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Exposure to Umbelliferone Reduces Ralstonia solanacearum Biofilm Formation, Transcription of Type III Secretion System Regulators and Effectors and Virulence on Tobacco.

ABSTRACT: Ralstonia solanacearum is one of the most devastating phytopathogens and causes bacterial wilt, which leads to severe economic loss due to its worldwide distribution and broad host range. Certain plant-derived compounds (PDCs) can impair bacterial virulence by suppressing pathogenic factors of R. solanacearum. However, the inhibitory mechanisms of PDCs in bacterial virulence remain largely unknown. In this study, we screened a library of coumarins and derivatives, natural PDCs with fused benzene and ?-pyrone rings, for their effects on expression of the type III secretion system (T3SS) of R. solanacearum. Here, we show that umbelliferone (UM), a 7-hydroxycoumarin, suppressed T3SS regulator gene expression through HrpG-HrpB and PrhG-HrpB pathways. UM decreased gene expression of six type III effectors (RipX, RipD, RipP1, RipR, RipTAL, and RipW) of 10 representative effector genes but did not alter T2SS expression. In addition, biofilm formation of R. solanacearum was significantly reduced by UM, though swimming activity was not affected. We then observed that UM suppressed the wilting disease process by reducing colonization and proliferation in tobacco roots and stems. In summary, the findings reveal that UM may serve as a plant-derived inhibitor to manipulate R. solanacearum T3SS and biofilm formation, providing proof of concept that these key virulence factors are potential targets for the integrated control of bacterial wilt.

SUBMITTER: Yang L

PROVIDER: S-EPMC5492427 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Exposure to Umbelliferone Reduces Ralstonia solanacearum Biofilm Formation, Transcription of Type III Secretion System Regulators and Effectors and Virulence on Tobacco.

Yang Liang L Li Shili S Qin Xiyun X Jiang Gaofei G Chen Juanni J Li Bide B Yao Xiaoyuan X Liang Peibo P Zhang Yong Y Ding Wei W

Frontiers in microbiology 20170630

Ralstonia solanacearum is one of the most devastating phytopathogens and causes bacterial wilt, which leads to severe economic loss due to its worldwide distribution and broad host range. Certain plant-derived compounds (PDCs) can impair bacterial virulence by suppressing pathogenic factors of R. solanacearum. However, the inhibitory mechanisms of PDCs in bacterial virulence remain largely unknown. In this study, we screened a library of coumarins and derivatives, natural PDCs with ...[more]

PMID: 28713361

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Project description:Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found that R. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lacking fliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphA cassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.

Project description:UnlabelledPlants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule.ImportancePlant pathogens such as the bacterial wilt agent Ralstonia solanacearum threaten food and economic security by causing significant losses for small- and large-scale growers of tomato, tobacco, banana, potato, and ornamentals. Like most plants, these crop hosts use salicylic acid (SA) both indirectly as a signal to activate defenses and directly as an antimicrobial chemical. We found that SA inhibits growth of R. solanacearum and induces a general stress response that includes repression of multiple bacterial wilt virulence factors. The ability to degrade SA reduces the pathogen's sensitivity to SA toxicity and increases its virulence on tobacco.

Project description:Ralstonia solanacearum species complex (RSSC) posses extremely abundant type III effectors (T3Es) that are translocated into plant cells via a syringe-like apparatus assembled by a type III secretion system (T3SS) to subvert host defense initiated by innate immunity. More than 100 T3Es are predicted among different RSSC strains, with an average of about 70 T3Es in each strain. Among them, 32 T3Es are found to be conserved among the RSSC and hence called the core T3Es. Here, we genetically characterized contribution of abundant T3Es to virulence of a Japanese RSSC strain OE1-1 toward host plants. While all the T3Es members of AWR family contributed slightly to virulence, those of the GALA, HLK, and SKWP families did not influence full virulence of OE1-1. Mutant OE1-1D21E (with deletion of all 21 T3Es members of four families) exhibited slightly impaired virulence, while mutant OE1-1D36E (deleting all 21 T3Es of 4 families and 15 core T3Es) exhibited substantially reduced virulence. Mutant OE1-1D42E (deleting all 21 T3Es of 4 families, 15 core T3Es and 6 extended core T3Es) failed to cause any disease on tobacco plants with leaf infiltration but retained faint virulence on tobacco plants with petiole inoculation. The proliferation of mutant OE1-1D42E in tobacco stems was substantially impaired with about three orders of magnitude less than that of OE1-1, while no impact in tobacco leaves if directly infiltrated into leaves. On the contrary, the OE1-1D42E mutant retained faint virulence on eggplants with leaf infiltration but completely lost virulence on eggplants with root-cutting inoculation. The proliferation of OE1-1D42E mutant both in eggplant leaves and stems was substantially impaired. Intriguingly, mutant OE1-1D42E still caused necrotic lesions in tobacco and eggplant leaves, indicating that some other than the 42 removed effectors are involved in expansion of necrotic lesions in host leaves. All taken together, we here genetically demonstrated that all the core and extended core T3Es are nearly crucial for virulence of OE1-1 toward host plants and provided currently a kind of T3Es-free strain that enables primary functional studies of individual T3Es in host cells.

Dataset Information

Exposure to Umbelliferone Reduces Ralstonia solanacearum Biofilm Formation, Transcription of Type III Secretion System Regulators and Effectors and Virulence on Tobacco.

Publications

Exposure to Umbelliferone Reduces <i>Ralstonia solanacearum</i> Biofilm Formation, Transcription of Type III Secretion System Regulators and Effectors and Virulence on Tobacco.

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