Reviewer
Priyanka Das
  • Bose Institute
Research fields
  • Plant Science
A Novel Method for Measuring Mitochondrial Respiratory Parameters in Wheat Paleae (Paleae Superior) Using the XF24 Analyzer
Authors:  Daniel Schniertshauer and Jörg Bergemann, date: 08/05/2023, view: 249, Q&A: 0

Understanding the influence of secondary metabolites from fungi on the mitochondria of the host plant during infection is of great importance for the knowledge of fungus–plant interactions in general; it could help generate resistant plants in the future and in the development of specifically acting plant protection products. For this purpose, it must first be possible to record the mitochondrial parameters in the host plant. As of the date of this protocol, no measurements of mitochondrial respiration parameters have been performed in wheat paleae. The protocol shown here describes the measurements using the XF24 analyzer, which measures the rate of oxygen consumption in the sample by changes in the fluorescence of solid-state fluorophores. This procedure covers the preparation of samples for the XF24 analyzer and the measurement of mitochondrial parameters by adding specific mitochondrial inhibitors. It also shows the necessary approach and steps to be followed to obtain reliable, reproducible results. This is a robust protocol that allows the analysis of mitochondrial respiration directly in the wheat paleae. It demonstrates an important add-on method to existing screenings and also offers the possibility to test the effects of early infection of plants by harmful fungi (e.g., Fusarium graminearum) on mitochondrial respiration parameters.


Key features

• This protocol offers the possibility of testing the effects of early infection of plants by pathogens on mitochondrial respiration parameters.

• This protocol requires a Seahorse XF24 Flux Analyzer with Islet Capture Microplates and the Seahorse Capture Screen Insert Tool.


Graphical overview


PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System
Authors:  Naim Hassan, Farhana Easmin, Keisuke Ekino and Satoshi Harashima, date: 07/05/2021, view: 3477, Q&A: 0

Nowadays, CRISPR (clustered regularly interspaced short palindromic repeats) and the CRISPR-associated protein (Cas9) system play a major role in genome editing. To target the desired sequence of the genome successfully, guide RNA (gRNA) is indispensable for the CRISPR/Cas9 system. To express gRNA, a plasmid expressing the gRNA sequence is typically constructed; however, construction of plasmids involves much time and labor. In this study, we propose a novel procedure to express gRNA via a much simpler method that we call gRNA-TES (gRNA-transient expression system). This method employs only PCR, and all the steps including PCR and yeast transformation can be completed within 1 day. In comparison with the plasmid-based gRNA delivery system, the performance of gRNA-TES is more effective, and its total time and cost are significantly reduced.

Transient Expression Assay in Strawberry Fruits
Authors:  Mengting Pi, Qi Gao and Chunying Kang, date: 06/05/2019, view: 6995, Q&A: 0
Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, virus-induced gene silencing (VIGS) and Agrobacterium tumefaciens-mediated transformation in fruit have been widely used as the transient expression approaches. Unlike VIGS, the latter one can be utilized not only for gene knock-down, but also for overexpression and knock-out. Here, we show the procedures of transiently expressing the 35S::FveMYB10 construct into fruit of the white-fruited F. vesca accession Yellow Wonder. As a master regulator of anthocyanin production, overexpressing FveMYB10 will cause fruit coloration, which was observed at one week post infiltration. We also exhibit the previous results of knocking down Reduced Anthocyanin in Petioles (RAP), encoding an anthocyanin transporter, by RNAi in fruit of the strawberry cultivar ‘Sweet Charlie’. Overall, Agrobacterium-mediated transient transformation in strawberry fruit is a quick and versatile approach for studying gene functions in fruit ripening.
Knock-in Blunt Ligation Utilizing CRISPR/Cas9
Authors:  Jonathan M. Geisinger and Michele P. Calos, date: 03/05/2017, view: 9769, Q&A: 0
The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.
Antifungal and Zearalenone Inhibitory Activity of Ocimum sanctum L. Essential Oil on Fusarium graminearum Determined by UHPLC and RT-qPCR
Fusarium graminearum has been given special attention in the context of agricultural commodities due to its ability to grow in diverse climatic conditions, and to produce different mycotoxins including zearalenone (ZEA) and type-B trichothecenes, which cause ill health effects on humans, animals and plants. The application of synthetic antifungal agents for the control of F. graminearum result in negative health impacts in livestock and humans and the upsurge of resistant organisms as well. Therefore, there is a need to propose proper food grain management practices, including the application of herbal antifungal and mycotoxin controlling agents, to reduce the growth of toxigenic F. graminearum as well as the production of ZEA in agricultural commodities. Ocimum sanctum also known as Holy Basil or Tulsi is widely used as a medicinal plant in Ayurveda. The current protocol demonstrates to quantify the antifungal activity of O. sanctum L. essential oil (OSEO) as reflected by the decreased F. graminearum growth and ZEA production. Antifungal activities of OSEO are carried out by micro well dilution method and further validated quantitatively by scanning electron microscopic methods. Effects of OSEO on ZEA production is analysed by Quantitative reverse transcription PCR (RT-qPCR) and Ultra high performance liquid chromatography (UHPLC) methods from a broth culture of F. graminearum. Anti-mycotoxic efficacy of OSEO is assessed directly on F. graminearum inoculated maize grains. The protocol efficiently assessed the activity of OSEO as an herbal antagonistic agent against fungal infestation and ZEA production by F. graminearum. The protocol can be used to test a wide variety of herbal compounds for antifungal activity against F. graminearum or with modifications on other mycotoxigenic fungi, an important intervention in food safety and processing industries where the fungal infestation is a major concern.
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