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

DEOXYNIVALENOL (DON) 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   Inexpensive / easy to use  Conservation of biochemical processes

Yeast Genomic Screening on DON Pin (2X) 1536 / plate Singer Robot YPD + TI ug/mL DON Grow 30°C, 2-6 days Photograph and quantify growth Collection in 96 well format Repeat 2 times

Screening Results – Example ATG4 TI + DON TI DMSO

Screening Results – Example RPL27A DMSO TI TI + DON

Screening Results – Top Strains GENEESSENTIAL?DESCRIPTION A VTH1N Putative membrane glycoprotein ECM15N Non-essential protein of unknown function ABF1Y DNA binding protein with possible chromatin-reorganizing activity BCY1Y Regulatory subunit of the cyclic AMP-dependent protein kinase (PKA) ARC18N Subunit of the ARP2/3 complex; required for motility & integrity of cortical actin patches ARC35Y Subunit of the ARP2/3 complex; required for motility & integrity of cortical actin patches AYT1N Acetyltransferase; catalyzes trichothecene 3-O-acetylation to form less toxic compound 3 ATG4N Cysteine protease required for autophagy KRE9Y Glycoprotein involved in cell wall beta-glucan assembly GCD10Y Subunit of tRNA (1-methyladenosine) methyltransferase with Gcd14p MMF1Y Mitochondrial protein involved in maintenance of the mitochondrial genome RPS30BN Protein component of the small (40S) ribosomal subunit GSP1Y GTP binding protein (mammalian Ranp homolog) RPS3Y Protein component of the small (40S) ribosomal subunit YBL044WN Putative protein of unknown function B RPL12BN Protein component of the large (60S) ribosomal subunit RPL5Y Protein component of the large (60S) ribosomal subunit UBP13N Putative ubiquitin-specific protease RPL27AN Protein component of the large (60S) ribosomal subunit RPL39N Protein component of the large (60S) ribosomal subunit GENEESSENTIAL?DESCRIPTION VTH1NPutative membrane glycoprotein ECM15NGene of unknown function ABF1YDNA binding protein ARC18N Required for integrity of cortical actin patches ARC35Y Required for integrity of cortical actin patches AYT1NAcetyltransferase ATG4NCysteine protease required for autophagy KRE9Y Glycoprotein: cell-wall β-glucan assembly UBP13NPutative ubiquitin-specific protease RPL27ANComponent of (60S) ribosomal subunit RPL39NComponent of (60S) ribosomal subunit

Serial Dilution Dot Assay Results (Growth on TI ug/mL DON) DMSOTITI + DON WT ARC35 AYT1 ATG4 RPL27A RPL39 68h5d

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

 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

 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 WheatMaize (68 unique) Barley (2 unique) Fg genes detected by 96hai

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 vitro 1.  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).

 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: F 6 seed of (B73 X CO441) lines. - Summer 2010 – begin phenotyping silk and kernel resistance. Defining resistance in maize B73CO441

20 UninoculatedInoculated Arabidopsis is susceptible to F. graminearum

21 DMSO Chemical compounds A C B

22