scholarly journals Real-time monitoring of Ralstonia solanacearum infection progress in tomato and Arabidopsis using bioluminescence imaging technology

Plant Methods ◽  
2022 ◽  
Vol 18 (1) ◽  
Author(s):  
Cuihong Xu ◽  
Lingkun Zhong ◽  
Zeming Huang ◽  
Chenying Li ◽  
Jiazhang Lian ◽  
...  

Abstract Background Ralstonia solanacearum, one of the most devastating bacterial plant pathogens, is the causal agent of bacterial wilt. Recently, several studies on resistance to bacterial wilt have been conducted using the Arabidopsis-R. solanacearum system. However, the progress of R. solanacearum infection in Arabidopsis is still unclear. Results We generated a bioluminescent R. solanacearum by expressing plasmid-based luxCDABE. Expression of luxCDABE did not alter the bacterial growth and pathogenicity. The light intensity of bioluminescent R. solanacearum was linearly related to bacterial concentrations from 104 to 108 CFU·mL−1. After root inoculation with bioluminescent R. solanacearum strain, light signals in tomato and Arabidopsis were found to be transported from roots to stems via the vasculature. Quantification of light intensity from the bioluminescent strain accurately reported the difference in disease resistance between Arabidopsis wild type and resistant mutants. Conclusions Bioluminescent R. solanacearum strain spatially and quantitatively measured bacterial growth in tomato and Arabidopsis, and offered a tool for the high-throughput study of R. solanacearum-Arabidopsis interaction in the future.

2022 ◽  
Vol 12 ◽  
Author(s):  
Dylan R. Zeiss ◽  
Paul A. Steenkamp ◽  
Lizelle A. Piater ◽  
Ian A. Dubery

Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most destructive bacterial plant pathogens. This is linked to its evolutionary adaptation to evade host surveillance during the infection process since many of the pathogen’s associated molecular patterns escape recognition. However, a 22-amino acid sequence of R. solanacearum-derived cold shock protein (csp22) was discovered to elicit an immune response in the Solanaceae. Using untargeted metabolomics, the effects of csp22-elicitation on the metabolome of Solanum lycopersicum leaves were investigated. Additionally, the study set out to discover trends that may suggest that csp22 inoculation bestows enhanced resistance on tomato against bacterial wilt. Results revealed the redirection of metabolism toward the phenylpropanoid pathway and sub-branches thereof. Compared to the host response with live bacteria, csp22 induced a subset of the discriminant metabolites, but also metabolites not induced in response to R. solanacearum. Here, a spectrum of hydroxycinnamic acids (especially ferulic acid), their conjugates and derivatives predominated as signatory biomarkers. From a metabolomics perspective, the results support claims that csp22 pre-treatment of tomato plants elicits increased resistance to R. solanacearum infection and contribute to knowledge on plant immune systems operation at an integrative level. The functional significance of these specialized compounds may thus support a heightened state of defense that can be applied to ward off attacking pathogens or toward priming of defense against future infections.


2010 ◽  
Vol 23 (8) ◽  
pp. 1042-1052 ◽  
Author(s):  
Jennifer Colburn-Clifford ◽  
Caitilyn Allen

Ralstonia solanacearum race 3 biovar 2 (R3bv2) is an economically important soilborne plant pathogen that causes bacterial wilt disease by infecting host plant roots and colonizing the xylem vessels. Little is known about R3bv2 behavior in the host rhizosphere and early in bacterial wilt pathogenesis. To explore this part of the disease cycle, we used a novel taxis-based promoter-trapping strategy to identify pathogen genes induced in the plant rhizosphere. This screen identified several rex (root exudate expressed) genes whose promoters were upregulated in the presence of tomato root exudates. One rex gene encodes an assembly protein for a high affinity cbb3-type cytochrome c oxidase (cbb3-cco) that enables respiration in low-oxygen conditions in other bacteria. R3bv2 cbb3-cco gene expression increased under low-oxygen conditions, and a cbb3-cco mutant strain grew more slowly in a microaerobic environment (0.5% O2). Although the cco mutant could still wilt tomato plants, symptom onset was significantly delayed relative to the wild-type parent strain. Further, the cco mutant did not colonize host stems or adhere to roots as effectively as wild type. These results suggest that R3bv2 encounters low-oxygen environments during its interactions with host plants and that the pathogen depends on this oxidase to help it succeed in planta.


2000 ◽  
Vol 1 (3) ◽  
pp. 179-185 ◽  
Author(s):  
J. Tans-Kersten ◽  
J. Gay ◽  
C. Allen

2004 ◽  
Vol 17 (12) ◽  
pp. 1376-1384 ◽  
Author(s):  
Alison E. Robertson ◽  
W. Patrick Wechter ◽  
Timothy P. Denny ◽  
Bruce A. Fortnum ◽  
Daniel A. Kluepfel

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 ≈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 ≈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 wild-type 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.


mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Jonathan M. Jacobs ◽  
Annett Milling ◽  
Raka M. Mitra ◽  
Clifford S. Hogan ◽  
Florent Ailloud ◽  
...  

ABSTRACTDuring bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression ofpopS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in theR. solanacearumspecies complex. The phylogeny ofpopSmirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. ApopSmutant ofR. solanacearumUW551 had reduced virulence on agriculturally importantSolanumspp., including potato and tomato plants. However, thepopSmutant had wild-type virulence on a weed host,Solanum dulcamara, suggesting that some species can avoid the effects of PopS. ThepopSmutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, thepopSmutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated thepopSmutant virulence defect. Finally, thepopSmutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which areR. solanacearum’s natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed inNicotianaleaf tissue, unlike the AvrE homolog DspEPccfrom the necrotrophPectobacterium carotovorumsubsp.carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophicR. solanacearum.IMPORTANCEThe type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We characterized the biology of PopS, the only AvrE homolog made by the bacterial wilt pathogenRalstonia solanacearum. To our knowledge, this is the first study ofR. solanacearumeffector function in roots and stems, the natural infection sites of this pathogen. Unlike the functionally redundantR. solanacearumeffectors studied to date, PopS is required for full virulence and wild-type colonization of two natural crop hosts.R. solanacearumis a biotrophic pathogen that causes a nonnecrotic wilt. Consistent with this, PopS suppressed plant defenses but did not elicit cell death, unlike AvrE homologs from necrosis-causing plant pathogens. We propose that AvrE family effectors have functionally diverged to adapt to the necrotic or nonnecrotic lifestyle of their respective pathogens.


2007 ◽  
Vol 73 (12) ◽  
pp. 3779-3786 ◽  
Author(s):  
Enid T. Gonz�lez ◽  
Darby G. Brown ◽  
Jill K. Swanson ◽  
Caitilyn Allen

ABSTRACT 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.


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.


2020 ◽  
Vol 33 (3) ◽  
pp. 462-473 ◽  
Author(s):  
April M. MacIntyre ◽  
John X. Barth ◽  
Molly C. Pellitteri Hahn ◽  
Cameron O. Scarlett ◽  
Stéphane Genin ◽  
...  

The xylem-dwelling plant pathogen Ralstonia solanacearum changes the chemical composition of host xylem sap during bacterial wilt disease. The disaccharide trehalose, implicated in stress tolerance across all kingdoms of life, is enriched in sap from R. solanacearum–infected tomato plants. Trehalose in xylem sap could be synthesized by the bacterium, the plant, or both. To investigate the source and role of trehalose metabolism during wilt disease, we evaluated the effects of deleting the three trehalose synthesis pathways in the pathogen: TreYZ, TreS, and OtsAB, as well as its sole trehalase, TreA. A quadruple treY/treS/otsA/treA mutant produced 30-fold less intracellular trehalose than the wild-type strain missing the trehalase enzyme. This trehalose-nonproducing mutant had reduced tolerance to osmotic stress, which the bacterium likely experiences in plant xylem vessels. Following naturalistic soil-soak inoculation of tomato plants, this triple mutant did not cause disease as well as wild-type R. solanacearum. Further, the wild-type strain out-competed the trehalose-nonproducing mutant by over 600-fold when tomato plants were coinoculated with both strains, showing that trehalose biosynthesis helps R. solanacearum overcome environmental stresses during infection. An otsA (trehalose-6-phosphate synthase) single mutant behaved similarly to ΔtreY/treS/otsA in all experimental settings, suggesting that the OtsAB pathway is the dominant trehalose synthesis pathway in R. solanacearum.


2017 ◽  
Vol 5 (40) ◽  
Author(s):  
Madeline M. Hayes ◽  
April M. MacIntyre ◽  
Caitilyn Allen

ABSTRACT Ralstonia solanacearum is a globally distributed plant pathogen that causes bacterial wilt diseases of many crop hosts, threatening both sustenance farming and industrial agriculture. Here, we present closed genome sequences for the R. solanacearum type strain, K60, and the cool-tolerant potato brown rot strain R. solanacearum UW551, a highly regulated U.S. select agent pathogen.


2021 ◽  
Vol 37 (2) ◽  
pp. 177-193
Author(s):  
C.O Ojesola ◽  
A.K Akintokun ◽  
P.O Akintokun ◽  
A.R Oloyede

Tomato (Lycopersicon esculentum, Mill) is a rich source of vitamins, minerals and lycopene, which has many health benefits. However, its production is hampered by bacterial wilt caused by Ralstonia solanacearum resulting in significant yield losses. Use of chemicals in the control of plant pathogens has detrimental effects on humans and the environment in terms of leaving residues in soil which later find their way into underground waters. Therefore, it is desirable to find an alternative to chemical control of this bacterial pathogen. This study investigates the potential of native Bacillus thuringiensis (Bt) for biological control of Ralstonia solanacearum (Rs) under laboratory conditions. B. thuringiensis was isolated from cultivated soil, non- cultivated soil, stagnant water, sawdust, horse dung, grain dust, dead leaves and poultry manure. R. solanacearum was isolated from stem exudates of bacterial wilt infected plants and its pathogenicity assay was carried out using 2-week-old seedlings of Beske tomato variety. The Bt and R. solanacearum isolates were then characterized phenotypically. Bt isolates were further identified using endospore and parasporal staining techniques. All the Bt isolates were tested for in-vitro antagonistic activity on R. solanacearum using agar well diffusion method. Isolates Bt2, Bt16, Bt17, Bt32 and Bt34 were confirmed as Bacillus thuringiensis while isolate Rs was confirmed as R. solanacearum. Beske showed wilting symptoms from the fourth day of inoculation and eventual death of seedlings. The zone of inhibition exhibited ranged from 0.0 mm to 20.0 mm. Keywords: Bacillus thuringiensis, In-vitro, Bacterial wilt, Ralstonia solanacearum, Tomato


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