Transcript 5. DNA barcoding and new sequencing technologies

Defining the Fusarium/host
interaction through genomics
and proteomics
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
2
DEOXYNIVALENOL (DON)
OH
O
O
O
OH
HO
• Trichothecene mycotoxin
• 322 Da
• Different forms affect
cytoxicity
– 15-Acetyl DON
– 3-Acetyl DON
• Important in pathogen
virulence
Cellular Effects of DON
Inhibits protein synthesis
Binds 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 fungi
Haploid or diploid
5 um diameter
Eukaryote
Saccharomyces cerevisiae as a Model System
1997 – first eukaryotic organism sequenced
6200 ORF’s
Saccharomyces Genome Database
http://www.yeastgenome.org
Inexpensive / easy to use
Conservation of biochemical processes
Yeast Genomic Screening on DON
Collection in 96 well format
Pin (2X) 1536 / plate
Singer Robot
YPD + TI + 125 ug/mL DON
Grow 30°C, 2-6 days
Photograph and quantify growth
Repeat 2 times
Screening Results – Example ATG4
DMSO
TI
TI + DON
Screening Results – Example RPL27A
DMSO
TI
TI + DON
Screening Results – Top Strains
GENE
VTH1
ESSENTIAL?
N
DESCRIPTION
Putative membrane glycoprotein
ECM15
ABF1
ARC18
ARC35
N
Y
N
Y
Gene of unknown function
DNA binding protein
AYT1
ATG4
KRE9
N
N
Y
Acetyltransferase
Cysteine protease required for autophagy
UBP13
RPL27A
RPL39
N
N
N
Putative ubiquitin-specific protease
Component of (60S) ribosomal subunit
Component of (60S) ribosomal subunit
Required for integrity of cortical actin patches
Required for integrity of cortical actin patches
Glycoprotein: cell-wall β-glucan assembly
Serial Dilution Dot Assay Results
(Growth on TI + 175 ug/mL DON)
68h
DMSO
WT
ARC35
AYT1
ATG4
RPL27A
RPL39
5d
TI
TI + DON
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
FUTURE WORK
Screen yeast knock-out collections on other
Fusarium mycotoxins
 Select genes for altered expression in plants
Fusarium systems biology pipeline
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).
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.
Wheat
Maize (68 unique)
3500.0
6979 2311
2389
2164
4597
2166
4602
Intensity (Fg minus mock)
3000.0
2500.0
oligo1-Zm
2000.0
oligo2-Zm
oligo3-Zm
1500.0
oligo1-Ta
oligo2-Ta
1000.0
oligo3-Ta
500.0
Barley (2 unique)
Fg genes detected by 96hai
0.0
1d
2d
-500.0
Tim e post inoculation
4d
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).
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).
B73
CO441
 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.
Arabidopsis is susceptible to F. graminearum
Uninoculated
Inoculated
20
DMSO
Chemical compounds
DMSO
A
B
C
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