Fig. 1
Schematic representation of the locus for TSI and confocal analysis of Fg transformants. A The TSI locus 1 is located adjacent to a micro-region within chromosome 1 between the genes designated FGRAMPH1_01G06815 and FGRAMPH1_01G06817. At this location, there is an intergenic region of 2.7 kb where the insertion of the expression cassette can occur. To allow integration of the expression cassette in the locus, a vector system was developed based on the split-marker technique. Three recombination events allow insertion of the cassette into the TSI locus 1. Primer combinations P5–P6, P7–P8, P9–P10 and P11–P12 are used to confirm correct cassette integration. B A vector system based on the Golden Gate approach was developed to allow cassette integration into the TSI locus 1. PCR fragments amplified by primer combinations P1–P2 and P3–P4 are used for fungal transformation. C Confocal images of strains confirming expression of a non-secreted version of mCherry (PtrpC-mCherry-TtrpC) and a secreted version (PtrpC-SPOSP24-mCherry-TtrpC). Fluorescence emissions were detected in 16 h old spore germlings. The wild type strain PH-1 was used as the control to set the confocal conditions. D IGV screenshot showing the reads aligned to the LB and RB of the TSI locus 1 in the wild type strain PH-1 and the transformed strains. Numbers in brackets indicate the range of read depth coverage per bp. E IGV screenshot displaying reads aligned to the expression cassettes from the transformed strains and the genes flanking the TSI locus 1. F Bar graphs represent the average read depth values for each section of the expression cassette and for the two genes (FGRAMPH1_01G06815 and FRGAMPH1_01G06817) flanking the TSI locus 1 for both transformants. Values above the bars are the ratio value calculated as the average read depth value of each section divided by the average read depth value from both flanking genes. Error bars represent standard deviation (SD)