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Functional Genomics applied to the study of resistance against Powdery Mildew in grapevine

Publication . Pimentel, Diana; Fortes, Ana Margarida; Granell, Antonio

Grapevine (Vitis vinifera L.) is one of the most valuable non-climacteric fruit crops worldwide and is susceptible to several pathogens. Powdery Mildew (PM) is one of the most widespread diseases and is caused by the biotrophic fungus Erysiphe necator. This pathogen relies on the host metabolism to complete its life cycle. PM can affect all green tissues, such as leaves and green berries, frequently resulting in a negative effect on grape production. Despite the several studies performed so far, the mechanisms behind grapevine defense are very complex, and responses against PM remain unclear in infected fruits. In order to study the mechanisms involved in grape berries response to PM infection in the early stages of ripening, naturally infected and control grapes from ‘Carignan’ variety were collected at green (EL33) and véraison (EL35) stages and metabolic, transcriptomic and hormonal changes upon PM infection were analyzed. Results demonstrated that PM-susceptible grape berries were able to induce defense mechanisms and accumulate defense-associated metabolites, such as resveratrol, catechins, gallic acid, and long-chain saturated fatty acids, which could be explored as markers of infection at earlier ripening stages on field conditions. Induction of defenses was also previously observed in leaves, but certain responses seem to be organ-specific, such as the reprogramming of fatty acid metabolism and isoprenoid biosynthesis. This study was the first to quantify jasmonates’ levels in PM infected berries suggesting an involvement of specific jasmonates in response to PM. These growth regulators are classically associated with response to necrotrophic fungi. Some LATERAL ORGAN BOUNDARIES (LOB) domain (LBD) and GRAS genes were responsive to powdery mildew and/ or modulated at véraison stage. LBD constitute a family of plant-specific transcription factors with important roles in several plant processes. In this work, a genome-wide analysis was performed to identify and map the LBD genes in the grapevine genome. Fifty LBD genes were identified and grouped in two classes. Expression profiling suggests the involvement of LBD transcription factors in grapevine development, berry ripening and stress responses. GRAS transcription factor family has also been reported as involved in multiple processes; however, their role in fruit ripening is poorly studied. Tomato SlGRAS10 and its grapevine ortholog VviPAT6 were previously suggested as putative regulators of fruit ripening in both climacteric and non-climacteric plants. In this work, preliminary studies and targeted mutagenesis using CRISPR-Cas9 technology were developed in order to study the role of SlGRAS10 in fruit ripening.

2020-05Doctoral thesis

Open access

Remodelling of grape cell wall upon infection with biotrophic and necrotrophic pathogens

Publication . Santos, Helena; Fortes, Ana Margarida; Moore, John Paul

Grapes are fruits highly susceptible to various diseases and stresses, including powdery mildew, esca disease, and grey mould. These conditions significantly impact grape quality, yield production, and aroma profiles. The infection strategy by the biotrophic fungus Erysiphe necator prioritises the development of appressoria for cell penetration of photosynthetic tissues using mechanical force, while the hemibiotrophic Botrytis cinerea secretes cell wall degrading enzymes to access the sugar-rich and ripe tissues. On the other hand, esca infects the grapevine trunk, leading to xylem destruction. The diverse responses of grapes to these different infection strategies often involve the cell wall, a critical component in grape development and defence that undergoes dynamic changes during different developmental stages and in response to pathogens. This thesis aims to investigate cell wall alterations in grapes, in the context of diverse fungal diseases, and their association with disease susceptibility, defensive responses, ontogenic resistance, and quality attributes. Grape cultivars were selected based on their susceptibility to the corresponding disease. Carignan exhibits high susceptibility to E. necator at the green stage, while more tolerant at veraison. Aragones (Tempranillo in Spain) is a cultivar susceptible to esca disease that exhibits either symptomatic or asymptomatic esca phenotypes in leaves, but often with limited symptoms in grapes. Trincadeira is highly susceptible to B. cinerea, as it displays infection symptoms from green to ripe, while Syrah, considered as tolerant to the fungus, shows symptoms mostly at the ripe stage. Two approaches were used, namely Gas Chromatography for the analysis of cell wall monosaccharide composition, and Comprehensive Microarray Polymer Profiling for cell wall polysaccharide evaluation. This provided a combined analysis of the putative grape cell wall architecture and composition during ripening and diseases incidence. These studies were complemented with transcriptional profiling regarding E. necator infection in grapes, and immunofluorescence studies with a xyloglucan-binding probe concerning Trincadeira and Syrah green grapes infected with B. cinerea. The cell wall studies on E. necator revealed significant differences in cell wall responses between green and veraison grapes. Results suggested increased callose levels and low degree of methylesterification in homogalacturonan in green grapes, while veraison grapes showed an increase in glucuronic acid. Regarding esca disease, grapes from symptomatic plants exhibited an increase in partially methylated homogalacturonan and a reduction in xyloglucans, mannans, and arabinogalactan proteins. Cultivar–specific differences in cell wall composition were observed in Trincadeira and Syrah samples, with higher abundance of arabinose and lower glucose in Syrah. Immunofluorescence images of Trincadeira grapes acquired using xyloglucan-binding probe displayed a putative B. cinerea infection, with intercellular hyphae development at the grape epidermis. These experiments highlight the diverse cell wall remodelling under different biotic stresses and reinforce its importance in grape defence strategies. The consequences of these diseases are most notable in the impact in grape aroma components, ranging from new compounds or content changes in certain compounds synthesised by the plant, to the release of fungal metabolites with off-flavours. Hence, this thesis also aimed to investigate the volatile organic compounds profile of Trincadeira, a cultivar classified as non-aromatic, and its changes under infection with B. cinerea. Headspace-–SPME Gas Chromatography was used for the analysis, identifying various volatile organic compounds, including alcohols, ester acetates, and fatty acid esters. Significant differences in volatile composition were observed between healthy and infected grapes, indicating amyl alcohol and 2-trans-hexenal as putative markers of infection, and providing insights into the impact of B. cinerea on grape quality and health. In conclusion, this comprehensive thesis contributes to decipher the pivotal role of the grape cell wall in response to various fungal diseases. These investigations into cell wall alterations contribute to a deeper understanding of the intricate interplay between the grape cell wall and biotic stresses, ultimately paving the way for the development of innovative strategies to enhance grapevine health, grape quality (with preserved aroma), and overall resilience against pathogens.

2024-01Doctoral thesis

Open access

Hormone Changes in Tolerant and Susceptible Grapevine Leaves Under Powdery Mildew Infection

Publication . Amaro, Rute; Diniz, Inês; Santos, Helena; Pimentel, Diana; Rego, Cecilia; Mithofer, Axel; Fortes, Ana Margarida

The biotrophic fungus Erysiphe necator causes powdery mildew (PM) in grapevine. Phytohormones are major modulators of defensive responses in plants but the analysis of the hormonome associated with grapevine tolerance and susceptibility against this pathogen has not been elucidated. In this study, changes in hormonal profiling were compared between a tolerant (Vitis rupestris × riparia cv. 101-14 Millardet et de Grasset) and a susceptible (Vitis vinifera cv. Aragonêz) species upon E. necator infection. Control and PM-infected leaves were collected at 0, 6, 24, 96 h post-infection (hpi), and analysed through LC-MS/MS. The results showed a distinct constitutive hormonome between tolerant and susceptible species. Constitutive high levels of salicylic acid (SA) and indole-3- acetic acid together with additional fast induction of SA within the first 6 hpi as well as constitutive low levels of jasmonates and abscisic acid may enable a faster and more efficient response towards the PM. The balance among the different phytohormones seems to be species-specific and fundamental in providing tolerance or susceptibility. These insights may be used to develop strategies for conventional breeding and/or editing of genes involved in hormonal metabolism aiming at providing a durable resistance in grapevine against E. necator

2022Journal article

Open access

Volatile Metabolism of Wine Grape Trincadeira: Impact of Infection with Botrytis cinerea

Publication . Santos, H.; Augusto, C.; Reis, P.; Rego, Cecilia; Figueiredo, A.C.; Fortes, A.M.

The aroma of grapes is cultivar dependent and is influenced by terroir, vineyard practices, and abiotic and biotic stresses. Trincadeira is a non-aromatic variety associated with low phenolic content and high sugar and organic acid levels. This cultivar, widely used in Portuguese wines, presents high susceptibility to Botrytis cinerea. This work aimed to characterise the volatile profile of Trincadeira grapes and how it changes under infection with B. cinerea. Thirty-six volatile organic compounds were identified, from different functional groups, namely alcohols, ester acetates, fatty acid esters, fatty acids, aldehydes, and products of the lipoxygenase pathway. Both free and glycosidic volatile organic compounds were analysed by Gas Chromatography and Gas Chromatography coupled to Mass Spectrometry for component quantification and identification, respectively. A multivariance analysis showed a clear discrimination between healthy and infected grapes with 2-trans-hexenal and isoamyl-acetate among the compounds identified as negative and positive markers of infection, respectively. Ester acetates such as 2-phenylethyl acetate, isoamyl acetate, and 2-methylbutyl acetate were present in higher contents in infected samples, whereas the contents of several fatty acid esters, such as ethyl decanoate and ethyl dodecanoate, decreased. These data were integrated with quantitative PCR data regarding genes involved in volatile metabolism and showed up-regulation of a gene coding for Hydroperoxide Lyase 2 in infected grapes. Altogether, these changes in volatile metabolism indicate an impact on the grape quality and may be related to defence against B. cinerea. The presence/absence of specific compounds might be used as infection biomarkers in the assessment of Trincadeira grapes’ quality

2022Journal article

Open access