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…

Aromatic maturity is a cornerstone of terroir expression in red wine

In this video recording of the IVES science meeting 2023, Stéphanie Marchand (University of Bordeaux, ISVV, INRAE, UMR 1366 OENOLOGIE, Villenave d’Ornon, France) speaks about the aromatic maturity as a cornerstone of terroir expression in red wine. This presentation is based on an original article accessible for free on OENO One.

Agronomic behavior of three grape varieties in different planting density and irrigation treatments

In the O Ribeiro Denomination of Origin, there is a winemaking tradition of growing vines under a high-density plantation framework (8,920 vines/ha) and maintaining its vegetative cycle under rainfed conditions.
Currently, viticulture is advancing to plantation frames in which the density is considered medium (5,555 vines/ha), thus allowing mechanized work to be carried out for vineyard management operations. Although, the application of irrigation applied proportionally to the needs of the vegetative cycle of the vine, is a factor that increasingly helps a good development of the vine compared to the summer period, with increasingly uncertain weather forecasts.

Vineyard Landscape in Vale dos Vinhedos: Pressure and Protection

The region with Origin Indication Vale dos Vinhedos (IPVV) is located in the cities of Bento Gonçalves, Garibaldi and Monte Belo do Sul. It was established in November 2002, through an act of Instituto Nacional de Propriedade Industrial, according to Resolução INPI Nº. 75, of 2000, and Law Nº. 9.279 of 1996. The changes on the grape growing and wine making in the last 15 years, with the expansion of grapevines Vitis viníferas cultivation, the installation of several modern family wine companies, making wine with competitive quality, among other aspects, have enabled the implementation of concepts of Geographical Indications.

Retrospective analysis of our knowledge regarding the genetics of relevant traits for rootstock breeding 

Rootstocks were the first sustainable and environmentally friendly strategy to cope with a major threat for Vitis vinifera cultivation. In addition to providing Phylloxera resistance, they play an important role in protecting against other soil-borne pests, such as nematodes, and in adapting V. vinifera to limiting abiotic conditions. Today viticulture has to adapt to ongoing climate change whilst simultaneously reducing its environmental impact. In this context, rootstocks are a central element in the development of agro-ecological practices that increase adaptive potential with low external inputs. Despite the apparent diversity of the Vitis genus, only few rootstock varieties are used worldwide and most of them have a very narrow genetic background. This means that there is considerable scope to breed new, improved rootstocks to adapt viticulture for the future.

Wine aging : a bottleneck Story ?

The sporadic oxidation of white wines remains an open question, making wine shelf life a subjective debate. Through a multidisciplinary synoptic approach performed as a remarkable case study on aged bottles of white wine, this work unraveled a yet unexplored route for uncontrolled oxidation.