Dnase digestion

Total RNA was extracted from embryos using Trizol (Invitrogen) according to the manufacturer´s protocol. For one biological replicate 20 ul embryos were collected. A total of 1 ug from each RNA sample was subjected to DNase digestion (Sigma) and cDNA synthesis (Applied Biosystems) according to the manufacturer´s protocol. Samples were confirmed for quality and quantity using agarose gel electorphoresis and Nanodrop. Out of 200 μl (10x diluted) total cDNA, 2 μl was subsequently used for RT-qPCR in a total volume of 20 μl using CFX96 (BioRad instruments, Sweden) with EvaGreen supermix (Solis BioDyne). Resulting Ct-values were converted into raw quantities with the ΔCt method using Genorm, which were used to calculate the geometric mean of four reference genes ß-tubulin, RpL32 28SrRNA and GAPDH, serving as normalization factors for each cDNA sample. The stability measure M [55 (link)], for the mean of the reference genes was below 1.5 for all the conditions tested. Relative expression levels were calculated where expression in control embryos derived from mothers with only the TubGal4 transgene was set to 100%.

Lung cells were isolated by collagenase and DNase digestion (Sigma Aldrich). Splenocytes were mashed through 100 µm nylon cell strainers and erythrocytes were lysed in ACK buffer. Lung cells were washed with Hank’s Balanced Salt Solution (HBSS) and splenocytes were washed with PBS. Lung cells and splenocytes were pre-incubated with the 2.4 G2 antibody to block Fc receptor binding, followed by incubation with various cell surface Abs: CD3 (17-A2), NK1.1 (PK136), B220 (RA3-6B2), F4/80 (BM8), MHCII (M5/114.15.2), (eBioscience, San Diego, CA), Siglec F (E50-2440), CD11c (N418), CD11b (M1/70), Gr-1 (RB6-8C5) and Ly6G (1A8) (BD Biosciences, San Jose, CA). Cells were fixed with BD Cytofix and analysed on BD LSRII.

Leaf tissue samples (100 mg) for RNA isolation were collected from each genotype at 0, 24 and 48 h post inoculation (hpi). Total RNA was isolated as described in Spectrum™ Plant Total RNA kit protocol with on-column DNase digestion (Sigma-Aldrich, USA). RNA samples were purified using RNA clean and concentration TM-25 (ZYMO RESEARCH) and the quality was determined both by NanoDrop and Agilent 2100 Bioanalyzer. A total RNA (~ 3 μg) for each sample was used to prepare mRNA-seq library according to TrueSeq RNA Sample Prep Kit protocol (Illumina). Library quality control and quantification were performed with an Experion DNA 1 K Chip (Bio-Rad) and a Qubit fluorometer (Invitrogen), respectively. For each library, more than 100 million, 100-bp paired-end sequences were generated (Table 1) using an Illumina HiSeq 2500.