Fig. 4

The CRISPR-Cas system regulates SPI-1, and SPI-2 genes expression. (A) The bacterial strains were cultivated in conditions (1:100 dilution in LB, followed by 8 h incubation) that promote SPI-1 activation. Subsequently, qRT-PCR was conducted on isolated RNA samples to assess the expression levels of key SPI-1 components, including transcriptional regulator-hilA, and SPI-1 effectors- sipA, sipD, sopB. (B) The bacterial strains were cultivated in SPI-2 inducing conditions (MgM- MES media) for 5 h, and qRT-PCR was performed from isolated RNA to check expression of SPI-2 effectors- pipB2 and spiC, SPI-2 encoded transcriptional regulator-ssrB, and SPI-3 encoded protein-mgtC. Relative expression of the gene was calculated using the 2 ^–ΔΔCt method and normalized to the reference gene, rpoD. One-way ANOVA (Dunnett’s multiple comparison test) was used to determine significant differences between the WT and knockout strains, in at least three independent experiments, with at least 3 replicates in each. Error bars indicate SD. Statistical significance is shown as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p < 0.0001; and ns, not significant