Defining the Defining the FusariumFusarium/host /host interaction through genomics interaction through genomics

and proteomicsand proteomics

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Fusarium graminearum

Broad host fungal pathogen causing fusarium head blight in wheat, barley, and oats and gibberella ear rot in maize.

Reduced grain yield and quality. Mycotoxin deposition. (Deoxynivalenol-DON) • food and feed safety issues

• potential export barrier

DEOXYNIVALENOL (DON)• Trichothecene mycotoxin• 322 Da• Different forms affect

cytoxicity– 15-Acetyl DON– 3-Acetyl DON

• Important in pathogen virulence

OO

OH

OOH

HO

OO

OH

OOH

HO

Cellular Effects of DON

Inhibits protein synthesisBinds to ribosomal protein

L3 (RPL3)Blocks peptidyl

transferase?

Objective:

Find genes, whose altered expression in plants, will increase resistance to Fusarium graminearum

Saccharomyces cerevisiae

Single cellular fungiHaploid or diploid5 um diameterEukaryote

Saccharomyces cerevisiae as a Model System

1997 – first eukaryotic organism sequenced6200 ORF’sSaccharomyces Genome Database

http://www.yeastgenome.orgInexpensive / easy to useConservation of biochemical processes

Yeast Genomic Screening on DON

Pin (2X) 1536 / plate

Singer RobotYPD + TI + 125 ug/mL DON

Grow 30°C, 2-6 days

Photograph and quantify growth

Collection in 96 well format

Repeat 2 times

Screening Results – Example ATG4

TI + DON

TI

DMSO

Screening Results – Example RPL27A

DMSO

TI

TI + DON

Screening Results – Top StrainsGENE ESSENTIAL? DESCRIPTION

VTH1 N Putative membrane glycoproteinECM15 N Gene of unknown functionABF1 Y DNA binding protein

ARC18 N Required for integrity of cortical actin patches

ARC35 Y Required for integrity of cortical actin patches

AYT1 N AcetyltransferaseATG4 N Cysteine protease required for autophagyKRE9 Y Glycoprotein: cell-wall β-glucan assembly

UBP13 N Putative ubiquitin-specific proteaseRPL27A N Component of (60S) ribosomal subunitRPL39 N Component of (60S) ribosomal subunit

Serial Dilution Dot Assay Results (Growth on TI + 175 ug/mL DON)

DMSO TI TI + DON

WT

ARC35

AYT1

ATG4

RPL27A

RPL39

68h 5d

CONCLUSIONS

Potential mycotoxin target genes discovered:Expected – eg. RPL39, RPL27A, AYT1Novel – eg. ARC35, ATG4,

Screening method flags both “hits” and “suppressors”:

Hits - deleted genes give DON hypersensitivitySuppressors – deleted genes suppress DON

cytotoxicity

FUTURE WORK

Screen yeast knock-out collections on other Fusarium mycotoxins

Select genes for altered expression in plants

Fusarium systems biology pipeline

Tri1 (unlinked to trichothecene gene cluster)1

Butenolide gene cluster – fg08079 encodes a P450 required for butenolide synthesis.2

Clm1 – encoding an enzyme required for culmorin synthesis.3

1 McCormick et al. Appl. Environ. Microbiol. (2004).2 Harris et al. Fungal Genet. Biol. (2007).3 McCormick et al. Appl. Environ. Microbiol. (2010).

Identification of genes involved in mycotoxin synthesis

Whole gene set expression profiling conducted using Agilent 4X44K array platform - up to three 60mers representing each predicted F. graminearum gene.

monitoring impact of Fusarium regulatory genes. monitoring in planta expression – wheat, barley, maize.

Fusarium transcriptomics

Wheat Maize (68 unique)

Barley (2 unique)

6979 2389

4602

21644597

2311

2166

Fg genes detected by 96hai

Fusarium proteomics Using non-gel-based quantitative proteomics technology

(iTRAQ), we monitored 435 Fusarium proteins ID over a time course during which mycotoxin synthesis was induced in vitro1.

The quantitative data of 130 proteins were ID as statistically significant (ANOVA, p<0.05). Many of these proteins are potentially involved in pathogenicity.

1 Taylor et al. Proteomics (2008).

Gene expression profiling (55K oligomer arrays) and quantitative protein profiling (iTRAQ):

- B73 (susceptible)- CO441 (silk & kernel resistance; Reid et al., 2003).

Construction of recombinant inbred line by single seed descent: F6 seed of (B73 X CO441) - 414 lines.

- Summer 2010 – begin phenotyping silk and kernel resistance.

Defining resistance in maize

B73 CO441

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Uninoculated Inoculated

Arabidopsis is susceptible to F. graminearum

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DMSO DMSOChemical compounds

A

C

B

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