondria

+

FUM14

OH

HO

O

HO

O

CH3CH3

O

CH3CH3NH2

CH3CH3NH2

OH

OHOH

OH

CH3CH3NH2

OH OH

OH

FUM1

FUM6

FUM13

FUM8

HO

O

CH3CH3

OH

OH

FUM2

Acetate

HFB3

HFB4

S OH

O OOHO

CoA

+

CH3CH3NH2

OH O

O

OH

OH

O O

O O

OH

OH

O

O

OHFB3

CH3CH3NH2

OH O

O

OH

OH

O O

O O

OH

OH

O

O

FUM14

FB4

OH

O

NH2

+

CH3CH3NH2

OH O

O

OH

OH

O O

O O

OH

OH

O

O

OH

OH

FUM3

FB1

CH3CH3NH2

OH O

O

OH

OH

O O

O O

OH

OH

O

O

OH

FB2

FUM3

1.1. pHpH

2.2. Nutritional source Nutritional source egeg. nitrogen, carbon, etc.. nitrogen, carbon, etc.

3.3. Elemental availability Elemental availability egeg. phosphorus, selenium. phosphorus, selenium

silica, cobalt, magnesiumsilica, cobalt, magnesium

4.4. TemperatureTemperature

5.5. Other physiological factors Other physiological factors egeg. volatile compounds. volatile compounds

6.6. Biotic factors Biotic factors egeg. competing flora/fauna. competing flora/fauna

THE “AGROECOSYSTEM”THE “AGROECOSYSTEM”

Complex environmental and ecological factors affecting A.flavus and aflatoxin contamination process:

Not Dealing with Obligate Fungal Not Dealing with Obligate Fungal

Parasites (requiring their host plantParasites (requiring their host plant

to complete their life cycle)to complete their life cycle)

*Mycotoxin producing fungi occupy broad ecological niches and range from being pathogenic, to opportunistic parasites, to saprophytes living on dead organic material in the soil.

*Resistance to infection by these fungi in plants likely involves several resistance genes that need to be coordinately expressed.

Mycotoxin Problems Fit Two Criteria Mycotoxin Problems Fit Two Criteria

Pointing Toward a Genomics Pointing Toward a Genomics

Approach For Finding Solutions:Approach For Finding Solutions:

1. Complexity of Plant-Fungus Interaction Requires a Global Approach in Identifying and Monitoring Key Genes Involved.

2. Some Benchmark Studies are Available Providing Knowledge of the Biology, Biochemistry and Genetics of Mycotoxins/Mycotoxin-Producing Fungi.

Provides an “anchor” for comprehensive genomics investigations.

A GENOMICS A GENOMICS

approach toapproach to

Mycotoxin ControlMycotoxin Control

Selected Fungal Genome ProjectsSelected Fungal Genome ProjectsSelected Fungal Genome Projects

1,2,71,2,7Whole genome sequencingWhole genome sequencingAspergillus Aspergillus flavusflavus

6,86,8Public, Private, ESTPublic, Private, EST’’s and whole genome in s and whole genome in

progressprogressFusarium graminearumFusarium graminearum

1,5,7,8,91,5,7,8,9Public, Public, PrivatePrivate, EST, EST’’s s andand wholewhole genomegenomeFusarium Fusarium verticillioidesverticillioides

44Public, ESTPublic, EST’’ssFusarium sporotrichioidesFusarium sporotrichioides

1,2,31,2,3Public, expressed sequence tags (ESTPublic, expressed sequence tags (EST’’s)s)Aspergillus Aspergillus flavusflavus

SourcesSourcesSourcesFungusFungusFungus StatusStatusStatus

1 1 USDA, Agricultural Research ServiceUSDA, Agricultural Research Service--funded genomicsfunded genomics

22 North Carolina State Univ.North Carolina State Univ.

33 Univ. of Oklahoma Advanced Center for Genome Tech./ The Univ. ofUniv. of Oklahoma Advanced Center for Genome Tech./ The Univ. ofWisconsinWisconsin

44 ACGT/ Texas A&M Univ.ACGT/ Texas A&M Univ.

55 Purdue Univ.Purdue Univ.

66 Michigan State Univ./ Purdue Univ./ ARS Cereal Disease LabMichigan State Univ./ Purdue Univ./ ARS Cereal Disease Lab

77 The Institute for Genomic Research, MarylandThe Institute for Genomic Research, Maryland

88 Syngenta, Inc. (4X)Syngenta, Inc. (4X)

99 DuPont, Inc. (1X)DuPont, Inc. (1X)

Aspergillus flavusAspergillus flavusgenomics project;genomics project;

Emphasis on Emphasis on

identifyingidentifying

Expressed Sequence Expressed Sequence

Tags (EST’s)Tags (EST’s)

Yu, J., Whitelaw, C. A., Nierman, W. C.,

Bhatnagar, D., Cleveland, T. E. 2004.

FEMS Microbiology Letters 237:333-340.

√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√Potato dextrose Potato dextrose

BrothBroth

√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√PMS(peptone) PMS(peptone)

liquidliquid

√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√GMS(glucose) GMS(glucose)

liquidliquid

√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√√Wheat bran Wheat bran

solidsolid

96 96

hrshrs72 72

hrshrs48 48

hrshrs24 24

hrshrs18 18

hrshrsCulture mediumCulture medium

Normalized cDNA libraryNormalized cDNA libraryMade from mycelia of Aspergillus Made from mycelia of Aspergillus flavusflavus NRRLNRRL 33573357

Annotation Annotation �� Gene IndexGene Index

Total clones sequenced:Total clones sequenced: 26,11026,110

Total usable ESTs:Total usable ESTs: 22,03722,037

Unique ESTs:Unique ESTs: 7,2187,218

UniquesUniques::

7,2187,218USDA-ARS-SRRC Cooperative Agreement with The Institute for Genomics Research (TIGR)

Genes of Interest in EST Databases:

A. Mycotoxin biochemical pathwaysA. Mycotoxin biochemical pathways

B.B. Virulence / pathogenicityVirulence / pathogenicity

C.C. Fungal development/Fungal development/sporulationsporulation

D. Regulatory / signal transductionD. Regulatory / signal transduction

Individual gene discovery process greatly accelerated:

Genes of Interest IdentifiedA. Aflatoxin pathway genes

2.2. regulatory regulatory

genes:genes:

aflRaflRaflJaflJ

1.1. structural genes:structural genes:pksA pksA verBverBnornor--1 1 verAverAfasfas--1 1 avfAavfAfasfas--22 omtBomtBadhA adhA omtAomtAestA estA ordAordAnorA norA vbsvbsavnA avnA cypXcypXcypAcypA moxYmoxY

3.3. toxin related:toxin related:

toxin transportertoxin transporter

sugar transportersugar transporter

lipase genelipase gene

TT--2 toxin biosynthesis:2 toxin biosynthesis:

TRI 8 TRI 8 homologhomolog

Genes of Interest IdentifiedB. Virulence/pathogenicity

Hydrolytic enzymesHydrolytic enzymes

AmylaseAmylase

CellulaseCellulase

Chitinase Chitinase

ChitosanaseChitosanase

pectin pectin methylesterasemethylesterase

EndoglucanaseEndoglucanase C precursorC precursor

GlucoamylaseGlucoamylase S1/S2 precursorS1/S2 precursor

b b ––1,31,3--glucanase precursorglucanase precursor

1,41,4--bb--DD--glucan glucan cellobiohydrolasecellobiohydrolase A precursorA precursor

Highly similar to glycogen Highly similar to glycogen debranchingdebranching enzymeenzyme

XyloglucanXyloglucan--specific endospecific endo--bb--1,41,4--glucanase precursorglucanase precursor

Function unknownFunction unknown

Pathogenicity protein (Pathogenicity protein (MagnaportheMagnaporthe griseagrisea))Parasitic phaseParasitic phase--specific protein PSPspecific protein PSP--11

SporulationSporulation::

ConidiationConidiation--specific protein 8specific protein 8

ConidiationConidiation--specific protein 10specific protein 10

ConidiophoreConidiophore development protein development protein hymAhymA

Spore coat protein SP96Spore coat protein SP96

Growth/cell wallGrowth/cell wall

Cell cycle control proteinCell cycle control protein

Cell division cycle 2 homologCell division cycle 2 homolog

Growth differentiation factorGrowth differentiation factor--77

Cell wall anchor domain protein Cell wall anchor domain protein

Cell wall chitin biosynthesisCell wall chitin biosynthesis

Cell wall organization and biogenesisCell wall organization and biogenesis

Cell wallCell wall--plasma membrane linker proteinplasma membrane linker protein

Genes of Interest IdentifiedC. Fungal development/sporulation

Genes of Interest IdentifiedD. Regulatory/signal transduction

Transcriptional RegulatorTranscriptional RegulatorDNA binding proteinDNA binding protein

RNA binding proteinRNA binding protein

Zinc Finger protein 2Zinc Finger protein 2

Ring finger proteinRing finger protein

GTPGTP--binding proteinbinding protein

Positive regulatorPositive regulator

Transcription regulatorTranscription regulator

Transcription factor GATATranscription factor GATA

Transcription factor SOXTranscription factor SOX--88

bHLHbHLH transcription factor Cph2transcription factor Cph2

19S 19S proteasomeproteasome regulatory regulatory

particleparticle

Gastrula zinc finger protein Gastrula zinc finger protein

XLCGF9.1XLCGF9.1

Signal transduction/Signal transduction/

Receptor/Receptor/

Global regulatorGlobal regulatorTransducinTransducin a subunita subunit

Signal recognition particle protein Sec65Signal recognition particle protein Sec65

bb--transducintransducin family (WDfamily (WD--40 repeat)40 repeat)

proteinprotein

Probable osmotic sensitivity map kinaseProbable osmotic sensitivity map kinase

MitogenMitogen--activated protein kinase kinaseactivated protein kinase kinase

Multifunctional Multifunctional cyclincyclin--dependent kinasedependent kinase

PHO85PHO85

Dual specificity protein kinase;Dual specificity protein kinase;

Negative regulator of filamentous growthNegative regulator of filamentous growth

& flocculation& flocculation

Putative Putative abscisicabscisic acid response proteinacid response protein

AuxinAuxin--repressed 12.5 kDa proteinrepressed 12.5 kDa protein

Transmembrane receptorTransmembrane receptor

cAMPcAMP receptorreceptor

Aspergillus Aspergillus flavus microarrayflavus microarray

Cooperative Agreement with The Institute for Genomics Research (TIGR)

5002 element

Microarray ApplicationsMicroarray Applications

� Identify genes involved in aflatoxin formation

� Identify regulatory factors

� Host plant-fungus interactions

� Environmental influence on the fungus

� Ecological significance of A. flavus

� Evolutionary significance of aflatoxin

Understanding aflatoxin biosynthesis Understanding aflatoxin biosynthesis and identifying critical vulnerabilities in the and identifying critical vulnerabilities in the process that can be targeted for interruptionprocess that can be targeted for interruption

A. flavusA. flavus Whole Genome SequencingWhole Genome Sequencing

1.1. $500,000 USDA/NSF Grant$500,000 USDA/NSF Grant

2.2. USDA internal fundingUSDA internal funding

PI:PI: Prof. Gary Payne, Prof. Gary Payne, NCSUNCSU

CoCo--PI:PI: Prof. Ralph Dean, Prof. Ralph Dean, NCSUNCSU

CoCo--PI:PI: Dr. William Dr. William NiermanNierman, , TIGRTIGR

Steering Committee:Steering Committee:

Prof. Joan Bennett, Prof. Joan Bennett, Tulane Univ.Tulane Univ.Prof. Nancy Keller, Prof. Nancy Keller, Univ. Univ. WiscWisc..--MadisonMadisonDr. Thomas. E. Cleveland, Dr. Thomas. E. Cleveland, USDA/ARS/SRRCUSDA/ARS/SRRCDr. Deepak Bhatnagar,Dr. Deepak Bhatnagar, USDA/ARS/SRRCUSDA/ARS/SRRCDr. Jiujiang Yu, Dr. Jiujiang Yu, USDA/ARS/SRRCUSDA/ARS/SRRCDr. Charles Dr. Charles WoloshukWoloshuk Purdue UniversityPurdue University

Progress of sequencingProgress of sequencing

�� 5 X coverage5 X coverage

�� ScaffoldsScaffolds 1717

�� Contigs in scaffoldsContigs in scaffolds 29552955

�� Scaffold spanScaffold span 33.1 Mb33.1 Mb

�� Predicted genesPredicted genes 13,77813,778

�� Optical Map being generatedOptical Map being generated

Fumonisin Genomics Group

USDA ARS NCAUR


Robert ButchkoRobert Butchko


Ronald PlattnerRonald Plattner


Collaborators

The Institute for Genomic Research

Christopher Town Catherine Whitelaw

Foo Cheung Li Zheng

Purdue University USDA ARS Athens

Charles Woloshuk Anthony Glenn

Genomics of Genomics of Fusarium Fusarium verticillioidesverticillioides

1809318093

91209120

1286112861

76477647

Total Total

ESTsESTs

12531253FumonisinFumonisin--nonproducingnonproducing mutant mutant

90 hours in FB production medium90 hours in FB production mediumFumonisin IIIFumonisin III

57357396 hours in FB production medium96 hours in FB production mediumFumonisin IIFumonisin II

67367348 & 72 h in FB production medium48 & 72 h in FB production mediumFumonisin IFumonisin I

67067024 hours in FB production medium 24 hours in FB production medium No fumonisinNo fumonisin

Unique Unique

SequencesSequencesGrowth ConditionsGrowth ConditionsLibraryLibrary

Fusarium verticillioidesFusarium verticillioides EST ProjectEST Project

Goal: Identify genes involved in fumonisin biosynthesis

347347

1764817648

156156

1095310953

96039603

Total Total

ESTsESTs

2626

17801780

8080

20572057

846846

Unique Unique

SequencesSequences

Aqueous extracts of maize seedlingAqueous extracts of maize seedlingMaize IMaize I

Exposure to antimicrobial compound Exposure to antimicrobial compound

(BOA) produced by maize(BOA) produced by maizeMaize VMaize V

Autoclaved corn mealAutoclaved corn mealMaize IVMaize IV

Developing maize kernelsDeveloping maize kernelsMaize IIIMaize III

Excised maize seedling tissueExcised maize seedling tissueMaize IIMaize II

Growth ConditionGrowth ConditionLibraryLibrary

Fusarium verticillioidesFusarium verticillioides EST ProjectEST Project

Goal: Identify genes involved in maize-F. verticillioides interaction

Summary EST DataSummary EST Data

Total cDNA clones sequenced: 86,428

Total Unique ESTs: 11,126

(= Unique Genes)

http://www.tigr.org/tdb/tgi/cw/cwgi2/

EST approach

•“in silico subtraction”

•24 hour EST library vs. 96 hour EST library

•Identified 30 EST based on BLAST sequence similarity that fall into the categories of

•transcription factors•zinc binuclear DNA binding proteins•DNA binding proteins•regulatory factors

EST no. first descriptor Expression In Gz? F G H I J K L M N O

2087 “transcription factor” FUM-like ++ ABSENT

1809 “transcriptional activator” FUM-like ++ PRESENT 0 4 0 2 0 0 0 0 2 7

2258 “Zinc” FUM-like + ABSENT 0 12 0 2 0 0 0 12 5 4

2814 “transcriptional activator” FUM-like + PRESENT 0 1 1 0 0 0 0 0 0 0

1997 “DNA binding” FUM-like + PRESENT 0 1 1 0 0 0 0 4 0 0

1785 “Zinc” FUM-like + PRESENT 1 3 0 0 0 0 0 0 4 1

1438 “Zinc” FUM-like + PRESENT 0 2 0 0 0 0 0 0 0 0

2226 “transcription factor” FUM-like + PRESENT 2 5 4 4 0 0 0 3 5 2

1573 “transcription factor” FUM-like + PRESENT

2177 “DNA binding” FUM-like ABSENT

2893 “Zinc” FUM-like PRESENT 0 1 0 1 0 0 0 0 0 0

3009 “DNA binding” FUM-like PRESENT 0 4 5 8 0 0 0 19 8 9

3224 “Zinc” FUM-like PRESENT

2946 “transcription factor” FUM-like PRESENT 0 1 0 3 0 0 0 1 0 6

3128 “DNA binding” FUM-like PRESENT





EST approach

•Confirmed the transcript pattern of expression of each of the EST’s of interest

•Range of gene expression assayed by total RNA slot blot

•constitutive

•FUM-like

EST approach

2087 – FUM-like

2177 – FUM-like

FUM3 (old FUM9)

2817 – constitutive

Days 1 2 3 4 5

EST approach

•How else can the number of potential “regulatory” genes be narrowed down?

•FUM-like gene expression pattern?18

•Are they present in Fusarium graminearum?3 of the 18 are absent from F. graminearum

•Plan to disrupt each one to see if there is an affect on fumonisin production

Gene Disruption Constructs

GenomicDNAEST

PlasmidDNA

GeneticinEST

Gene Disruption Constructs

GenomicDNA

PlasmidDNA

EST

GeneticinEST

EST Geneticin EST

Utilize a FUM1::GUS fusion strain

GUS expression follows FUM gene cluster expression

Can use a blue/white screen to look for mutantsThat have lost the ability to activate the FUMGene cluster and thus the FUM1::GUS fusion Construct

Gene Disruption Strategy

Use of a FUM1::GUS containing strain for screening disruption mutants for affects on FUMgene cluster activity

1

2

1 – positive control2 – negative control

Each colony represents a different mutant with a disruption of a potential regulatory gene identified from a screen of the EST library

•Deletion analysis has allowed the functional characterization of seven genes in the pathway

•We have identified biochemical intermediates in the fumonisin pathway

•We have been able to place genes at particular steps in the biosynthetic pathway

•We have been able to reconcile naturally occurring mutants with genes in the FUM cluster

Summary

Whole genome sequencing ofWhole genome sequencing ofFusarium graminearumFusarium graminearum

The second plant pathogenic fungus sequencedThe second plant pathogenic fungus sequenced

Funded by the NSF/USDA NRI Microbial Genome ProgramFunded by the NSF/USDA NRI Microbial Genome Program

http://www.broad.mit.edu/annotation/fungi/fusarium/

The The Fusarium graminearumFusarium graminearum genome project represents a partnership genome project represents a partnership between the Broad Institute and the International between the Broad Institute and the International GibberellaGibberella zeaezeaeGenomics Consortium (IGGR). The main collaborators of Fusarium Genomics Consortium (IGGR). The main collaborators of Fusarium genome project are:genome project are:

Dr. Dr. JinrongJinrong XuXu: : Purdue Univ., USAPurdue Univ., USADr. H. Corby Dr. H. Corby KistlerKistler: : USDA/ARS, USAUSDA/ARS, USADr. Frances Trail:Dr. Frances Trail: Michigan State Univ., USAMichigan State Univ., USA

-- 10 x coverage 10 x coverage

-- at MIT, WIat MIT, WI--CGRCGR

-- 1st release, May, 1st release, May, 20032003

(DNA sequence (DNA sequence

assembly)assembly)

-- 2nd release, Oct., 20032nd release, Oct., 2003

(Automated annotation)(Automated annotation)

http://www.broad.mit.edu/annotation/fungi/fusarium/

Acknowledgement: this slides is provided by Prof. Jinrong Xu, Purdue Univ., USA

The genetic mapThe genetic map�� > 99.8% of assembled contigs > 99.8% of assembled contigs anchored to the genetic map.anchored to the genetic map.–– 237 genetic markers237 genetic markers

–– 164 sequence tagged sites164 sequence tagged sites

–– ca. 70 kb not linked to mapca. 70 kb not linked to map

�� All but three genetic markers coAll but three genetic markers co--linear linear with the assembly. with the assembly. –– minor disagreements among closely linked minor disagreements among closely linked genes.genes.

�� Four major blocks of scaffolds joined Four major blocks of scaffolds joined by genetic linkage groupsby genetic linkage groups–– In agreement with four chromosomes In agreement with four chromosomes detected detected cytologicallycytologically..

Jurgenson et al., 2002. Genetics 160:1451-1460 Lee et al., poster 372 Gale et al., 2nd release. 2004Gale et al., 2nd release. 2004


GEN-AU / MIPS F. graminearum Genome Database

– Funded by the Austrian Federal Ministry for Education, Science and Culture to Gerhard Adam.

– Conducted at MIPS (Ulrich Gueldener, et al.)�First release – February, 2004

�Advanced annotation, Synteny viewer, etc

�PEDANT database (w/ ORPHEUS)

http://mips.gsf.de/genre/proj/fusarium/

13759 genes

Munich Information Center for Protein Sequences

Classification of 13759 predicted Classification of 13759 predicted ORFsORFs

ORF Classification

1 known protein

2 strong similarity to known protein

3 similarity to known protein

4 similarity to unknown protein

5 strong similarity to EST

6 no similarity

Number of ORFs

53

1714

4225

3097

265

4405

2312 predicted to be secreted proteins

Broad (Whitehead) Institute – more EST sequencingto improve annotation


...the exciting new world of genomics

Mycotoxin Mycotoxin RiskRisk FactorsFactors

ChemicaltreatmentsVariety

...

Previous, previouscrop

Previous crop

Cultivation

Interactions

Customer Needs and Expectations:

YIELD

Fast Drydown Standability

YIELD YIELD

view A–A

A A

Illumination Fiber

Reflectance Fiber

Corn Kernel

Fiber Optic

to Spectrometer

Fiber Optic

from Light Source

Reflectance Sampling ApparatusReflectance Sampling Apparatus

Control

Fusarium verticillioides

Diplodia maydis

A. Flavus (BGYF)

Fusarium graminearum

Trichoderma viride

Disease Resistance Breeding

Challenges:Germplasm

Multiple Mycotoxins

Inexpensive and reliable detection methods

Reproducible screening techniques

10 year cycle to develop and commercialize a corn hybrid

Alternative Breeding Strategies:

Stress Tolerance

Foliar Disease Resistance

“Genetically Enhanced” ResistanceAntifungal Genes

Insect Resistance GenesMycotoxin Degradation Genes

Mycotoxin Formation Inhibitor Genes

Immunolocalization of glutamine synthetase in kernels

Non-specific antibody GSp1-specific antibody

Tissue specificity

Disease responsive

Stress Responsive

Developmental

Regulatory sequence motifs

Transcriptional sequence motifs

CATT box

TATA box

5’ Ex In Ex

promoter

Transcriptional

start siteAUG

ExI InEx

3’ UTR

5’ UTR

Translation

terminator

Transcription

terminator

3’

Regulation of Gene Expression-

Potential Sites Of Action

**

*

**

Potential regulatory influence

Fumonisin B1

Deoxynivalenol

Zearaleone

a genomic approach to mycotoxin control - cnrmycoglobe.ispa.cnr.it/brussel/usa-kendra.pdf · a...

Documents