terclim by ICS banner
IVES 9 IVES Conference Series 9 Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Abstract

Cis-acting regulatory elements are DNA sequences that can be bound by transcription factors to regulate the expression of genes in a condition-dependent and tissue-specific way. It is nowadays possible to search for DNA motives and sequences that a given transcription factor is binding or at least can, but it is still hard to have a glance at all the transcription factors that are contemporaneously located at the same locus. Inspired by an existing technique that uses the CRISPR-Cas system in mammal cells, we are trying to develop a protocol to study such regulation in Vitis vinifera. Using the highly sequence-specific binding capacity of a catalytically inactive Cas9 protein (dCas9), our idea is to set up a system to target a desired sequence and precipitate all the crosslinked proteins and distantly interacting chromatin at this locus and analyze them. After conducting preliminary assays on protoplast system, we got introduced to the CRISPR-FISH technique, that uses dCas9 and a fluorescent guide to label telomeres on nuclei directly isolated from a small quantity of fixed leaves with a very simple procedure. We used part of such technique to easily obtain the chromatin that was needed for our trials and eventually performed the pull-down of the targeted DNA sequences directly on these nuclei after transforming them with the dCas9 complex. Sequencing the obtained fragments allows to verify the specificity of the tool. Far from having eliminated the idea of using protoplasts as model system, we proceed developing both approaches in parallel.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Poster

Authors

Aurélien Devillars1*, Silvia Farinati1, Edoardo Bertini2, Chiara Fattorini2, Sara Lissandrini2, Adriana Fernanda Soria Garcia1, Bhanu Prakash Potlapalli3, Andreas Houben3, Sara Zenoni2 and Alessandro Vannozzi1

1 Department of Agriculture Food Natural Resources Animals and Environment, University of Padova, Agripolis, Legnaro, Italy
2 Department of Biotechnology, University of Verona, Verona, Italy
3 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany

Contact the author*

Keywords

Molecular Biology, Grapevine, Gene Regulation, CRISPR-Cas9, Protoplasts

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

YEAST-PRODUCED VOLATILES IN GRAPE BASED SYSTEM MODEL ACTING AS ANTIFUNGAL BIOAGENTS AGAINST PHYTOPATHOGEN BOTRYTIS CINEREA

Botrytis cinerea Pers., the causal agent of grey mould disease, is responsible for substantial economic losses, as it causes reduction of grape and wine quality and quantity. Exploitation of antagonistic yeasts is a promising strategy for controlling grey mould incidence and limiting the usage of synthetic fungicides. In our previous studies, 119 different indigenous yeasts were screened for putative multidimensional modes of action against filamentous fungus B. cinerea [1]. The most promissing biocontrol yeast was Pichia guilliermondii ZIM624, which exhibited several anatagonistic traits (production of cell wall degrading enzymes, chitinase and β-1,3-glucanase; demonstration of in vitro inhibitory effect on B. cinerea mycelia radial growth; production of antifungal volatiles, assimilation of a broad diversity of carbon sources, contributing to its competitivnes in inhabiting grapes in nature).

Impact of heating must before fermentation on Chardonnay wines

Prefermentation steps of white winemaking are very important for controlling the stability and the sensory attributes of wines. Usually musts are clarified by cold settling to prevent the start of the fermentation, before racking big lees and thus limiting the appearance of vegetable or reduction off flavour while favouring an aromatic expression with low turbidity. Besides, to reach the protein stability, some white wines further require a bentonite fining, sometimes associated with negative effects on the sensory quality. This study aims to know the impact of musts heating after pressing on a Chardonnay wine in northern conditions by comparison with a classic cold racking of the must.

Using elicitors in different grape varieties. Effect over their phenolic composition

Phenolic compounds are very important in crop plants and have been the subject of a large number of studies. Three main reasons can be cited for optimizing the level of phenolic compounds in crop plants: their physiological role in plants, their technological significance for food processing, and their nutritional characteristics1 Indeed, an enormous diversity of phenolic antioxidants is found in fruits and vegetables, and their presence and roles can be affected or modified by several pre- and postharvest cultural practices and/or food processing technologies (Ruiz-García et al. 2012, Goldman et al. 1999, Tudela et al. 2002). In winegrapes, the technological importance of phenolic compounds, mainly flavonoids, is well-known.

BORDEAUX RED WINES WITHOUT ADDED SULFITES SPECIFICITIES: COMPOSITIONAL AND SENSORY APPROACHES TOWARDS HIGHLIGHTING AND EXPLAI-NING THEIR SPECIFIC FRUITINESS AND COOLNESS

With the development of naturality expectations, wines produced without any addition of sulfur dioxide (SO₂) become very popular for consumers and such wines are increasingly present on the market. Recent studies also showed that Bordeaux red wines without added SO₂ could be differentiated from a sensory point of view from similar wines produced with SO₂¹. Thus, the aim of the current study was to characterize from a sensory point of view, specific aromas of wines without added SO₂ and to identify compounds involved.

Conventional and alternative pest management strategies: a comparative proteomic study on musts

In a context of sustainable agriculture, “agroecological immunity” is an emerging concept to reduce the use of chemical pesticides to protect crops against pathogens. This alternative strategy aims to combine different levers including the use of “bio”solutions. These include biocontrol products, some of which being plant defense elicitors, as well as products authorized in organic farming such as copper or sulfur. In vineyards, depending on climate conditions, powdery and downy mildews can be devastating diseases.