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

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 ORF’s • 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 30°C, 2-6 days Photograph and quantify growth Repeat 2 times

9. DMSO TI TI + DON Screening Results – Example ATG4

10. DMSO TI TI + DON Screening Results – Example RPL27A

11. Screening Results – Top Strains

12. DMSO TI TI + DON 68h 5d WT ARC35 AYT1 ATG4 RPL27A RPL39 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. Wheat Maize (68 unique) 6979 2311 2389 2164 4597 2166 4602 Barley (2 unique) Fg genes detected by 96hai 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, p<0.05). Many of these proteins are potentially involved in pathogenicity. 1Taylor 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 Chemical compounds DMSO A B C