COMPARATIVE FUNGAL GENOMICS : KNOWN KNOWNS & KNOWN UNKNOWNS. Josh Herr, Plant Biology Interdepartmental Program Problems, hurdles, help wanteds… No developed fungal genome browser? Gbrowse? Data can be acquired – which format to use? raw nucleotide, GFF3, PEP, GBK Which genes to compare? (remember: annotation is a problem) ex: aquaporin genes, fruitbody morphogenesis? How do we get a grip on purifying (or other?) selective forces in the fungi? How do we work with the data? BLAST and then Galaxy, but what else? Whole genome MUSCLE alignments? COMPARATIVE FUNGAL GENOMICS : KNOWN KNOWNS & KNOWN UNKNOWNS. Josh Herr, Plant Biology Interdepartmental Program COMPARATIVE GENOMICS OF SOME PLANT GENES RESPONDING TO FUNGI: AN EXAMPLE FROM THE ALLENE OXIDE SYNTHASE GENE FAMILY. Josh Herr, Plant Biology Interdepartmental Program Modified from Finlay 2008 Photo: David Read Induced Systemic Resistance (ISR) From: Harman et al 2004 Nat. Rev. Microbiol. From: Schilmiller & Howe (2005) Allene Oxide Synthase (AOS) AOS is a member in a large Cytochrome p450 enzyme class responsible for fatty acid metabolism. Member of the CYP74 family of oxygenbinding Heme-like lipoxygenase proteins. Best characterized member of this group is the thromboxane synthase enzyme in Humans which mediates blood platelet aggregation. Using RT-PCR to measure gene expression in plants with mycorrhizae. Extract RNA from Poplar trees with and without symbiotic fungi on roots. Using RT-PCR to measure gene expression in plants with mycorrhizae. Extract RNA from Poplar trees with and without symbiotic fungi on roots. Make cDNA library. Measure gene expression. Characterization of AOS gene family Itoh et al 2002 describe 3 proteins in gene family (AOS1, AOS2, HPL) in Arabidopsis. Numerous studies have cited AOS, but no phylogeny exists in plants. Research Goal: To determine the number of homologs of AOS in plants and understand the gene evolution, eventually designing primers to address gene expression across this family. Characterization of AOS gene family (con’t) BLAST known AOS sequence across plant genomes. Take close hits, align, and construct phylogeny. Determine synteny for AOS family genes across the plant lineage (problem: only whole genome alignments are Arabidopsis, Oryza, and Populus.) Eventually develop primers to determine gene expression of individual members of this family. HPL (hydroperoxide lyase) DEL (divinyl ether synthase) AOS3 (allene oxide synthase 3) AOS2 (allene oxide synthase 3) AOS1 (allene oxide synthase 1) Basal gene: hydroperoxide lyase (HPL) AOS3 / divinyl ester synthase (DES) AOS2 AOS1 Distant relative is conserved: thromoxane synthase. Conclusions? There are at least three paralogous members of the AOS gene family (AOS1, AOS2, DES/AOS3, and HPL(?). These genes appeared to have diverged in the basal plants (i.e. orthologous gene alignment across plants). Next goal is to design primers to study gene expression of the individual members of this gene family. Questions?