A key antisense sRNA modulates the oxidative stress response and virulence in Xanthomonas oryzae pv. oryzicola
Fig 1
sRNA Xonc3711 binds Hfq and targets the Xoc_3982 mRNA in Xoc BLS256.
(a) Interaction between Xonc3711 and Hfq protein by EMSA. Lane 1 contains 3’-biotinylated Xonc3711; lane 2, contains biotinylated Xonc3711 and Hfq protein; and lane 3, consists of biotinylated Xonc3711, Hfq protein, and unlabeled Xonc3711. (b) Northern blot analysis of Xonc3711 expression in wild-type Xoc BLS256, ΔHfq, ΔXonc3711 and ΔXoc_3982 strains grown to OD600 = 1.0 in NB. 5S rRNA was used as a loading control, and Image J was used to calculate expression levels. The intensity of the band in the first lane (WT) was normalized to a value of 100. (c) qRT-PCR analysis of xoc_3982 expression in Xoc BLS256 overexpressing Xonc3711 (Xonc3711OE), wild-type BLS256 (WT) and the ΔXonc3711 mutant. Expression levels of target genes were calculated relative to rpoD using the ΔΔCT method, where CT is the threshold cycle. Four independent biological replicates were carried out in this study (Wilcoxon-Mann-Whitney test). (d) Northern (upper two panels) and western (lower two panels) blot analysis of Xoc_3982 mRNA and protein levels, respectively, in BLS256 (WT), ΔXonc3711, and Δ3982 strains. In lanes labeled with (+), Xonc3711 was overexpressed from the pHM1::Xonc3711 construct. Expression of 5S rRNA and levels of RNAP were used as loading controls for northern and western blots, respectively. Values above each band represent band intensity and were calculated using Image J software. Band intensity in the first lane was normalized as 100. ND, not detected.