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Biosystems and Integrative Sciences Institute
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Nitrogen Acquisition and Transport in the Ectomycorrhizal Symbiosis—Insights from the Interaction between an Oak Tree and Pisolithus tinctorius
Publication . Sebastiana, Mónica; Serrazina, Susana; Monteiro, Filipa; Wipf, Daniel; Fromentin, Jérome; Teixeira, Rita; Malhó, Rui; Courty, Pierre-Emmanuel
In temperate forests, the roots of various tree species are colonized by ectomycorrhizal
fungi, which have a key role in the nitrogen nutrition of their hosts. However, not much is known
about the molecular mechanisms related to nitrogen metabolism in ectomycorrhizal plants. This
study aimed to evaluate the nitrogen metabolic response of oak plants when inoculated with the
ectomycorrhizal fungus Pisolithus tinctorius. The expression of candidate genes encoding proteins
involved in nitrogen uptake and assimilation was investigated in ectomycorrhizal roots. We found
that three oak ammonium transporters were over-expressed in root tissues after inoculation, while
the expression of amino acid transporters was not modified, suggesting that inorganic nitrogen
is the main form of nitrogen transferred by the symbiotic fungus into the roots of the host plant.
Analysis by heterologous complementation of a yeast mutant defective in ammonium uptake and
GFP subcellular protein localization clearly confirmed that two of these genes encode functional am-
monium transporters. Structural similarities between the proteins encoded by these ectomycorrhizal
upregulated ammonium transporters, and a well-characterized ammonium transporter from E. coli,
suggest a similar transport mechanism, involving deprotonation of NH4+, followed by diffusion of
uncharged NH3 into the cytosol. This view is supported by the lack of induction of NH4+ detoxifying
mechanisms, such as the GS/GOGAT pathway, in the oak mycorrhizal roots
Grapevine subtilisin-like proteases and Plasmopara viticola serine protease inhibitors in the defence – offense interaction
Publication . Gouveia, Catarina; Figueiredo, Andreia; Malhó, Rui; Buchholz, Guenther
Grapevine (Vitis vinifera L. subsp. sativa) is one of the most cultivated fruit crops worldwide. However, is highly susceptible to several pathogens which poses a severe threat to production yields. One of the most devastating diseases is the grapevine downy mildew caused by the obligate biotrophic oomycete Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni. This pathogen was introduced in Europe in the mid19th century and led to the near-collapse of European viticulture only some years later. To manage this disease several preventive applications of pesticides are used but more sustainable approaches such as breeding for resistance are being conducted. As such, most of the research in the field of grapevine-pathogen interaction is still focused on increasing knowledge of how the plant activates defence pathways. However, the success of the breeding programs depends also on a deep knowledge of this pathosystem. Not only on the identification of strong resistance-associated candidate genes for introgression from the resistance carriers and hybrids, but as well as on the interacting players in the plant-pathogen offense-defence, as plant defence proteins/ enzymes and pathogen effectors. The activation of the defence pathways and therefore the establishment of an incompatible interaction depends on the early recognition of pathogens. Among the groups of molecules, that play a role in pathogen recognition and signalling cascades, we focused on plant proteases, particularly in serine proteases, or the subtilisin-like proteases (SBT, subtilases) family. In grapevine, some subtilases were observed to have an enhanced gene expression upon infection with P. viticola in the tolerant cultivar “Regent” and susceptible cultivar “Trincadeira”, as VviSBT4.19. Moreover, some subtilases, as VviSBT5.3, were found to have their expression induced not only in response to the pathogen but also by jasmonic acid, a phytohormone involved in the signalling pathway in biotic stress response. Focusing on the pathogen, apoplastic effectors such as protease inhibitors are necessary for a successful invasion and counter defence. In this context, one hypothesis is that P. viticola may secrete protease inhibitors to block host serine proteases. This doctoral project’s main aim is to understand the role of subtilases in grapevine resistance and the role of serine-protease inhibitors secreted by P. viticola as pathogenicity effectors, particularly to understand the potential crosstalk between grapevine and P. viticola focusing on host subtilases and pathogen serine proteases inhibitors. Several objectives were defined for the analysis of host serine proteases as the analysis of VviSBT4.19 and VviSBT5.3 gene expression in V. vinifera genotypes with different tolerance and Rpv background in a time-course inoculation with P. viticola. Stable transformation of Solanum lycopersicum with constructs for overexpression of VviSBT4.19 and SlSBT4.14, a tomato homolog of VviSBT4.19 to access gene function, and characterization of these Solanum lycopersicum transformed lines. For the analysis of serine protease inhibitors, we identified P. viticola protease inhibitors from the serine family, performed gene expression analysis of these inhibitors and analysis of their subcellular localization and their effect on plant defence mechanism by agroinfiltration in N. benthamiana. Gene expression analysis of two candidate subtilases, VviSBT4.19 and VviSBT5.3a, was performed in seven cultivars with different levels of susceptibility to P. viticola (Chapter II). Two Vitis vinifera susceptible genotypes: “Chardonnay” and “MuellerThurgau” and five tolerant genotypes “Calardis blanc” (Rpv3–1, Rpv3–2), “Cabernet blanc” (Rpv3–1), “Regent” (Rpv3–1), “Solaris” (Rpv10, Rpv3-3) and “Sauvignac” (Rpv12, Rpv 3 − 1) were analysed. Moreover, two P. viticola isolates with different degrees of virulence were tested, avrRpv3 + which is an isolate unable to overcome Rpv3-based resistance and NW-10/16 which was isolated from ‘Regent’ in October 2016 in Neustadt/Weinstr. (Germany). Not only NW-10/16 can overcome the resistance observed in Rpv3 genotypes when infected with the avirulent isolate avrRpv3+, in the susceptible cultivars, “Chardonnay” NW-10/16 also exhibits an increase in sporulation. This higher sporulation on susceptible cultivars might indicate that the isolate NW-10/16 presents a higher aggressiveness. The subtilases display different patterns of expression with the VviSBT4.19 exhibiting only one peak of expression, while VviSBT5.3a presents a bimodal behaviour. Nevertheless, both subtilases presented a more pronounced response to the more aggressive P. viticola isolate, with the difference in gene expression being also dependent on the grapevine cultivar. Our results support the hypothesis that the two subtilases are involved in the early events of the defence mechanisms, being their transcription highly activated in response to a more aggressive P. viticola isolate. The P. viticola isolates were further characterized in Chapter III. The interaction of these isolates with 5 grapevine genotypes with different Rpv loci was analysed. In this chapter, P. viticola development, aggressiveness and the expression of serine protease inhibitors was assessed. The aim was to distinguish two P. viticola isolates through their infection strategy and growth stages, and possible determine if a different gene expression of serine protease inhibitors could be a cause of one of the causes of the increased aggressiveness. The results showed that the isolate NW-10/16 was able to overcome Rpv3 resistance but not Rpv12 resistance. Moreover, it can be considered more aggressive with increased sporulation and increased hyphae growth, with an acceleration in hyphae growth after 36 hours post inoculation. P. viticola serine protease inhibitors gene family was characterized with eight genes encoding for five proteins. From these five serine protease inhibitors, two present highly dynamic pattern of expression depending on the isolate and cultivar. The Pvit020s, a serine protease inhibitor gene that has three identical copies in the genome, plus one containing a mutation which alters 1 aminoacid, present a later peak of expression in the susceptible cultivar but earlier in tolerant cultivars. While Pvit026 has an both a higher expression level and an earlier peak of expression for NW-10/16 compared to avrRpv3+. When comparing the gene expression of both isolates, NW-10/16 presented a higher expression for Pvit026 in all cultivars and Pvit544 in the tolerant and resistant cultivars. The expression patterns indicate a role of serine proteases inhibitors in the pathogen’s counterattack against plant defence. Moreover, we can hypothesize that Pvit026 may be involved in increased aggressiveness, while Pvit544 might have a role as a facilitator to overcome resistance. The function of subtilisin-like serine protease VviSBT4.19 was studied in Chapter IV with the stable transformation of Solanum lycopersicum to obtain transgenic plant lines overexpressing the grapevine VviSBT4.19. To compare, the homolog subtilisinlike serine protease gene from tomato, SlSBT4.14 was also cloned and used for stable transformation. Cotyledons of S. lycopersicum “Hellfrucht” were co-incubated with A. tumefaciens AGL1 for stable transformation. No difference in calli morphology was observed between constructs (T-DNA containing VviSBT4.19, SlSBT4.14 or none). However, calli transformed with the transgene VviSBT4.19 presented a slightly faster regeneration. Three generations of transgenic plant lines were analyzed. For each construct two transgenic plant lines were studied. Morphologically, the overexpression of VviSBT4.19 altered the angle of the tomato leaves, which tended to grow with a vertical orientation. And plants overexpressing SlSBT4.14 tended to be shorter. Another major difference was found in flowering and fruit development. Plants overexpressing VviSBT4.19 and SlSBT4.14 tended to flower later with some not producing fruits and seeds. The resistance to Phytophthora infestans was evaluated for the transgenic plant lines and plants overexpressing SlSBT4.14 were found to be more susceptible. And plants overexpressing VviSBT4.19 tend to present a lower pathogen sporulation, and therefore tend to be less susceptible to P. infestans infection. These results indicate that both subtilases have possible functions in biotic stress response as well as plant development and reproduction. Moreover, the overexpression of VviSBT4.19 and SlSBT4.14 resulted in very different phenotypes regarding growth and biotic stress response, further indicating that these subtilases may participate in different mechanisms. Though the exact mechanism of action remains uncertain. As it was possible to determine in which plant processes these subtilases have probable functions, new experiments can be design and carried out more specifically, and thus further elucidating the subtilases roles in these processes. This thesis is divided in four chapters. The first chapter was written as a review article and the 3 following chapters were written as scientific articles and each has its own abstract, introduction, materials and methods, results and discussion, conclusion, acknowledgments and references.
Genome-wide identification of epigenetic regulators in Quercus suber L.
Publication . Silva, Helena G.; Sobral, Rómulo S.; Magalhães, Alexandre P.; Morais-Cecilio, Leonor; Costa, M.Manuela R.
Modifications of DNA and histones, including methylation and acetylation, are critical
for the epigenetic regulation of gene expression during plant development, particularly during
environmental adaptation processes. However, information on the enzymes catalyzing all these
modifications in trees, such as Quercus suber L., is still not available. In this study, eight DNA
methyltransferases (DNA Mtases) and three DNA demethylases (DDMEs) were identified in Q. suber.
Histone modifiers involved in methylation (35), demethylation (26), acetylation (8), and deacetylation
(22) were also identified in Q. suber. In silico analysis showed that some Q. suber DNA Mtases,
DDMEs and histone modifiers have the typical domains found in the plant model Arabidopsis, which
might suggest a conserved functional role. Additional phylogenetic analyses of the DNA and histone
modifier proteins were performed using several plant species homologs, enabling the classification of
the Q. suber proteins. A link between the expression levels of each gene in di erent Q. suber tissues
(buds, flowers, acorns, embryos, cork, and roots) with the functions already known for their closest
homologs in other species was also established. Therefore, the data generated here will be important
for future studies exploring the role of epigenetic regulators in this economically important species
Sea Urchin Inspired Adhesive Proteins – Expression and Purification of Nectin Structural Domains
Publication . Santos, Mariana Rodrigues dos; Henriques, Bárbara J.; Santos, Romana Lopes Almeida
Atualmente, há uma grande procura por adesivos biológicos que sejam eficazes em ambientes aquosos,
para aplicações nas áreas da biotecnologia e da biomedicina (por exemplo, para a produção de adesivos
cirúrgicos ou como promotores de adesão celular para culturas de células e tecidos in vitro).
Na natureza, este tipo de adesivos está muito presente em animais marinhos que usam a adesão para a
sua locomoção e alimentação, tais como as estrelas-do-mar e os ouriços-do-mar. Estes animais possuem
órgãos especializados para a adesão, os pés ambulacrários, que são constituídos por um caule cilíndrico,
responsável pela mobilidade e flexibilidade do pé, e um disco adesivo, que interage com o substrato e é
responsável pela produção de secreções adesivas. Os pés ambulacrários possuem uma epiderme duoglandular com células adesivas e de-adesivas, que produzem secreções adesivas e de-adesivas,
respetivamente. Estas secreções permitem que os ouriços-do-mar se liguem reversivelmente ao
substrato, deixando fixo ao mesmo um círculo de material adesivo. No caso dos ouriços-do-mar,
encontra-se descrito que as secreções adesivas são constituídas por uma grande fração inorgânica,
glícidos, lípidos e proteínas.
Estudos prévios sobre o ouriço-do-mar Paracentrotus lividus identificaram a Nectina-2 como sendo
uma proteína importante no processo de adesão destes organismos, uma vez que está sobre-expressa
tanto nos pés ambulacrários como nas secreções adesivas. A Nectina-2 é uma proteína de elevado peso
molecular (tem mais de 100 kDa) composta por seis domínios discoidina (DS), que lhe permitem ligarse a moléculas com resíduos de galactose e N-acetilglucosamina. Considera-se que estes domínios sejam
os principais responsáveis pelas propriedades adesivas da Nectina. Para além disso, esta proteína possui
um péptido sinal (com 24 resíduos de aminoácidos), uma sequência LDT, que se pensa ser responsável
por manter a interface entre o pé ambulacrário e a secreção adesiva (através de interações com recetores
de integrina do tipo α4/β7 nas células da epiderme do disco), e um possível local de sulfatação na Tyr77
(importante local para modificações pós-traducionais para proteínas que passam pela via secretora). A
Nectina-2 é ainda passível de sofrer modificações pós-traducionais, tais como fosforilação e
glicosilação.
Considerando que a produção da proteína Nectina-2, em larga escala, será um processo moroso e
provavelmente pouco rentável a nível industrial devido ao seu elevado tamanho e à sua complexa
estrutura, o principal objetivo deste trabalho é encontrar uma combinação mais simples de domínios DS
que tenha propriedades adesivas comparáveis à proteína completa e com estabilidade adequada à sua
produção em larga escala.
Deste modo, este trabalho consistiu na otimização dos processos de expressão e purificação de diferentes
construtos de Nectina-2, e posterior caracterização bioquímica e estrutural. Para alcançar estes objetivos,
foram desenhados seis constructos que foram inseridos em dois vetores diferentes [pHTP1 e pET28b(+)]
(obtido como serviço) para expressão de proteínas recombinantes em E. coli. Dos seis constructos,
quatro foram expressos com sucesso (constructos C1, C2, C5 e C6). Os constructos inseridos no vetor
pHTP1 (constructos C1 e C2) tiveram um nível de expressão de proteína mais elevado que os restantes.
Seguidamente testaram-se protocolos de purificação para os diferentes constructos, usando diferentes
técnicas cromatográficas. Para os constructos C1 e C6 não foi possível estabelecer protocolos de
purificação, pois os protocolos testados falharam na obtenção de proteína pura. Neste sentido no futuro
será necessário realizar mais testes. O constructo C2 foi purificado com sucesso através do uso de
cromatografia de afinidade (His-trap), tendo-se obtido uma proteína com um grau de pureza de 90-95%.
Através de estudos de dicroísmo circular e espectroscopia de fluorescência, percebemos que o constructo
purificado tem um fold do tipo α/β (o que vai de encontro às previsões obtidas com o software
AlphaFold) e os seus resíduos de triptofano estão parcialmente expostos ao solvente.
Para estudar a estabilidade conformacional do constructo C2 em diferentes ambientes, semelhantes aos
que a Nectina-2 está exposta quando expressa nos ouriços-do-mar, recorremos a estudos da desnaturação térmica. Deste modo, as condições estudadas foram água do mar artificial (ASW1 - 223 mM NaCl, 30
mM MgCl2, 5 mM KCl, 5 mM CaCl2, 12 mM NaHCO3, 5 mM Hepes pH 8) ou elevada concentração
de NaCl (25 mM Tris pH 8, 5% glicerol, 600 mM NaCl) para simular o ambiente marinho, e pH baixo
(78 mM tampão Na+
/K+
de fosfato, pH 5.5) para simular o interior dos grânulos secretores, onde
proteínas adesivas, como a Nectina-2, são armazenadas antes da sua secreção.
Os espectros de emissão de fluorescência dos triptofanos obtidos para o constructo C2, no estado nativo,
nos diversos ambientes foram muito semelhantes ao obtido na presença de tampão Tris. Não obstante,
após desnaturação térmica na presença de água do mar, no estado desnaturado, observou-se um red shift
no máximo de emissão comparativamente ao tampão Tris. Globalmente estes dados sugerem que
embora a água do mar artificial possa não causar alterações conformacionais instantâneas na proteína,
parece influenciar a sua conformação quando exposta a condições desnaturantes.
A fluorescência dos triptofanos foi também usada para seguir a estabilidade térmica da proteína. Os
resultados obtidos mostraram que, na maioria das condições estudadas, este processo é cooperativo, e
que a temperatura de desnaturação aparente (Tm
app) é muito semelhante entre condições (entre 55 e 56
°C). A única exceção foi para o tampão com pH 5.5, neste caso a transição apresentada correspondia a
um processo não cooperativo (e por isso não pode ser diretamente comparada com as restantes
condições) obtendo-se uma Tm
app ligeiramente superior às restantes (58 °C).
Outra técnica utilizada para estudar a estabilidade térmica do constructo purificado foi a fluorescência
de varrimento diferencial (DSF), recorrendo ao fluoróforo SYPRO Orange, cuja fluorescência aumenta
na presença de ambientes apolares, tais como as regiões hidrófobas de uma proteína desnaturada.
Novamente, obteve-se um processo de desnaturação cooperativo nas condições estudadas. O facto de os
valores de Tm
app obtidos na presença de água do mar artificial e de tampão Tris terem sido diferentes (53
e 56 °C, respetivamente), corrobora a influência da água do mar no processo de desnaturação do
constructo. Para além disso, o facto de o valor de Tm
app ser inferior na presença da água do mar artificial
significa que esta condição tem um efeito desestabilizador no constructo, pelo menos quando se
considera a exposição de resíduos hidrófobos.
Curiosamente, os valores de Tm
app obtidos por fluorescência e por DSF na presença de água do mar
foram diferentes (56 e 53 °C, respetivamente). Isto pode indicar que, durante o processo de desnaturação
térmica, a exposição de resíduos hidrófobos acontece a temperaturas mais baixas do que as alterações
ao ambiente circundante dos triptofanos.
Seguidamente ainda avaliámos a propensão para este constructo agregar em diferentes condições, uma
vez que, em estudos anteriores, foi demonstrado que o adesivo secretado por P. lividus comporta-se
como um amiloide funcional. Estes ensaios foram realizados seguindo a dispersão de luz ao longo do
tempo, à temperatura ambiente, de uma solução do constructo C2 em tampão Tris versus água do mar
artificial (ASW2 - 445 mM NaCl, 60 mM MgCl2, 10 mM KCl, 10 mM CaCl2, 24 mM NaHCO3, 10 mM
Hepes pH 8). Interessantemente, em água do mar houve formação de precipitados. Embora estes dados
sejam muito preliminares, mais uma vez indicam que a água do mar provoca alterações conformacionais
na proteína alvo.
No geral, os resultados obtidos neste trabalho poderão vir a contribuir para uma melhor compreensão
dos mecanismos de adesão de P. lividus e para o desenvolvimento de novos bioadesivos inspirados em
ouriços-do-mar com potencial para aplicações nas áreas da biomedicina ou biotecnologia.
Epileptiform activity influences theta-burst induced LTP in the adult hippocampus: a role for synaptic lipid raft disruption in early metaplasticity?
Publication . Carvalho Rosa, José D.; Rodrigues, Nádia C.; Cruz, Armando; Vaz, Sandra H.; Cunha-Reis, Diana
Non-epileptic seizures are identified as a common epileptogenic trigger. Early metaplasticity following seizures may contribute to epileptogenesis by abnormally altering synaptic strength and homeostatic plasticity. We now studied how in vitro epileptiform activity (EA) triggers early changes in CA1 long-term potentiation (LTP) induced by theta-burst stimulation (TBS) in rat hippocampal slices and the involvement of lipid rafts in these early metaplasticity events. Two forms of EA were induced: (1) interictal-like EA evoked by Mg2+ withdrawal and K+ elevation to 6 mM in the superfusion medium or (2) ictal-like EA induced by bicuculline (10 μM). Both EA patterns induced and LTP-like effect on CA1 synaptic transmission prior to LTP induction. LTP induced 30 min post EA was impaired, an effect more pronounced after ictal-like EA. LTP recovered to control levels 60 min post interictal-like EA but was still impaired 60 min after ictal-like EA. The synaptic molecular events underlying this altered LTP were investigated 30 min post EA in synaptosomes isolated from these slices. EA enhanced AMPA GluA1 Ser831 phosphorylation but decreased Ser845 phosphorylation and the GluA1/GluA2 ratio. Flotillin-1 and caveolin-1 were markedly decreased concomitantly with a marked increase in gephyrin levels and a less prominent increase in PSD-95. Altogether, EA differentially influences hippocampal CA1 LTP thorough regulation of GluA1/GluA2 levels and AMPA GluA1 phosphorylation suggesting that altered LTP post-seizures is a relevant target for antiepileptogenic therapies. In addition, this metaplasticity is also associated with marked alterations in classic and synaptic lipid raft markers, suggesting these may also constitute promising targets in epileptogenesis prevention.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
UIDP/04046/2020