scholarly journals The In Planta Transcriptome of Ralstonia solanacearum: Conserved Physiological and Virulence Strategies during Bacterial Wilt of Tomato

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Jonathan M. Jacobs ◽  
Lavanya Babujee ◽  
Fanhong Meng ◽  
Annett Milling ◽  
Caitilyn Allen

ABSTRACTPlant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogenRalstonia solanacearumis well adapted to it, growing to 108to 109 CFU/g tomato stem. To better understand howR. solanacearumsucceeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinctR. solanacearumstrains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 108 CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly alteredin plantaversus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in thescrABYcluster. A UW551scrAmutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed hostSolanum dulcamara. FunctionalscrAcontributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose.scrAexpression was induced by sucrose, but to a much greater degree by growthin planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulatedin plantaat high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle.IMPORTANCERalstonia solanacearumis a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions thatR. solanacearumexpresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen’s dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further,R. solanacearumwas highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Beth L. Dalsing ◽  
Alicia N. Truchon ◽  
Enid T. Gonzalez-Orta ◽  
Annett S. Milling ◽  
Caitilyn Allen

ABSTRACTGenomic data predict that, in addition to oxygen, the bacterial plant pathogenRalstonia solanacearumcan use nitrate (NO3−), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3−, corresponding to R. solanacearum's optimal NO3−concentration for anaerobic growthin vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). TheΔnarG,ΔaniA, andΔnorBmutants grew poorly on NO3−compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3−respiration directly enhanced growth, AniA-dependent NO2−reduction did not. NO2−and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3−acts as a TEA, but the resulting NO2−and NO likely do not. None of the mutants grew as well as the wild typein planta, and strains lacking AniA (NO2−reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3−to respire, grow, and cause disease. Degradation of NO2−and NO is also important for successful infection and depends on denitrification and NO detoxification systems.IMPORTANCEThe plant-pathogenic bacteriumRalstonia solanacearumcauses bacterial wilt, one of the world's most destructive crop diseases. This pathogen's explosive growth in plant vascular xylem is poorly understood. We used biochemical and genetic approaches to show that R. solanacearum rapidly depletes oxygen in host xylem but can then respire using host nitrate as a terminal electron acceptor. The microbe uses its denitrification pathway to detoxify the reactive nitrogen species nitrite (a product of nitrate respiration) and nitric oxide (a plant defense signal). Detoxification may play synergistic roles in bacterial wilt virulence by converting the host's chemical weapon into an energy source. Mutant bacterial strains lacking elements of the denitrification pathway could not grow as well as the wild type in tomato plants, and some mutants were also reduced in virulence. Our results show how a pathogen's metabolic activity can alter the host environment in ways that increase pathogen success.


2012 ◽  
Vol 56 (12) ◽  
pp. 6147-6153 ◽  
Author(s):  
Susan E. Puckett ◽  
Kaleb A. Reese ◽  
Georgi M. Mitev ◽  
Valerie Mullen ◽  
Rudd C. Johnson ◽  
...  

ABSTRACTPeptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)3RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is β-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential geneacpP(which encodes acyl carrier protein). The MIC of (RFF)3RXB-AcpP was 2.5 μM (14 μg/ml) inEscherichia coliW3110. The rate of spontaneous resistance ofE. colito (RFF)3RXB-AcpP was 4 × 10−7mutations/cell division. A spontaneous (RFF)3RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)3RXB-AcpP was 40 μM (224 μg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence ofacpPwas identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)3RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation insbmA, which encodes an active transport protein. In separate experiments, a (RFF)3RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped tosbmA. Genetic complementation of PR200.1 or RR3 withsbmArestored susceptibility to (RFF)3RXB-AcpP. Deletion ofsbmAcaused resistance to (RFF)3RXB-AcpP. We conclude that resistance to (RFF)3RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations insbmA. The data further suggest that (RFF)3R-XB PPMOs may be transported across the plasma membrane by SbmA.


2001 ◽  
Vol 183 (12) ◽  
pp. 3597-3605 ◽  
Author(s):  
Julie Tans-Kersten ◽  
Huayu Huang ◽  
Caitilyn Allen

ABSTRACT 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 thatR. 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 lackingfliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphAcassette 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.


2017 ◽  
Vol 5 (14) ◽  
Author(s):  
Deju Chen ◽  
Bo Liu ◽  
Yujing Zhu ◽  
Jieping Wang ◽  
Zheng Chen ◽  
...  

ABSTRACT An avirulent strain of Ralstonia solanacearum FJAT-1458 was isolated from a living tomato. Here, we report the complete R. solanacearum FJAT-1458 genome sequence of 6,059,899 bp and 5,241 genes. This bacterial strain is a potential candidate as a biocontrol agent in the form of a plant vaccine for bacterial wilt.


2016 ◽  
Vol 82 (22) ◽  
pp. 6498-6506 ◽  
Author(s):  
Eric L. Bruger ◽  
Christopher M. Waters

ABSTRACTCommunication has been suggested as a mechanism to stabilize cooperation. In bacteria, chemical communication, termed quorum sensing (QS), has been hypothesized to fill this role, and extracellular public goods are often induced by QS at high cell densities. Here we show, with the bacteriumVibrio harveyi, that QS provides strong resistance against invasion of a QS defector strain by maximizing the cellular growth rate at low cell densities while achieving maximum productivity through protease upregulation at high cell densities. In contrast, QS mutants that act as defectors or unconditional cooperators maximize either the growth rate or the growth yield, respectively, and thus are less fit than the wild-type QS strain. Our findings provide experimental evidence that regulation mediated by microbial communication can optimize growth strategies and stabilize cooperative phenotypes by preventing defector invasion, even under well-mixed conditions. This effect is due to a combination of responsiveness to environmental conditions provided by QS, lowering of competitive costs when QS is not induced, and pleiotropic constraints imposed on defectors that do not perform QS.IMPORTANCECooperation is a fundamental problem for evolutionary biology to explain. Conditional participation through phenotypic plasticity driven by communication is a potential solution to this dilemma. Thus, among bacteria, QS has been proposed to be a proximate stabilizing mechanism for cooperative behaviors. Here, we empirically demonstrate that QS inV. harveyiprevents cheating and subsequent invasion by nonproducing defectors by maximizing the growth rate at low cell densities and the growth yield at high cell densities, whereas an unconditional cooperator is rapidly driven to extinction by defectors. Our findings provide experimental evidence that QS regulation prevents the invasion of cooperative populations by QS defectors even under unstructured conditions, and they strongly support the role of communication in bacteria as a mechanism that stabilizes cooperative traits.


2017 ◽  
Vol 5 (23) ◽  
Author(s):  
Adam Kotorashvili ◽  
Galina Meparishvili ◽  
Giorgi Gogoladze ◽  
Nato Kotaria ◽  
Maka Muradashvili ◽  
...  

ABSTRACT Ralstonia solanacearum, the causative agent of bacterial wilt, is a devastating bacterial plant pathogen with a wide range of hosts. We report here the first draft genome sequences for three strains of Ralstonia solanacearum isolated from infected potato, tomato, and pepper plants in Georgia.


2015 ◽  
Vol 197 (13) ◽  
pp. 2122-2128 ◽  
Author(s):  
Rebecca L. Scholz ◽  
E. Peter Greenberg

ABSTRACTMany bacteria produce secreted iron chelators called siderophores, which can be shared among cells with specific siderophore uptake systems regardless of whether the cell produces siderophores. Sharing secreted products allows freeloading, where individuals use resources without bearing the cost of production. Here we show that theEscherichia colisiderophore enterochelin is not evenly shared between producers and nonproducers. Wild-typeEscherichia coligrows well in low-iron minimal medium, and an isogenic enterochelin synthesis mutant (ΔentF) grows very poorly. The enterochelin mutant grows well in low-iron medium supplemented with enterochelin. At high cell densities the ΔentFmutant can compete equally with the wild type in low-iron medium. At low cell densities the ΔentFmutant cannot compete. Furthermore, the growth rate of the wild type is unaffected by cell density. The wild type grows well in low-iron medium even at very low starting densities. Our experiments support a model where at least some enterochelin remains associated with the cells that produce it, and the cell-associated enterochelin enables iron acquisition even at very low cell density. Enterochelin that is not retained by producing cells at low density is lost to dilution. At high cell densities, cell-free enterochelin can accumulate and be shared by all cells in the group. Partial privatization is a solution to the problem of iron acquisition in low-iron, low-cell-density habitats. Cell-free enterochelin allows for iron scavenging at a distance at higher population densities. Our findings shed light on the conditions under which freeloaders might benefit from enterochelin uptake systems.IMPORTANCESociality in microbes has become a topic of great interest. One facet of sociality is the sharing of secreted products, such as the iron-scavenging siderophores. We present evidence that theEscherichia colisiderophore enterochelin is relatively inexpensive to produce and is partially privatized such that it can be efficiently shared only at high producer cell densities. At low cell densities, cell-free enterochelin is scarce and only enterochelin producers are able to grow in low-iron medium. Because freely shared products can be exploited by freeloaders, this partial privatization may help explain how enterochelin production is stabilized inE. coliand may provide insight into when enterochelin is available for freeloaders.


2021 ◽  
Author(s):  
Ebrahim Osdaghi

Abstract Ralstonia solanacearum is included in the A2 (high risk) list of quarantine organisms by the European and Mediterranean Plant Protection Organization (EPPO). EPPO Code for R. solanacearum is RALSSO, while the phytosanitary categorization of the species in EPPO A2 list is no.58, EU: I/A2 (EPPO, 2018). Bacterial wilt disease was first reported in southern USA in the late nineteenth century on tomato plants (Smith, 1896). Infected plant materials (e.g. potato tubers) transmit the pathogen over long distances; hence, quarantine inspections and plant sanitary practices are the cornerstone of disease management (EPPO, 2018). R. solanacearum strains in the race 3 group are a select agent under the US Agricultural Bioterrorism Protection Act of 2002 (USDA, 2005). Peculiarly, the organism, if not yet already present in North America in pelargonium (Strider et al., 1981), was introduced with cuttings of this host by American companies producing these cuttings for their markets in countries like Kenya and Guatemala (Norman et al., 1999, 2009; Kim et al., 2002; Williamson et al., 2002; O'Hern, 2004). A similar situation led to introductions of the pathogen from Kenya into some northern European nurseries. Once the source (contaminated surface water) was recognized and proper control measures (use of deep soil water, disinfection of cutting producing premises and replacement of mother stock), the problem was solved and the disease in greenhouses eradicated (Janse et al., 2004). Similarly race 1 has been introduced into greenhouses with ornamental plants (rhizomes, cuttings or fully grown plants) such as Epipremnum, Anthurium, Curcuma spp. and Begonia eliator from tropical areas (Norman and Yuen, 1998, 1999; Janse et al., 2006; Janse, 2012). Introduction can and did occur from Costa Rica and the Caribbean, Indonesia, Thailand and South Africa. However, this idea of placing pathogens on bioterrorist list for unclear and perhaps industry-driven reasons and its effects, is strongly opposed in a recent publication from leading phytobacteriologists. This is because R. solanacearum is an endemic pathogen, causing endemic disease in most parts of its geographic occurrence, moreover normal quarantine regulations are already in place where the disease is not present or only sporadically and are thought to be more efficient and less damaging to trade and research than placing this pathogen on select agent lists and treating it as such (Young et al., 2008). Peculiarly, it has been used in the control of a real invasive species, the weed kahili ginger (Hedychium gardenarium) in tropical forests in Hawaii. This is not without risks because strains occurring on this weed host were thought to be non-virulent, but later appeared to be virulent on many edible and ornamental ginger species as well (Anderson and Gardner, 1999; Paret et al., 2008). Another threat for these countries could be strains belonging to race 1, biovar 1 (phylotype I) that have already been reported from field-grown potatoes in Portugal (Cruz et al., 2008).


2014 ◽  
Vol 27 (3) ◽  
pp. 277-285 ◽  
Author(s):  
Andrea Paola Zuluaga Cruz ◽  
Virginia Ferreira ◽  
María Julia Pianzzola ◽  
María Inés Siri ◽  
Núria S. Coll ◽  
...  

Several breeding programs are under way to introduce resistance to bacterial wilt caused by Ralstonia solanacearum in solanaceous crops. The lack of screening methods allowing easy measurement of pathogen colonization and the inability to detect latent (i.e., symptomless) infections are major limitations when evaluating resistance to this disease in plant germplasm. We describe a new method to study the interaction between R. solanacearum and potato germplasm that overcomes these restrictions. The R. solanacearum UY031 was genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. Colonization of this reporter strain on different potato accessions was followed using life imaging. Bacterial detection in planta by this nondisruptive system correlated with the development of wilting symptoms. In addition, we demonstrated that quantitative detection of the recombinant strain using a luminometer can identify latent infections on symptomless potato plants. We have developed a novel, unsophisticated, and accurate method for high-throughput evaluation of pathogen colonization in plant populations. We applied this method to compare the behavior of potato accessions with contrasting resistance to R. solanacearum. This new system will be especially useful to detect latency in symptomless parental lines before their inclusion in long-term breeding programs for disease resistance.


mSystems ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Divya Balasubramanian ◽  
Preethi T. Ragunathan ◽  
Jingyi Fei ◽  
Carin K. Vanderpool

ABSTRACT sRNAs are ubiquitous and versatile regulators of bacterial gene expression. A number of well-characterized examples in E. coli are highly conserved and present in the E. coli core genome. In contrast, the sRNA DicF (identified over 20 years ago but remaining poorly characterized) is encoded by a gene carried on a defective prophage element in many E. coli genomes. Here, we characterize DicF in order to better understand how horizontally acquired sRNA regulators impact bacterial gene expression and physiology. Our data confirm the long-hypothesized DicF-mediated regulation of ftsZ, encoding the bacterial tubulin homolog required for cell division. We further uncover DicF-mediated posttranscriptional control of metabolic gene expression. Ectopic production of DicF is highly toxic to E. coli cells, but the toxicity is not attributable to DicF regulation of ftsZ. Further work is needed to reveal the biological roles of and benefits for the host conferred by DicF and other products encoded by defective prophages. Hundreds of small RNAs (sRNAs) have been identified in diverse bacterial species, and while the functions of most remain unknown, some regulate key processes, particularly stress responses. The sRNA DicF was identified over 25 years ago as an inhibitor of cell division but since then has remained uncharacterized. DicF consists of 53 nucleotides and is encoded by a gene carried on a prophage (Qin) in the genomes of many Escherichia coli strains. We demonstrated that DicF inhibits cell division via direct base pairing with ftsZ mRNA to repress translation and prevent new synthesis of the bacterial tubulin homolog FtsZ. Systems analysis using computational and experimental methods identified additional mRNA targets of DicF: xylR and pykA mRNAs, encoding the xylose uptake and catabolism regulator and pyruvate kinase, respectively. Genetic analyses showed that DicF directly base pairs with and represses translation of these targets. Phenotypes of cells expressing DicF variants demonstrated that DicF-associated growth inhibition is not solely due to repression of ftsZ, indicating that the physiological consequences of DicF-mediated regulation extend beyond effects on cell division caused by reduced FtsZ synthesis. IMPORTANCE sRNAs are ubiquitous and versatile regulators of bacterial gene expression. A number of well-characterized examples in E. coli are highly conserved and present in the E. coli core genome. In contrast, the sRNA DicF (identified over 20 years ago but remaining poorly characterized) is encoded by a gene carried on a defective prophage element in many E. coli genomes. Here, we characterize DicF in order to better understand how horizontally acquired sRNA regulators impact bacterial gene expression and physiology. Our data confirm the long-hypothesized DicF-mediated regulation of ftsZ, encoding the bacterial tubulin homolog required for cell division. We further uncover DicF-mediated posttranscriptional control of metabolic gene expression. Ectopic production of DicF is highly toxic to E. coli cells, but the toxicity is not attributable to DicF regulation of ftsZ. Further work is needed to reveal the biological roles of and benefits for the host conferred by DicF and other products encoded by defective prophages.


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