A Genomic Approach to. Mycotoxin Control

Transcription

1 A Genomic Approach to Mycotoxin Control Deepak Bhatnagar David Kendra United States Department of Agriculture Agricultural Research Service

2 MYCTXINS F U.S. INTEREST In U.S.A. mycotoxins of major concern include: Toxin Aflatoxins Trichothecenes Fumonisins Commodities Affected Corn, cottonseed, peanuts, treenuts Corn, wheat, barley Corn Producing Fungi Aspergillus flavus Aspergillus parasiticus Fusarium graminearum Fusarium verticillioides

3 MYCTXINS F U.S. INTEREST The following mycotoxins are also receiving significant attention: Patulin Toxin chratoxin A Affected Commodities Wheat Barley, oats, corn, others Apples, wheat straw residue Producing Fungi Aspergillus ochraceus Penicillium verrucosium Aspergillus spp. Penicillium spp. Zearalenone Cyclopiazonic acid Corn, hay Corn, kodo millet Fusarium graminearum Penicillium cyclopium Aspergillus flavus

4 REGULATIN The U.S. Food and Drug Administration has issued regulatory limits for aflatoxins. Current aflatoxin action levels Food and Feed Human foods (except milk) Milk Animal feeds (except as listed below) Cottonseed meal (as a feed ingredient) Corn and peanut products for breeding beef cattle, swine and mature poultry Corn and peanut meal for finishing swine Corn and peanut meal for feedlot beef cattle Corn for immature animals and dairy cattle Food and Drug Administration (1996) Action levels (µg/kg, ppb)

5 REGULATIN FDA has issued guidelines or advisories for other mycotoxins: Deoxynivalenol Fumonisins (B1, B2, B3) Patulin* chratoxin A 1 mg/kg (ppm( ppm) ) for finished wheat products for human use 5-10 ppm for other food and feed 2-4 ppm for human foods ppms for animal feeds 50 µg /kg (ppb) in apple juice (none), recommendation: good agricultural and manufacturing practices *Patulin Technical Symposium (FDA), Feb.18-19, 19, 2003, rlando, Florida

6 ECNMIC IMPACT F MYCTXIN CNTAMINATIN U.S. Food and Drug Administration (based on sum of the value of food and feed losses) estimated annual crop losses of: $932 million per year (direct costs) $466 million per year (mitigating costs) $6 million per year (livestock costs) from contamination with 3 mycotoxins: Aflatoxins,, Fumonisins, Deoxynionlenol *Reviewed in U.S. Council for Agricultural Science and Technology Task Force Report, 2003.

7 MYCTXIN CNTAMINATIN 1. Post-Harvest 2. Pre-Harvest

8 Post-Harvest Contamination can be managed by Pre-Harvest Control

9 Strategies to Control Pre-Harvest Mycotoxin Contamination: 1. Cultural Practices 2. Enhancing host resistance - Plant breeding / Genetic Engineering 3. Biological Control 4. Understanding basic biology / toxin producing ability of the fungus

10 For Control of toxin formation it is important to know: 1. How are toxins synthesized by the producing organism 2. Where are they produced in the cells of the producing organism 3. What environmental factors affect the synthesis of the toxin and the mechanism of the effect

11 Mycotoxin Biosynthesis: Chemistry of toxin formation - Precursor Identification Biochemical Studies - Enzyme Studies Genetics - Gene Cloning

12 USDA/ARS Southern Regional Research Center New rleans, LA Aflatoxin Biosynthesis Group Deepak Bhatnagar Jeffrey Cary Perng-Kuang Chang Thomas E. Cleveland Kenneth Ehrlich Jiujiang Yu

13 Collaborative Effort Deepak Bhatnagar / Thomas E. Cleveland USDA-ARS-SRRC Gary Payne N. C. State Univ. Joan Bennett Tulane University John Linz / Frances Trail Michigan State Univ. Nancy Keller Univ. Wisconsin-Madison Fun Sun Chu Univ. Wisconsin-Madison Charles Woloshuk Purdue Univ.

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15 USDA/ ARS/National Center for Agriculture Utilization Research, Peoria, IL Trichothecene Biosynthesis Group Nancy Alexander Daren Brown Anne Desjardins David Kendra Susan McCormick Ron Plattner Robert Proctor (Rex Dyer - Post-doc)

16 Core Fusarium trichothecene gene cluster 0 kb orff orfe orfd orfc orfb orfa methytransferase unknown unknown beta-1,3-glucanosyltransferase acetylesterase tyrosinase known role unknown role no role and examined not examined TRI8 TRI7 TRI3 TRI4 TRI6 TRI5 TRI10 TRI9 TRI11 TRI12 Similar to extracellular lipase from Candida albicans Acetylesterase 15-decalonectrin 15-0-acetyltransferase Cytochrome P450 DNA Binding Positive Acting Transcription Factor Trichodiene Synthase (sequiterpene cyclase) Regulation Unknown P450 or isotrichodermin C-15 hydroxylase (CYP 65A1 Trichothecene Efflux Pump orfg orfh orfi orfj orfk orfl deacetylase unknown dehydrogenase NADH-cytochrome b5 reductase cytochrome P450 unknown

17 Trichothecenes biosynthesis

18 USDA/ ARS/National Center for Agriculture Utilization Research, Peoria, IL Fumonisin Biosynthesis Group Daren Brown Robert Butchko Anne Desjardins David Kendra Ronald Plattner Robert Proctor

19 Fumonisin Biosynthetic Gene Cluster FUM1 FUM6 FUM7 FUM8 FUM3 FUM10 FUM11 FUM2 FUM13 FUM14 FUM15 FUM16 FUM17 FUM18 FUM19 0 kb scale Gene Name FUM1 FUM6 FUM7 FUM8 FUM3 FUM10 FUM11 FUM2 FUM13 FUM14 FUM15 FUM16 FUM17 FUM18 FUM19 Predicted Function Based on Sequence Similarity Polyketide synthase Cytochrome P450 Monooxygenase Dehydrogenase Aminotransferase Dioxygenase Fatty Acid-CoA Synthase Tricarboxylate Transporter Cytochrome P450 Monooxygenase Short-chain Dehydrogenase/Reductase Peptide Synthetase Condensation Domain Cytochrome P450 Monooxygenase Fatty Acid-CoA Synthetase Longevity Assurance Factor Longevity Assurance Factor ABC Transporter

20 Proposed Fumonisin H Biosynthetic Pathway HFB FUM2 3 HFB 4 FB 3 S CoA NH 2 H FB3 H + NH 2 H + H FUM14 H H NH 2 H NH HFB4 2 H NH HFB3 2 CH 3 H FUM1 FUM8 FUM6 FUM13 CH 3 H H Acetate H CH 3 CH 3 CH 3 H CH 3 H CH 3 H H H H H CH 3 CH 3 CH 3 CH 3 CH 3 NH 2 H + FB4 S Mitochondria CoA FUM3 H FUM14 H H H FUM10 FUM7 CH 3 H FB 4 H FUM11 CH 3 H H H H H FB 1 H NH 2 FUM3 H FB1 H CH 3 H H H CH 3 H NH 2 H H FB2 CH 3 FB 2 CH 3 H H H H

21 1. ph THE AGRECSYSTEM Complex environmental and ecological factors affecting A.flavus and aflatoxin contamination process: 2. Nutritional source eg.. nitrogen, carbon, etc. 3. Elemental availability eg.. phosphorus, selenium silica, cobalt, magnesium 4. Temperature 5. ther physiological factors eg.. volatile compounds 6. Biotic factors eg.. competing flora/fauna

22 Not Dealing with bligate Fungal Parasites (requiring their host plant 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.

23 Mycotoxin Problems Fit Two Criteria Pointing Toward a Genomics 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.

24 A GENMICS approach to Mycotoxin Control

25 Selected Fungal Genome Projects Fungus Aspergillus flavus Aspergillus flavus Status Public, expressed sequence tags (EST s) Whole genome sequencing Sources 1,2,3 1,2,7 Fusarium sporotrichioides Fusarium verticillioides Public, EST s Public, Private,, EST s and whole genome 4 1,5,7,8,9 Fusarium graminearum Public, Private, EST s s and whole genome in progress 1 USDA, Agricultural Research Service-funded genomics 2 North Carolina State Univ. 3 Univ. of klahoma Advanced Center for Genome Tech./ The Univ. of Wisconsin 4 ACGT/ Texas A&M Univ. 5 Purdue Univ. 6 Michigan State Univ./ Purdue Univ./ ARS Cereal Disease Lab 7 The Institute for Genomic Research, Maryland 8 Syngenta, Inc. (4X) 9 DuPont, Inc. (1X) 6,8

26 Aspergillus flavus genomics project; Emphasis on identifying Expressed Sequence Tags (EST s) Yu, J., Whitelaw, C. A., Nierman, W. C., Bhatnagar, D., Cleveland, T. E FEMS Microbiology Letters 237:

27 Normalized cdna library Made from mycelia of Aspergillus flavus NRRL 3357 Culture medium Wheat bran solid GMS(glucose) liquid PMS(peptone) liquid Potato dextrose Broth 18 hrs 24 hrs 48 hrs 72 hrs 96 hrs

28 Annotation Gene Index Total clones sequenced: 26,110 Total usable ESTs: 22,037 Unique ESTs: 7,218 Uniques: 7,218 USDA-ARS-SRRC Cooperative Agreement with The Institute for Genomics Research (TIGR)

29 Genes of Interest in EST Databases: Individual gene discovery process greatly accelerated: A. Mycotoxin biochemical pathways B. Virulence / pathogenicity C. Fungal development/sporulation D. Regulatory / signal transduction

30 Genes of Interest Identified A. Aflatoxin pathway genes 1. structural genes: pksa nor-1 fas-1 fas-2 adha esta nora avna cypa verb vera avfa omtb omta orda vbs cypx moxy 2. regulatory genes: aflr aflj 3. toxin related: toxin transporter sugar transporter lipase gene T-2 2 toxin biosynthesis: TRI 8 homolog

31 Genes of Interest Identified B. Virulence/pathogenicity Hydrolytic enzymes Amylase Cellulase Chitinase Chitosanase pectin methylesterase Endoglucanase C precursor Glucoamylase S1/S2 precursor b 1,3-glucanase precursor 1,4-b-D-glucan cellobiohydrolase A precursor Highly similar to glycogen debranching enzyme Xyloglucan-specific endo-b-1,4 1,4-glucanase precursor Function unknown Pathogenicity protein ( (Magnaporthe grisea) Parasitic phase-specific specific protein PSP-1

32 Genes of Interest Identified C. Fungal development/sporulation Sporulation: Conidiation-specific protein 8 Conidiation-specific protein 10 Conidiophore development protein hyma Spore coat protein SP96 Growth/cell wall Cell cycle control protein Cell division cycle 2 homolog Growth differentiation factor-7 Cell wall anchor domain protein Cell wall chitin biosynthesis Cell wall organization and biogenesis Cell wall-plasma membrane linker protein

33 Genes of Interest Identified D. Regulatory/signal transduction Transcriptional Regulator DNA binding protein RNA binding protein Zinc Finger protein 2 Ring finger protein GTP-binding protein Positive regulator Transcription regulator Transcription factor GATA Transcription factor SX-8 bhlh transcription factor Cph2 19S proteasome regulatory particle Gastrula zinc finger protein XLCGF9.1 Signal transduction/ Receptor/ Global regulator Transducin a subunit Signal recognition particle protein Sec65 b-transducin family (WD-40 repeat) protein Probable osmotic sensitivity map kinase Mitogen-activated protein kinase kinase Multifunctional cyclin-dependent kinase PH85 Dual specificity protein kinase; Negative regulator of filamentous growth & flocculation Putative abscisic acid response protein Auxin-repressed 12.5 kda protein Transmembrane receptor camp receptor

34 Aspergillus flavus microarray 5002 element Cooperative Agreement with The Institute for Genomics Research (TIGR)

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

36 A. flavus Whole Genome Sequencing 1. $500,000 USDA/NSF Grant PI: Prof. Gary Payne, Co-PI: Prof. Ralph Dean, Co-PI: Dr. William Nierman, Steering Committee: NCSU NCSU TIGR Prof. Joan Bennett, Prof. Nancy Keller, Dr. Thomas. E. Cleveland, Dr. Deepak Bhatnagar, Dr. Jiujiang Yu, Dr. Charles Woloshuk 2. USDA internal funding Tulane Univ. Univ. Wisc.-Madison USDA/ARS/SRRC USDA/ARS/SRRC USDA/ARS/SRRC Purdue University

37 Progress of sequencing 5 X coverage Scaffolds 17 Contigs in scaffolds 2955 Scaffold span 33.1 Mb Predicted genes 13,778 ptical Map being generated

38 Genomics of Fusarium verticillioides Fumonisin Genomics Group USDA ARS NCAUR Daren Brown Robert Butchko David Kendra Ronald Plattner Robert Proctor Collaborators The Institute for Genomic Research Christopher Town Catherine Whitelaw Foo Cheung Li Zheng Purdue University USDA ARS Athens Charles Woloshuk Anthony Glenn

39 Fusarium verticillioides EST Project Goal: Identify genes involved in fumonisin biosynthesis Library Growth Conditions Total ESTs Unique Sequences No fumonisin 24 hours in FB production medium Fumonisin I 48 & 72 h in FB production medium Fumonisin II 96 hours in FB production medium Fumonisin III Fumonisin-nonproducing nonproducing mutant 90 hours in FB production medium

40 Fusarium verticillioides EST Project Goal: Identify genes involved in maize-f. verticillioides interaction Library Growth Condition Total ESTs Unique Sequences Maize I Aqueous extracts of maize seedling Maize II Excised maize seedling tissue Maize III Developing maize kernels Maize IV Autoclaved corn meal Maize V Exposure to antimicrobial compound (BA) produced by maize

41 Summary EST Data Total cdna clones sequenced: 86,428 Total Unique ESTs: 11,126 (= Unique Genes)

42 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

43 EST no. first descriptor Expression In Gz? F G H I J K L M N 2087 transcription factor FUM-like ++ ABSENT 1809 transcriptional activator FUM-like ++ PRESENT Zinc FUM-like + ABSENT transcriptional activator FUM-like + PRESENT DNA binding FUM-like + PRESENT Zinc FUM-like + PRESENT Zinc FUM-like + PRESENT transcription factor FUM-like + PRESENT transcription factor FUM-like + PRESENT 2177 DNA binding FUM-like ABSENT 2893 Zinc FUM-like PRESENT DNA binding FUM-like PRESENT Zinc FUM-like PRESENT 2946 transcription factor FUM-like PRESENT DNA binding FUM-like PRESENT 2344 Zinc FUM-like PRESENT Zinc FUM-like PRESENT Zinc FUM-like PRESENT Zinc FUM-like PRESENT

44 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

45 EST approach Days FUM3 (old FUM9) 2087 FUM-like 2177 FUM-like 2817 constitutive

46 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

47 Gene Disruption Constructs EST Genomic DNA EST Geneticin Plasmid DNA

48 Gene Disruption Constructs EST Genomic DNA EST Geneticin Plasmid DNA EST Geneticin EST

49 Gene Disruption Strategy Utilize a FUM1::GUS fusion strain GUS expression follows FUM gene cluster expression Can use a blue/white screen to look for mutants That have lost the ability to activate the FUM Gene cluster and thus the FUM1::GUS fusion Construct

50 Use of a FUM1::GUS containing strain for screening disruption mutants for affects on FUM gene cluster activity 2 Each colony represents a different mutant with a disruption of a potential regulatory gene identified from a screen of the EST library 1 1 positive control 2 negative control

51 Summary 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

52 Whole genome sequencing of Fusarium graminearum The second plant pathogenic fungus sequenced Funded by the NSF/USDA NRI Microbial Genome Program The Fusarium graminearum genome project represents a partnership between the Broad Institute and the International Gibberella zeae Genomics Consortium (IGGR). The main collaborators of Fusarium genome project are: Dr. Jinrong Xu: Dr. H. Corby Kistler: Dr. Frances Trail: Purdue Univ., USA USDA/ARS, USA Michigan State Univ., USA

53 x coverage - at MIT, WI-CGR - 1st release, May, 2003 (DNA sequence assembly) - 2nd release, ct., 2003 (Automated annotation) Acknowledgement: this slides is provided by Prof. Jinrong Xu, Purdue Univ., USA

54 The genetic map > 99.8% of assembled contigs anchored to the genetic map. 237 genetic markers 164 sequence tagged sites ca. 70 kb not linked to map All but three genetic markers co-linear with the assembly. minor disagreements among closely linked genes. Four major blocks of scaffolds joined by genetic linkage groups In agreement with four chromosomes detected cytologically. Jurgenson et al., Genetics 160: Lee et al., poster 372 Gale et al., 2nd release Acknowledgement: this slides is provided by Prof. Jinrong Xu, Purdue Univ., USA

55 Munich Information Center for Protein Sequences genes 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/ RPHEUS)

56 Classification of predicted RFs RF 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 RFs predicted to be secreted proteins Broad (Whitehead) Institute more EST sequencing to improve annotation Acknowledgement: this slides is provided by Prof. Jinrong Xu, Purdue Univ., USA

57 ...the exciting new world of genomics

58 Mycotoxin Risk Factors Previous, previous crop Previous crop Cultivation Variety Chemical treatments... Interactions

59 Customer Needs and Expectations: YIELD YIELD YIELD Fast Drydown Standability

60 Reflectance Sampling Apparatus Illumination Fiber Reflectance Fiber Corn Kernel A A view A A Fiber ptic to Spectrometer Fiber ptic from Light Source

61 Control A. Flavus (BGYF) Fusarium verticillioides Fusarium graminearum Diplodia maydis Trichoderma viride

62 Challenges: Germplasm Disease Resistance Breeding Multiple Mycotoxins Inexpensive and reliable detection methods Reproducible screening techniques 10 year cycle to develop and commercialize a corn hybrid

63 Alternative Breeding Strategies: Stress Tolerance Foliar Disease Resistance Genetically Enhanced Resistance Antifungal Genes Insect Resistance Genes Mycotoxin Degradation Genes Mycotoxin Formation Inhibitor Genes

64 Immunolocalization of glutamine synthetase in kernels Non-specific antibody GSp1-specific antibody

65 Regulation of Gene Expression- Potential Sites f Action promoter Transcriptional start site AUG * * * 5 Ex In Ex I Ex In Ex Translation terminator 3 UTR * 3 Regulatory sequence motifs * Tissue specificity Disease responsive Stress Responsive Developmental 5 UTR Potential regulatory influence Transcriptional sequence motifs CATT box TATA box Transcription terminator

66 Deoxynivalenol Fumonisin B 1 Zearaleone

67