Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi

Nair, Rakesh; Raina, Sheetal; Keshavarz, Tajalli; Kerrigan, Mark J.P.

doi:10.1016/j.funbio.2010.12.012

Cited by 17 publications

(11 citation statements)

References 40 publications

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“…In plants, the apical growth is controlled by ROS-dependent Ca 2+ influx and gradient concentration formation at the apex[ 30 ]. To test whether this mechanism also has specific effects on the establishment of polarized penetration peg formation, we detected the free calcium in the cytoplasm of hyphopodia with Fluo-4AM, an intracellular calcium indicator[ 38 ]. As expected, V592 and complemented strains Vd Δnoxb / VdNoxB and Vd Δpls1/VdPls1 sets up a tip-high Ca 2+ gradient in the hyphopodium upon Fluo-4AM treatment ( Fig 6A ), whereas Vd Δnoxb and Vd Δpls1 fail to accumulate detectable Ca 2+ in the cytoplasm of the hyphopodium ( Fig 6A ).…”

Section: Resultsmentioning

confidence: 99%

Hyphopodium-Specific VdNoxB/VdPls1-Dependent ROS-Ca2+ Signaling Is Required for Plant Infection by Verticillium dahliae

2016

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Verticillium dahliae is a phytopathogenic fungus obligate in root infection. A few hyphopodia differentiate from large numbers of hyphae after conidia germination on the root surface for further infection. However, the molecular features and role of hyphopodia in the pathogenicity of V. dahliae remain elusive. In this study, we found that the VdPls1, a tetraspanin, and the VdNoxB, a catalytic subunit of membrane-bound NADPH oxidases for reactive oxygen species (ROS) production, were specifically expressed in hyphopodia. VdPls1 and VdNoxB highly co-localize with the plasma membrane at the base of hyphopodia, where ROS and penetration pegs are generated. Mutant strains, VdΔnoxb and VdΔpls1, in which VdPls1 and VdNoxB were deleted, respectively, developed defective hyphpodia incapable of producing ROS and penetration pegs. Defective plasma membrane localization of VdNoxB in VdΔpls1 demonstrates that VdPls1 functions as an adaptor protein for the recruitment and activation of the VdNoxB. Furthermore, in VdΔnoxb and VdΔpls1, tip-high Ca2+ accumulation was impaired in hyphopodia, but not in vegetative hyphal tips. Moreover, nuclear targeting of VdCrz1 and activation of calcineurin-Crz1 signaling upon hyphopodium induction in wild-type V. dahliae was impaired in both knockout mutants, indicating that VdPls1/VdNoxB-dependent ROS was specifically required for tip-high Ca2+ elevation in hyphopodia to activate the transcription factor VdCrz1 in the regulation of penetration peg formation. Together with the loss of virulence of VdΔnoxb and VdΔpls1, which are unable to initiate colonization in cotton plants, our data demonstrate that VdNoxB/VdPls1-mediated ROS production activates VdCrz1 signaling through Ca2+ elevation in hyphopodia, infectious structures of V. dahliae, to regulate penetration peg formation during the initial colonization of cotton roots.

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Section: Resultsmentioning

confidence: 99%

Hyphopodium-Specific VdNoxB/VdPls1-Dependent ROS-Ca2+ Signaling Is Required for Plant Infection by Verticillium dahliae

2016

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“…This disparity is in large part due to the lack of robust tools for imaging subcellular Ca 2+ changes over time at the single cell level, which is required to correlate the function and activity of various Ca 2+ signaling proteins with Ca 2+ signatures. Although fluorescent dyes as Ca 2+ indicators have been used in a few fungi (Nair et al, 2011;Read et al, 1992;Silverman-Gavrila and Lew, 2003), their utility is limited due to several technical difficulties and low resolution. The advent of gene cloning has resulted in several types of protein-based Ca 2+ sensors as alternatives to fluorescent dyes (Demaurex, 2005;Knight et al, 1991b;Zhao et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca2+ signatures associated with polarized growth, development, and pathogenesis

Kim

Czymmek

Patel

et al. 2012

Fungal Genetics and Biology

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“…Changes in intracellular calcium were determined using a fluorescence plate (BMG LabTech, Ortenberg, Germany) reader in conjunction with Fluo-4 AM (Invitrogen, Paisley, UK), as previously described. 13 , 21 , 22 Chondrocytes were incubated with 3 µM Fluo-4 AM (Invitrogen) for 30 minutes at 37 °C, media replaced with BPS, and initial calcium measurement recorded prior to inducing a rise in [Ca 2+ ] i by loading 50 µM REV5901 (Cambridge Biosciences, Cambridge, UK) and observing changes in [Ca 2+ ] i for 5 minutes (excitation at 494 nm and emission at 520 nm). All experiments were performed at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Quantification of Changes in Morphology, Mechanotransduction, and Gene Expression in Bovine Articular Chondrocytes in Response to 2-Dimensional Culture Indicates the Existence of a Novel Phenotype

Qusous

Kerrigan

2012

CARTILAGE

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Objective:Matrix-induced autologous chondrocyte implantation (ACI) offers a potential solution for cartilage repair but is currently hindered by loss of the chondrocyte differentiated phenotype. To further our understanding of the mechanism of dedifferentiation, changes in the phenotype in relation to mechanotransduction were recorded in response to monolayer culture.Methods:Bovine cartilage explants were excised and chondrocytes cultured for 9 days (P1), 14 days (P2), and 21 (P3) days. Changes in morphology and regulatory volume increase (RVI; a mechanotransduction response) were determined by the expression of key genes by RT-PCR and confocal microscopy, respectively.Results:A loss of a differentiated phenotype was observed in P1 with a reduction in sphericity and an overall increase in cell volume from 474.7 ± 32.1 µm3 to 725.2 ± 35.6 µm3. Furthermore, the effect of 2-dimensional (2-D) culture-induced dedifferentiation on mechanotransduction was investigated, whereby RVI and Gd3+-sensitive REV5901-induced calcium rise were only observed in 2-D cultured chondrocytes. A significant up-regulation of types I and II collagens and Sox9 was observed in P1 chondrocytes and no further significant change in type I collagen but a return to baseline levels of type II collagen and Sox9 upon further culture.Conclusion:These data indicated the presence of an intermediate, mesodifferentiated phenotype and highlight the importance of mechanotransduction as a marker of the chondrocytic cell type.

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Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi

Cited by 17 publications

References 40 publications

Hyphopodium-Specific VdNoxB/VdPls1-Dependent ROS-Ca2+ Signaling Is Required for Plant Infection by Verticillium dahliae

Hyphopodium-Specific VdNoxB/VdPls1-Dependent ROS-Ca2+ Signaling Is Required for Plant Infection by Verticillium dahliae

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca2+ signatures associated with polarized growth, development, and pathogenesis

Quantification of Changes in Morphology, Mechanotransduction, and Gene Expression in Bovine Articular Chondrocytes in Response to 2-Dimensional Culture Indicates the Existence of a Novel Phenotype

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