The integration host factor regulates multiple virulence pathways in bacterial pathogen Dickeya zeae MS2

Abstract

Dickeya zeae is an aggressive bacterial phytopathogen that infects a wide range of host plants. It has been reported that integration host factor (IHF), a nucleoid-associated protein consisting of IHFα and IHFβ subunits, regulates gene expression by influencing nucleoid structure and DNA bending. To define the role of IHF in the pathogenesis of D. zeae MS2, we deleted either and both of the IHF subunit encoding genes ihfA and ihfB, which significantly reduced the production of cell wall-degrading enzymes (CWDEs), an unknown novel phytotoxin and the virulence factor-modulating (VFM) quorum-sensing (QS) signal, cell motility, biofilm formation, and thereafter the infection ability towards both potato slices and banana seedlings. To characterize the regulatory pathways of IHF protein associated with virulence, IHF binding sites (consensus sequence 5'-WATCAANNNNTTR-3') were predicted and 272 binding sites were found throughout the genome. The expression of 110 tested genes was affected by IHF. Electrophoretic mobility shift assay (EMSA) showed direct interaction of IhfA protein with the promoters of vfmE, speA, pipR, fis, slyA, prtD, hrpL, hecB, hcp, indA, hdaA, flhD, pilT, gcpJ, arcA, arcB, and lysR. This study clarified the contribution of IHF in the pathogenic process of D. zeae by controlling the production of VFM and putrescine QS signals, phytotoxin, and indigoidine, the luxR-solo system, Fis, SlyA, and FlhD transcriptional regulators, and secretion systems from type I to type VI. Characterization of the regulatory networks of IHF in D. zeae provides a target for prevention and control of plant soft rot disease.

Keywords: Dickeya zeae; DNA binding; cell motility; cell wall-degrading enzymes; integration host factor; transcriptional regulator; virulence.

FIGURE 1

Electrophoretic mobility shift assay of IhfA protein with predicted gene promoters. 1 nM of labelled probe of C1O30_RS12170, vfmE, speA, speC, pipR, fis, slyA, prtD, gspC, hrpL, hecB, hcp, indA, hdaA, flhD, pilT, gcpJ, arcA, arcB, and lysR genes was incubated with 0–5 μM IhfA. 100 nM unlabelled corresponding probes were used as the specific competitors before adding 1 nM of the labelled probes in different reactions. The positions of free DNA and of IhfA–DNA complexes are shown

FIGURE 2

IHF regulates the production of VFM rather than the AHL quorum‐sensing (QS) signal. (a) AHL signal diffusion assay of Dickeya zeae MS2, ∆ihfA, and ∆ihfB. Strains were spotted on the top of the agar strips and Agrobacterium tumefaciens CF11 (tra‐lacZ) was used as a biosensor for the AHL QS signal (Hussain et al., 2008). (b) VFM QS signal detection of D. zeae MS2, ∆ihfA, ∆ihfB, ∆ihfAB, and complemented strains ∆ihfA::ihfA and ∆ihfB::ihfB. Different concentrations of bacterial supernatants were co‐cultured with the reporter strain in medium supplemented with X‐Gal at 30°C with 200 rpm for 12 h (Lv et al., 2019). CK, negative control. Experiments were repeated three times in triplicate

FIGURE 3

IHF regulates the production of cell wall‐degrading enzymes and phytotoxin. (a) IHF contributes to cell wall degrading enzymatic activities. (b) IHF significantly modulates phytotoxin production of Dickeya zeae MS2. The results were averaged over the three replicates and the error bars represent standard deviations. *p < 0.05, ***p < 0.0001; ns, not significant (Student's t test) (n = 3 independent experiments)

FIGURE 4

IHF regulates cell motility and biofilm formation of Dickeya zeae MS2. (a) Swarming, swimming, and twitching motilities of MS2 and its derivatives. (b) Nonadherent biofilms of MS2 and its derivatives formed at the air/liquid interface. (c) Attached biofilms of MS2 and its derivatives measured by crystal violet staining. The experimental results were averaged over the three replicates and the error bars represent standard deviations (SD). *p < 0.05, **p < 0.001, ***p < 0.0001; ns, not significant (Student's t test) (n = 3 independent experiments)

FIGURE 5

Deletion of ihf genes and gcpJ decreases c‐di‐GMP concentration in Dickeya zeae MS2. c‐di‐GMP concentrations of mutants and complemented stains were normalized to that of the wild‐type MS2, which was set to 100%. The experimental results were averaged over the three replicates and the error bars represent standard deviations (SD). **p < 0.001, ***p < 0.0001, ns, not significant (Student's t test) (n = 3 independent experiments)

FIGURE 6

Deletion of ihf genes reduces the virulence of Dickeya zeae MS2. (a) Virulence of MS2 and ihf mutants on banana seedlings. Each banana seedling was inoculated with 200 μl of fresh bacterial culture (OD₆₀₀ 1.0) and kept in an incubator at 28°C with 12‐h alternating light–dark cycles for 14 days. Three biological replicates were set for each treatment. The virulence scoring method was described by Feng et al. (2019). (b) Virulence of MS2 and ihf mutants on potato slices. Each potato slice was inoculated with 2 μl of bacterial culture (OD₆₀₀ 1.0) and kept at 28°C under moist conditions. The area of lesions was measured using ImageJ v. 1.52a after 24 h. CK, negative control. ***p < 0.0001; ns, not significant (Student's t test) (n = 3 independent experiments)

FIGURE 7

The regulatory network of IHF in Dickeya zeae MS2. IHF directly binds to the promoter of vfmE and affects the biosynthesis of VFM quorum‐sensing (QS) signal (A), directly interacts with the promoter of speA and indirectly interacts with speC to modulate the intracellular putrescine signal level (B), directly binds to the promoter region of the luxR‐solo homologue pipR (C), regulates the production of cell wall‐degrading enzymes (CWDEs) via multiple regulatory pathways including IHF‐VfmE, IHF‐Fis/IHF‐Fis‐VfmE (D), IHF‐ArcAB‐Fis (E), IHF‐SlyA (F), IHF‐PrtD (T1SS)/IHF‐GspC (type II secretion system) (G), directly binds to the indA promoter to regulate production of indigoidine (H), regulates the production of phytotoxin by direct binding to the hdaA promoter (I), directly binds the hrpL promoter and acts as an enhancer to activate hrpL expression, thus affecting type III secretion system effector secretion to the extracellular environment (J), and directly binds to hecB and hcp (k). IHF regulates cell motility and biofilm formation by directly binding to the flhD promoter (L), the c‐di‐GMP encoding gene gcpJ, and the pilT (M)