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.
Fig 2.
Analysis of the Xonc3711-xoc_3982 RNA interaction.
(a) Nucleotide sequence of BLS256 xoc_3982 mRNA from its initiation codon (+3) to +1245 in the CDS, with those of the predicted export signal sequence in blue font. Blue panel: The location of point mutations in Xoc_3982*; Green panel: The location of point mutations in Xonc3711*. (b) Schematic showing Xoc_3982::gfp translational fusions. Two fusions were constructed that varied in the number of nucleotides in the coding sequence. Constructs X+3 and X+1242 contained three and 1242 nucleotides of the Xoc_3982 coding sequence fused to GFP, respectively. (c) Northern blot showing Xonc3711 expression levels in the presence of reporter constructs X+3 and X+1242 (upper two panels). The lower two panels show Xoc_3982GFP proteins detected by western blotting. GFP fusion proteins were detected using antibodies directed against GFP (1:2000, mouse anti-GFP; Roche). (d) Interaction of Xonc3711 sRNA and full-length xoc_3982 mRNA in electrophoretic mobility shift assays. Lane 1, biotinylated Xonc3711; lane 2, biotinylated Xonc3711 and 40 μM xoc_3982 mRNA; lane 3, biotinylated Xonc3711 and 20 μM xoc_3982 mRNA; and lane 4, biotinylated Xonc3711, xoc_3982 mRNA, and unlabeled Xonc3711.
Fig 3.
Validation of the Xonc3711-xoc_3982 RNA interaction in vivo and requirement of RNase E for degradation.
(a) Compensatory point mutations validate the Xonc3711-xoc_3982 RNA interaction in vivo. All mutations were constructed in the Xoc_3982::gfp fusion strain, and asterisks denote point mutations. Lanes: 1, deletion mutant ΔXonc3711; 2, Xoc_3982::gfp fusion strain (labeled ‘Xonc3711’); 3, Xonc3711*; 4, ΔXonc3711-3982*; 5, 3982*; and 6, Xonc3711*-3982*. Upper three panels show northern blots using Xonc3711 or Xonc3711*, xoc_3982, and 5S rRNA as probes. The lower two panels show Xoc_3982GFP or Xoc_3982*GFP protein levels as determined by immunoblotting with mouse anti-GFP antisera; RNAP was used as a loading control. (b) RNase E is essential for xoc_3982 repression by Xonc3711. Upper two panels show northern blot analysis of Xonc3711 expression in the ΔRNaseEC mutant, the wild-type BL256, and the ΔXonc3711 and ΔRNaseECΔXonc3711 mutants. The lower two panels show western blot analysis of Xoc_3982GFP production. GFP fusion proteins were detected using anti-GFP antisera. 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.
Fig 4.
Mutations in Xonc3711, xoc_3982, and fliC decrease tolerance to oxidative stress.
Panels: (a) growth in NB and (b) NB supplemented with 0.1 mM H2O2. Strains evaluated for growth included wild-type (WT) Xoc BL256, ΔXonc3711, Xonc3711OE, ΔfliC, and ΔXoc_3982 in NB. Strains were grown in quadruplicate to the mid-exponential phase, diluted to OD600 = 0.1, and transferred to fresh NB or NB with 0.1 mM H2O2; growth was measured every 15 min for 48 h in a Bioscreen C apparatus at 28°C. Error intervals (shaded regions) indicate means ± SE (n = 4). (c) Volcano plot showing the FDR P values and fold-change of ΔXonc3711 versus the WT by RNA-Seq. Red and blue dots indicate upregulated and downregulated genes, respectively, with FDR P < 0.01. (d) Expression levels of fliC, fliF, fliM, flgA, flhA, and flhB in WT, ΔXonc3711, and ΔXonc3711-pXonc3711. Expression levels of flagella-related 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 experiment.
Fig 5.
Mutations in Xonc3711 and xoc_3982 reduce flagella numbers and length.
Ultrastructure of (a) wild-type Xoc BL526; (b) ΔXonc3711, (c) ΔfliC, and (d) ΔXoc_3982. (e) Length of flagella (μM) produced by WT, ΔXonc3711, ΔXonc3711-pXonc3711, and ΔXoc_3982. Flagella were randomly measured from 20 parallel views by transmission electron microscopy. **, significant at P < 0.01, Wilcoxon rank-sum test.
Fig 6.
Xonc3711 contributes to biofilm formation.
Confocal laser scanning microscopy of fluorescence in (a) GFP-labeled wild-type Xoc BL256 and (b) GFP-labeled ΔXonc3711. (c) Fluorescence in GFP-labeled Xoc WT, ΔXonc3711, and the complemented strain, ΔXonc3711-pXonc3711. Bacteria were grown under static conditions at 28°C for 96 h on glass coverslips. Biofilms were fixed, and fluorescence intensity was measured from 20 parallel replicates. **, P<0.01, Wilcoxon rank-sum test.
Fig 7.
Virulence and in planta growth of Xoc strains in rice cv. Yuanfengzao.
Virulence was assessed by inoculating six-week-old susceptible rice plants. (a) Leaves (n = 11) were inoculated with needleless syringes, and lesion lengths were evaluated 14 d after inoculation. Results indicate means ± SD. ANOVA was performed with Dunnett’s multiple-comparison post-hoc correction as compared with the WT (*, P < 0.05; **, P < 0.01). (b) Symptoms on rice leaves inoculated with Xoc WT, ΔXonc3711, Xonc3711OE, Xoc_3982, ΔXopC2, and C-ΔXopC2.
Fig 8.
Xoc_3982 recognizes a conserved motif and regulates effector gene xopC2.
(a) A potential Xoc_3982 binding motif was identified by MEME analysis of ChIP-seq peak regions. Representative sequences potentially bound by Xoc_3982 are listed. A conserved sequence in the promoter of the five genes is shown in red. (b) Interaction of the wild-type xopC2 promoter, the Xoc_3982 protein, and a xopC2 promoter mutant in electrophoretic mobility shift assays. Lane 1, 3’-biotinylated xopC2 promoter; lane 2, Xoc_3982 and a 3’-biotinylated random sequence (5’-TGTACAGTGATCAGTACAGG-3’); lane 3, 3’-biotinylated xopC2 promoter and Xoc_3982; lane 4, mutated xopC2 promoter and Xoc_3982. (c) qRT-PCR analysis of xopC2 expression levels in ΔXonc3711, WT BL256 and Δxoc_3982.
Fig 9.
Proposed model for Xonc3711-mediated regulation of flagella production and virulence.
Additional copies of the sRNA Xonc3711 transcript are produced during oxidative stress. Hfq-dependent Xonc3711 targets xoc_3982 mRNA, and Xonc3711-induced degradation of xoc_3982 mRNA is dependent on Rnase E; this results in the expression of flagella-associated genes though an unknown molecule. The DNA-binding protein Xoc_3982 interacts with the promoter of xopC2, repressing its transcription. When repression is lifted, XopC2 traverses the T3SS, enters the plant cell, and interacts with proteins that enhance the infection process.