scholarly journals An Innovative Root Inoculation Method to StudyRalstonia solanacearumPathogenicity in Tomato Seedlings

2018 ◽  
Vol 108 (4) ◽  
pp. 436-442 ◽  
Author(s):  
N. Singh ◽  
T. Phukan ◽  
P. L. Sharma ◽  
K. Kabyashree ◽  
A. Barman ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Ralstonia Solanacearum ◽  
Virulence Genes ◽  
Wild Type ◽  
Gus Staining ◽  
Tomato Seedlings ◽  
Inoculation Method ◽  
Virulence Assay ◽  
Bacterial Inoculum ◽  
Adult Plants

In this study, we report Ralstonia solanacearum pathogenicity in the early stages of tomato seedlings by an innovative root inoculation method. Pathogenicity assays were performed under gnotobiotic conditions in microfuge tubes by employing only 6- to 7-day-old tomato seedlings for root inoculation. Tomato seedlings inoculated by this method exhibited the wilted symptom within 48 h and the virulence assay can be completed in 2 weeks. Colonization of the wilted seedlings by R. solanacearum was confirmed by using gus staining as well as fluorescence microscopy. Using this method, mutants in different virulence genes such as hrpB, phcA, and pilT could be clearly distinguished from wild-type R. solanacearum. The method described here is economic in terms of space, labor, and cost as well as the required quantity of bacterial inoculum. Thus, the newly developed assay is an easy and useful approach for investigating virulence functions of the pathogen at the seedling stage of hosts, and infection under these conditions appears to require pathogenicity mechanisms used by the pathogen for infection of adult plants.

scholarly journals Loss of Virulence of the Phytopathogen Ralstonia solanacearum Through Infection by φRSM Filamentous Phages

2012 ◽  
Vol 102 (5) ◽  
pp. 469-477 ◽  
Author(s):  
Hardian S. Addy ◽  
Ahmed Askora ◽  
Takeru Kawasaki ◽  
Makoto Fujie ◽  
Takashi Yamada
Keyword(s):  
Ralstonia Solanacearum ◽  
Virulence Genes ◽  
Host Cells ◽  
Extracellular Polysaccharides ◽  
Twitching Motility ◽  
Wild Type ◽  
Tomato Plants ◽  
Type Iv ◽  
Infected Cells ◽  
Filamentous Phages

φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.

scholarly journals Ralstonia solanacearum Needs Motility for Invasive Virulence on Tomato

2001 ◽  
Vol 183 (12) ◽  
pp. 3597-3605 ◽  
Author(s):  
Julie Tans-Kersten ◽  
Huayu Huang ◽  
Caitilyn Allen
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Soft Agar ◽  
Cell Protein ◽  
In Planta ◽  
Tomato Plants ◽  
Bacterial Wilt Disease ◽  
Virulence Assay ◽  
Flagellar Filament

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.

scholarly journals ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1080-1092 ◽  
Author(s):  
A. A. Bartosik ◽  
J. Mierzejewska ◽  
C. M. Thomas ◽  
G. Jagura-Burdzy
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescence Microscopy ◽  
Bacterial Growth ◽  
Complete Loss ◽  
Physiological Parameters ◽  
Wild Type ◽  
Partial Removal ◽  
Partner Protein ◽  
The One ◽  
Mutant Phenotypes

Deletions leading to complete or partial removal of ParB were introduced into the Pseudomonas aeruginosa chromosome. Fluorescence microscopy of fixed cells showed that ParB mutants lacking the C-terminal domain or HTH motif formed multiple, less intense foci scattered irregularly, in contrast to the one to four ParB foci per cell symmetrically distributed in wild-type P. aeruginosa. All parB mutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation of parA of P. aeruginosa. As complete loss of ParA destabilized its partner ParB it was unclear deficiency of which protein is responsible for the mutant phenotypes. Analysis of four parB mutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all four parB mutants demonstrate the same defects it can be concluded that either ParB, or ParA and ParB in combination, plays an important role in nucleoid distribution, growth and motility in P. aeruginosa.

scholarly journals NtRNF217, Encoding a Putative RBR E3 Ligase Protein of Nicotiana tabacum, Plays an Important Role in the Regulation of Resistance to Ralstonia solanacearum Infection

2021 ◽  
Vol 22 (11) ◽  
pp. 5507
Author(s):  
Ying Liu ◽  
Yuanman Tang ◽  
Xi Tan ◽  
Wei Ding
Keyword(s):  
Nicotiana Tabacum ◽  
Ralstonia Solanacearum ◽  
Plant Immunity ◽  
E3 Ligase ◽  
Marker Genes ◽  
Susceptible Cultivar ◽  
Wild Type ◽  
Disease Symptoms ◽  
Short Time ◽  
Resistance To Ralstonia Solanacearum

E3 ubiquitin ligases, the most important part of the ubiquitination process, participate in various processes of plant immune response. RBR E3 ligase is one of the E3 family members, but its functions in plant immunity are still little known. NtRNF217 is a RBR E3 ligase in tobacco based on the sequence analysis. To assess roles of NtRNF217 in tobacco responding to Ralstonia solanacearum, overexpression experiments in Nicotiana tabacum (Yunyan 87, a susceptible cultivar) were performed. The results illuminated that NtRNF217-overexpressed tobacco significantly reduced multiplication of R. solanacearum and inhibited the development of disease symptoms compared with wild-type plants. The accumulation of H2O2 and O2− in NtRNF217-OE plants was significantly higher than that in WT-Yunyan87 plants after pathogen inoculation. The activities of CAT and SOD also increased rapidly in a short time after R. solanacearum inoculation in NtRNF217-OE plants. What is more, overexpression of NtRNF217 enhanced the transcript levels of defense-related marker genes, such as NtEFE26, NtACC Oxidase, NtHIN1, NtHSR201, and NtSOD1 in NtRNF217-OE plants after R. solanacearum inoculation. The results suggested that NtRNF217 played an important role in regulating the expression of defense-related genes and the antioxidant enzymes, which resulted in resistance to R. solanacearum infection.

Ralstonia solanacearum preferential colonization in the shoot apical meristem explains its pathogenicity pattern in tomato seedlings

2020 ◽  
Vol 69 (7) ◽  
pp. 1347-1356
Author(s):  
Kristi Kabyashree ◽  
Rahul Kumar ◽  
Piyali Sen ◽  
Siddhartha S. Satapathy ◽  
Suvendra K. Ray
Keyword(s):  
Ralstonia Solanacearum ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Tomato Seedlings

scholarly journals Pyramiding Unmarked Deletions in Ralstonia solanacearum Shows That Secreted Proteins in Addition to Plant Cell-Wall-Degrading Enzymes Contribute to Virulence

2005 ◽  
Vol 18 (12) ◽  
pp. 1296-1305 ◽  
Author(s):  
Huanli Liu ◽  
Shuping Zhang ◽  
Mark A. Schell ◽  
Timothy P. Denny
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Ralstonia Solanacearum ◽  
Plant Cell Wall ◽  
Secreted Proteins ◽  
Cellulolytic Enzymes ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Tomato Plants ◽  
Degrading Enzymes

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 (δpehA, δpehB, δpehC, δpme, and δegl) and one inactivated allele (cbhA::aphA-3) resulted in 15 mutants missing one to six CWDE. In soil-drench inoculation assays, virulence of mutants lacking only pectic enzymes (PehA, PehB, 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.

scholarly journals Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association

2003 ◽  
Vol 77 (21) ◽  
pp. 11651-11660 ◽  
Author(s):  
Mariam Andrawiss ◽  
Yasuhiro Takeuchi ◽  
Lindsay Hewlett ◽  
Mary Collins
Keyword(s):  
Fluorescence Microscopy ◽  
Murine Leukemia Virus ◽  
Fluorescent Proteins ◽  
Leukemia Virus ◽  
Particle Formation ◽  
Membrane Association ◽  
Wild Type ◽  
Particle Assembly ◽  
Gag Assembly ◽  
Murine Leukemia

ABSTRACT In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology 216:418-424, 1996; K. Alin and S. P. Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding.

scholarly journals Long-range cooperative binding of kinesin to a microtubule in the presence of ATP

2005 ◽  
Vol 168 (5) ◽  
pp. 691-696 ◽  
Author(s):  
Etsuko Muto ◽  
Hiroyuki Sakai ◽  
Kuniyoshi Kaseda
Keyword(s):  
Fluorescence Microscopy ◽  
Long Range ◽  
State Transition ◽  
Atp Hydrolysis ◽  
Cooperative Binding ◽  
Adjacent Region ◽  
Wild Type ◽  
Active Involvement ◽  
Latex Beads ◽  
Close Proximity

Interaction of kinesin-coated latex beads with a single microtubule (MT) was directly observed by fluorescence microscopy. In the presence of ATP, binding of a kinesin bead to the MT facilitated the subsequent binding of other kinesin beads to an adjacent region on the MT that extended for micrometers in length. This cooperative binding was not observed in the presence of ADP or 5′-adenylylimidodiphosphate (AMP-PNP), where binding along the MT was random. Cooperative binding also was induced by an engineered, heterodimeric kinesin, WT/E236A, that could hydrolyze ATP, yet remained fixed on the MT in the presence of ATP. Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction. These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end. Thus, our study highlights the active involvement of MTs in kinesin motility.

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