Figure 1

Experimental approach used to identify and characterize genes that confer resistance to chitosan oligosaccharide (COS). Three chemogenomic assays were used: Haploinsufficiency profiling (HIP), homozygous profiling (HOP) and multicopy suppression profile (MSP). (1) Heterozygous, homozygous deletion pools and multicopy suppression pool were grown competitively in the presence of COS-5.44. If a gene is required to grow in the presence of COS, the corresponding deletion strain will grow more slowly and therefore will be underrepresented. Cells overexpressing a gene that suppresses sensitivity will growth faster and will be overrepresented in the MSP pool. (2) Genomic DNA was isolated from cells prior to and after the HIP-HOP assays, and plasmid purification from the COS treated MSP pool was carried out. (3) Barcodes were PCR amplified for HIP-HOP assays as well as the plasmid inserts of MSP. (4) PCR products (barcodes and plasmid inserts) were hybridized to a TAG4 array. Intensity of treatment samples is compared with intensity of a control sample to determine relative abundance (~ fitness). (5) Sensitive deletion strains and constructed overexpressing strains were individually confirmed. (6) Five resistant overexpressing strains that were also sensitive as deletion strains were selected for transcriptome analysis. Overexpressing strains and vector control were grown in the presence of COS (112.5 μg/ml) and cells were harvested before COS treatment and after 60 min of COS treatment. (7) RNA was isolated from harvested cells, cDNA synthesized and labelled with fluorescent dyes. (8) Labelled samples were hybridized to expression microarrays. Transcriptional changes were indentified by differential expression analysis. Figure modified from Ericson et al. (2010).