Inoculum production, Macroconidia of the single spore F. graminearum isolate IFA 65 were grown on synthetic nutrient agar medium Spezieller NAhrstoffarmer Agar at 20 C under cool white and near UV light illumination. After seven days macroconidia were collected by centrifuga tion and washed in double distilled water. For the inocu lations 10 ml stock solutions of the inoculum were stored at ?80 C until use. Inoculation and sampling, Dream and Lynx wheat plants were grown in the greenhouse. After vernalisa tion at 4 C for eight weeks with a 16 8 h day night light regime, plants were cultivated at day night tem peratures of 22 18 C with a photoperiod of 16 8 h. At early anthesis single floret inoculation with the F. graminearum strain IFA 65 was carried out by pipetting 10 ul of the fungal Inhibitors,Modulators,Libraries suspension between the palea and lemma of each floret.
Control plants were inocu lated Inhibitors,Modulators,Libraries with distilled water instead of the macroconidia suspension. Eight florets per spike were inoculated. Greenhouse day temperature was increased to 24 C to ensure optimum infection conditions. Tissues of inocu lated florets and a part of the attached rachis of Dream and Lynx spikes were collected. Six plants per genotype treatment timepoint were sampled. Samples were immediately frozen in liquid nitrogen and stored at ?80 C. For the microarray analysis three replications were made for each inoculation treatment and samples were collected at 32 and 72 h after inocu lation. For the qPCR analysis samples were col lected at 8, 24, 32, 48, 72, and 96 hai. Sumai 3 and Florence Aurore wheat plants were grown under open air conditions.
At early anthesis, spikes were spray inoculated with Brefeldin_A 2 ml of the F. grami nearum macroconidia Inhibitors,Modulators,Libraries suspension or distilled water according to. For qPCR analysis whole spikes of treated cv. Sumai 3 and cv. Florence Aurore plants were collected at 0, 8, 32, 48, 72, 96, 120 and 336 hai. Four plants per genotype treatment time point were sampled. All samples were immediately fro zen in liquid nitrogen and stored at ?80 C. RNA extraction and cDNA synthesis For cv. Dream and cv. Lynx, floret tissue of six wheat heads per genotype, treatment and sampling timepoint were pooled prior to RNA extraction in order to reduce the biological variation between the samples. Accordingly, for cv. Sumai 3 and cv.
Florence Aurore spike tissue of four wheat plants per genotype, treatment and sampling timepoint were pooled prior to RNA extraction. Total RNA was extracted from fine ground samples using the guanidinium thiocyanate phenol chloroform method as described by. Subsequently, a DNase digest Inhibitors,Modulators,Libraries was per formed according to manufacturers instructions. RNA was further purified using phenol chloroform extrac tion. RNA quantity and quality were evaluated using ND 1000 spectrophotometer meas urement and agarose gel electrophoresis. cDNA was synthesised with 1. 2 ug total RNA and 0.