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IFAFS Meeting Gene Expression – Disease and Water Deficit John Davis

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 /

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IFAFS Meeting Gene Expression – Disease and Water Deficit John Davis

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

  2. 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 / re-rack to unigene set UGA (root library, 10k ESTs) 4/02 – UF (DD), UGA (SSH) done array construction / use / analysis / reduction UF (~300 for routine arrays) 1/03 – UF, UGA (~300 for routine arrays) done ID candidates Team 3/03 Team now – 12/02 UF (greenhouse trials) 2-10/02 (tissue) 1-8/03 (arrays) UF (PC R vs. S, FR R vs. S) UF, UGA (Asheville clones) UF, UGA (re-inoc’s at UF) ongoing 9/02 – 9/03 – biological experiments Materials Flow, Plants to Candidate Genes Step or Process Water DeficitStatus DiseaseStatus

  3. Membrane Preparation cDNAs from differential display PCR to increase amount of DNA DNA purification, concentration Spot on membrane with arrayer Denature, fix DNA to membrane This membrane has 384 spots from 96 cDNAs (4 spots/cDNA)

  4. Probe Preparation and Array Hybridization Extract total RNA (2 or more samples) Label mRNA with radioisotope (e.g. 32P) Purify, quantify probe 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

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

  6. Responses to Pathogen Challenge: Growth chamber and greenhouse materials Cronartium challenge Fusarium challenge Susceptible Water Resistant Susceptible Water Small Rough Large Susceptible Resistant

  7. Disease: An Intimate Interaction Between Two Organisms • 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 • Biotrophic pathogen tendencies • obligate (host tissues must live) • genetically rich interactions • are common after co-evolution • host defense pathways not • induced (or actively suppressed) … expect host response pathways to be distinct

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

  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

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