Transcript Membrane Preparation

IFAFS Meeting
Gene Expression – Disease and Water Deficit
John Davis
Materials Flow, Plants to Candidate Genes
Step or Process
treated plant material
Water Deficit
Status
UF (control, WD, WD/recovery) 2/02
Disease
Status
UF (lab), UGA (greenhouse)
done
cDNA cloning / sequence /
UGA (root library, 10k ESTs)
re-rack to unigene set
4/02 –
UF (DD), UGA (SSH)
done
PCR inserts, purify
UC-D (384-well, ~3,000)
10/02
UF, UGA (96-well format)
done
array construction / use /
analysis / reduction
UF (~300 for routine arrays)
1/03 –
UF, UGA (~300 for routine arrays)
done
UF (greenhouse trials)
biological experiments
ID candidates
Team
2-10/02 (tissue) UF (PC R vs. S, FR R vs. S)
1-8/03 (arrays) UF, UGA (Asheville clones)
UF, UGA (re-inoc’s at UF)
3/03
Team
ongoing
9/02 –
9/03 –
now – 12/02
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)
Extract total RNA
(2 or more samples)
Label mRNA with radioisotope (e.g.
Probe Preparation and
Array Hybridization
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
Relative
Gene
Sequence
Expression Designation Similarity
Gene expression arrays
300
200
pi059
pathogen response
protein (potato)
pi073
chitinase
pi213
phenylalanine
ammonia-lyase
pi148
transcription
factor
100
0
600
300
0
50
25
0
30
20
10
0
30
20
pi125
10
0
No pathogen challenge Pathogen challenge
225
150
pi107
75
0
Up-regulated after challenge
375
Down-regulated after challenge
125
Non-regulated (control) gene
200
250
pi117
0
pi171
100
se
ea
is
D
H
ea
l
th
y
d
0
Pine ESTs
(unknown
function)
Responses to Pathogen Challenge:
Growth chamber and greenhouse materials
Fusarium challenge
Susceptible
Susceptible
Water
Resistant
Resistant
Cronartium challenge
Susceptible
Small
Rough
Water
Large
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
Diagram of two major pathways thought to be
involved in regulating resistance responses
EXTERNAL
FACTORS
pathogen
(Resistance Gene Action)
INTERNAL
PROCESSES
IN RESISTANT
TREES
[SA]
[JA]
biotrophic
necrotrophic
SA-dependent
gene expression
JA-dependent
gene expression
SA- and/or JA-dependent
mechanisms of disease
resistance
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