Transcript Comparative Genomics of Plant Genes Responding to Fungi

COMPARATIVE FUNGAL
GENOMICS :
KNOWN KNOWNS
&
KNOWN UNKNOWNS.
Josh Herr, Plant Biology Interdepartmental Program
Problems, hurdles, help wanteds…
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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.
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Using RT-PCR to measure gene
expression in plants with mycorrhizae.
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Extract RNA from Poplar trees with and without
symbiotic fungi on roots.
Using RT-PCR to measure gene
expression in plants with mycorrhizae.
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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.
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Characterization of AOS gene family (con’t)
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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?
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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?