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

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.

scholarly journals Intracellular Macromolecular Mobility Measured by Fluorescence Recovery after Photobleaching with Confocal Laser Scanning Microscopes

2004 ◽  
Vol 15 (10) ◽  
pp. 4749-4760 ◽  
Author(s):  
José Braga ◽  
Joana M.P. Desterro ◽  
Maria Carmo-Fonseca
Keyword(s):  
Aqueous Solution ◽  
Diffusion Coefficients ◽  
Laser Scanning ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Confocal Laser Scanning ◽  
Fluorescence Recovery ◽  
Confocal Laser ◽  
Wide Range ◽  
Green Fluorescent

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22°C reduces the dextran diffusion rates by ∼30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.

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

2012 ◽  
Vol 102 (1) ◽  
pp. 23-31 ◽  
Author(s):  
L. Chalupowicz ◽  
E.-M. Zellermann ◽  
M. Fluegel ◽  
O. Dror ◽  
R. Eichenlaub ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wall Thickening ◽  
Apical Region ◽  
In Planta ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp

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.

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

Plant Disease ◽  
2018 ◽  
Vol 102 (12) ◽  
pp. 2421-2429 ◽  
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.

scholarly journals Novel Aspects of Tomato Root Colonization and Infection by Fusarium oxysporum f. sp. radicis-lycopersici Revealed by Confocal Laser Scanning Microscopic Analysis Using the Green Fluorescent Protein as a Marker

2002 ◽  
Vol 15 (2) ◽  
pp. 172-179 ◽  
Author(s):  
Anastasia L. Lagopodi ◽  
Arthur F. J. Ram ◽  
Gerda E. M. Lamers ◽  
Peter J. Punt ◽  
Cees A. M. J. J. Van den Hondel ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusarium Oxysporum ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Microscopic Analysis ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Tomato Root ◽  
Green Fluorescent

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F. oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.

scholarly journals 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

2002 ◽  
Vol 15 (10) ◽  
pp. 1014-1024 ◽  
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.

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

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.

scholarly journals Biocontrol of Soft Rot: Confocal Microscopy Highlights Virulent Pectobacterial Communication and Its Jamming by Rhodococcal Quorum-Quenching

2019 ◽  
Vol 32 (7) ◽  
pp. 802-812 ◽  
Author(s):  
Andrea Chane ◽  
Corinne Barbey ◽  
Magalie Robert ◽  
Annabelle Merieau ◽  
Yoan Konto-Ghiorghi ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Rhodococcus Erythropolis ◽  
Soft Rot ◽  
Quorum Quenching ◽  
Laser Scanning Microscopy ◽  
Green Fluorescent Protein Gene ◽  
Confocal Laser ◽  
Plant Colonization

Confocal laser-scanning microscopy was chosen to observe the colonization and damage caused by the soft rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection, or degradation. The dual-colored pathogen and biocontrol strains were used to coinoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls, as well as a concomitant rhodococcal quorum-quenching response, at both the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.

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