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Bacteriophytochromes from Pseudomonas syringae pv. tomato DC3000 modulate the early stages of plant colonization during bacterial speck disease

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Living organisms have evolved the ability to perceive and respond to light of different wavelengths within the visible spectrum by the generation of photoreceptor proteins. Recent studies revealed the participation of these proteins in the virulence of plant pathogenic bacteria. Pseudomonas syringae pv. tomato DC3000 (Pto) is responsible for the bacterial speck, which affects tomato crops. Pto genome contains two genes encoding red/far-red light photoreceptors (BphP1: PSPTO_1902 and BphP2: PSPTO_2652). This work demonstrates the participation of Pto phytochromes and light in the bacterial physiology and during the interaction with tomato plants. We found that Pto phytochromes are implicated in the control of some features related with the bacteria capability to enter into the plant apoplast and cause bacterial speck disease, such as motility, biofilm formation, adhesion and emulsification capability. Red light and bacteriophytochromes are important during the early colonization stage of tomato phyllosphere, affecting Pto virulence. In addition, the development of disease symptoms in infiltrated leaflets is affected by light, which may be the consequence of type-two secretion system regulation.

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Acknowledgments

We thank, Rodrigo Vena (IBR-CONICET) for microscopic analyses, the Statistics Department (Cátedra de Estadística Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario) for their assistance in the statistical analysis of the data and the staff from the English Department of the Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR) for the language correction of the manuscript. The anti-flagellin antibodies were kindly provided by the laboratory of Dr. Eleonora García Véscovi.

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Correspondence to Elena G. Orellano.

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Electronic supplementary material

Fig. S1
figure8

Adhesion and biofilm formation by P. syringae pv. tomato DC3000 exposed to white light, red light and dark conditions. (A) Representative photograph of Pto WT, bphP1::Spr, cbphP1::Spr, bphP2::Gnr and cbphP2::Gnr adhered to the abaxial leaf surface and stained with Crystal violet dye. KB medium was taken as control. The order of inoculation is indicated at the left side of the panel. Dashed lines in the leaves indicate the inoculated area. (B) Representative photographs of biofilm formation assay. Pto strains were statically grown on glass flasks for 24 h at 28 °C. Biofilm formation levels on the air-liquid-glass interface were determined by Crystal violet staining. (D) Representative photographs of green fluorescent protein (GFP)-labeled bacteria were visualized under confocal laser scanning microscopy (CLSM) after three days of bacterial growth. Scale bars 50 μm. (PNG 2087 kb)

Fig S2
figure9

Biosurfactant production of P. syringae pv. tomato DC3000 exposed to white light, red light and dark conditions. Representative photograph of Pto WT, bphP1::Spr, cbphP1::Spr, bphP2::Gnr and cbphP2::Gnr biosurfactant production. (PNG 1948 kb)

Fig. S3
figure10

Effect of phytochrome genes disruption of P. syringae pv. tomato DC3000 and light on the epiphytic fitness. (A) Representative photograph of disease symptoms on tomato leaflets inoculated by dipping with Pto WT, bphP1::Spr, cbphP1::Spr, bphP2::Gnr and cbphP2::Gnr strains and exposed to white light, red light and darkness. (PNG 1130 kb)

Fig. S4
figure11

Bacterial growth curves in KB liquid medium. Growth curves of Pto WT, bphP1::Spr, cbphP1::Spr, bphP2::Gnr and cbphP2::Gnr strains in fresh KB medium considering the optical density at 600 nm (OD600) under (A) white light, (B) red light and (C) darkness, and the CFU/mL under (D) white light, (E) red light and (F) darkness, as a function of time. Data are represented as the mean ± standard error of two independent experiments. (PNG 407 kb)

Fig. S5
figure12

Expression of bacteriophytochrome genes in WT and complemented strains. (A) Amplified products of the phytochrome genes by semiquantitative RT-PCR using RNA preparation from WT and complemented strains. As a control for constitutive bacterial expression a fragment of 16S rRNA was simultaneously amplified. (PNG 45 kb)

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Appendix S1

. Statistical analysis for Figs. 1B, 5B and 6B. (DOCX 16 kb)

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Moyano, L., Carrau, A., Petrocelli, S. et al. Bacteriophytochromes from Pseudomonas syringae pv. tomato DC3000 modulate the early stages of plant colonization during bacterial speck disease. Eur J Plant Pathol (2020) doi:10.1007/s10658-019-01918-5

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Keywords

  • Bacteriophytochromes
  • Light
  • Virulence
  • Pseudomonas syringae
  • Plant colonization
  • Bacterial speck disease