Red dots indicate the upregulated genes with FDR P < 0.01, while blue dots indicate downregulated genes with FDR P < 0.01. (a) Volcano plot showing the FDR P-value and fold change of S128P versus WT. The upregulation of a siderophore biosynthesis gene cluster, which was detected by RNA-seq, contributes to a reduction in the Fenton reaction. Each suspension was subjected to a 10-fold serial dilution, and 5 μl drops from the 10 −1–10 −6 dilutions were placed on NB agar with cephalexin. Three leaves, each infected with one strain of Xoc, were excised around the inoculation site and then crushed with sterile beads. (c) Population dynamics of WT silent, S128P, Δ tadA, tadA OE and WT pHM1 cells in planta (means ± SD). (b) Symptoms on rice leaves inoculated with the WT silent, S128P mutant, Δ tadA, tadA OE and WT pHM1. The asterisks (***) indicate significant differences between the data obtained for Δ tadA, S128P and tadA OE as compared to the WT silent strain ( P < 0.001 ANOVA with Dunnett’s multiple test post-hoc correction compared with WT silent). Values represent mean lesion length ± SD. (a) Leaves ( n = 12) were inoculated with needleless syringes and evaluated for lesion length 14 days after inoculation. Virulence was assessed by inoculating six-week-old susceptible Zhenshan 97 rice plants with Xoc strains. Virulence and in planta growth in rice cv. Biofilms were fixed, and fluorescence intensity was randomly measured from 12 parallel replicates. Bacteria were grown under static conditions at 28☌ for 96 h on glass coverslips. Panel (d) shows fluorescence intensity in GFP-labeled Xoc WT silent, S128P, and Δ fliC. Statistically significant differences occurred when the mean of one sample did not lie within the 95% CI.Ĭonfocal laser scanning microscopy of fluorescence in GFP-labeled (a) Xoc WT silent, (b) S128P mutant, and (c) Δ fliC mutant. Error bars show the 95% CI around the mean for each sample. Measurement of filament length (C) and radii (D) from 120 cells of WT silent and the S128P mutant. The carbons of the D1 residues are colored green in the WT model (left) and with cyan in the mutant (right), whereas D2 residues are colored yellow in both models. The loss of several critical H bonds in the Ser128Pro mutant (right) may disrupt the H-bonding network surrounding Ser128 (left), which stabilizes the D1-D2 interface. (B) Comparison of hydrogen bonding around the mutation site. The serine residue (Ser128) is depicted using spheres. The backbone is colored green, yellow and magenta at the D1, D2 and D3 domains of the model, respectively, and with orange for the template. aeruginosa flagellin 4NX9 (PDB ID: 1IO1) as the template. Homology modeling of FliC and measurement of flagellar filament length and radius. Error intervals (shaded regions) indicate mean ± SE of n = 4 replicates. Panels: (a) growth in NB (b) growth in NB supplemented with 0.1 mM H 2O 2. Strains were grown in quadruplicate to mid-exponential phase, diluted to OD 600 = 0.1, transferred to fresh NB and placed in a Bioscreen C apparatus at 28☌. Growth of Xoc WT silent, S128P, Δ tadA, Δ fliC, tadA OE and WT pHM1 in NB medium. (e) Peaks that were called at fliC in the iRIP-seq data. (d) KEGG analysis of genes that were enriched in iRIP-seq. (c) Percentage of reads used for peak calling in iRIP-seq. All sites share a conserved TACG motif that is identical to the four nucleotide sequence recognized by TadA. (b) Conserved motif in A-to-I edited sites based on RNA-seq.
Chromatograms show edited sites in gDNA and cDNA, and the percentage of edited sequence as determined by pyrosequencing is indicated.
Samples were exposed to 0.1 mM H 2O 2 and then subjected to Sanger sequencing. PCR fragments were obtained from genomic DNA or cDNA (mRNA) using primers described in Methods. (a) Sequence analysis of DNA (gDNA) and/or cDNA in edited regions of wild-type (WT), ΔtadA, and tadA oe. Analysis of the fliC (S128P) A-to-I RNA editing event by Sanger sequencing, RNA-seq and iRIP-seq.
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