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. 2022 Jun;23(6):870-884.
doi: 10.1111/mpp.13200. Epub 2022 Mar 7.

Cyclic di-GMP modulates sessile-motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors

Yufan Chen  1   2 , Mingfa Lv  1   2 , Zhibin Liang  1   2 , Zhiqing Liu  1   2 , Jianuan Zhou  1   2 , Lian-Hui Zhang  1   2
Affiliations

Cyclic di-GMP modulates sessile-motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors

Yufan Chen et al. Mol Plant Pathol. 2022 Jun.

Abstract

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice-growing countries. We showed recently that the universal bacterial second messenger c-di-GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c-di-GMP receptor domain, known as the PilZ-domain. By deleting all the genes encoding c-di-GMP-degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c-di-GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild-type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c-di-GMP, which together play a critical role in regulating the c-di-GMP-associated functionality. The findings from this study fill a gap in our understanding of how c-di-GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.

Keywords: Dickeya oryzae; PilZ domain receptor; c-di-GMP; sessile-motile transition; virulence.

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Conflict of interest statement

Authors declare that there are no known conflicts of interest associated with this paper.

Figures

FIGURE 1
FIGURE 1
Domain structure and sequence alignment of the PilZ domains in Dickeya oryzae EC1. (a) Predicted domain architectures of BcsA and YcgR. Domains of the proteins were predicted using the simplified modular architecture research tool (SMART) and represented using DOG 2.0 software (Ren et al., 2009). TM, predicted transmembrane domain; Glyco_tranf_, this domain is found in several members of glycosyl transferases that transfer the sugar moiety from UDP‐glucose; Cellulose_synt, cellulose biosynthetic process protein; YcgR, involved in the flagellar motor function and a member of the flagellar regulon family. (b) Conserved residues implicated in c‐di‐GMP binding function of the PilZ domain are shown with red and black backgrounds, indicating ≥75% and 100% homology, respectively. Asterisks indicate conserved motifs and residues that are essential for c‐di‐GMP binding: an arginine‐rich RxxxR motif and a (D/N)xSxxG motif, where x represents any residue. The reference YcgR and BcsA homolog sequences used in the alignment are EC_YCGR (UniProt entry P76010) and EC_BCSA (UniProt entry P37653) from Escherichia coli, PA4608 (UniProt entry Q9HVI1) and PA3353 (UniProt entry Q9HYP3) from Pseudomonas aeruginosa PAO1, VC0395_0091 (UniProt entry A0A0H3ADR8) from Vibrio cholerae, XC2317 (UniProt entry A0A0H2X9P4) from Xanthomonas campestris pv. campestris, STM1798 (UniProt entry Q8ZP19) from Salmonella typhimurium, and CC_1509 (UniProt entry Q9A7X0) from Caulobacter crescentus
FIGURE 2
FIGURE 2
Motile and sessile phenotypes of Dickeya oryzae EC1 and its derivatives. (a) Swimming motility. (b) Swarming motility. (c) Biofilm formation in SOBS medium. (d) Quantification of cellulose expression level using calcofluor white stain. Dotted lines indicate the level of the wild type (WT). Experiments were performed in triplicate. The same lowercase letters above the bars indicate no significant difference and different letters indicate significant differences (p < 0.05 as determined by one‐way analysis of variance with multiple comparison test)
FIGURE 3
FIGURE 3
Motile and sessile phenotypes of Dickeya oryzae EC1 and derivatives. (a) Swimming motility. (b) Swarming motility. (c) Biofilm formation in SOBS medium. (d) Quantification of cellulose expression using calcofluor white stain. Dotted lines indicate the level of the wild type (WT). Experiments were performed in triplicate. The same lowercase letters indicate no significant difference and different letters indicate significant differences (p < 0.05 as determined by one‐way analysis of variance with multiple comparison test)
FIGURE 4
FIGURE 4
Motile and sessile phenotypes of Dickeya oryzae EC1 and its derivatives. (a) Swimming motility. (b) Swarming motility. (c) Biofilm formation in SOBS medium. (d) Quantification of cellulose expression using calcofluor white stain. Dotted lines indicate the level of the wild type (WT). Experiments were performed in triplicates. The same lowercase letters indicate samples with no significant difference and different letters indicate significant differences (p < 0.05 as determined by one‐way analysis of variance with multiple comparison test)
FIGURE 5
FIGURE 5
Rice seed germination and bacterial invasion assays of Dickeya oryzae EC1 and its derivatives. (a) Virulence assay on rice seed germination. Five concentrations of bacterial cultures were co‐cultured with 15 rice seeds at room temperature for 6 h before washing with sterile water. Rice seeds were grown at 28°C under 16‐h light and 8‐h dark for 1 week. Inhibition rate of mutant strains at each inoculum concentration was compared with each other by two‐way analysis of variance with multiple comparisons (see Table S3 for details). (b) Rice seeds were infiltrated with EC1, 7ΔPDE, 7ΔPDEΔycgR, 7ΔPDEΔbcsA, and 7ΔPDEΔycgRΔbcsA carrying GFP (green fluorescent protein) plasmid for 6 h and transferred to moistened filter papers at 28°C for 40 h before fluorescence microscopy observation. Green and red fluorescence photographs were merged using LAS‐X software (Leica). The RFP channel was used to visualize the rice seed based on the autofluorescent properties of plant cells, which distinguished plant tissues from the bacterial cells expressing GFP fluorescence. Bars = 500 μm. (c) Bacterial cfu counting after inoculation. Experiments were performed at least twice and the wild‐type EC1 value was used as control. ns, p > 0.05; ***p < 0.001 as determined by one‐way analysis of variance with multiple comparison test
FIGURE 6
FIGURE 6
Isothermal titration calorimetry (ITC) analysis of YcgR, YcgRR124D, BcsA(PilZ) and BcsA(PilZ)R556D with c‐di‐GMP. (a) Binding affinity of 10 μM YcgR to 250 μM c‐di‐GMP. Upper panels and lower panels indicate raw data and integrated heat values, respectively. (b) Binding affinity of 24 μM YcgRR124D to 600 μM c‐di‐GMP. (c) Binding affinity of 10 μM BcsA(PilZ) to 500 μM c‐di‐GMP. (d) Binding affinity of 10 μM BcsA(PilZ)R556D to 500 μM c‐di‐GMP. The experiment was repeated at least twice with similar results
FIGURE 7
FIGURE 7
Swimming motility and seed germination inhibition ratio of Dickeya oryzae EC1 (WT) and its derivatives. (a) Swimming motility. (b) Seed germination inhibition ratio. Experiments were performed at least twice. The same lowercase letters indicate samples with no significant difference and different letters indicate significant differences (p < 0.05 as determined by one‐way analysis of variance with multiple comparison test)
FIGURE 8
FIGURE 8
A schematic diagram of the regulatory network of two PilZ‐type c‐di‐GMP receptors in Dickeya oryzae EC1. An increase in intracellular c‐di‐GMP concentration promotes binding of cytoplasmic protein YcgR to c‐di‐GMP through its PilZ domain, thus regulating bacterial motility and attachment‐related phenotypes. BcsA, involved in bacterial cellulose synthesis, binds to c‐di‐GMP through its PilZ domain to modulate bacterial cellulose and biofilm production
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References

    1. Alm, R.A. , Bodero, A.J. , Free, P.D. & Mattick, J.S. (1996) Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa . Journal of Bacteriology, 178, 46–53. - PMC - PubMed
    1. Amikam, D. & Galperin, M.Y. (2006) PilZ domain is part of the bacterial c‐di‐GMP binding protein. Bioinformatics, 22, 3–6. - PubMed
    1. Baraquet, C. , Murakami, K. , Parsek, M.R. & Harwood, C.S. (2012) The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c‐di‐GMP. Nucleic Acids Research, 40, 7207–7218. - PMC - PubMed
    1. Campoccia, D. , Montanaro, L. & Arciola, C.R. (2021) Extracellular DNA (eDNA). A major ubiquitous element of the bacterial biofilm architecture. International Journal of Molecular Sciences, 22, 9100. - PMC - PubMed
    1. Cheang, Q.W. , Xin, L. , Chea, R.Y.F. & Liang, Z.X. (2019) Emerging paradigms for PilZ domain‐mediated c‐di‐GMP signaling. Biochemical Society Transactions, 47, 381–388. - PubMed

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