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…

Possible toxicological risk arising from contamination of grapes and derivatives by emerging mycotoxins: patulin

Following the acquired awareness of the presence of ochratoxin A in grape derivatives, actions were undertaken to contain this contamination, and attempts were made to evaluate the presence of any other molecule belonging to this class.

Read More

Identification of arbuscular mycorrhizal fungi species preferentially associated with grapevine roots inoculated with commercial bioinoculants 

Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with plant roots and can help plants acquire nutrients from the soil in exchange for photosynthetic carbon. Commercial bioinoculants containing AMF are widely available and represent a potential opportunity to reduce the dependence of grapevines on agrochemicals. However, which commercially available AMF species colonize vine roots and affect vine growth remains unknown. The aim of this study was to identify the AMF species from commercial bioinoculants that colonize grapevine roots using high-throughput sequencing, and to evaluate the performance of five commercial bioinoculants and their effects on own-rooted Cabernet sauvignon.

Read More

Modelling grape and wine quality through PLS Spline statistical method

Started in 1994, this project intends to explain quality of grapes and wines using data of soil, climate and vineyard that are currently used in field trials.

Read More

Metatranscriptomic analysis of “aszú” berries: the potential role of the most important species of the grape microbiota in the aroma of wines with noble rot

Botrytis cinerea has more than 1200 host plants and is one of the most important plant pathogens in viticulture. Under certain environmental conditions, it can lead to the development of a noble rot, which results in a specific metabolic profile, altering physical texture and chemical composition. The other microbes involved in this process and their functional genes are poorly characterised. We have generated metatranscriptomic [1,2] and DNA metabarcoding data from three months of the Furmint grape variety, representing the four phases of noble rot, from healthy berries to completely dried berries.

Read More

Response of different nitrogen supplementation on Saccharomyces cerevisiae metabolic response and wine aromatic profile

The wine yeast Saccharomyces cerevisiae can highly affect wine aromatic profile by producing and/or mediating the release of a whole range of metabolites (such as thiols, esters, and terpenes), which in turn contribute to enhanced aroma and flavor. These metabolites depend on yeast metabolism activated during fermentation which can constitute the ‘’metabolic footprint’’ of the yeast strain that carried out the process.

Read More