Colonization and Movement of GFP-Labeled Clavibacter michiganensis subsp. michiganensis During Tomato Infection

The vascular pathogen Clavibacter michiganensis subsp. michiganensis is responsible for bacterial wilt and canker of tomato. Pathogenicity of this bacterium is dependent on plasmid-borne virulence factors and serine proteases located on the chromosomal chp/tomA pathogenicity island (PAI). In this study, colonization patterns and movement of C. michiganensis subsp. michiganensis during tomato infection was examined using a green fluorescent protein (GFP)-labeled strain. A plasmid expressing GFP in C. michiganensis subsp. michiganensis was constructed and found to be stable in planta for at least 1 month. Confocal laser-scanning microscopy (CLSM) of inoculated stems showed that the pathogen extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem. Acropetal movement of the wild-type strain C. michiganensis subsp. michiganensis NCPPB382 (Cmm382) in tomato resulted in an extensive systemic colonization of the whole plant reaching the apical region after 15 days, whereas Cmm100 (lacking the plasmids pCM1 and pCM2) or Cmm27 (lacking the chp/tomA PAI) remained confined to the area surrounding of the inoculation site. Cmm382 formed biofilm-like structures composed of large bacterial aggregates on the interior of xylem walls as observed by CLSM and scanning electron microscopy. These findings suggest that virulence factors located on the chp/tomA PAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates.

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Verticillium Wilt on Fiber Flax: Symptoms and Pathogen Development In Planta

Plant Disease ◽

10.1094/pdis-01-18-0139-re ◽

2018 ◽

Vol 102 (12) ◽

pp. 2421-2429 ◽

Cited By ~ 5

Author(s):

Adrien Blum ◽

Mélanie Bressan ◽

Abderrakib Zahid ◽

Isabelle Trinsoutrot-Gattin ◽

Azeddine Driouich ◽

...

Keyword(s):

Cell Differentiation ◽

Verticillium Wilt ◽

Laser Scanning ◽

Fluorescent Protein ◽

Lateral Roots ◽

Laser Scanning Microscopy ◽

Root Tip ◽

Confocal Laser ◽

In Planta ◽

Fiber Flax

Fiber flax (Linum usitatissimum L.), an important crop in Normandy (France), is increasingly affected by Verticillium wilt caused by the soilborne fungus Verticillium dahliae. This disease leads to nonnegligible yield losses and depreciated fibers that are consequently difficult to upgrade. Verticillium wilt is a major threat to a broad range of agriculture. In this study, susceptible fiber flax cultivar Adélie was infected by VdLu01 (isolated from fiber flax, this study) or green fluorescent protein-tagged VdLs17 (transformed and provided by the department of Plant Pathology, University of California, Davis). Between 3 and 4 weeks postinoculation, wilting symptoms on leaves were first observed, with acropetal growth during the following weeks. Pathogen development was tracked by confocal laser-scanning microscopy during the asymptomatic and symptomatic stages. First, conidia germination led to the development of hyphae on root epidermis; more particularly, on the zone of cell differentiation and around emerging lateral roots, while the zone of cell division and the root tip were free of the pathogen. At 3 days postinoculation, the zone of cell differentiation and lateral roots were embedded into a fungal mass. Swelling structures such as appressoria were observed at 1 week postinoculation. At 2 weeks postinoculation and onward, the pathogen had colonized xylem vessels in roots, followed by the stem and, finally, leaves during the symptomatic stage. Additionally, observations of infected plants after retting in the field revealed microsclerotia embedded inside the bast fiber bundle, thus potentially contributing to weakening of fiber. All of these results provide a global account of V. dahliae development when infecting fiber flax.

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A Gene in the Pseudomonas syringae pv. tomato Hrp Pathogenicity Island Conserved Effector Locus, hopPtoA1, Contributes to Efficient Formation of Bacterial Colonies in Planta and Is Duplicated Elsewhere in the Genome

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.10.1014 ◽

2002 ◽

Vol 15 (10) ◽

pp. 1014-1024 ◽

Cited By ~ 33

Author(s):

J. L. Badel ◽

A. O. Charkowski ◽

W.-L. Deng ◽

A. Collmer

Keyword(s):

Pseudomonas Syringae ◽

Laser Scanning ◽

Fluorescent Protein ◽

Pathogenicity Island ◽

Effector Proteins ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

In Planta ◽

Tomato Dc3000 ◽

Green Fluorescent

The ability of Pseudomonas syringae to grow in planta is thought to be dependent upon the Hrp (type III secretion) system and multiple effector proteins that this system injects into plant cells. ORF5 in the conserved effector locus of the P. syringae pv. tomato DC3000 Hrp pathogenicity island was shown to encode a Hrp-secreted protein and to have a similarly secreted homolog encoded in an effector-rich pathogenicity island located elsewhere in the genome. These putative effector genes were designated hopPtoA1 and hopPtoA2, respectively. DNA gel blot analysis revealed that sequences hybridizing with hopPtoA1 were widespread among P. syringae pathovars, and some strains, like DC3000, appear to have two copies of the gene. uidA transcriptional fusions revealed that expression of hopPtoA1 and hopPtoA2 can be activated by the HrpL alternative sigma factor. hopPtoA1 and hopPtoA1/hopPtoA2 double mutants were not obviously different from wild-type P. syringae pv. tomato DC3000 in their ability to produce symptoms or to increase their total population size in host tomato and Arabidopsis leaves. However, confocal laser-scanning microscopy of GFP (green fluorescent protein)-labeled bacteria in Arabidopsis leaves 2 days after inoculation revealed that the frequency of undeveloped individual colonies was higher in the hopPtoA1 mutant and even higher in the hopPtoA1/hopPtoA2 double mutant. These results suggest that hopPtoA1 and hopPtoA2 contribute redundantly to the formation of P. syringae pv. tomato DC3000 colonies in Arabidopsis leaves.

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ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology

10.1101/153163 ◽

2017 ◽

Author(s):

Valentina De Col ◽

Philippe Fuchs ◽

Thomas Nietzel ◽

Marlene Elsässer ◽

Chia Pao Voon ◽

...

Keyword(s):

Laser Scanning ◽

Fluorescent Protein ◽

Research Training ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

In Planta ◽

Science Center ◽

Living Plant

AbstractGrowth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here we establish live MgATP2− assessment in plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2− changes in planta. A MgATP2− map of the Arabidopsis seedling highlights different MgATP2− concentrations between tissues and in individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.One-sentence SummarySensing of MgATP2− by fluorimetry and microscopy allows dissection of ATP fluxes of isolated organelles, and dynamics of cytosolic MgATP2−in vivo.Funding AgenciesThis work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Emmy-Noether programme (SCHW1719/1-1; M.S. and GR4251/1-1; C.G.), the Research Training Group GRK 2064 (M.S.; A.J.M.), the Priority Program SPP1710 (A.J.M.) and a grant (SCHW1719/5-1; M.S.) as part of the package PAK918. The Seed Fund grant CoSens from the Bioeconomy Science Center, NRW (A.J.M.; M.S.) is gratefully acknowledged. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-002 13). A.Co. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca through the FIRB 2010 programme (RBFR10S1LJ_001) and Piano di Sviluppo di Ateneo 2015 (Università degli Studi di Milano). M.Z. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca (Italy) through the PRIN 2010 programme (PRIN2010CSJX4F). S.W. and T.N. received travel support by the Deutscher Akademischer Austauschdienst (DAAD). V.D.C. was supported by the European Social Fund, Operational Programme 2007/2013, and an Erasmus+ Traineeship grant. M.D.F was supported by The Human Frontier Science Program (RPG0053/2012), and the Leverhulme Foundation (RPG-2015-437). I.M.M. was supported by a grant from the Danish Council for Independent Research - Natural Sciences. V.C.P. was supported by the Innovation and Technology Fund (Funding Support to Partner State Key Laboratories in Hong Kong) of the HKSAR.AbbreviationsAAC – ADP/ATP carrier; AK – adenylate kinase; cAT – carboxyatractyloside; CCCP – carbonyl cyanide m-chlorophenyl hydrazone; CFP – cyan fluorescent protein; CLSM – confocal laser scanning microscopy; ETC – electron transport chain; FRET – Förster Resonance Energy Transfer; LSFM – light sheet fluorescence microscopy.

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Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

Microorganisms ◽

10.3390/microorganisms9020295 ◽

2021 ◽

Vol 9 (2) ◽

pp. 295

Author(s):

Yvann Bourigault ◽

Andrea Chane ◽

Corinne Barbey ◽

Sylwia Jafra ◽

Robert Czajkowski ◽

...

Keyword(s):

Laser Scanning ◽

Fluorescent Protein ◽

Infected Plant ◽

Soft Rot ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Cell Physiology ◽

In Planta ◽

Reporter System ◽

Cell Wall Lysis

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

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Potential role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress

Journal of Cell Science ◽

10.1242/jcs.114.9.1643 ◽

2001 ◽

Vol 114 (9) ◽

pp. 1643-1653 ◽

Cited By ~ 5

Author(s):

Z. Dastoor ◽

J.L. Dreyer

Keyword(s):

Oxidative Stress ◽

Laser Scanning ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Apoptotic Cells ◽

Transfected Cells ◽

And Oxidative Stress

Recent studies indicating a role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in apoptosis or oxidative stress has been reported. Using confocal laser-scanning microscopy, we have investigated the cellular distribution of GAPDH in central nervous system (CNS)-derived cells (neuroblastoma mNB41A3), in non-CNS derived cells (R6 fibroblast) and in an apoptosis-resistant Bcl2 overexpressing cell line (R6-Bcl2). Induction of apoptosis by staurosporine or MG132 and oxidative stress by H(2)O(2) or FeCN enhanced the nuclear translocation of endogenous GAPDH in all cell types, as detected by immunocytochemistry. In apoptotic cells, GAPDH expression is three times higher than in non-apoptotic cells. Consistent with a role for GAPDH in apoptosis, overexpression of a GAPDH-green fluorescent protein (GAPDH-GFP) hybrid increased nuclear import of GAPDH-GFP into transfected cells and the number of apoptotic cells, and made them more sensitive to agents that induce apoptosis. Bcl2 overexpression prevents nuclear translocation of GAPDH and apoptosis in untransfected cells, but not in transfected cells that overexpress GAPDH-GFP. Our observations indicate that nuclear translocation of GAPDH may play a role in apoptosis and oxidative stress, probably related to the activity of GAPDH as a DNA repair enzyme or as a nuclear carrier for pro-apoptotic molecules.

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Postharvest Handling Conditions Affect Internalization of Salmonella in Baby Spinach during Washing

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-539 ◽

2013 ◽

Vol 76 (7) ◽

pp. 1145-1151 ◽

Cited By ~ 13

Author(s):

VICENTE M. GÓMEZ-LÓPEZ ◽

ALICIA MARÍN ◽

ANA ALLENDE ◽

LARRY R. BEUCHAT ◽

MARÍA I. GIL

Keyword(s):

Green Fluorescent Protein ◽

Laser Scanning ◽

Fluorescent Protein ◽

Negative Temperature ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Temperature Differential ◽

Postharvest Handling ◽

Green Fluorescent ◽

Spinach Leaves

Internalization of foodborne pathogens in fruits and vegetables is an increasing safety concern. The aim of this research was to assess the potential for internalization of an enteric pathogen (Salmonella enterica serotype Typhimurium) in a leafy vegetable (baby spinach) during washing as influenced by three postharvest handling conditions: (i) illumination, (ii) negative temperature differential, and (iii) relative humidity (RH). To compare these potential postharvest handling conditions, leaves were exposed to different levels of illumination (0, 1,000, and 2,000 lx), temperature differential (5, 11, 14, 20, and 26uC), and RH (99, 85, and 74%) for a short time before or during washing. Washing of baby spinach was carried out in water containing green fluorescent protein–tagged Salmonella Typhimurium (6.5 log CFU/ml) at 5uC for 2 min, followed by surface disinfection with chlorine (10,000 μg/ml) for 1 min, two rinses in water for 10 s, and spin drying for 15 s. Internalization was assessed by enumerating the pathogen on Salmonella-Shigella agar and by confocal laser scanning microscopy. Illumination of spinach leaves before and during washing and a negative temperature differential during washing did not significantly (P > 0.05) increase the number of internalized bacteria. However, exposure of leaves to low-RH conditions before washing, which reduced the tissue water content, decreased internalization of Salmonella compared with internalization in baby spinach exposed to high RH (P ≤ 0.05). Green fluorescent protein–tagged Salmonella Typhimurium was visualized by confocal laser scanning microscopy at a depth of up to 30 μm beneath the surface of spinach leaves after exposure to a high inoculum level (8 log CFU/ml) for an extended time (2 h). Results show that internalization of Salmonella into baby spinach leaves can occur but can be minimized under specific postharvest handling conditions such as low RH.

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Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

The Journal of Cell Biology ◽

10.1083/jcb.136.1.71 ◽

1997 ◽

Vol 136 (1) ◽

pp. 71-80 ◽

Cited By ~ 112

Author(s):

Erik A.C. Wiemer ◽

Thibaut Wenzel ◽

Thomas J. Deerinck ◽

Mark H. Ellisman ◽

Suresh Subramani

Keyword(s):

Mammalian Cells ◽

Laser Scanning ◽

Fluorescent Protein ◽

Time Lapse ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Small Subset ◽

Subcellular Compartment ◽

Directional Movement ◽

Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

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Calpurnia aurea (Aiton) Benth Extracts Reduce Quorum Sensing Controlled Virulence Factors in Pseudomonas aeruginosa

Molecules ◽

10.3390/molecules25102283 ◽

2020 ◽

Vol 25 (10) ◽

pp. 2283

Author(s):

Sekelwa Cosa ◽

Jostina R. Rakoma ◽

Abdullahi A. Yusuf ◽

Thilivhali E. Tshikalange

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Docking ◽

Quorum Sensing ◽

Virulence Factors ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Promising Alternative ◽

The Individual

Pseudomonas aeruginosa is the causative agent of several life-threatening human infections. Like many other pathogens, P. aeruginosa exhibits quorum sensing (QS) controlled virulence factors such as biofilm during disease progression, complicating treatment with conventional antibiotics. Thus, impeding the pathogen’s QS circuit appears as a promising alternative strategy to overcome pseudomonas infections. In the present study, Calpurnia aurea were evaluated for their antibacterial (minimum inhibitory concentrations (MIC)), anti-quorum sensing/antivirulence (AQS), and antibiofilm potential against P. aeruginosa. AQS and antivirulence (biofilm formation, swimming, and swarming motility) activities of plant extracts were evaluated against Chromobacterium violaceum and P. aeruginosa, respectively. The in vitro AQS potential of the individual compounds were validated using in silico molecular docking. Acetone and ethanolic extracts of C. aurea showed MIC at 1.56 mg/mL. The quantitative violacein inhibition (AQS) assay showed ethyl acetate extracts as the most potent at a concentration of 1 mg/mL. GCMS analysis of C. aurea revealed 17 compounds; four (pentadecanol, dimethyl terephthalate, terephthalic acid, and methyl mannose) showed potential AQS through molecular docking against the CviR protein of C. violaceum. Biofilm of P. aeruginosa was significantly inhibited by ≥60% using 1-mg/mL extract of C. aurea. Confocal laser scanning microscopy correlated the findings of crystal violet assay with the extracts significantly altering the swimming motility. C. aurea extracts reduced the virulence of pseudomonas, albeit in a strain- and extract-specific manner, showing their suitability for the identification of lead compounds with QS inhibitory potential for the control of P. aeruginosa infections.

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Migration of Salmonella Enteritidis Phage Type 30 through Almond Hulls and Shells

Journal of Food Protection ◽

10.4315/0362-028x-71.2.397 ◽

2008 ◽

Vol 71 (2) ◽

pp. 397-401 ◽

Cited By ~ 29

Author(s):

MICHELLE D. DANYLUK ◽

MARIA T. BRANDL ◽

LINDA J. HARRIS

Keyword(s):

Green Fluorescent Protein ◽

Laser Scanning ◽

Salmonella Enteritidis ◽

Fluorescent Protein ◽

Phage Type ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Aqueous Environment ◽

Serovar Typhimurium ◽

Green Fluorescent

The ability of Salmonella to migrate from an external aqueous environment through the almond hull and shell, and to colonize the kernel, was evaluated in two ways. First, the outer surface of shell halves from five varieties of almonds that differed in shell hardness were placed in contact with a suspension of Salmonella enterica serovar Enteritidis phage type 30 for 24hat24°C. Salmonella Enteritidis was isolated from the inside of these almond shells in 46 and 100% of the samples, by direct swabbing of the inner surface of the shell and by enrichment from the swab, respectively. These findings suggested that hardness of the shell is not a significant factor in the migration of the pathogen through that tissue. In addition, both motile and nonmotile strains of S. enterica serovar Typhimurium migrated through the almond shells to the same extent under the conditions of this assay, indicating that bacterial migration through the wet shell may be a passive process. Second, whole almonds (intact hull, shell, and kernel) were soaked for 24 to 72 h at 24°C in a suspension of Salmonella Enteritidis phage type 30 labeled with the green fluorescent protein. Green fluorescent protein–labeled Salmonella cells were observed on the outer and inner surfaces of both the almond hull and shell, and on the kernel, by confocal laser scanning microscopy. Our data provide direct evidence that wet conditions allow for Salmonella migration through the hull and shell and onto the almond kernel, thus providing a means by which almond kernels may become contaminated in the field.

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Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation

Journal of Medical Microbiology ◽

10.1099/jmm.0.45539-0 ◽

2004 ◽

Vol 53 (7) ◽

pp. 679-690 ◽

Cited By ~ 98

Author(s):

Andres Plata Stapper ◽

Giri Narasimhan ◽

Dennis E. Ohman ◽

Johnny Barakat ◽

Morten Hentzer ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Laser Scanning ◽

Fluorescent Protein ◽

Cell System ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Alginate Production ◽

Green Fluorescent ◽

Biofilm Development

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+ PAOmucA22 and Alg− PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (bip) and Community Statistics (comstat) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

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