ifafs meeting gene expression – disease and water deficit john davis
DESCRIPTION
IFAFS Meeting Gene Expression – Disease and Water Deficit John Davis. treated plant material. UF (control, WD, WD/recovery). 2/02. UF (lab), UGA (greenhouse). done. PCR inserts, purify. UC-D (384-well, ~3,000). 10/02. UF, UGA (96-well format). done. cDNA cloning / sequence / - PowerPoint PPT PresentationTRANSCRIPT
IFAFS MeetingGene Expression – Disease and Water Deficit
John Davis
Materials Flow, Plants to Candidate Genes
Water Deficit Status Disease StatusStep or Process
treated plant material UF (control, WD, WD/recovery) UF (lab), UGA (greenhouse)2/02 done
cDNA cloning / sequence / re-rack to unigene set UGA (root library, 10k ESTs) UF (DD), UGA (SSH)4/02 – done
PCR inserts, purify UC-D (384-well, ~3,000) UF, UGA (96-well format)10/02 done
array construction / use / analysis / reduction
UF (~300 for routine arrays) UF, UGA (~300 for routine arrays)1/03 – done
biological experimentsUF (greenhouse trials) UF (PC R vs. S, FR R vs. S)
UF, UGA (Asheville clones)UF, UGA (re-inoc’s at UF)
2-10/02 (tissue)1-8/03 (arrays)
ongoing9/02 – 9/03 –
ID candidates Team now – 12/02Team 3/03
cDNAs from differential display
PCR to increase amount of DNA
Denature, fix DNA to membrane
Membrane Preparation
Spot on membrane with arrayer
DNA purification, concentration
This membrane has 384 spotsfrom 96 cDNAs (4 spots/cDNA)
Extract total RNA (2 or more samples)
Label mRNA with radioisotope (e.g. 32P)
Bathe each membrane with its respective probe,then wash away excess
Quantify signal from each cDNAExpose to film: semi-quantitativeCount in phosphorimager: quantitative
Purify, quantify probe
Probe Preparation and Array Hybridization
pi059 pathogen response protein (potato)
pi073 chitinase
pi125
pi213 phenylalanine ammonia-lyase
pi148 transcription factor
pi107
pi117
pi171
Pine ESTs(unknownfunction)
WaterPath.
Relative Gene SequenceExpression Designation Similarity
No pathogen challenge Pathogen challenge
Up-regulated after challenge
Down-regulated after challenge
Non-regulated (control) gene
Gene expression arrays
Responses to Pathogen Challenge:Growth chamber and greenhouse materials
WaterSusceptible Resistant
Small
Fusarium challenge Cronartium challenge
Rough LargeSusceptible Resistant
WaterSusceptible
Disease: An Intimate Interaction Between Two Organisms
Biotrophic pathogen tendencies• obligate (host tissues must live)• genetically rich interactions are common after co-evolution• host defense pathways not induced (or actively suppressed)
Necrotrophic pathogen tendencies• facultative (host tissues must die)• pathogens tend to be more uniform w/respect to virulence• disease state is associated with stress / defense responses
… expect host response pathways to be distinct
pathogen
JA-dependent gene expression
SA-dependentgene expression
EXTERNALFACTORS
INTERNAL PROCESSESIN RESISTANTTREES
[JA] [SA]
SA- and/or JA-dependentmechanisms of disease resistance
(Resistance Gene Action)
Diagram of two major pathways thought to be involved in regulating resistance responses
biotrophic necrotrophic
Criteria for Selecting Candidate Genes
Disease• differentially regulated by R and S within pathosystem• differentially regulated by pathogen type within R and S• transgene effects on disease expression in models• > likelihood of large effects based on presumed function
Water Deficit• differentially regulated by drought and recovery treatments• differentially regulated in contrasting genotypes• transgene effects on drought tolerance in models• > likelihood of large effects based on presumed function