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…

The moment of preharvest elicitor application influence its final effect on winegrapes quality

Phenolic compounds are secondary metabolites of grapes. Plants produce a wide variety of this type of metabolites through diverse biosynthesis pathways and their production is sometimes a response to external stimuli, either environmental or biotic stresses. Some of them may act as chemical defenses against pathogens or herbivores and their synthesis is increased when the attack exists. However, it is remarkable that the synthesis of these interesting compounds can be activated even when the stimulus is not present, with the use of elicitors. These are substances that when applied exogenously trigger the biosynthetic pathways conducting to the synthesis of these defense compounds.

Les outils pour favoriser le renouvellement des générations en viticulture

French lawmakers have chosen the family-type winegrowing business as the benchmark for drafting the legal framework for winegrowing businesses and winegrowers. In france (source: msa), in 2022, there were 1,444 new winegrowers, an increase of 3% compared with 2021, representing 10% of new farm managers. The retention rate for winegrowers is 75% (up 13% on 2021), compared with 77% for all agricultural sectors (stable).

Rationalizing The Wine Nucleophilic Competition For Quinone Addition

loss and color browning which lead to wine unacceptance by consumers. These changes are mainly driven by the consumption of oxygen by polyphenols leading to the production of quinones which are oxidant compounds. Quinones can react with numerous nucleophilic compounds notably aromatic thiols, decreasing the aromatic bouquet of the wine.

PROFILING OF LIPIDS IN WINES FROM MONOCULTURE FERMENTATION WITH INDIGENOUS METSCHNIKOWIA YEAST SPECIES

Lipids are a diverse group of organic compounds essential for living systems. They are vital compounds for yeast which makes them an important modulator of yeast metabolism in alcoholic fermentation. This study presents a comprehensive lipidome analysis of wine samples from the Vitis vinifera L., Maraština. The fermentation trails were set up in monoculture with different indigenous yeast strains selected from a collection of native yeasts established at the Institute for Adriatic Crops and Karst Reclamation in 2021, previously isolated from Croatian Maraština grapes: Metschnikowia pulcherrima, Metshnikowia sinensis/shanxiensis , and Metschnikowia chyrsoperlae.

Aroma accumulation trends during berry development and selection of grape aroma candidate genes suitable for functional characterization

Grape flavour management in the vineyard requires knowledge of the derivation of individual flavour and aroma characteristics and the effects that different concentrations and interactions between these compounds have on flavour potential.