Metabolic activity in filamentous fungi can be analysed by flow cytometry

Bradner, J.R.; Nevalainen, Helena

doi:10.1016/s0167-7012(03)00043-5

Cited by 43 publications

(18 citation statements)

References 19 publications

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“…Hence, a fluorescence assay with DHE was performed (Figure 3). Interaction of cytosolic DHE when oxidized by ROS (singlet oxygen, hydroxyl radicals, superoxide, peroxides and hydro peroxides) produces ethidium, which intercalates with DNA and emits bright red fluorescence at 605 nm [48]. Induction of oxidative stress after FA and H 2 O 2 detected by DHE was observed (Figure 3).…”

Section: Discussionmentioning

confidence: 91%

Antifungal Potential of Terpenes from Spondias Purpurea L. Leaf Extract against Moniliophthora Perniciosa that causes Witches Broom Disease of Theobroma Cacao

Pungartnik¹

2017

IJCAM

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The present in vitro study evaluates antifungal properties of Spondias purpurea plant extract against Moniliophthora perniciosa. Fractions of Ethanol Crude Extracts (CE) from S. purpurea were tested for their antifungal activity. CE inhibited mycelial growth of M. perniciosa by 60%at a dose of 10 mg/mL, while survival of broken hyphae of the fungus was inactivated by 90% at a dose of 8 mg/mL. Fractionation of CE yielded a terpene containing sub fraction (FA) with the highest fungicidal activity. FA acted as oxidant and its application induced oxidative stress in M. perniciosa that may be the responsible for the observed induced cell death. The chromatographic profile obtained with FA of S. purpurea revealed a variety of terpenes, which included Spathulenol (14.2%), Linolenic acid (8.4%), trans Caryophyllene (6.9%), and Alpha-muurolene (6.9%).This study describes for the first time effects of terpenes of S. purpurea related to induced oxidative stress in fungus. S. purpurea, therefore, may be a natural source for isolation of antifungal compounds against M. perniciosa. This characteristic may be useful in application as botanical fungicide in biological control.

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Section: Discussionmentioning

confidence: 91%

Antifungal Potential of Terpenes from Spondias Purpurea L. Leaf Extract against Moniliophthora Perniciosa that causes Witches Broom Disease of Theobroma Cacao

Pungartnik¹

2017

IJCAM

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“…Then, the M. perniciosa hyphae pretreated with the TcPR-4b protein were incubated with 10 mM of dihydroethidium (DHE; Sigma) at 25°C for 30 min. The chemical reaction involved the DHE reaction with O 2 - , forming ethidium that intercalated with DNA and then emitted red fluorescence [93-95]. The hyphae were mounted on slides and observed under fluorescence microscope BX51 (Olympus).…”

Section: Methodsmentioning

confidence: 99%

The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

et al. 2014

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BackgroundThe production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b.ResultsTcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae.ConclusionTo our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively.

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“…Reports also indicate that HE can be oxidized by other oxidants, including ONOO - and the Fenton's reagent, forming a fluorescent product, suggesting that HE is not specific for detecting the superoxide radical anion [50]. These reports concluded that HE is a non-specific probe for ROS [51,52]. However, further research, as outlined below, indicated that this appears not to be the case.…”

Section: Evolution Of He From a Vital Dye To Superoxide Probementioning

confidence: 99%

Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: Another inconvenient truth

Zielonka

Kalyanaraman

2010

Free Radical Biology and Medicine

454

399

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Hydroethidine (or dihydroethidium) (HE) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E+). In biological systems, another red fluorescent product, ethidium (E+), is also formed, usually at a much higher concentration than 2-OH-E+. In this article, we have reviewed the methods to selectively detect the superoxide-specific product (2-OH-E+) and the factors affecting its levels in cellular and biological systems. The most important conclusion of the present review is that it is nearly impossible to assess the intracellular levels of the superoxide specific product, 2-OH-E+, using the confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E+, in order to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX Red ®) with superoxide is similar to the reactivity of HE with superoxide and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or Mito-SOX) as well.

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Metabolic activity in filamentous fungi can be analysed by flow cytometry

Cited by 43 publications

References 19 publications

Antifungal Potential of Terpenes from Spondias Purpurea L. Leaf Extract against Moniliophthora Perniciosa that causes Witches Broom Disease of Theobroma Cacao

Antifungal Potential of Terpenes from Spondias Purpurea L. Leaf Extract against Moniliophthora Perniciosa that causes Witches Broom Disease of Theobroma Cacao

The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: Another inconvenient truth

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