Pseudomonas bacteriocin syringacin M released upon desiccation suppresses the growth of sensitive bacteria in plant necrotic lesions

Abstract

Bacteriocins are regarded as important factors mediating microbial interactions, but their exact role in community ecology largely remains to be elucidated. Here, we report the characterization of a mutant strain, derived from Pseudomonas syringae pv. tomato DC3000 (Pst), that was incapable of growing in plant extracts and causing disease. Results showed that deficiency in a previously unannotated gene saxE led to the sensitivity of the mutant to Ca²⁺ in leaf extracts. Transposon insertions in the bacteriocin gene syrM, adjacent to saxE, fully rescued the bacterial virulence and growth of the ΔsaxE mutant in plant extracts, indicating that syrM-saxE encode a pair of bacteriocin immunity proteins in Pst. To investigate whether the syrM-saxE system conferred any advantage to Pst in competition with other SyrM-sensitive pathovars, we compared the growth of a SyrM-sensitive strain co-inoculated with Pst strains with or without the syrM gene and observed a significant syrM-dependent growth reduction of the sensitive bacteria on plate and in lesion tissues upon desiccation-rehydration treatment. These findings reveal an important biological role of SyrM-like bacteriocins and help to understand the complex strategies used by P. syringae in adaptation to the phyllosphere niche in the context of plant disease.

Figure 1

Pst mutant 25D12 was sensitive to plant extracts and unable to cause disease. For the growth in leaf extracts assay, wild‐type Pst, Pst ^{▵sax AB /F} and 25D12 strains were inoculated into the 100% Arabidopsis (A) and tomato (C) leaf extracts at OD ₆₀₀ = 0.001. Samples were collected at 0 and 1 day postinoculation (dpi) for colony counts. For the growth in planta assay, bacterial strains were infiltrated into Arabidopsis (B) and tomato (D) leaves at OD ₆₀₀ = 0.001. Samples were taken at 0 and 3 dpi for colony counts. Arabidopsis (E) and tomato (F) plants were sprayed with the bacteria at OD ₆₀₀ = 0.2, and disease symptoms were photographed 4 days after inoculation. Non‐sprayed plants were used as control. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates statistical significance (t‐test, P < 0.01).

Figure 2

The saxE gene was required for Pst growth in leaf extracts and in planta. A. A schematic diagram of the complementary tests pinpointing the minimal region required for rescuing the growth of 25D12 mutant in plant leaf extracts. Filled arrows indicate genes annotated by the sequencing project marked with locus tags on the top. The filled triangle shows the insertion site of Ω‐Km transposon in mutant 25D12. Genomic DNA fragments downstream the transposon insertion site were tested for the ability to confer tolerance to leaf extracts. B. The minimal region that protected 25D12 in leaf extracts also rescued the growth of the mutant on Arabidopsis plant. Bacterial inocula were infiltrated into Arabidopsis leaves at OD ₆₀₀ = 0.001. Samples were taken at 0 and 3 dpi for colony counts. The saxE orf was required for bacterial virulence on Arabidopsis (C) and tomato (D) plants. Wild‐type Pst, Pst ^▵saxE and the complemented strain Pst ^{▵saxE‐saxEhis} were infiltrated into Arabidopsis (C) and tomato (D) leaves at OD ₆₀₀ = 0.001. Samples were taken at 0 and 3 dpi for colony counts. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference (t‐test, P < 0.01). ns indicates no significant difference (t‐test, P > 0.05).

Figure 3

Inhibitory effect of Ca²⁺ on the growth of strains deficient in the saxE gene. A. Ca²⁺ is the inhibitory factor that suppressed the growth of Pst ^▵saxE. Bacteria were inoculated into the King's B (KB) liquid medium supplemented with 1 mM CaCl₂ or MgCl₂ at OD ₆₀₀ = 0.001. Samples were collected at 0 and 1 dpi for colony counts. B. A schematic diagram of Ω‐Km transposon insertion events that suppressed the Pst ^▵saxE phenotype. Filled triangles indicate the insertion sites that were able to restore the growth of Pst ^▵saxE in the presence of Ca²⁺. The empty and the dashed arrows denote the gene PSPTO_0572 (syrM) and the deleted saxE gene respectively. C. Deletion of syrM abolished the growth inhibitory effect of Ca²⁺ on Pst ^▵saxE. Bacteria were inoculated into the KB liquid medium supplemented with 1 mM CaCl₂ at OD ₆₀₀ = 0.001, and samples were collected at 0 and 1 dpi for colony counts. D. Ca²⁺ and Mg²⁺ enhanced inhibitory activity of bacteriocin SyrM on the sensitive P. syringae pv. lachrymans‐8 (Psl). Purified SyrM (1 μM) was mixed with 100 μl KB liquid cultures supplemented with 1 mM CaCl₂ or MgCl₂ inoculated by Psl at OD ₆₀₀ = 0.0002. Samples were collected at 0 and 16 h postinoculation (hpi) for colony counts. Plain KB medium was used as control. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference (t‐test, P < 0.01). ns indicates no significant difference (t‐test, P > 0.05).

Figure 4

The saxE gene encodes an immunity protein of the bacteriocin SyrM. A. Amino acid sequence and the predicted secondary structure of SaxE protein. The coils above the amino acids denote the helices predicted by TMHMM (http://www.cbs.dtu.dk/services/ TMHMM/). B. Gel blot assay of the SaxE protein. Proteins extracted from Pst ^▵saxE bacteria harbouring the pME6012 expressing wild‐type SaxEhis (WT) or mutated SaxEhis(ins) (mutant) protein were used for blot assay detected with the his‐tag antibody. C, D. Purified SyrM protein inhibited the growth of Psl. Five microlitres of serial‐diluted SyrM protein solutions was applied on a plate overlaid with soft agar inoculated with Psl‐pME6012 (C) or Psl‐pME6012‐saxEhis (D). The plates were incubated at 28°C and photographed 1 day after inoculation. E. SyrM is released upon desiccation treatment. Pst‐pME6012‐syrMhis (OD ₆₀₀ = 0.01) was applied onto filter paper discs placed on KB plates and cultured at 28°C for 16 h. The discs were desiccated for 4 h (+), and non‐treated samples were used as control (−). Bacteria were washed off from the discs and spun, and the resulting supernatant samples, together with the purified SyrM protein, were used for gel blot assay with the his‐tag antibody.

Figure 5

SyrM‐mediated competition between Pst and Psl on plate culture. A. Desiccation treatment reduced the number of viable bacteria on filter paper discs. Wild‐type Pst (OD ₆₀₀ = 0.01) was applied onto filter paper discs placed on plates and cultured for 16 h. The inoculated filter paper discs were desiccated for 4 h. Desiccated and untreated paper discs were collected for bacterial counts. B. Total RNA samples were prepared from bacteria on paper discs collected in (A) and subjected to the quantitative real‐time PCR assay of relative transcript levels of syrM. The recA was used as the reference gene. C, D. SyrM‐mediated competition depended on dehydration and rehydration treatments. The SyrM‐sensitive Psl were mixed at 1:1 ratio with wild‐type Pst (C) and Pst ^▵syrM (D) strains, respectively, and co‐cultured at the same conditions as in (A). After desiccation treatment, the paper discs were rehydrated and cultured to 48 h. Untreated samples were used as controls. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference (t‐test, P < 0.01). * indicates significant difference (t‐test, P < 0.05). ns indicates no significant difference.

Figure 6

SyrM suppressed the rebound of Psl population during the rehydration of desiccated leaf lesions. A. Ca²⁺ released at the late stage of plant–bacteria interaction. Pst was infiltrated into tomato leaves at OD ₆₀₀ = 0.001. Leaves were collected at 0 and 2 dpi for detection of the concentration of free Ca²⁺ in the intercellular fluids. B. Pst infection promoted the growth of Psl on tomato plants. Pst, Psl and a 1:1 mixture of Pst and Psl were infiltrated into tomato leaves at OD ₆₀₀ = 0.0002 respectively. C. Bacterial population in leaf lesions fluctuated widely during a dehydration–rehydration regimen. Pst was infiltrated into tomato leaves at OD ₆₀₀ = 0.001. At 2 dpi, the inoculated leaf lesions were collected and desiccated for 2 day. After dehydration treatment, the desiccated leaf lesions were rehydrated for 2 day. The rehydrated leaf lesions were then treated again as the same desiccation–rehydration treatment. D. syrM is required for Pst to limit the growth of Psl in rehydrated lesion tissues. The 1:1 mixtures of Pst/Psl and Pst ^▵syrM/Psl were infiltrated into tomato leaves respectively. At 2 dpi, the leaf lesions were collected and treated the same as in (C). Lesion samples were taken at 2 and 6 dpi for bacterial counts. The ratio above the columns is means of the ratio between two pathovars in lesion samples. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference (t‐test, P < 0.01). ns indicates no significant difference.