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