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

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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.

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Relative gene expression analysis of key genes correlated to TTSS and QS of Psn23.Relative mRNA levels of hrpA, hrpL, hrpV, hrpRS, rpoN, lon, psnI, psnR genes of Psn23, grown in MM supplemented with the polyphenolic extracts TV, VN, FO or FC compared to levels in MM alone (untreated). The data are expressed as the average of three replicates ± SD. Asterisks indicate significant differences compared with the untreated sample at P <0.05.
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pone.0163357.g002: Relative gene expression analysis of key genes correlated to TTSS and QS of Psn23.Relative mRNA levels of hrpA, hrpL, hrpV, hrpRS, rpoN, lon, psnI, psnR genes of Psn23, grown in MM supplemented with the polyphenolic extracts TV, VN, FO or FC compared to levels in MM alone (untreated). The data are expressed as the average of three replicates ± SD. Asterisks indicate significant differences compared with the untreated sample at P <0.05.

Mentions: To determine the effect of these polyphenolic extracts on the modulation of TTSS and QS pathways of P. savastanoi pv. nerii, we performed a gene expression analysis by real-time PCR. The whole sequence and organization of the TTSS cluster of Psn23 has been previously reported [29], and the QS genomic organization is available as well (GenBank Accession Number FR717654). To induce in vitro the expression of genes related to the TTSS, Psn23 was grown on MM, which is considered to mimic plant apoplastic conditions [41]. This medium was supplemented with 100 μM of VN, TV, FO or FC. The expression of the gene hrpA, coding for the main component of TTSS pilus, was evaluated as well as that of several genes known to be involved in TTSS regulation, such as hrpL, hrpV, hrpRS, rpoN and lon [42]. As shown in Fig 2, all the polyphenolic extracts here examined strongly reduce hrpA mRNA levels.


Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of Pseudomonas savastanoi by Polyphenols Extracts from Vegetable Residues
Relative gene expression analysis of key genes correlated to TTSS and QS of Psn23.Relative mRNA levels of hrpA, hrpL, hrpV, hrpRS, rpoN, lon, psnI, psnR genes of Psn23, grown in MM supplemented with the polyphenolic extracts TV, VN, FO or FC compared to levels in MM alone (untreated). The data are expressed as the average of three replicates ± SD. Asterisks indicate significant differences compared with the untreated sample at P <0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163357.g002: Relative gene expression analysis of key genes correlated to TTSS and QS of Psn23.Relative mRNA levels of hrpA, hrpL, hrpV, hrpRS, rpoN, lon, psnI, psnR genes of Psn23, grown in MM supplemented with the polyphenolic extracts TV, VN, FO or FC compared to levels in MM alone (untreated). The data are expressed as the average of three replicates ± SD. Asterisks indicate significant differences compared with the untreated sample at P <0.05.
Mentions: To determine the effect of these polyphenolic extracts on the modulation of TTSS and QS pathways of P. savastanoi pv. nerii, we performed a gene expression analysis by real-time PCR. The whole sequence and organization of the TTSS cluster of Psn23 has been previously reported [29], and the QS genomic organization is available as well (GenBank Accession Number FR717654). To induce in vitro the expression of genes related to the TTSS, Psn23 was grown on MM, which is considered to mimic plant apoplastic conditions [41]. This medium was supplemented with 100 μM of VN, TV, FO or FC. The expression of the gene hrpA, coding for the main component of TTSS pilus, was evaluated as well as that of several genes known to be involved in TTSS regulation, such as hrpL, hrpV, hrpRS, rpoN and lon [42]. As shown in Fig 2, all the polyphenolic extracts here examined strongly reduce hrpA mRNA levels.

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