Fig. 2

Transcription and translation analyses of the Mr-OPY2 gene. a Representative Northern blot image of RNA isolated from undifferentiated mycelia grown in SDY or hemolymph (HE), and from germlings differentiating appressoria on locust wings (AP). The uncropped Northern blot is shown in Supplementary Fig. 11a. b 5′RACE with the RLM RT-PCR kit. The uncropped image of the agarose gel is shown in Supplementary Fig. 11b. c A diagrammatic representation of differences between the two mRNA variants of Mr-OPY2. Note: the two transcripts share the same major ORF but they have different 5′UTR. d Representative western blot image and analysis to quantify Mr-OPY2 levels in ΔMr-OPY2 and WT grown in SDY or hemolymph (HE) (Upper panel). The OPY2 levels of hemolymph-grown WT mycelium (1.4) is calculated relative to growth in SDY which is set at 1. ΔMr-OPY2 is included as a negative control. Lower panel: a portion of the loading control gel (for the Western blot analysis in upper panel) stained with Coomassie Brilliant Blue. M: Protein ladder (Thermo scientific, USA). The uncropped images of the western blot and the SDS-PAGE gel are shown in Supplementary Fig. 11c, d. e Immunohistochemical staining of the Mr-OPY2 protein in germlings differentiating appressoria against a plastic surface, and in non-differentiating germlings from 1/2 SDY shake cultures. Scale bar represents 10 μm. FITC: Fluorescein isothiocyanate; Merge: FITC and bright field pictures are merged; AP: appressorium. Note: no fluorescent signal in ΔMr-OPY and a weak signal in WT grown in 1/2 SDY. Images are representative of at least three independent experiments for each condition