Defining the Fusarium /host interaction through genomics and proteomics
-
Upload
ivor-cervantes -
Category
Documents
-
view
59 -
download
2
description
Transcript of Defining the Fusarium /host interaction through genomics and proteomics
![Page 1: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/1.jpg)
Defining the Defining the FusariumFusarium/host /host interaction through genomics interaction through genomics
and proteomicsand proteomics
![Page 2: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/2.jpg)
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
![Page 3: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/3.jpg)
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
![Page 4: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/4.jpg)
Cellular Effects of DON
Inhibits protein synthesisBinds to ribosomal protein
L3 (RPL3)Blocks peptidyl
transferase?
![Page 5: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/5.jpg)
Objective:
Find genes, whose altered expression in plants, will increase resistance to Fusarium graminearum
![Page 6: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/6.jpg)
Saccharomyces cerevisiae
Single cellular fungiHaploid or diploid5 um diameterEukaryote
![Page 7: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/7.jpg)
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
![Page 8: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/8.jpg)
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
![Page 9: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/9.jpg)
Screening Results – Example ATG4
TI + DON
TI
DMSO
![Page 10: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/10.jpg)
Screening Results – Example RPL27A
DMSO
TI
TI + DON
![Page 11: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/11.jpg)
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
![Page 12: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/12.jpg)
Serial Dilution Dot Assay Results (Growth on TI + 175 ug/mL DON)
DMSO TI TI + DON
WT
ARC35
AYT1
ATG4
RPL27A
RPL39
68h 5d
![Page 13: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/13.jpg)
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
![Page 14: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/14.jpg)
FUTURE WORK
Screen yeast knock-out collections on other Fusarium mycotoxins
Select genes for altered expression in plants
![Page 15: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/15.jpg)
Fusarium systems biology pipeline
![Page 16: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/16.jpg)
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
![Page 17: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/17.jpg)
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
![Page 18: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/18.jpg)
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).
![Page 19: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/19.jpg)
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
![Page 20: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/20.jpg)
20
Uninoculated Inoculated
Arabidopsis is susceptible to F. graminearum
![Page 21: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/21.jpg)
21
DMSO DMSOChemical compounds
A
C
B
![Page 22: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/22.jpg)
22
![Page 23: Defining the Fusarium /host interaction through genomics and proteomics](https://reader036.fdocuments.us/reader036/viewer/2022070402/56813792550346895d9f3629/html5/thumbnails/23.jpg)