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.

Download the abstract

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…

A procedure for the zoning of grapevine in a hilly area (Collio, North-Eastern Italy) using simulation models and GIS

The zoning of grapevine in a hilly area should consider the variability of the environmental characteristics due to topography. Since soil and climate data are usually available as point data

Read More

Evaluation of uhph treatment as an alternative to heat treatment prior to the use of proteolytic enzymes on must to achieve protein stability in wine

There are currently enzyme preparations on the market with specific protease activities capable of degrading unstable must proteins and preventing turbidity in white and rosé wines. The main drawback is the need to heat the must at 75ºc for 1-2 minutes to denature the proteins and facilitate enzyme action.

Read More

Grape phylloxera leaf-feeding populations in commercial vineyards – a new biotype ?

Grape Phylloxera (Daktulosphaira vitifoliae Fitch) ordinarily has great difficulty establishing leaf galls on the European Grapevine (VitisviniferaL.). Yet populations of leaf-feeding Phylloxera are increasingly being observed throughout commercial vineyards world-wide. Effective plant protection strategies including quarantine actions are currently missing to fight, grape phylloxera populations in affected vineyards and combat linked negative effects on vines and yield. Contrary to the otherwise mandatory continuous infestation pressure from externally established populations (e.g. from populations developed on rootstock foliage or other interspecific hybrids, these leaf-feeding populations seem to establish themselves annually.

Read More

A multivariate clustering approach for a gis based territorial characterization of the montepulciano d’abruzzo DOCG “Colline Teramane”

The aim of the project was to characterize the Premium Denomination of Guaranteed Origin (DOCG) “Colline Teramane” wine-growing region and to delineate and define homogeneous zones (terroir units) within it, by applying a multivariate clustering approach combined with geomatics.

Read More

Impact of some agronomic practices on grape skins anthocyanin content

Wine colour is the first quality characteristic to be assessed, especially regarding red wines. Anthocyanins are very well known to be the main responsible compounds for red wine colour. Red cultivars can synthesize and accumulate anthocyanins in berry skin to express their colour. However, anthocyanin accumulation is often influenced by a series of factors, such as genetic regulation, phytohormones, environmental conditions and viticultural management.

Read More