Two Host-Induced Ralstonia solanacearum Genes, acrA and dinF, Encode Multidrug Efflux Pumps and Contribute to Bacterial Wilt Virulence

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10⁷ CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.     

Using the Ralstonia solanacearum Tat secretome to identify bacterial wilt virulence factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.     

Ralstonia solanacearum genes induced during growth in tomato: an inside view of bacterial wilt

The phytopathogen Ralstonia solanacearum has over 5000 genes, many of which probably facilitate bacterial wilt disease development. Using in vivo expression technology (IVET), we screened a library of 133 200 R. solanacearum strain K60 promoter fusions and isolated approximately 900 fusions expressed during bacterial growth in tomato plants. Sequence analysis of 307 fusions revealed 153 unique in planta-expressed (ipx) genes. These genes included seven previously identified virulence genes (pehR, vsrB, vsrD, rpoS, hrcC, pme and gspK) as well as seven additional putative virulence factors. A significant number of ipx genes may reflect adaptation to the host xylem environment; 19.6%ipx genes are predicted to encode proteins with metabolic and/or transport functions, and 9.8%ipx genes encode proteins possibly involved in stress responses. Many ipx genes (18%) encode putative transmembrane proteins. A majority of ipx genes isolated encode proteins of unknown function, and 13% were unique to R. solanacearum. The ipx genes were variably induced in planta; beta-glucuronidase reporter gene expression analysis of a subset of 44 ipx fusions revealed that in planta expression levels were between two- and 37-fold higher than in culture. The expression of many ipx genes was subject to known R. solanacearum virulence regulators. Of 32 fusions tested, 28 were affected by at least one virulence regulator; several fusions were controlled by multiple regulators. Two ipx fusion strains isolated in this screen were reduced in virulence on tomato, indicating that gene(s) important for bacterial wilt pathogenesis were interrupted by the IVET insertion; mutations in other ipx genes are necessary to determine their roles in virulence and in planta growth. Collectively, this profile of ipx genes suggests that in its host, R. solanacearum confronts and overcomes a stressful and nutrient-poor environment.     

Evaluation of bacterial antagonists of Ralstonia solanacearum,causal agent of bacterial wilt of potato

Bacterial wilt of potato caused by Ralstonia solanacearum is one of the most destructive diseases in Kurdistan province, Iran. The objective of the present study was to evaluate antagonistic effects of some rhizobacteria isolated from the rhizosphere of potato plants against R. solanacearum, the agent of potato bacterial wilt. A total of 52 rhizobacteria were isolated and screened for in vitro antagonistic activity against R. solanacearum. Seven isolates with inhibiting effects of the pathogen were identified by phenotypic properties and partial sequencing of 16s rRNA as Pseudomonas fluorescens Pf11, P. fluorescens Pf16, Pseudomonas putida Pp17, Paenibacillus sp. Pb28 and Enterobacter sp. En38, Pseudomonas fluorescens Pp23 and Serratia sp. Se40. Strains Pf11, Pf16, Pp17 and Pb28 significantly inhibited the growth of the pathogen. Strains En38, Pp23 and Se40 showed a moderate or weak inhibition. During greenhouse study, strains were evaluated for their effects in reducing of disease and increasing biomass of potato plants. In according to greenhouse experiment results, isolates Pb28, Pp17 and Pf11significantly reduced disease by 55.56%, 51.50% and 38.58%, respectively. In addition, plant biomass significantly increased in plants treated with Pb28, Pp17, Pf11 and Pf16, compared to the control. Therefore, this study shows that these four strains have potential to be used as biocontrol agents against R. solanacearum. To confirm their effectiveness as commercial biocontrol agent, it is necessary to evaluate their efficiency in the field conditions in the next studies.     

细菌耐多药外排泵的研究进展

近年来,由于抗生素的不合理及广泛使用,全球多重耐药菌和广泛耐药菌不断出现。关于细菌耐药机制中外排泵及生物膜形成的研究越来越受关注。研究发现,外排泵与生物膜形成有密切联系,不同细菌的不同外排泵对生物膜形成的影响各异,而生物膜形成又影响外排泵基因表达。本文就细菌耐多药外排泵的研究进展进行综述。     

Unravelling physiological signatures of tomato bacterial wilt and xylem metabolites exploited by Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum uses plant resources to intensely proliferate in xylem vessels and provoke plant wilting. We combined automatic phenotyping and tissue/xylem quantitative metabolomics of infected tomato plants to decipher the dynamics of bacterial wilt. Daily acquisition of physiological parameters such as transpiration and growth were performed. Measurements allowed us to identify a tipping point in bacterial wilt dynamics. At this tipping point, the reached bacterial density brutally disrupts plant physiology and rapidly induces its death. We compared the metabolic and physiological signatures of the infection with drought stress, and found that similar changes occur. Quantitative dynamics of xylem content enabled us to identify glutamine (and asparagine) as primary resources R. solanacearum consumed during its colonization phase. An abundant production of putrescine was also observed during the infection process and was strongly correlated with in planta bacterial growth. Dynamic profiling of xylem metabolites confirmed that glutamine is the favoured substrate of R. solanacearum. On the other hand, a triple mutant strain unable to metabolize glucose, sucrose and fructose appears to be only weakly reduced for in planta growth and pathogenicity.     

Effect of temperature and resistance of tobacco cultivars to the progression of bacterial wilt,caused by Ralstonia solanacearum

Plant and Soil - Resistant cultivars are one of the most effective control measures used against bacterial wilt, caused by the soilborne bacterium Ralstonia solanacearum. We aimed to determine the...     

Relationship between avirulence gene (avrA) diversity in Ralstonia solanacearum and bacterial wilt incidence

Bacterial wilt, caused by Ralstonia solanacearum, is a serious disease of tobacco in North and South Carolina. In contrast, the disease rarely occurs on tobacco in Georgia and Florida, although bacterial wilt is a common problem on tomato. We investigated whether this difference in disease incidence could be explained by qualitative characteristics of avirulence gene avrA in the R. solanacearum population in the southeastern United States. Sequence analysis established that wild-type avrA has a 792-bp open reading frame. Polymerase chain reaction (PCR) amplification of avrA from 139 R. solanacearum strains generated either 792-bp or approximately 960-bp DNA fragments. Strains that elicited a hypersensitive reaction (HR) on tobacco contained the 792-bp allele, and were pathogenic on tomato and avirulent on tobacco. All HR-negative strains generated a approximately 960-bp DNA fragment, and wilted both tomato and tobacco. The DNA sequence of avrA in six HR-negative strains revealed the presence of one of two putative miniature inverted-repeat transposable elements (MITEs): a 152-bp MITE between nucleotides 542 and 543, or a 170-bp MITE between nucleotides 461 and 462 or 574 and 575. Southern analysis suggested that the 170-bp MITE is unique to strains from the southeastern United States and the Caribbean. Mutated avrA alleles were present in strains from 96 and 75% of North and South Carolina sites, respectively, and only in 13 and 0% of the sites in Georgia and Florida, respectively. Introduction of the wildtype allele on a plasmid into four HR-negative strains reduced their virulence on both tobacco and tomato. Inactivation of avrA in an HR-positive, avirulent strain, resulted in a mutant that was weakly virulent on tobacco. Thus, the incidence of bacterial wilt of tobacco in the southeastern United States is partially explained by which avrA allele dominates the local R. solanacearum population.     

Effect of temperature,cultivars, injury of root and inoculums load of Ralstonia solanacearum to cause bacterial wilt of tomato

Bacterial wilt, caused by Ralstonia solanacearum , is responsible for severe losses in tomato crops in the world. In the present study, the effect of temperature, cultivars of tomato, injury of root system and inoculums load of R. solanacearum to cause bacterial wilt disease under control conditions was undertaken. Three strains UTT-25, HPT-3 and JHT-5 of R. solanacearum were grown at 5–40?°C in vitro to study, the effect of temperature on the growth of bacteria and maximum growth was found at 30?°C after 72?h in all the strains. Twenty-one days old seedlings of two cultivars of tomato i.e. N-5 (moderately resistant) and Pusa Ruby (highly susceptible) were transplanted into the pots and inoculated with R. solanacearum strain UTT-25 (5 × 10⁸?cfu/ml), mechanically injured and uninjured roots of the plant. The plants were allowed to grow at 20, 25, 30 and 35?°C at National Phytotron Facility, IARI, New Delhi to study the effect of temperature on intensity of bacterial wilt disease. Maximum wilt disease intensity was found 98.73 and 95.9 % in injured roots of Pusa Ruby and N-5 cultivars of tomato at 35?°C on 11th days of inoculation, respectively. However, no wilt disease was observed in both the cultivars at 20?°C up to 60?days. For detection of R. solanacearum from asymptomatic tomato plants, hrpB-based sequence primers (Hrp_rs2F and Hrp_rs2R) amplified at 323?bp was used in bio-PCR to detect R. solanacearum from crown, mid part of stem and upper parts of the plant. Another experiment was conducted to find out the inoculum potential of R. solanacearum strain UTT-25 to cause bacterial wilt in susceptible cultivar Pusa Ruby. The bacteria were inoculated at concentration of bacterial suspension 10 to 10¹⁰?cfu/ml in injured and uninjured roots of the plants separately and injured root accelerated wilt incidence and able to cause wilt disease 63.3% by 100?cfu/ml of R. solanacearum, while no disease appeared at 10?cfu/ml on the 11th day of inoculation in injured and uninjured roots of the plant.     

Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribute to virulence

Ralstonia solanacearum, like many phytopathogenic bacteria, makes multiple extracellular plant cell-wall-degrading enzymes (CWDE), some of which contribute to its ability to cause wilt disease. CWDE and many other proteins are secreted to the milieu via the highly conserved type II protein secretion system (T2SS). R. solanacearum with a defective T2SS is weakly virulent, but it is not known whether this is due to absence of all the CWDE or the loss of other secreted proteins that contribute to disease. These alternatives were investigated by creating mutants of wild-type strain GMI1000 lacking either the T2SS or up to six CWDE and comparing them for virulence on tomato plants. To create unmarked deletions, genomic regions flanking the target gene were polymerase chain reaction (PCR)-amplified, were fused using splice overlap extension PCR, were cloned into a suicide plasmid harboring the sacB counter-selectable marker, and then, were site-specifically introduced into the genome. Various combinations of five deletions (delta pehA, delta pehB, delta B, PehC, and Pme) was not statistically different from GMI1000, but all the mutants lacking one or both cellulolytic enzymes (Egl or CbhA) wilted plants significantly more slowly than did the wild type. The GMI-6 mutant that lacks all six CWDE was more virulent than the mutant lacking only its two cellulolytic enzymes, and both were significantly more virulent than the T2SS mutant (GMI-D). Very similar results were observed in wounded-petiole inoculation assays, so GMI-6 and GMI-D appear to be less capable of colonizing tomato tissues after invasion. Because the T2SS mutant was much less virulent than the sixfold CWDE mutant, we conclude that other secreted proteins contribute substantially to the ability of R. solanacearum GMI1000 to systemically colonize tomato plants.     

A somatic hybrid between Solanum integrifolium and Solanum violaceum that is resistant to bacterial wilt caused by Ralstonia solanacearum

Protoplast fusion was performed between a commercial rootstock variety of Solanum integrifolium that is widely used in the cultivation of edible eggplant, and Solanum violaceum, a wild eggplant species tolerant to bacterial wilt. One of the somatic hybrid lines (27–14) obtained showed morphological traits intermediate between those of its parents. Its hybrid nature was confirmed by analysis of random amplified polymorphic DNA and restriction fragment length polymorphism patterns. Microscopic observation revealed that the line was amphidiploid. The hybrid line and both parent species were infected with a virulent strain of Ralstonia solanacearum by the root cutting method. Line 27–14 developed much less severe symptoms of bacterial wilt than the susceptible S. integrifolium. Twenty days after inoculation, the bacterial density in the shoot apex tissue was significantly lower in the hybrid than in S. integrifolium, suggesting that the multiplication of bacteria was inhibited in line 27–14.     

Characterisation and diversity of Indian isolates of Ralstonia solanacearum causing bacterial wilt of Capsicum annuum L.

Genetic diversity of 13 isolates of Ralstonia solanacearum causing bacterial wilt in hot pepper and bell pepper (Capsicum annuum L.) from 6 states of India was assessed. All isolates of R. solanacearum belonged to biovar 3, race 1 and phylotype I. These isolates consisted of 4 distinct DNA types at 75% similarity coefficient using ERIC, BOX and REP-PCRs techniques. Multilocus sequence analysis of hrpB, fliC and egl genes of 6 isolates of R. solanacearum along with 2 out group bacteria was done and they showed high level of variability within these three regions of the genome involving in pathogenicity.     

prhKLM genes of Ralstonia solanacearum encode novel activators of hrp regulon and are required for pathogenesis in tomato

Burkholderia pseudomallei, the causative agent of melioidosis, exploits the Bsa type III secretion system (T3SS) to deliver effector proteins into host cells. These effectors manipulate host cell functions; thus, contributing to the ability of the bacteria to evade the immune response and cause disease. Only two Bsa-secreted effectors have been conclusively identified to date. Here, we report the identification of the third B. pseudomallei type III secreted effector protein, designated BopC. BopC is encoded by the bpss1516 gene abutting bpss1517, which encodes its putative chaperone. The genes are located in the close proximity to the bsa T3SS gene cluster of B. pseudomallei K96243 (Fig. 1). BopC was secreted into culture supernatant by the wild-type B. pseudomallei strain, but its secretion was abolished in the bsaZ T3SS mutant. Using pull down and co-purification assays, we confirmed that BopC interacts with its putative chaperone, BPSS1517, in vitro. Furthermore, the first 20 N-terminal amino acids of BopC were found to be sufficient to mediate the T3SS-dependent translocation of a reporter protein from a heterologous enteropathogenic Escherichia coli host into mammalian cells. Finally, bopC mutant was found to be less invasive than the wild-type strain in the epithelial cells.     

Bacterial multidrug efflux pumps: Mechanisms,physiology and pharmacological exploitations

Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.     

Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solanacearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 x 10(5) in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.