tometer. Purified RNA extracts were checked at random for DNA contamination by using RNA extracts as templates in qPCR assays. High quality RNA was reverse transcribed with SuperScript VILO cDNA Synthesis Kit according to the manufacturer’s protocol. Thereafter, the obtained cDNA was split for 
bacterial and plant gene expression analysis. Analysis of E. amylovora gene expression Quantitative real-time PCR assays for E. amylovora genes hrpL, hrpA, hrpN, dspA/E, amsG, recA, and gyrA were established and purchase LY-2835219 optimized. Primer sequences were derived from available sequence information in GenBank: hrpL, hrpA, hrpN, dspA/E, amsG, recA and gyrA. To optimize primer concentrations, each primer was tested in 50 nM steps in a concentration range from 50 to 600 nM and annealing temperatures increasing in 1uC steps deviating from the calculated melting temperature by maximally 3uC. Genespecific PCR products spanning the sequence targeted by the qPCR-primers with 104 copies per reaction were used as templates during optimization. Specific amplification of E. amylovora targets was verified by the presence of a specific PCR product in cDNA template from inoculated flowers and absence in mock-inoculated flowers as determined by agarose gel electrophoresis. For analysis, the cDNA transcripts were amplified in a Mastercycler ep realplex with standard reaction conditions: 5 ml cDNA-sample with a final 1:5 dilution were used in a standard 20 ml-qPCR reaction with Power SYBR Green PCR Master Mix and specific primers. Cycling parameters included a 10 min initial denaturation at 95uC followed by 45 cycles consisting of denaturation at 95uC for 15 sec, annealing at primer specific temperature for 25 sec and amplification at 65uC for 25 sec with signal detection. A melting curve analysis was included at the end of each run with a temperature ramp from 60uC to 95uC in 20 min to determine specificity of amplified qPCR products. Each sample was analyzed for bacterial gene expression in triplicate. Quantification of the absolute copy numbers was performed with standard curves of defined dilutions of gene-specific PCR-products in each qPCR run. Data were analyzed with Eppendorf realplex 2.0 software and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189214 qPCR efficiencies were between 0.851.14 and standard curve R2 values above 0.98. No template controls included in each run were always negative. Transcript values were normalized against expression of two standard reference genes: recA encoding recombinase A and gyrA encoding gyrase subunit A. Plant gene expression analysis Expression of the pathogen related protein-1 and a gene encoding for the putative proteinase inhibitor Miraculin were determined by qPCR as described in Milcevicova et al. . For normalization, actin and GAPDH were selected as reference genes. Analysis of relative gene expression between inoculated und uninoculated flower samples was done with the software REST 2008. Results The transcriptional timing and coordination of the type III secretion system of E. amylovora was investigated for the first time at the site of primary infection, in flowers still attached to the tree. Without wounding, single flowers of the susceptible apple cv. Golden Delicious were manually inoculated with approximately 108 bacterial cells. Bacterial suspension was placed in two droplets at the stigmas and close to the hypanthium. Subsequently, the expression of selected genes essential for type III secretion and necessary for bacterial virulence was monitored. These genes compri
