Expression analysis of Fusarium genes involved in mycotoxin analysis - experiments explained Erik Lysøe Fusarium, genes, and toxins 2008
Problem toxins in food and feed Approx. 400 known mycotoxins Fusarium toxins in cereals: Trichothecenes: - Deoxynivalenol (DON) - Nivalenol (NIV) - T-2 toxin - HT-2 toxin Zearalenone (ZON) Fumonisin Others: Aflatoxin - Aspergillus - Cereals (maize, sorghum, pearl millet, rice, wheat), oilseeds (peanut, soybean, sunflower, cotton), spices (chile peppers, black pepper, coriander, turmeric, ginger), and tree nuts (almond, pistachio, walnut, coconut, brazil nut). Ergotoxin - Claviceps purpurea - Cereals, rye (most common), triticale, wheat and barley. It affects oats only rarely. Ochratoxin A - Aspergillus, Penicillium - 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 t gene encoding a multifunctional l enzyme catalyzing N-methyldepsipeptide tid formation in Fusarium 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 humans and animals. Inhibition of ribosomal protein synthesis Two known gene clusters 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 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 F. graminearum 3 replicate experiments
Real-time experiments Trizol RNA extraction Quality, gel Concentration, spectrophotometer or fluorometer DNAse treatment Clean-up of total RNA RNEasy column Quality and concentration, Bioanalyzer cdna synthesis Real-time PCR using SybrGreen Calculating primer efficiency Analyse results
Real-time experiments Pipetting of cdna, primers, water and SybrGreen Analyse results with Pfaffl method 1. Make standard d curve to calculate l primer efficiency i (E value) 2. Combine housekeeping genes into one value using BestKeeper 3. Analyse fold changes using REST program and Erik s graph maker
Using Excel
1. BestKeeper with example: 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 regulated target genes and sample integrity: BestKeeper - Excel-based tool using gp pair-wise correlations. Biotechnol. Lett. 26, 509-515. 2. REST with example: 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 target genes and sample integrity: BestKeeper - Excel-based tool using gp pair-wise correlations. Biotechnol. Lett. 26, 509-515. 3. Erik s real-time graph maker with example http://fou02.bioforsk.no/fusarium/files/real-time-pcr-graph-maker-(eriklysoe).xls If time, also learn the 2 ΔΔC T(Livak) method, example found in the lecture Introduction to real-time PCR All real-time files available on: http://fou02.bioforsk.no/fusarium/files
BestKeeper
REST http://fou02.bioforsk.no/fusarium/files/rest-example.xls
Expression pattern in a gene cluster using REST - normalizing against one gene (FG12056, WT, 7d)
Erik s graph maker http://fou02.bioforsk.no/fusarium/files/real-time-pcr-graph-maker-(eriklysoe).xls Single CT values for all replicates Single CT values for all replicates Advantages: Standard deviation for the fold changes differences on the replicate runs Standard/log plot with error bars Minimum, maximum and median values Whiskers plot (Box plot)