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Defining the Fusarium/host interaction through genomics and proteomics

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Defining the Fusarium/host interaction through genomics and proteomics – PowerPoint PPT presentation

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Title: Defining the Fusarium/host interaction through genomics and proteomics


1
Defining the Fusarium/host interaction through
genomics and proteomics
2
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

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

4
Cellular Effects of DON
  • Inhibits protein synthesis
  • Binds to ribosomal protein L3 (RPL3)
  • Blocks peptidyl transferase?

5
Objective
  • Find genes, whose altered expression in plants,
    will increase resistance to Fusarium graminearum

6
Saccharomyces cerevisiae
  • Single cellular fungi
  • Haploid or diploid
  • 5 um diameter
  • Eukaryote

7
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

8
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
9
Screening Results Example ATG4
10
Screening Results Example RPL27A
11
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
12
Serial Dilution Dot Assay Results (Growth on TI
175 ug/mL DON)
13
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

14
FUTURE WORK
  • Screen yeast knock-out collections on other
    Fusarium mycotoxins
  • Select genes for altered expression in plants

15
Fusarium systems biology pipeline
16
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).

17
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.

18
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).

19
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
20
Arabidopsis is susceptible to F. graminearum
Uninoculated
Inoculated
21
DMSO
DMSO
Chemical compounds
A
B
C
22
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23
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