Expression analysis of Fusarium
ggenes involved in mycotoxin
y
analysis
y
- experiments explained
Erik Lysøe – Fusarium, genes, and toxins 2008
Problem toxins in food and feed
Æ Approx. 400 known mycotoxins
Fusarium toxins in cereals:
•
Trichothecenes:
-
•
•
Deoxynivalenol (DON)
Nivalenol (NIV)
T-2
T
2 toxin
HT-2 toxin
Zearalenone (ZON)
Fumonisin
Others:
•
Aflatoxin - Aspergillus
-
•
Ergotoxin - Claviceps purpurea
-
•
Cereals, rye (most common), triticale, wheat and barley. It affects oats only
rarely.
Ochratoxin A - Aspergillus, Penicillium
-
•
Cereals
C
l (maize,
( i
sorghum,
h
pearll millet,
ill
rice,
i
wheat),
h
) oilseeds
il
d ((peanut, soybean,
b
sunflower, cotton), spices (chile peppers, black pepper, coriander, turmeric,
ginger), and tree nuts (almond, pistachio, walnut, coconut, brazil nut).
Cereals, coffee, beer, dried fruit, wine, cocoa and nuts.
Patulin - Penicillium, Aspergillus and Byssochlamys
-
Apples
Genes involved in mycotoxin synthesis
• Trichothecenes
• Zearalenone
• Enniatin
– Haese, A., Schubert M., Herrmann M. and Zocher R. (1993). Molecular characterization of the enniatin
– synthetase
th t
gene encoding
di a multifunctional
ltif
ti
l enzyme catalyzing
t l i N-methyldepsipeptide
N
th ld i
tid formation
f
ti in
i Fusarium
F
i
– scirpi. Molecular Microbiology, 7, 905-914
• Fusarin C
– Song, Z.S., Cox R.J., Lazarus C.M. and Simpson T.J. (2004). Fusarin C biosynthesis in Fusarium moniliforme
– and Fusarium venenatum. Chembiochem, 5, 1196-1203
• Aurofusarin
Trichothecenes
• The Fusarium toxins most strongly assosiated with chronic and fatal toxicoses in
h
humans
and
d animals.
l
• Inhibition of ribosomal protein synthesis
• Two
T
known
k
gene clusters
l
• Two single genes
Tri5 – Trichodiene synthase
T i h th
Trichothecene
genes hi
highly
hl expressed
d in
i planta
l t
Trichothecene genes
Wheat
Barley
CMC
Myro
Conidia
Asco
CM,ND,CD
Perithecia
Conidia
Asco
CM,ND,CD
Perithecia
Aurofusarin genes
Wheat
Barley
CMC
Myro
Real-time
Real
time experiments
• Trichothecene primers
– ELTri5a
– ELTri6a (regulator)
• Aurofusarin primers
– ELAurR1a (regulator)
• Housekeeping genes
– UBC
– EF1α
– Btub
• Samples
– Wheat control, 2d, 4d and 6d after inoculation
with
i h F.
F graminearum
i
– 3 replicate
li
experiments
i
Real-time
Real
time experiments
• Trizol RNA extraction
– Quality
Quality, gel
– Concentration, spectrophotometer or
fluorometer
• DNAse treatment
• Clean
Clean-up
up of total RNA
– RNEasy column
– Quality and concentration, Bioanalyzer
• cDNA synthesis
• Real-time PCR using
g SybrGreen
y
• Calculating primer efficiency
• Analyse results
R l ti
Real-time
experiments
i
t
• Pipetting of cDNA, primers, water and SybrGreen
• Analyse results with Pfaffl method
1 Make
1.
M k standard
d d curve to calculate
l l
primer
i
efficiency
ffi i
(E value)
l )
2. Combine housekeeping
p gg
genes into one value using
g BestKeeper
p
3. Analyse fold changes using REST program and Erik’s graph maker
Using Excel
1. BestKeeper
p with example:
p
http://fou02.bioforsk.no/Fusarium/Files/Bestkeeper-example-UBC-EF1a.xls
Pfaffl, M.W., Tichopad, A., Prgomet, C., Neuvians, T.P., 2004. Determination of stable housekeeping genes, differentially
g
target
g ggenes and sample
p integrity:
g y BestKeeper
p - Excel-based tool usingg ppair-wise correlations. Biotechnol. Lett. 26,,
regulated
509-515.
2. REST with example:
p
http://fou02.bioforsk.no/Fusarium/Files/REST-example.xls
Pfaffl, M.W., Tichopad, A., Prgomet, C., Neuvians, T.P., 2004. Determination of stable housekeeping genes, differentially
regulated
g
target
g ggenes and sample
p integrity:
g y BestKeeper
p - Excel-based tool usingg ppair-wise correlations. Biotechnol. Lett. 26,,
509-515.
3. Erik’s real-time ggraph
p maker with example
p
http://fou02.bioforsk.no/Fusarium/Files/Real-time-PCR-graph-maker-(ErikLysoe).xls
ΔΔCT (Livak) method
If time
time, also learn the 2–ΔΔC
method, example found in the
lecture “Introduction to real-time PCR”
All real-time files available on:
http://fou02.bioforsk.no/Fusarium/Files
B tK
BestKeeper
REST
• http://fou02.bioforsk.no/Fusarium/Files/REST-example.xls
Expression pattern in a gene cluster using REST
- normalizing against one gene (FG12056,
(FG12056 WT
WT, 7d)
Erik’ss graph maker
Erik
http://fou02.bioforsk.no/Fusarium/Files/Real-time-PCR-graph-maker-(ErikLysoe).xls
Single CT values for
all replicates
Single
Si
l CT values
l
for
f
all replicates
Advantages:
• Standard deviation for the fold changes differences on
th replicate
the
li t runs
¾ Standard/log plot with error bars
• Minimum,
Minimum maximum and median values
¾ Whiskers plot (Box plot)