Analyse fonctionnelle et étude de la régulation de gènes candidats sous-jacents au QTL GpaVspl impliqué dans la résistance au nématode à kyste Globodera pallida chez la pomme de terre

Les nématodes à kystes sont l un des bioagresseurs causant le plus de dégâts sur les cultures de pommes de terre. La résistance trouvée chez l'accession spl88S.329.18, issue de l'espèce Solanum sparsipilum est caractérisée par un déterminisme oligogénique avec un QTL à localisé sur le chromosome V (GpaVspl) et un QTL mineur localisé sur le chromosome XI(GpaXIspl). Pour obtenir une résistance de haut niveau, l'effet du QTL GpaVspl, doit êtrecomplémenté par celui du QTL à effet faible GpaXIspl. Par génomique comparative, le locusGpaV a été localisé dans un intervalle compris entre 16 et 60 kb sur les génomes de la tomateet des espèces apparentées à la pomme de terre, Solanum demissum et Solanum phureja. Deuxgènes ont été annotés dans cet intervalle sur les génomes de la tomate et de S. demissum : lepremier appartient à la famille des TIR-NBS-LRR (TNL), famille de gènes de résistanceclassiques, et le second appartient à la famille des mitochondrial, transcription terminationfactor (mTERF), dont l implication dans des mécanismes de résistance n a jamais étédémontrée.Les objectifs de ma thèse étaient d'identifier le(s) gène(s) responsable(s) de la résistance àG. pallida, conférée par le locus GpaVspl, et d'étudier sa régulation. Suite à la publication de laséquence du génome de S. phureja, en 2011, nous avons mis en évidence que le locus GpaVétait dupliqué chez S. phureja et que cette duplication était également présente chezS. sparsipilum. Les quatre gènes annotés au locus GpaVspl ont été nommésSpl_mTERF18430, Spl_TNL18429, Spl_mTERF18453 et Spl_TNL18428.L'effet des deux gènes Spl_mTERF18430 et Spl_TNL18428 sur la résistance à G. pallida aété analysé via des expériences de transformation génétique suivies par des tests de résistancesur les plantes transformées. Un effet partiel du gène Spl_TNL18428 sur la résistance àG. pallida a été mis en évidence par complémentation de plantes sensibles. Aucun effetsignificatif n'a été détecté pour le gène Spl_mTERF18430. Des expériences d'extinctiongénique suggèrent que le deuxième gène TIR-NBS-LRR, Spl_TNL18429, qui est égalementexprimé dans les racines et qui présente un fort pourcentage d'identité de séquence avec legène Spl_TNL18428, pourrait également être impliqué dans la résistance à G. pallida.L'expression du gène rapporteur GFP, placé sous le contrôle du promoteur du gèneSpl_TNL18428, est fortement induite dans les cellules situées autour du syncytium. Cecirenforce l'hypothèse d'une implication du gène Spl_TNL18428 dans la résistance à G. pallida,car la localisation de l'expression de la GFP est similaire à celle de la nécrose, qui estcaractéristique de la réaction développée par les plantes résistantes autour du syncytium induitpar les nématodes.En tenant compte des données bibliographiques récentes, montrant que plusieurs gènes NBSLRRpeuvent être indispensables à l'expression d'une résistance, nos résultats suggèrent queles deux gènes Spl_TNL18428 et Spl_TNL18429 sont nécessaires à l'expression de larésistance à G. pallidaCyst nematodes are one of the pests that cause the most damage to potato cultures. Resistance found in the accession spl88S.329.18 in Solanum sparsipilum is characterized by oligogenic determinism with a strong effect QTL on chromosome V (GpaVspl) and a minor effect QTL on chromosome XI (GpaXIspl). To obtain a high level of resistance, the effect of QTL GpaVspl must be complemented by the low effect QTL GpaXIspl. By comparative genomics, the GpaV locus was located in a range between 16 and 60 kb in genomes of tomato and potato related species: Solanum demissum and Solanum phureja. Two genes were annotated: the first belonging to the TIR -NBS -LRR gene family (TNL) and the second one belonging to the mitochondrial transcription termination factor family (mTERF). The effect of both genes -Spl_TNL18428 and Spl_mTERF18430- on resistance to G. pallida were analyzed via genetic transformation experiments followed by resistance tests on transformed plants. A partial effect of Spl_TNL18428 on resistance to G. pallida was identified by complementation of susceptible plants. Gene silencing experiments suggested that Spl_TNL18429, which occurs in roots and presents a high percentage of sequence identity with the gene Spl_TNL18428, is also involved in resistance to G. pallida. The expression of the GFP reporter gene, under the control of the Spl_TNL18428 gene promoter, is strongly induced in cells located around the syncytium. This strengthens the hypothesis of an involvement of Spl_TNL18428 gene in resistance to G. pallida, because the location of GFP expression is similar to necrosis, which is characteristic of resistant plants. Taking into account that recent data showing that several NBS-LRR genes may be essential for the expression of resistance, our results suggest that both Spl_TNL18428 and Spl_TNL18429 genes are necessary for the expression of resistance to G. pallidaAVIGNON-Bib. numérique (840079901) / SudocSudocFranceF

Flashes of UV-C light: An innovative method for stimulating plant defences.

Leaves of lettuce, pepper, tomato and grapevine plants grown in greenhouse conditions were exposed to UV-C light for either 60 s or 1 s, using a specific LEDs-based device, and wavelengths and energy were the same among different light treatments. Doses of UV-C light that both effectively stimulated plant defences and were innocuous were determined beforehand. Tomato plants and lettuce plants were inoculated with Botrytis cinerea, pepper plants with Phytophthora capsici, and grapevine with Plasmopara viticola. In some experiments we investigated the effect of a repetition of treatments over periods of several days. All plants were inoculated 48 h after exposure to the last UV-C treatment. Lesions on surfaces were measured up to 12 days after inoculation, depending on the experiment and the pathogen. The results confirmed that UV-C light stimulates plant resistance; they show that irradiation for one second is more effective than irradiation for 60 s, and that repetition of treatments is more effective than single light treatments. Moreover a systemic effect was observed in unexposed leaves that were close to exposed leaves. The mechanisms of perception and of the signalling and metabolic pathways triggered by flashes of UV-C light vs. 60 s irradiation exposures are briefly discussed, as well as the prospects for field use of UV-C flashes in viticulture and horticulture

Assessing the effects of water deficit on photosynthesis using parameters derived from measurements of leaf gas exchange and of chlorophyll a fluorescence

Water deficit (WD) is expected to increase in intensity, frequency and duration in many parts of the world as a consequence of global change, with potential negative effects on plant gas exchange and growth. We review here the parameters that can be derived from measurements made on leaves, in the field, and that can be used to assess the effects of WD on the components of plant photosynthetic rate, including stomatal conductance, mesophyll conductance, photosynthetic capacity, light absorbance, and efficiency of absorbed light conversion into photosynthetic electron transport. We also review some of the parameters related to dissipation of excess energy and to rerouting of electron fluxes. Our focus is mainly on the techniques of gas exchange measurements and of measurements of chlorophyll a fluorescence (ChlF), either alone or combined. But we put also emphasis on some of the parameters derived from analysis of the induction phase of maximal ChlF, notably because they could be used to assess damage to photosystem II. Eventually we briefly present the non-destructive methods based on the ChlF excitation ratio method which can be used to evaluate non-destructively leaf contents in anthocyanins and flavonols

Prospects for using environmental levers in greenhouses to stimulate plant defences and the quality of fruits and vegetables

The environment of greenhouses is commonly modified to reduce the pressure of pests and diseases, but it can also be exploited to stimulate natural defences of plants. In addition to genetic influences, several agronomic factors can be used as levers to increase the concentrations of secondary metabolites involved in the defence of fruit and vegetable crops. Many secondary metabolites involved in plant defences are also phytonutrients, i.e., compounds that have beneficial effects on human health, including protection against cardiovascular diseases, cancers, and metabolic or neurodegenerative diseases. Nitrogen deprivation, photooxidative stress and exposure to specific wavelengths such as those supplied by UV radiations are well known for stimulating the production of secondary metabolites in a large range of plants. We propose here a review of factors meeting the specifications of organic farming, which are both effective and applicable in greenhouse conditions. Among all these factors, mild stress and innovative covering materials emerge as especially promising

Effet de traitements UV-C sur la protection du fraisier et des fruits après récolte contre différents agents pathogènes

National audienceLes agents pathogènes fongiques ont un impact majeur sur le fraisier (Fragaria x ananassa) et des traitements fongicides fréquents sont nécessaires pour limiter les pertes économiques. Il est donc important de trouver des solutions alternatives aux produits phytosanitaires. Les rayonnements UV-C (254 nm) ont montré un potentiel intéressant de protection contre des agents pathogènes, suite à des traitements durant la croissance des plantes (Vasquez et al. 2017. Sci. Hort. 222, 32-39), ou en post récoltes (Jin et al. 2017. Sci. Hort. 225:106-111). Au cours de ce travail, nous avons testé l’hypothèse qu’il est possible de diminuer la sensibilité du fraisier et des fraises produites, vis-à-vis de différents agents pathogènes par l’application d’UV-C en cours de culture. Pour cela, trois modalités de traitement des fraisiers ont été réalisées : avant floraison, après floraison, et pendant toute la durée de la culture. Le développement de trois agents pathogènes a été suivi en cours de culture (Botrytis cinerea et Podosphera aphanis) et en post-récolte sur fruits (B. cinerea et Rhizopus sp.). Les traitements UV-C ont un effet significatif sur l’incidence de l’oïdium en cours de culture en diminuant d’environ 50% le nombre de feuilles attaquées par rapport au contrôle non traité. Ils ont également un effet protecteur des fruits en post-récolte contre les attaques de Rhizopus, en réduisant d’environ 75% le nombre de fruits naturellement contaminés. L’effet vis-à-vis de B. cinerea est plus contrasté : une efficacité de protection de 20% est observée sur feuilles détachées après inoculation artificielle, et aucun effet bénéfique n’est observé sur les fruits. Les implications possibles de ces résultats seront discutées

Prospects for using environmental levers in greenhouses to stimulate plant defences and the quality of fruits and vegetables.

The environment of greenhouses is commonly modified to reduce the pressure of pests and diseases, but it can also be exploited to stimulate natural defences of plants. In addition to genetic influences, several agronomic factors can be used as levers to increase the concentrations of secondary metabolites involved in the defence of fruit and vegetable crops. Many secondary metabolites involved in plant defences are also phytonutrients, i.e., compounds that have beneficial effects on human health, including protection against cardiovascular diseases, cancers, and metabolic or neurodegenerative diseases. Nitrogen deprivation, photooxidative stress and exposure to specific wavelengths such as those supplied by UV radiations are well known for stimulating the production of secondary metabolites in a large range of plants. We propose here a review of factors meeting the specifications of organic farming, which are both effective and applicable in greenhouse conditions. Among all these factors, mild stress and innovative covering materials emerge as especially promising

Assessing the Effects of Water Deficit on Photosynthesis Using Parameters Derived from Measurements of Leaf Gas Exchange and of Chlorophyll a Fluorescence

Water deficit (WD) is expected to increase in intensity, frequency and duration in many parts of the world as a consequence of global change, with potential negative effects on plant gas exchange and growth. We review here the parameters that can be derived from measurements made on leaves, in the field, and that can be used to assess the effects of WD on the components of plant photosynthetic rate, including stomatal conductance, mesophyll conductance, photosynthetic capacity, light absorbance, and efficiency of absorbed light conversion into photosynthetic electron transport. We also review some of the parameters related to dissipation of excess energy and to rerouting of electron fluxes. Our focus is mainly on the techniques of gas exchange measurements and of measurements of chlorophyll a fluorescence (ChlF), either alone or combined. But we put also emphasis on some of the parameters derived from analysis of the induction phase of maximal ChlF, notably because they could be used to assess damage to photosystem II. Eventually we briefly present the non-destructive methods based on the ChlF excitation ratio method which can be used to evaluate non-destructively leaf contents in anthocyanins and flavonols

Prospects for Increasing the Efficacy of Plant Resistance Inducers Stimulating Salicylic Acid

Systemic acquired resistance is a powerful mechanism, based on the salicylic acid (SA) signaling pathway, which allows plants to resist to a wide range of pathogens. High SA, moreover, plays a key role in plant tolerance to abiotic stress. It seems, therefore, desirable to supply analogs of SA or stimulate the production of endogenous SA. Unfortunately, the chemical substances or physical means used for this effect often display a variable efficacy. After providing a review of them, we defend three major ideas: (i) plant resistance inducers (PRIs) must be combined for higher efficacy, notably for exploiting synergic effects between the SA and other signaling pathways, (ii) disease pressure can be reduced by exploiting the fungicidal properties displayed by some PRIs, (iii) biostimulants and crop management techniques should be used to ensure that plants have the resources they need to synthesize the compounds and structures required for efficient and lasting resistance. Some PRIs could also be used for their biostimulant effects in stress conditions. It could be concluded that holistic approaches which jointly address the issues of defense and tolerance stimulation, disease pressure and resource availability in plants are the ones that will allow for substantial reduction in fungicide use without sacrificing crop performance

Impact of UV-C radiation combined with biocontrol agents on the susceptibility of strawberry plants to <em>Botrytis cinerea</em>

International audienceIn order to enhance biocontrol efficacy against plant diseases, the combination of different control methods together with a given biocontrol agent can be achieved. In this study we have tested the effect of combining UV-C radiation with the application of biocontrol agents (Bacillus subtilis and Gliocladium catenulatum based products) against Botrytis cinerea on strawberry. We hypothesize that UV-C radiation applied on plants previously to biocontrol treatment, in addition to its induced resistance effect, will somehow disinfect leaves thus limiting the competition of inoculated biocontrol agents with the phyllosphere microorganisms and then promoting its development and its efficacy. We have shown that, despite the confirmed germicidal effect of UV-C radiation, this treatment applied on plants before treatment with biocontrol agent has no effect on the level of efficacy of the biocontrol agents or even it tends to lower their efficacy. Work is in progress to understand mechanisms involved and to evaluate the effect of this combination of treatments on other plant species