terclim by ICS banner
IVES 9 IVES Conference Series 9 Enhancing plant defense: carbon dots for efficient spray-induced gene silencing 

Enhancing plant defense: carbon dots for efficient spray-induced gene silencing 

Abstract

Ectopic RNA application for plant defense faces challenges in tree crops, including size, diffusion, and stability of active compounds such as ribonucleoproteins and nucleic acids. While existing strategies involve expressing dsRNA in transgenic plants targeting pathogens, our research strives to develop a transient RNAi system based on Spray-Induced Gene Silencing (SIGS). This approach aims to circumvent legal barriers and public concerns associated with genetically modified organisms (GMOs). Our strategy integrates SIGS with branched polyethyleneimine-functionalized Carbon Dots (bPEI-CDs) as nanocarriers, effectively addressing unique delivery challenges in plant defense as RNA stability and uptake enhancement. We developed a cost-effective microwave-assisted protocol for the synthesis of 10 nm monodispersed bPEI-CDs. These molecules showed over 50% dsRNA protection against RNase III degradation at a 200:1 (w/w) ratio. We further demonstrated their capability to enhance cellular uptake by successfully delivering fluorolabeled-dsRNAs (Cy3-RNA) complexed with CDs into intact plant tissue-cultured cells. Notably, Cy3-RNA sprayed with CD on the surface of greenhouse grapevine leaves through a low-pressure spray application penetrated stomata cells and neighboring cells. Ultimately, the ability of dsRNA complexed with CD to trigger RNA interference was confirmed using a 21 nucleotide-dsRNA targeting eGFP . Ongoing work quantitatively compares, through qPCR, the reduced expression of GFP on eGFP-expressing transgenic microvine leaves sprayed with CD-complexed and naked siRNA. This innovative approach, leveraging CDs, aims to tackle critical barriers in dsRNA delivery, particularly by addressing cell wall-related limitations in plant. This research marks a pivotal step in optimizing RNA-based grapevine defense strategies, and propelling sustainable viticulture practices forward.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Poster

Authors

Christian Mandelli1*, Laurent G. Deluc1,2

1 Department of Horticulture, Oregon State University, Corvallis, OR, United States
2 Oregon Wine Research Institute, Oregon State University, Corvallis, OR, United States

Contact the author*

Keywords

RNA interference, Spray-Induced Gene Silencing (SIGS), Carbon dots, dsRNA delivery, Sustainable agriculture

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

VineyardFACE: Investigation of a moderate (+20%) increase of ambient CO2 level on berry ripening dynamics and fruit composition

Climate change and rising atmospheric carbon dioxide concentration is a concern for agriculture, including viticulture. Studies on elevated carbon dioxide have already been on grapevines, mainly taking place in greenhouses using potted plants or using field grown vines under higher CO2 enrichment, i.e. >650 ppm. The VineyardFACE, located at Hochschule Geisenheim University, is an open field Free Air CO2 Enrichment (FACE) experimental set-up designed to study the effects of elevated carbon dioxide using field grown vines (Vitis vinifera L. cvs. Riesling and Cabernet Sauvignon). As the carbon dioxide fumigation started in 2014, the long term effects of elevated carbon dioxide treatment can be investigated on berry ripening parameters and fruit metabolic composition.
The present study aims to investigate the effect on fruit composition under a moderate increase (+20%; eCO2) of carbon dioxide concentration, as predicted for 2050 on both Riesling and Cabernet Sauvignon. Berry composition was determined for primary (sugars, organic acids, amino acids) and secondary metabolites (anthocyanins). Special focus was given on monitoring of berry diameter and ripening rates throughout three growing seasons. Compared to previous results of the early adaptative phase of the vines [1], our results show little effect of eCO2 treatment on primary metabolites composition in berries. However, total anthocyanins concentration in berry skin was lower for eCO2 treatment in 2020, although the ratio between anthocyanins derivatives did not differ.
[1] Wohlfahrt Y., Tittmann S., Schmidt D., Rauhut D., Honermeier B., Stoll M. (2020) The effect of elevated CO2 on berry development and bunch structure of Vitis vinifera L. cvs. Riesling and Cabernet Sauvignon. Applied Science Basel 10: 2486

Climate influence on the grapevine phenology and anthocyanins content in wines from the Skopje vineyard area, Republic of Macedonia

The phenological stages and the content of the anthocyanins of non-irrigated cultivars Blatina, Vranec, Kratoshija, Prokupec and Stanushina were study

Evidence for terroir effect associated with botrytisation relatively to compounds implicated in typical aromas of noble rot sweet wines

Recent studies have demonstrated the role of certain lactones, particularly 2-nonen-4-olide, and volatile thiols (3-sulfanylhexan-1-ol) in the over ripped aromas of noble rot sweet wines (Stamatopoulos et al. 2014ab). These compounds are partly formed during the maturation and under the activity of B. cinerea on grapes. This research was carried out in the vineyard of Sauternes with aim to better understand their genesis depending on the grape over-ripening on two different soil types during 3 vintages. Thus, the study was conducted, with the Sémillon grape, during vintages 2012, 2014 & 2015, at 4 stages of over-maturation of the grapes (healthy, pourri plein, pourri roti, pourri roti + 15 days) considering two vineyard plots with different soil characteristics (calcosol & peyrosol) planted with the 315 Sémillon clone and grafted on 101-14 rootstock respectively in 1981 and 1980 and cultivated with the same vineyard management. Volatile lactones were assayed by liquid-liquid extraction followed by GC/MS analysis and the precursors of 3-sulfanylhexanol by an adaptation of the method by Capone et al. 2010 (SPE-
UPLC/FTMS).

Time vs drought: leaf age rather than drought drives osmotic adjustment in V. vinifera cv. Pinot Noir

Global warming and increased frequency and/or severity of drought events are among the most threatening consequences of climate change for agricultural crops. In response to drought, grapevine (as many other plants) exhibits osmotic adjustment through active accumulation of osmolytes which in turn shift the leaf turgor loss point (TLP) to more negative values, allowing to maintain stomata opened at lower water potentials1. We investigated the capacity of Pinot noir leaves to modulate their osmotic potential as a function of: (i) time (seasonal osmoregulation), (ii) growing temperatures, and (iii) drought events, to enhance comprehension of the resilience of grapevines in drought conditions. We performed trails under semi-controlled field conditions, and in two different greenhouse chambers (20/15 °C vs 25/20 °C day/night). For two consecutive vegetative seasons, grafted potted grapevines (Pinot noir/SO4) were subjected to two different water regimes for at least 30 days: well-watered (WW) and water deficit (WD).

Reduction of the height of the canopy in fruit set and in pea size: vegetative, productive and maturation effects, in cv. Verdejo

Global warming is accelerating the technological ripening of the grape, with a loss of acidity, which requires that vineyard management can delay ripening to avoid it. The source-sink relation is essential for grape ripening, since it affects the distribution of photosynthates and substances derived from plant metabolism. A work is proposed to know the response of the vineyard to the drastic reduction of the foliar surface by trim down the shoots in cv.