Defining the Fusarium/host interaction through genomics and proteomics

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Defining the Fusarium/host interaction through
genomics and 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

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DEOXYNIVALENOL (DON)

• Trichothecene mycotoxin
• 322 Da
• Different forms affect cytoxicity
• 15-Acetyl DON
• 3-Acetyl DON
• Important in pathogen virulence

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Cellular Effects of DON

• Inhibits protein synthesis
• Binds to ribosomal protein L3 (RPL3)
• Blocks peptidyl transferase?

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Objective

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

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Saccharomyces cerevisiae

• Single cellular fungi
• Haploid or diploid
• 5 um diameter
• Eukaryote

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Saccharomyces cerevisiae as a Model System

• 1997 first eukaryotic organism sequenced
• 6200 ORFs
• Saccharomyces Genome Database
• http//www.yeastgenome.org
• Inexpensive / easy to use
• Conservation of biochemical processes

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Yeast Genomic Screening on DON
Collection in 96 well format
Pin (2X) 1536 / plate
Singer Robot
YPD TI 125 ug/mL DON
Grow 30C, 2-6 days
Photograph and quantify growth
Repeat 2 times
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Screening Results Example ATG4
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Screening Results Example RPL27A
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Screening Results Top Strains
GENE ESSENTIAL? DESCRIPTION
VTH1 N Putative membrane glycoprotein
ECM15 N Gene of unknown function
ABF1 Y DNA binding protein
ARC18 N Required for integrity of cortical actin patches
ARC35 Y Required for integrity of cortical actin patches
AYT1 N Acetyltransferase
ATG4 N Cysteine protease required for autophagy
KRE9 Y Glycoprotein cell-wall ß-glucan assembly
UBP13 N Putative ubiquitin-specific protease
RPL27A N Component of (60S) ribosomal subunit
RPL39 N Component of (60S) ribosomal subunit
GENE ESSENTIAL? DESCRIPTION
A VTH1 N Putative membrane glycoprotein
  ECM15 N Non-essential protein of unknown function
  ABF1 Y DNA binding protein with possible chromatin-reorganizing activity
  BCY1 Y Regulatory subunit of the cyclic AMP-dependent protein kinase (PKA)
  ARC18 N Subunit of the ARP2/3 complex required for motility integrity of cortical actin patches
  ARC35 Y Subunit of the ARP2/3 complex required for motility integrity of cortical actin patches
  AYT1 N Acetyltransferase catalyzes trichothecene 3-O-acetylation to form less toxic compound3
  ATG4 N Cysteine protease required for autophagy
  KRE9 Y Glycoprotein involved in cell wall beta-glucan assembly
  GCD10 Y Subunit of tRNA (1-methyladenosine) methyltransferase with Gcd14p
  MMF1 Y Mitochondrial protein involved in maintenance of the mitochondrial genome
  RPS30B N Protein component of the small (40S) ribosomal subunit
  GSP1 Y GTP binding protein (mammalian Ranp homolog)
  RPS3 Y Protein component of the small (40S) ribosomal subunit
YBL044W N Putative protein of unknown function
B RPL12B N Protein component of the large (60S) ribosomal subunit
  RPL5 Y Protein component of the large (60S) ribosomal subunit
  UBP13 N Putative ubiquitin-specific protease
  RPL27A N Protein component of the large (60S) ribosomal subunit
  RPL39 N Protein component of the large (60S) ribosomal subunit
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Serial Dilution Dot Assay Results (Growth on TI
175 ug/mL DON)
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CONCLUSIONS

• Potential mycotoxin target genes discovered
• Expected eg. RPL39, RPL27A, AYT1
• Novel eg. ARC35, ATG4,
• Screening method flags both hits and
  suppressors
• Hits - deleted genes give DON hypersensitivity
• Suppressors deleted genes suppress DON
  cytotoxicity

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FUTURE WORK

• Screen yeast knock-out collections on other
  Fusarium mycotoxins
• Select genes for altered expression in plants

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Fusarium systems biology pipeline
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Identification of genes involved in mycotoxin
synthesis

• 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).

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Fusarium transcriptomics

• 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.

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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, plt0.05). Many
  of these proteins are potentially involved in
  pathogenicity.
• 1 Taylor et al. Proteomics (2008).

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Defining resistance in maize

• 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.

B73
CO441
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Arabidopsis is susceptible to F. graminearum
Uninoculated
Inoculated
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DMSO
DMSO
Chemical compounds
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B
C
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