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Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of Pseudomonas savastanoi by Polyphenols Extracts from Vegetable Residues

View Article: PubMed Central - PubMed

ABSTRACT

Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as Olea europaea, Cynara scolymus, and Vitis vinifera were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. nerii strain Psn23 was assessed. Extract from green tea (Camellia sinensis) was used as a positive control. Collectively, the data obtained through gfp-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of Psn23, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed in planta by a strong reduction in the ability of Psn23 to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca2+-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.

No MeSH data available.


Related in: MedlinePlus

ELISA assay on Psn23 bacterial supernatant amended with polyphenolic extracts.Quantification of HrpA protein by ELISA assay on bacterial supernatant of wild type Psn23 grown on MM, or on MM amended with the polyphenolic extracts VN, TV, FO, or FC. As a negative control the ∆hrpA mutant was used. As a reference for quantification, a standard curve was established by a serial dilution of the Psn23 HrpA recombinant protein (117 pg/ml– 40 ng/ml). The data represent the means ± SD of three replicates. Statistically significant differences are represented by different letters above the bars (ANOVA and Tukey’s test, P < 0.05).
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pone.0163357.g004: ELISA assay on Psn23 bacterial supernatant amended with polyphenolic extracts.Quantification of HrpA protein by ELISA assay on bacterial supernatant of wild type Psn23 grown on MM, or on MM amended with the polyphenolic extracts VN, TV, FO, or FC. As a negative control the ∆hrpA mutant was used. As a reference for quantification, a standard curve was established by a serial dilution of the Psn23 HrpA recombinant protein (117 pg/ml– 40 ng/ml). The data represent the means ± SD of three replicates. Statistically significant differences are represented by different letters above the bars (ANOVA and Tukey’s test, P < 0.05).

Mentions: To confirm and directly verify these findings, the amount of HrpA produced by Psn23 in the supernatant was also quantified by ELISA, following or not treatment with the VN, TV, FO or FC extracts. The results obtained are consistent with those from Congo red assay. In particular, in the supernatants of untreated Psn23 cells a concentration of 33.93 ng/ml of HrpA protein was detected (Fig 4). In contrast, when TV or VN were supplemented to MM, the HrpA concentrations in the supernatants were 5.58 and 3.68 ng/ml, respectively, thus comparable to the levels detected for the ∆hrpA mutant (1.85 ng/ml). Following the treatment with FO or FC, HrpA concentrations of 10.87 and 21.30 ng/ml, respectively, were found (Fig 4). In conclusion, these findings demonstrate that the polyphenolic extracts obtained and tested in our work are able to interfere in a very specific manner with TTSS, as previously indicated by the results on gene expression analysis of this master pathogenicity system.


Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of Pseudomonas savastanoi by Polyphenols Extracts from Vegetable Residues
ELISA assay on Psn23 bacterial supernatant amended with polyphenolic extracts.Quantification of HrpA protein by ELISA assay on bacterial supernatant of wild type Psn23 grown on MM, or on MM amended with the polyphenolic extracts VN, TV, FO, or FC. As a negative control the ∆hrpA mutant was used. As a reference for quantification, a standard curve was established by a serial dilution of the Psn23 HrpA recombinant protein (117 pg/ml– 40 ng/ml). The data represent the means ± SD of three replicates. Statistically significant differences are represented by different letters above the bars (ANOVA and Tukey’s test, P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036890&req=5

pone.0163357.g004: ELISA assay on Psn23 bacterial supernatant amended with polyphenolic extracts.Quantification of HrpA protein by ELISA assay on bacterial supernatant of wild type Psn23 grown on MM, or on MM amended with the polyphenolic extracts VN, TV, FO, or FC. As a negative control the ∆hrpA mutant was used. As a reference for quantification, a standard curve was established by a serial dilution of the Psn23 HrpA recombinant protein (117 pg/ml– 40 ng/ml). The data represent the means ± SD of three replicates. Statistically significant differences are represented by different letters above the bars (ANOVA and Tukey’s test, P < 0.05).
Mentions: To confirm and directly verify these findings, the amount of HrpA produced by Psn23 in the supernatant was also quantified by ELISA, following or not treatment with the VN, TV, FO or FC extracts. The results obtained are consistent with those from Congo red assay. In particular, in the supernatants of untreated Psn23 cells a concentration of 33.93 ng/ml of HrpA protein was detected (Fig 4). In contrast, when TV or VN were supplemented to MM, the HrpA concentrations in the supernatants were 5.58 and 3.68 ng/ml, respectively, thus comparable to the levels detected for the ∆hrpA mutant (1.85 ng/ml). Following the treatment with FO or FC, HrpA concentrations of 10.87 and 21.30 ng/ml, respectively, were found (Fig 4). In conclusion, these findings demonstrate that the polyphenolic extracts obtained and tested in our work are able to interfere in a very specific manner with TTSS, as previously indicated by the results on gene expression analysis of this master pathogenicity system.

View Article: PubMed Central - PubMed

ABSTRACT

Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as Olea europaea, Cynara scolymus, and Vitis vinifera were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. nerii strain Psn23 was assessed. Extract from green tea (Camellia sinensis) was used as a positive control. Collectively, the data obtained through gfp-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of Psn23, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed in planta by a strong reduction in the ability of Psn23 to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca2+-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.

No MeSH data available.


Related in: MedlinePlus