Publicação

Lipid signalling in grapevine resistance against fungal pathogens

Detalhes bibliográficos
Resumo:	Grapevine (Vitis vinifera L) is one of the most economically important crops worldwide, mostly due to its uses for wine and table grape production. However, it is prone to several diseases. Downy and powdery mildews and grey mold, caused by Plasmopara viticola, Erisiphe necator and Botrytis cinerea, respectively, are among the most devastating ones. Disease control strategies include phytochemical applications every growing season, jeopardizing the sustainability of viticulture. Understanding the molecular processes behind disease resistance or susceptibility is vital to define alternative control strategies and select new disease resistance traits for breeding programs. The identification of molecular markers that allow discriminating tolerant and susceptible grapevine genotypes to their pathogens is an important step to help breeders select genotypes for crossings to produce hybrids with good winemaking and disease tolerance traits. Lipids and lipid-derived metabolites are not only major structural and metabolic constituents of the cell, but they also function as modulators of a multitude of signal transduction pathways evoked by biotic stresses. It has been proposed that specific fatty acids (FA) may be involved in plant resistance against pathogens with different colonization strategies (biotroph, hemibiotroph and necrotroph). Previous results indicate that the content of several FA suffers alterations at early time-points after grapevine inoculation with the biotrophic oomycete Plasmopara viticola. These alterations are linked with reactive oxygen species and Jasmonic acid (JA) associated signalling. Moreover, lipid molecules and their derivatives, including JA, when applied externally, can cause a modulation of the lipid and FA signalling mechanisms in a similar manner to the pathogen challenge. Plants that are exposed to these elicitor molecules show a quicker and more intense defence response upon contact with a pathogen. The extracellular matrix (ie apoplast) is the first battlefield where pathogen recognition occurs and secretion of both defence molecules and pathogen effectors take place. Therefore, the apoplast is one of the most important cell compartments in plant-pathogen interaction. Nonetheless, despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Because apoplastic fluid extraction from woody plants is a challenging task, studies regarding grapevine apoplast are still scarce to this day. There are two published studies on the grapevine leaf apoplast proteome and none on its metabolome. In this work, the problems raised above were addressed. The analysis of the constitutive lipid and FA composition of tolerant and susceptible grapevine genotypes to P. viticola was carried out, along with the expression analysis of FA desaturase (FAD) genes. These studies allowed to identify lipids and FA as potential biomarkers for tolerance or susceptibility to P. viticola. The saturated FA, mainly in monogalactosyldiacylglycerol and phosphatidyl choline are candidate tolerance biomarkers and the polyunsaturated linoleic acid (C18:2) as well as the plastidial lipids are candidate susceptibility biomarkers. Moreover, the higher expression levels of FAD4, FAD6 and FAD8 in susceptible genotypes suggest that they might also be considered as candidate biomarkers for susceptibility. The analysis of the total leaf FA composition revealed corroborating results in terms of FA saturation degree and FAD expression, and it is a more rapid and less costly approach (discussed in the chapters II and III). Due to the relevance of the JA mediated lipid signalling in the grapevine-P. viticola interaction, another question that raised was whether this mechanism would be conserved in the interaction with other pathogens with different invasion and/or lifestyles. Therefore, the FA modulation events, crucial for JA synthesis and signalling, were also addressed in the grapevine interaction with E. necator (biotroph, invading the plant leaf in the adaxial page from wound apertures) and B. cinerea (necrotroph). While the interaction with the biotrophs may trigger a higher synthesis of polyunsaturated FA (PUFA) at early time-points with a tendency to return to basal levels, the interaction with B. cinerea may trigger a later and more durable induction of PUFA synthesis. In all interactions, membrane fluidity modulation occurred, which may be crucial to maintain cellular function during infection (discussed in chapter IV). Since lipid molecules and JA showed previously to play important roles in the grapevine defence responses to P. viticola, the potential role of this molecule as a FA signalling trigger was studied. In fact, FA modulation after JA elicitation is similar to that described previously after P. viticola inoculation even in a susceptible cultivar, highlighting the potential of this molecule as an alternative to prevent grapevine diseases (discussed in chapter V). To uncover the lipid signalling events of the first moments of plant pathogen interaction, a thorough analysis of the apoplastic fluid is necessary. A new methodological approach to isolate grapevine leaf apoplast compatible with proteomic and lipidomic based studies was defined. The constitutive metabolome was assessed by FTICR-MS, which allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes. Among them, lipids are the most abundant molecular class. This methodology represents an optimization to the existing protocols and opens the way to study the lipid signalling events in the first battlefield of the grapevine-pathogen interaction (discussed in chapter VI). This work allowed to bring us a few steps closer to the complete disclosure of the grapevine lipid mediated defence mechanisms highlighting also candidate molecules to be used in future breeding programs for disease tolerance.
Autores principais:	Cavaco, Ana Rita Sebastião Mendes
Assunto:	Vitis vinifera lípidos viticultura marcadores míldio lipids viticulture biomarkers downy mildew
Ano:	2021
País:	Portugal
Tipo de documento:	tese de doutoramento
Tipo de acesso:	acesso aberto
Instituição associada:	Universidade de Lisboa
Idioma:	inglês
Origem:	Repositório da Universidade de Lisboa

Descrição
Resumo:	Grapevine (Vitis vinifera L) is one of the most economically important crops worldwide, mostly due to its uses for wine and table grape production. However, it is prone to several diseases. Downy and powdery mildews and grey mold, caused by Plasmopara viticola, Erisiphe necator and Botrytis cinerea, respectively, are among the most devastating ones. Disease control strategies include phytochemical applications every growing season, jeopardizing the sustainability of viticulture. Understanding the molecular processes behind disease resistance or susceptibility is vital to define alternative control strategies and select new disease resistance traits for breeding programs. The identification of molecular markers that allow discriminating tolerant and susceptible grapevine genotypes to their pathogens is an important step to help breeders select genotypes for crossings to produce hybrids with good winemaking and disease tolerance traits. Lipids and lipid-derived metabolites are not only major structural and metabolic constituents of the cell, but they also function as modulators of a multitude of signal transduction pathways evoked by biotic stresses. It has been proposed that specific fatty acids (FA) may be involved in plant resistance against pathogens with different colonization strategies (biotroph, hemibiotroph and necrotroph). Previous results indicate that the content of several FA suffers alterations at early time-points after grapevine inoculation with the biotrophic oomycete Plasmopara viticola. These alterations are linked with reactive oxygen species and Jasmonic acid (JA) associated signalling. Moreover, lipid molecules and their derivatives, including JA, when applied externally, can cause a modulation of the lipid and FA signalling mechanisms in a similar manner to the pathogen challenge. Plants that are exposed to these elicitor molecules show a quicker and more intense defence response upon contact with a pathogen. The extracellular matrix (ie apoplast) is the first battlefield where pathogen recognition occurs and secretion of both defence molecules and pathogen effectors take place. Therefore, the apoplast is one of the most important cell compartments in plant-pathogen interaction. Nonetheless, despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Because apoplastic fluid extraction from woody plants is a challenging task, studies regarding grapevine apoplast are still scarce to this day. There are two published studies on the grapevine leaf apoplast proteome and none on its metabolome. In this work, the problems raised above were addressed. The analysis of the constitutive lipid and FA composition of tolerant and susceptible grapevine genotypes to P. viticola was carried out, along with the expression analysis of FA desaturase (FAD) genes. These studies allowed to identify lipids and FA as potential biomarkers for tolerance or susceptibility to P. viticola. The saturated FA, mainly in monogalactosyldiacylglycerol and phosphatidyl choline are candidate tolerance biomarkers and the polyunsaturated linoleic acid (C18:2) as well as the plastidial lipids are candidate susceptibility biomarkers. Moreover, the higher expression levels of FAD4, FAD6 and FAD8 in susceptible genotypes suggest that they might also be considered as candidate biomarkers for susceptibility. The analysis of the total leaf FA composition revealed corroborating results in terms of FA saturation degree and FAD expression, and it is a more rapid and less costly approach (discussed in the chapters II and III). Due to the relevance of the JA mediated lipid signalling in the grapevine-P. viticola interaction, another question that raised was whether this mechanism would be conserved in the interaction with other pathogens with different invasion and/or lifestyles. Therefore, the FA modulation events, crucial for JA synthesis and signalling, were also addressed in the grapevine interaction with E. necator (biotroph, invading the plant leaf in the adaxial page from wound apertures) and B. cinerea (necrotroph). While the interaction with the biotrophs may trigger a higher synthesis of polyunsaturated FA (PUFA) at early time-points with a tendency to return to basal levels, the interaction with B. cinerea may trigger a later and more durable induction of PUFA synthesis. In all interactions, membrane fluidity modulation occurred, which may be crucial to maintain cellular function during infection (discussed in chapter IV). Since lipid molecules and JA showed previously to play important roles in the grapevine defence responses to P. viticola, the potential role of this molecule as a FA signalling trigger was studied. In fact, FA modulation after JA elicitation is similar to that described previously after P. viticola inoculation even in a susceptible cultivar, highlighting the potential of this molecule as an alternative to prevent grapevine diseases (discussed in chapter V). To uncover the lipid signalling events of the first moments of plant pathogen interaction, a thorough analysis of the apoplastic fluid is necessary. A new methodological approach to isolate grapevine leaf apoplast compatible with proteomic and lipidomic based studies was defined. The constitutive metabolome was assessed by FTICR-MS, which allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes. Among them, lipids are the most abundant molecular class. This methodology represents an optimization to the existing protocols and opens the way to study the lipid signalling events in the first battlefield of the grapevine-pathogen interaction (discussed in chapter VI). This work allowed to bring us a few steps closer to the complete disclosure of the grapevine lipid mediated defence mechanisms highlighting also candidate molecules to be used in future breeding programs for disease tolerance.