Lipid signalling in grapevine resistance against fungal pathogens

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

Sintase do óxido nítrico de Leishmania infantum: da sequência à estrutura

Tese de mestrado, Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2015O óxido nítrico (NO) tem um grande impacto nos seres vivos. No Homem, o NO participa em vários processos fisiológicos, como a regulação da pressão sanguínea, a neurotransmissão e a resposta imune. Nestes processos, o NO funciona como uma molécula sinalizadora. No entanto, em níveis mais elevados, este radical pode ter efeitos tóxicos nas células, fazendo parte da resposta de defesa contra mcirorganismos patogénicos. De facto, há um consenso de que, nas infeções por Leishmania e Trypanosoma cruzi, o NO produzido pelos macrófagos infetados medeia a morte dos parasitas. Em parasitas do género Leishmania, o NO é ainda importante na interação entre o parasita e o hospedeiro. Para além disso, sabe-se atualmente que a concentração de NO produzido pelos parasitas varia ao longo do seu ciclo de vida, sendo tanto maior quanto maior é a sua infetividade. Assim, o NO assume um papel importante no estabelecimento da infeção por Leishmania. O enzima sintase do óxido nítrico (NOS, EC: 1.14.13.39), que catalisa a reação de formação de NO a partir de L-arginina, é, portanto essencial para o parasita. Atualmente, sabe-se que não existe homologia de sequência entre o NOS de Leishmania infantum (LiNOS) e de humano. Neste sentido, o LiNOS surge como um potencial alvo terapêutico para o combate da leishmaniose visceral, doença pela qual o parasita é responsável. Neste projeto foi demonstrado, pela primeira vez, que o LiNOS expresso nativamente forma um homotetrâmero. Foi ainda possível concluir que o tetrâmero de LiNOS é estabilizado na presença de heme e de tetrahidrobiopterina. Através da análise da sequência da proteína em estudo, foi prevista a existência de uma região desordenada entre os resíduos 210 e 270 e de um segmento transmembranar entre os resíduos 424 e 446. Na análise das regiões desordenadas do LiNOS, foi também identificado um local de ligação a montante e/ou a jusante desta região, sendo esta ligação potencialmente estabelecida com a caveolina. A estrutura tridimensional do monómero de LiNOS foi ainda prevista por homologia, tendo sido possível prevê-la para a maioria da sequência (resíduos 95-584). Os resultados obtidos permitiram gerar informação crucial para dirigir os próximos passos experimentais no sentido da sua elucidação total, assim como de outros aspetos relacionados com a função.Nitric oxide (NO) exerts a great impact on living beings. On human cells, NO participates in several physiological processes, such as blood pressure regulation, neurotransmission and immune response. In these processes, NO works as a signaling molecule. However, when in higher levels, this radical can exert toxic effects on cells as part of the immune response against pathogenic microorganisms. In fact, that is a consensus that, in infections by Leishmania and Trypanosoma cruzi, NO produced by infected macrophages mediates parasite death. In Leishmania genus parasites, NO is also important for the parasite-host interaction. Besides, it is now known that the concentration of NO produced by the parasites varies during its life cycle and it increases as the parasite’s infectivity increases. Hence, NO plays an important role on the establishment of the infection by Leishmania. The enzyme nitric oxide synthase (NOS, EC: 1.14.13.39), wich catalizes the synthesis of NO from L-arginine, is essential for the parasite. At this time, it is known that Leishmania infanum NOS (LiNOS) has no sequence homology with human NOS. Therefore, LiNOS arises as a potential therapeutic target for the treatment of visceral leishmaniasis, the desiese for wich the parasite is responsible. It was established during this project, for the first time, that natively expressed LiNOS folds as a homotetramer. It was also possible to conclude that the tetramer is stabilized when in presence of heme and tetrahydrobiopterine. A protein sequence analysis revealed the existence of a disordered region between residues 210 and 270 and a transmembrane region between residues 424 and 446 was predicted. When analysing disordered regions in LiNOS a potential binding site upsteam and/or downstream the predicted region was identified and the bond might me established with caveoline. The tridimentional structure of LiNOS monomer was predicted by homology, and it was possible to predict it for the majority of the sequence (residues 95 to 584). The acquired results led to the generation of crutial information to lead the next experimental steps for the structure’s complete elucidation, as wel as other function related aspects