Functional characterization of a global virulence regulator Hfq and identification of Hfq-dependent sRNAs in the plant pathogen Pantoea ananatis

Shin, Giyoon; Schachterle, Jeffrey K.; Jeffrey K. Schachterle; Moleleki, Lucy Novungayo; Coutinho, Teresa A.; Sundin, George W.

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Functional characterization of a global virulence regulator Hfq and identification of Hfq-dependent sRNAs in the plant pathogen Pantoea ananatis

Shin, Giyoon; Schachterle, Jeffrey K.; Jeffrey K. Schachterle; Moleleki, Lucy Novungayo; Coutinho, Teresa A.; Sundin, George W.

URI: http://hdl.handle.net/2263/75783

Date: 2019-09-11

Abstract:

To successfully infect plant hosts, the collective regulation of virulence factors in a bacterial pathogen is crucial. Hfq is an RNA chaperone protein that facilitates the small RNA (sRNA) regulation of global gene expression at the post-transcriptional level. In this study, the functional role of Hfq in a broad host range phytopathogen Pantoea ananatis was determined. Inactivation of the hfq gene in P. ananatis LMG 2665T resulted in the loss of pathogenicity and motility. In addition, there was a significant reduction of quorum sensing signal molecule acyl-homoserine lactone (AHL) production and biofilm formation. Differential sRNA expression analysis between the hfq mutant and wild-type strains of P. ananatis revealed 276 sRNAs affected in their abundance by the loss of hfq at low (OD600 = 0.2) and high cell (OD600 = 0.6) densities. Further analysis identified 25 Hfq-dependent sRNAs, all showing a predicted Rho-independent terminator of transcription and mapping within intergenic regions of the P. ananatis genome. These included known sRNAs such as ArcZ, FnrS, GlmZ, RprA, RyeB, RyhB, RyhB2, Spot42, and SsrA, and 16 novel P. ananatis sRNAs. The current study demonstrated that Hfq is an important component of the collective regulation of virulence factors and sets a foundation for understanding Hfq-sRNA mediated regulation in the phytopathogen P. ananatis.

Description:

Figure S1 : Southern blot validation of hfq knock-out mutation in Pantoea ananatis. Genomic DNA of the wild-type (WT) and hfq mutant (1hfq) strains of P. ananatis LMG 2665T digested with EcoRI and HindIII restriction enzymes was hybridized to a DIG-labeled probe (a partial amplicon of kanamycin resistance gene). Positive detection of the antibiotic marker was observed in the 1hfq strains of P. ananatis LMG 2665T (lanes 2–8). WT of P. ananatis LMG 2665T DNA was used as a negative control (lane 1) whereas unlabeled probe was used as a positive control (lane 9).

Figure S2 : Colony PCR verification of hfq knock-out mutation in Pantoea ananatis. A colony PCR confirmation of insertion of kanamycin resistance gene in the hfq gene region using Test primers (Table 2) hfq mutant (1hfq) strains of P. ananatis LMG 2665T. L represents a molecular ladder and the sizes of its prominent bands 1, 3, and 6 kilo basepairs (kb) are indicated below. A wild-type (WT) colony of P. ananatis LMG 2665T was used as a negative control (lane 1; 500 bp). Insertion of kanamycin resistance marker is shown in colony PCRs of hfq mutant (1hfq) strains of P. ananatis LMG 2665T (lanes 2, 3, and 4; 1.5 kb).

Figure S3 : In vitro growth assay. Growths of wild-type (WT), hfq mutant (1hfq), and hfq complementing (1hfq pBBR1MCS::hfq) strains of Pantoea ananatis LMG 2665T in LB broth at 28 C. The growth was monitored for 20 h at optical density 600 nm (OD600) and the mean OD600 readings of the three replicates for each P. ananatis LMG 2665T strains were plotted. Solid line (yellow) represents WT, dashed line (purple) 1hfq, and dotted line (green) 1hfq pBBR1MCS::hfq. Asterisks denote significance differences (P < 0.05) in the absorbance of 1hfq relative to WT P. ananatis LMG 2665T.

Figure S4 : In planta growth assay. (A) Disease progression in onion scales inoculated with wild-type (WT), hfq mutant (1hfq), and hfq complementing [1hfq (pBBR1MCS::hfq)] strains of P. ananatis LMG 2665T, and incubated for 5 days post inoculation (dpi). (B) In planta populations of WT, 1hfq, and 1hfq (pBBR1MCS::hfq) strains of P. ananatis LMG 2665T in onion scales measured for 5 dpi. The mean CFUs of three replicates for each strain from two independent experiments were plotted. Solid line (yellow) represents WT, dashed line (purple) 1hfq, and dotted line (green) 1hfq (pBBR1MCS::hfq).

Figure S5 : Logarithmic plot of the number of putative small RNAs (sRNAs) identified in Pantoea ananatis LMG 2665T (pPAR sRNA) as a function of the threshold selected for calling sRNAs. This was generated by calling putative sRNAs across a range of thresholds using the custom script (see Supplementary Data Sheet S1 in the section “peak_ID.py”).

Figure S6 : In silico prediction of selected Pantoea ananatis sRNAs (pPAR sRNA) secondary structure. Secondary structures of P. ananatis LMG 2665T sRNAs (A) FnrS, (B) GlmZ, (C) pPAR 237, (D) pPAR 238, and (E) pPAR 395 were predicted based on a minimum free energy model provided by RNAfold (http://rna.tbi.univie.ac.at).

Figure S7 : Putative interaction of pPAR237 and pPAR238 to eanIR in Pantoea ananatis LMG 2665T. (A) Location of pPAR237 and pPAR238. In silico predicted interaction of pPAR237 (red) to eanIR (black): (B) eanI upstream sequence (energy: 8.62323 kcal/mol; hybridization energy: 23.5). (C) eanR coding sequence (energy: 13.63700 kcal/mol, hybridization energy: 39.4) and (D) in silico predicted interaction of pPAR238 (red) to eanI (black) upstream sequence (energy: 7.83954 kcal/mol, hybridization energy: 12.0).

Table S1 : Summary of sRNA sequencing reads obtained and filtered for use in sRNA identification.

Table S2 : A list of sRNAs identified, their genomic coordinates, sequences, and selected characteristics.

Table S3 : A list of sRNAs that has significant abundance difference between WT and hfq mutant strains of Pantoea ananatis.

Table S4 : A list of predicted targets of selected sRNAs.

Data Sheet S1 : A custom phython script compiled for bioinformatic analyses of sRNA sequencing data.

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