1. IFAFS Meeting Gene Expression – Disease and Water Deficit John Davis

2. Materials Flow, Plants to Candidate Genes Water Deficit StatusDisease Status Step or Process treated plant materialUF (control, WD, WD/recovery)UF (lab), UGA (greenhouse)2/02done cDNA cloning / sequence / re-rack to unigene set UGA (root library, 10k ESTs)UF (DD), UGA (SSH)4/02 –done PCR inserts, purifyUC-D (384-well, ~3,000)UF, UGA (96-well format)10/02done array construction / use / analysis / reduction UF (~300 for routine arrays)UF, UGA (~300 for routine arrays)1/03 –done biological experiments UF (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) ongoing 9/02 – 9/03 – ID candidatesTeamnow – 12/02Team3/03

3. 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 spots from 96 cDNAs (4 spots/cDNA)

4. Extract total RNA (2 or more samples) Label mRNA with radioisotope (e.g. 32 P) Bathe each membrane with its respective probe, then wash away excess Quantify signal from each cDNA Expose to film: semi-quantitative Count in phosphorimager: quantitative Purify, quantify probe Probe Preparation and Array Hybridization

5. pi059 pathogen response protein (potato) pi073 chitinase pi125 pi213 phenylalanine ammonia-lyase pi148 transcription factor pi107 pi117 pi171 Pine ESTs (unknown function) Water Path. Relative Gene Sequence Expression Designation Similarity No pathogen challengePathogen challenge Up-regulated after challenge Down-regulated after challenge Non-regulated (control) gene Gene expression arrays

6. Responses to Pathogen Challenge: Growth chamber and greenhouse materials WaterSusceptibleResistant Small Fusarium challenge Cronartium challenge RoughLarge SusceptibleResistant WaterSusceptible

7. 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

8. pathogen JA-dependent gene expression SA-dependent gene expression EXTERNAL FACTORS INTERNAL PROCESSES IN RESISTANT TREES [JA] [SA] SA- and/or JA-dependent mechanisms of disease resistance (Resistance Gene Action) Diagram of two major pathways thought to be involved in regulating resistance responses biotrophicnecrotrophic

9. 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