scholarly journals GR24, A Synthetic Strigolactone Analog, and Light Affect the Organization of Cortical Microtubules in Arabidopsis Hypocotyl Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuliya Krasylenko ◽  
George Komis ◽  
Sofiia Hlynska ◽  
Tereza Vavrdová ◽  
Miroslav Ovečka ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Genetic Manipulation ◽  
Cortical Microtubule ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cortical Microtubules ◽  
Wild Type ◽  
Structured Illumination Microscopy ◽  
Continuous Darkness ◽  
Microtubule Organization

Strigolactones are plant hormones regulating cytoskeleton-mediated developmental events in roots, such as lateral root formation and elongation of root hairs and hypocotyls. The latter process was addressed herein by the exogenous application of a synthetic strigolactone, GR24, and an inhibitor of strigolactone biosynthesis, TIS108, on hypocotyls of wild-type Arabidopsis and a strigolactone signaling mutant max2-1 (more axillary growth 2-1). Owing to the interdependence between light and strigolactone signaling, the present work was extended to seedlings grown under a standard light/dark regime, or under continuous darkness. Given the essential role of the cortical microtubules in cell elongation, their organization and dynamics were characterized under the conditions of altered strigolactone signaling using fluorescence microscopy methods with different spatiotemporal capacities, such as confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM). It was found that GR24-dependent inhibition of hypocotyl elongation correlated with changes in cortical microtubule organization and dynamics, observed in living wild-type and max2-1 seedlings stably expressing genetically encoded fluorescent molecular markers for microtubules. Quantitative assessment of microscopic datasets revealed that chemical and/or genetic manipulation of strigolactone signaling affected microtubule remodeling, especially under light conditions. The application of GR24 in dark conditions partially alleviated cytoskeletal rearrangement, suggesting a new mechanistic connection between cytoskeletal behavior and the light-dependence of strigolactone signaling.

scholarly journals Cortical microtubule remodelling during strigolactone- and light-mediated growth inhibition of Arabidopsis hypocotyls

2020 ◽  
Author(s):  
Yuliya A. Krasylenko ◽  
George Komis ◽  
Sonya Hlynska ◽  
Tereza Vavrdová ◽  
Miroslav Ovečka ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Genetic Manipulation ◽  
Cortical Microtubule ◽  
Hypocotyl Elongation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cortical Microtubules ◽  
Wild Type ◽  
Structured Illumination Microscopy ◽  
Strigolactone Signalling

AbstractStrigolactones are phytohormones involved in shoot branching and hypocotyl elongation. The latter phenomenon was addressed herein by the exogenous application of a synthetic strigolactone GR24 and an inhibitor of strigolactone biosynthesis TIS108 on hypocotyls of wild type Arabidopsis and a strigolactone signalling mutant max2-1 (more axillary growth 2-1). Owing to the interdependence between light and strigolactone signalling, the present work was extended to seedling cultivation under a standard light/dark regime, or under continuous darkness. Given the essential role of the cortical microtubules in cell elongation, their organization and dynamics were characterized under the conditions of altered strigolactone signalling using fluorescence microscopy methods with different spatiotemporal capacities such as confocal laser scanning microscopy and structured illumination microscopy. It was found that the strigolactone-dependent inhibition of hypocotyl elongation correlated with changes in cortical microtubule organization and dynamics, visualized in living wild type and max2-1 seedlings stably expressing genetically-encoded fluorescent molecular markers for microtubules. Quantitative analysis of microscopic datasets revealed that chemical and/or genetic manipulation of strigolactone signalling affected microtubule remodelling, especially under light conditions. The application of GR24 and TIS108 in dark conditions partially alleviated cytoskeletal rearrangement, suggesting a new mechanistic connection between the cytoskeletal behaviour and the light-dependence of strigolactone signalling.HighlightStrigolactones regulate organization and dynamics of cortical microtubules in hypocotyl cells, which contributes to the light-mediated inhibition of hypocotyl growth in Arabidopsis seedlings.

scholarly journals The PTS Components in Klebsiella pneumoniae Affect Bacterial Capsular Polysaccharide Production and Macrophage Phagocytosis Resistance

2021 ◽  
Vol 9 (2) ◽  
pp. 335
Author(s):  
Novaria Sari Dewi Panjaitan ◽  
Yu-Tze Horng ◽  
Chih-Ching Chien ◽  
Hung-Chi Yang ◽  
Ren-In You ◽  
...  
Keyword(s):  
Survival Rate ◽  
Klebsiella Pneumoniae ◽  
Laser Scanning ◽  
Bacterial Resistance ◽  
Capsular Polysaccharide ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Bacteria ◽  
Wild Type ◽  
Macrophage Phagocytosis

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

scholarly journals Effect of arsenite on swimming motility delays surface colonization in Herminiimonas arsenicoxydans

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2336-2342 ◽  
Author(s):  
M. Marchal ◽  
R. Briandet ◽  
S. Koechler ◽  
B. Kammerer ◽  
P. N. Bertin
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Wild Type Strain ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Arsenite Oxidase ◽  
Swimming Motility ◽  
In The Wild ◽  
Biofilm Development

Herminiimonas arsenicoxydans is a Gram-negative bacterium able to detoxify arsenic-contaminated environments by oxidizing arsenite [As(III)] to arsenate [As(V)] and by scavenging arsenic ions in an extracellular matrix. Its motility and colonization behaviour have been previously suggested to be influenced by arsenite. Using time-course confocal laser scanning microscopy, we investigated its biofilm development in the absence and presence of arsenite. Arsenite was shown to delay biofilm initiation in the wild-type strain; this was partly explained by its toxicity, which caused an increased growth lag time. However, this delayed adhesion step in the presence of arsenite was not observed in either a swimming motility defective fliL mutant or an arsenite oxidase defective aoxB mutant; both strains displayed the wild-type surface properties and growth capacities. We propose that during the biofilm formation process arsenite acts on swimming motility as a result of the arsenite oxidase activity, preventing the switch between planktonic and sessile lifestyles. Our study therefore highlights the existence, under arsenite exposure, of a competition between swimming motility, resulting from arsenite oxidation, and biofilm initiation.

scholarly journals Cell-to-Cell Signaling in Xylella fastidiosa Suppresses Movement and Xylem Vessel Colonization in Grape

2008 ◽  
Vol 21 (10) ◽  
pp. 1309-1315 ◽  
Author(s):  
Subhadeep Chatterjee ◽  
Karyn L. Newman ◽  
Steven E. Lindow
Keyword(s):  
Cell Signaling ◽  
Type Strain ◽  
Laser Scanning ◽  
Wild Type Strain ◽  
Xylella Fastidiosa ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Deficient Mutant ◽  
Wild Type ◽  
Wide Range

Cell-to-cell signaling mediated by a fatty acid diffusible signaling factor (DSF) is central to the regulation of the virulence of Xylella fastidiosa. DSF production by X. fastidiosa is dependent on rpfF and, although required for insect colonization, appears to reduce its virulence to grape. To understand what aspects of colonization of grape are controlled by DSF in X. fastidiosa and, thus, those factors that contribute to virulence, we assessed the colonization of grape by a green fluorescent protein–marked rpfF-deficient mutant. The rpfF-deficient mutant was detected at a greater distance from the point of inoculation than the wild-type strain at a given sampling time, and also attained a population size that was up to 100-fold larger than that of the wild-type strain at a given distance from the point of inoculation. Confocal laser-scanning microscopy revealed that approximately 10-fold more vessels in petioles of symptomatic leaves harbored at least some cells of either the wild type or rpfF mutant when compared with asymptomatic leaves and, thus, that disease symptoms were associated with the extent of vessel colonization. Importantly, the rpfF mutant colonized approximately threefold more vessels than the wild-type strain. Although a wide range of colony sizes were observed in vessels colonized by both the wild type and rpfF mutant, the proportion of colonized vessels harboring large numbers of cells was significantly higher in plants inoculated with the rpfF mutant than with the wild-type strain. These studies indicated that the hypervirulence phenotype of the rpfF mutant is due to both a more extensive spread of the pathogen to xylem vessels and unrestrained multiplication within vessels leading to blockage. These results suggest that movement and multiplication of X. fastidiosa in plants are linked, perhaps because cell wall degradation products are a major source of nutrients. Thus, DSF-mediated cell-to-cell signaling, which restricts movement and colonization of X. fastidiosa, may be an adaptation to endophytic growth of the pathogen that prevents the excessive growth of cells in vessels.

scholarly journals Visualization of Proteus mirabilis within the Matrix of Urease-Induced Bladder Stones during Experimental Urinary Tract Infection

2002 ◽  
Vol 70 (1) ◽  
pp. 389-394 ◽  
Author(s):  
Xin Li ◽  
Hui Zhao ◽  
C. Virginia Lockatell ◽  
Cinthia B. Drachenberg ◽  
David E. Johnson ◽  
...  
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Proteus Mirabilis ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Alizarin Red ◽  
Negative Mutant

ABSTRACT The virulence of a urease-negative mutant of uropathogenic Proteus mirabilis and its wild-type parent strain was assessed by using a CBA mouse model of catheterized urinary tract infection. Overall, catheterized mice were significantly more susceptible than uncatheterized mice to infection by wild-type P. mirabilis. At a high inoculum, the urease-negative mutant successfully colonized bladders of catheterized mice but did not cause urolithiasis and was still severely attenuated in its ability to ascend to kidneys. Using confocal laser scanning microscopy and scanning electron microscopy, we demonstrated the presence of P. mirabilis within the urease-induced stone matrix. Alizarin red S staining was used to detect calcium-containing deposits in bladder and kidney tissues of P. mirabilis-infected mice.

Colonization and bioherbicidal activity on green foxtail byPseudomonas fluorescensBRG100 in a pesta formulation

2012 ◽  
Vol 58 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Caressa J. Caldwell ◽  
Russell K. Hynes ◽  
Susan M. Boyetchko ◽  
Darren R. Korber
Keyword(s):  
Root Growth ◽  
Seed Coat ◽  
Laser Scanning ◽  
Doubling Time ◽  
Herbicidal Activity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Carbon Utilization ◽  
Green Foxtail

Pseudomonas fluorescens BRG100 produces secondary metabolites with herbicidal activity on green foxtail ( Setaria viridis ), an important weed pest in Canadian agriculture. Five gfp transformants of P. fluorescens BRG100 were compared with the wild-type isolate for green foxtail root herbicide activity, i.e., root growth suppression, doubling time, carbon utilization, and colonization of green foxtail root (proximal and distal regions). The most revealing comparison between the wild type and its gfp transformants was herbicidal activity on green foxtail. Herbicidal activity of transformant gfp-7 was not significantly different from the uninoculated control, suggesting that insertion of the gfp gene may have interfered with a gene, or genes, vital to the bioherbicide process. Doubling time, carbon utilization, and colonization of green foxtail did not differ to a great extent between the wild type and the gfp transformants, indicating their suitability as conservatively tagged organisms for subsequent colonization–herbicidal activity studies. Accordingly, a pesta granule formulation delivered transformant gfp-2 to the seed coat and roots of green foxtail. Epifluorescent and confocal laser scanning microscopy revealed the transformant gfp-2 colonized the ventral portion of the seed coat, root hairs, and all areas of the root except the root cap region, where gfp-2 presumably exerted herbicidal effects. These results suggest that P. fluorescens BRG100 has considerable potential as a bioherbicide because of its successful colonization and suppressive activity on green foxtail root growth.

Role of the luxS Gene in Initial Biofilm Formation by Streptococcus mutans

2015 ◽  
Vol 25 (1) ◽  
pp. 60-68 ◽  
Author(s):  
Zhiyan He ◽  
Jingping Liang ◽  
Zisheng Tang ◽  
Rui Ma ◽  
Huasong Peng ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Type Strain ◽  
Laser Scanning ◽  
Wild Type Strain ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Initial Biofilm

Quorum sensing (QS) is a process by which bacteria communicate with each other by secreting chemical signals called autoinducers (AIs). Among Gram-negative and Gram-positive bacteria, AI-2 synthesized by the LuxS enzyme is widespread. The aim of this study was to evaluate the effect of QS <i>luxS</i> gene on initial biofilm formation by <i>Streptococcus mutans</i>. The bacterial cell surface properties, including cell hydrophobicity (bacterial adherence to hydrocarbons) and aggregation, which are important for initial adherence during biofilm development, were investigated. The biofilm adhesion assay was evaluated by the MTT method. The structures of the 5-hour biofilms were observed by using confocal laser scanning microscopy, and QS-related gene expressions were investigated by real-time PCR. The <i>luxS</i> mutant strain exhibited higher biofilm adherence and aggregation, but lower hydrophobicity than the wild-type strain. The confocal laser scanning microscopy images revealed that the wild-type strain tended to form smaller aggregates with uniform distribution, whereas the <i>luxS</i> mutant strain aggregated into distinct clusters easily discernible in the generated biofilm. Most of the genes examined were downregulated in the biofilms formed by the <i>luxS</i> mutant strain, except the <i>gtfB </i>gene. QS <i>luxS</i> gene can affect the initial biofilm formation by <i>S. mutans.</i>

scholarly journals Essential Role for the Legionella pneumophila Rep Helicase Homologue in Intracellular Infection of Mammalian Cells

2000 ◽  
Vol 68 (12) ◽  
pp. 6970-6978 ◽  
Author(s):  
Omar S. Harb ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Intracellular Replication ◽  
Simultaneous Exposure ◽  
U937 Macrophage

ABSTRACT We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar torep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in therep helicase homologue mutant, rendering it defective in intracellular replication.

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