OENO IVAS 2019 banner
IVES 9 IVES Conference Series 9 OENO IVAS 9 OENO IVAS 2019 9 Grape and wine microorganisms: diversity and adaptation 9 Use of antisense RNA technology to modulate gene expression in Œnococcus oeni

Use of antisense RNA technology to modulate gene expression in Œnococcus oeni

Abstract

Œnococcus oeni is a wine-associated lactic acid bacterium performs the malolactic fermentation, which improves the taste and aromatic complexity of many wine. Although, wine exhibits harsh and challenging conditions (low pH, low temperature, nutrient-poor and presence of ethanol), O. oeni possesses a remarkable adaptability to those physiochemical conditions.

Mechanisms for responding to environmental changes are universally present in living beings and are essential for coping with the stress and for adapting to the new conditions. O. oeni tolerance to low pH and ethanol make its an interesting bacteria model for investigating stress response mechanism in lactic acid bacteria. However, lack of appropriate techniques to manipulate O. oeni genome has long delay molecular study of this fastidious bacterium. To get around the lack of genetic tool for gene replacement, we focused our work on gene inactivation by using antisense RNA approach to modulate gene expression. With the goal to understanding the function of O. oeni hsp genes in vivo, we produce antisense RNA targeting genes encoding: a small Hsp (hsp18)1, the master regulator of stress response (ctsR)2 and two caseinolytic protease L members of the HSP100 chaperone family (clpL1, clpL2). Thereby, we highlighted that in vivo inhibition of the expression of some of these genes strongly affects the survival of O. oeni in stress conditions.

This study presents an elegant approach providing access to an in vivo study of gene function in O. oeni.

References:

1. Darsonval, M., Msadek, T., Alexandre, H. & Grandvalet, C. The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium. Appl. Environ. Microbiol. 82, 18–26 (2016).

2. Darsonval, M., Julliat, F., Msadek, T., Alexandre, H. & Grandvalet, C. CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1. Front Microbiol 9, (2018).

DOI:

Publication date: June 10, 2020

Issue: OENO IVAS 2019

Type: Article

Authors

Cosette Grandvalet, Frédérique Julliat, Maud Darsonval, Tarek Msadek, Hervé Alexandre

UMR A 02.102 Procédés Alimentaires et Microbiologiques, AgroSup Dijon – Université de Bourgogne Franche-Comté, Dijon, FRANCE.
Unité de Biologie des Bactéries Pathogènes àGram Positif, Institut Pasteur, Paris, FRANCE.
CNRS ERL 6002, Paris, FRANCE.

Contact the author

Keywords

Oenococcus oeni, lactic acid bacteria, antisense RNA , stress response 

Tags

IVES Conference Series | OENO IVAS 2019

Citation

Related articles…

Understanding graft union formation by using metabolomic and transcriptomic approaches during the first days after grafting in grapevine

Since the arrival of Phyloxera (Daktulosphaira vitifolia) in Europe at the end of the 19th century, grafting has become essential to cultivate Vitis vinifera. Today, grafting provides not only resistance to this aphid, but it used to adapt the cultivars according to the type of soil, environment, or grape production requirements by using a panel of rootstocks. As part of vineyard decline, it is often mentioned the importance of producing quality grafted grapevine to improve vineyard longevity, but, to our knowledge, no study has been able to demonstrate that grafting has a role in this context. However, some scion/rootstock combinations are considered as incompatible due to poor graft union formation and subsequently high plant mortality soon after grafting. In a context of climate change where the creation of new cultivars and rootstocks is at the centre of research, the ability of new cultivars to be grafted is therefore essential. The early identification of graft incompatibility could allow the selection of non-viable plants before planting and would have a beneficial impact on research and development in the nursery sector. For this reason, our studies have focused on the identification of metabolic and transcriptomic markers of poor grafting success during the first days/week after grafting; we have identified some correlations between some specialized metabolites, especially stilbenes, and grafting success, as well as an accumulation of some amino acids in the incompatible combination. The study of the metabolome and the transcriptome allowed us to understand and characterise the processes involved during graft union formation.

A better understanding of the climate effect on anthocyanin accumulation in grapes using a machine learning approach

The current climate changes are directly threatening the balance of the vineyard at harvest time. The maturation period of the grapes is shifted to the middle of the summer, at a time when radiation and air temperature are at their maximum. In this context, the implementation of corrective practices becomes problematic. Unfortunately, our knowledge of the climate effect on the quality of different grape varieties remains very incomplete to guide these choices. During the Innovine project, original experiments were carried out on Syrah to study the combined effects of normal or high air temperature and varying degrees of exposure of the berries to the sun. Berries subjected to these different conditions were sampled and analyzed throughout the maturation period. Several quality characteristics were determined, including anthocyanin content. The objective of the experiments was to investigate which climatic determinants were most important for anthocyanin accumulation in the berries. Temperature and irradiance data, observed over time with a very thin discretization step, are called functional data in statistics. We developed the procedure SpiceFP (Sparse and Structured Procedure to Identify Combined Effects of Functional Predictors) to explain the variations of a scalar response variable (a grape berry quality variable for example) by two or three functional predictors (as temperature and irradiance) in a context of joint influence of these predictors. Particular attention was paid to the interpretability of the results. Analysis of the data using SpiceFP identified a negative impact of morning combinations of low irradiance (lower than about 100 μmol m−2 s−1 or 45 μmol m−2 s−1 depending on the advanced-delayed state of the berries) and high temperature (higher than 25oC). A slight difference associated with overnight temperature occurred between these effects identified in the morning.

Climate modeling at local scale in the Waipara winegrowing region in the climate change context

In viticulture, a warming climate can have a very significant impact on grapevine development and therefore on the quality and characteristics of wines across different spatial scales, ranging from global to local. In order to adapt wine-growing to climate change, global climate models can be used to define future scenarios, but only at the scale of major wine regions. Despite the huge progress made over the last ten years in terms of the spatial resolution of climate models (now downscaled to a few square kilometres), they are not yet sufficiently precise to account for the local climate variability associated with such parameters as local topography, in spite of these parameters being decisive for vine and wine characteristics. This study describes a method to downscale future climate scenarios to vineyard scale. Networks of data loggers have been used to collect air temperature at canopy level in the Waipara winegrowing region (New Zealand) over five growing seasons. These measurements allow the creation of fine-scale geostatistical models and maps of temperature (at 100 m resolution) for the growing season. In order to model climate change at pilot site scale, these geostatistical models have been combined with regional climate change predictions for the periods 2031-2050 and 2081-2100 based on the RCP8.5 climate change scenario. The integration of local climate variability with regionalized climate change simulations allows assessment of the impacts of climate change at the vineyard scale. The improved knowledge gained using this methodology results from the increased horizontal resolution that better addresses the concerns of winegrowers. The results provide the local winegrowers with information necessary to understand current processes, as well as historical and future viticulture trends at the scale of their site, thereby facilitating decisions about future response strategies.

Grape berry size is a key factor in determining New Zealand Pinot noir wine composition

Making high quality but affordable Pinot noir (PN) wine is challenging in most terroirs and New Zealand’s (NZ) situation is no exception. To increase the probability of making highly typical PN wines producers choose to grow grapes in cool climates on lower fertility soils while adopting labour intensive practices. Stringent yield targets and higher input costs necessarily mean that PN wine cost is high, and profitability lower, in line-priced varietal wine ranges. To understand the reasons why higher yielding vines are perceived to produce wines of lower quality we have undertaken an extensive study of PN in NZ. Since 2018, we established a network of twelve trial sites in three NZ regions to find individual vines that produced acceptable commercial yields (above 2.5kg per vine) and wines of composition comparable to “Icon” labels. Approximately 20% of 660 grape lots (N = 135) were selected from within a narrow juice Total Soluble Solids (TSS) range and made into single vine wines under controlled conditions. Principal Component Analysis of the vine, berry, juice and wine parameters from three vintages found grape berry mass to be most effective clustering variable. As berry mass category decreased there was a systematic increase in the probability of higher berry red colour and total phenolics with a parallel increase in wine phenolics, changed aroma fraction and decreased juice amino acids. The influence of berry size on wine composition would appear stronger than the individual effects of vintage, region, vineyard or vine yield. Our observations support the hypothesis that it is possible to produce PN wines that fall within an “Icon” benchmark composition range at yields above 2.5kg per vine provided that the Leaf Area:Fruit Weight ratio is above 12cm2 per g, mean berry mass is below 1.2g and juice TSS is above 22°Brix.

Assessing the relationship between cordon strangulation, dieback, and fungal trunk disease symptom expression

Grapevine trunk diseases including Eutypa dieback are a major factor in the decline of vineyards and may lead to loss of productivity, reduced income, and premature reworking or replanting. Several studies have yielded results indicating that vines may be more likely to express symptoms of vascular disease if their health is already compromised by stress. In Australia and many other wine-growing regions it is a common practice for canes to be wrapped tightly around the cordon wire during the establishment of permanent cordon arms. It is likely that this practice may have a negative effect on health and longevity, as older cordons that have been trained in this manner often display signs of decay and dieback, with the wire often visibly embedded within the wood of the cordon. It is possible that adopting a training method which avoids constriction of the vasculature of the cordon may help to limit the onset of vascular disease symptom expression. A survey was conducted during the spring of two consecutive growing seasons on vineyards in South Australia displaying symptoms of Eutypa lata infection when symptomless shoots were 50–100 cm long. Vines were assessed as follows: (i) the proportion of cordon exhibiting dieback was rated using a 0–100% scale; (ii) the proportion of canopy exhibiting foliar symptoms of Eutypa dieback was rated using a 0–100% scale; (iii) the severity of strangulation was rated using a 0–4 point scale. Images were also taken of each vine for the purpose of measuring plant area index (PAI) using the VitiCanopy App. The goal of the survey was to determine if and to what extent any correlation exists between severity of strangulation and cordon dieback, in addition to Eutypa dieback foliar symptom expression.