<p>As a consequence of the presence of both mitochondria and chloroplasts in plant cells there is a higher sorting requirement in a plant cell than that in a non-plant cell. Reflecting this, protein import to mitochondria and chloroplasts has been shown to be highly specific. However, there is a group of proteins which are encoded by a single gene in the nucleus, translated in the cytosol and targeted to both mitochondria and chloroplasts. These proteins are referred to as dual targeted proteins. The first protein shown to be dual targeted was pea glutathione reductase (GR). The focus of this thesis is the targeting properties of the dual targeted protein glutathione reductase.</p><p>In order to overcome the limitations with traditional in vitro import systems we have developed an import system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The chloroplastic precursor of the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) was mis-targeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual system. The dual GR reductase precursor was targeted to both mitochondria and chloroplasts in both the single and dual import system.</p><p>We have investigated the targeting and processing properties of the GR targeting signal. Using N-terminal truncations we have demonstrated that the GR targeting signal has a domain organisation. Our results show that GR has evolved a dual targeting signal with the C-terminal part being sufficient for chloroplast import, the internal part required for the mitochondrial import and the N-terminal part housing a “fine-tuning” function. Furthermore, we have constructed a range of point mutations on the GR signal sequence changing positive amino acid residues and stretches of hydrophobic amino acid residues. Overall single mutations had a greater effect on mitochondrial import compared to import into chloroplasts. We have also shown that the recognition of the GR processing site differs between MPP and SPP. Single amino acid substitutions in the vicinity of the processing site clearly affected processing by MPP while processing by SPP showed low sensitivity to single mutations.</p>

As a consequence of the presence of both mitochondria and chloroplasts in plant cells there is a higher sorting requirement in a plant cell than that in a non-plant cell. Reflecting this, protein import to mitochondria and chloroplasts has been shown to be highly specific. However, there is a group of proteins which are encoded by a single gene in the nucleus, translated in the cytosol and targeted to both mitochondria and chloroplasts. These proteins are referred to as dual targeted proteins. The first protein shown to be dual targeted was pea glutathione reductase (GR). The focus of this thesis is the targeting properties of the dual targeted protein glutathione reductase. In order to overcome the limitations with traditional in vitro import systems we have developed an import system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The chloroplastic precursor of the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) was mis-targeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual system. The dual GR reductase precursor was targeted to both mitochondria and chloroplasts in both the single and dual import system. We have investigated the targeting and processing properties of the GR targeting signal. Using N-terminal truncations we have demonstrated that the GR targeting signal has a domain organisation. Our results show that GR has evolved a dual targeting signal with the C-terminal part being sufficient for chloroplast import, the internal part required for the mitochondrial import and the N-terminal part housing a “fine-tuning” function. Furthermore, we have constructed a range of point mutations on the GR signal sequence changing positive amino acid residues and stretches of hydrophobic amino acid residues. Overall single mutations had a greater effect on mitochondrial import compared to import into chloroplasts. We have also shown that the recognition of the GR processing site differs between MPP and SPP. Single amino acid substitutions in the vicinity of the processing site clearly affected processing by MPP while processing by SPP showed low sensitivity to single mutations.

Na célula eucariota, os processos metabólicos estão compartimentalizados em organelas e proteínas sintetizadas no citosol são endereçadas para elas por meio de sistema celular específico. Devido a sobreposições funcionais entre organelas, uma dada proteína pode ser requerida em mais de um compartimento. É o caso de proteínas com duplo direcionamento, em que um gene nuclear é capaz de gerar produtos protéicos direcionados para mais de uma organela. Cerca de 60 proteínas de plantas já tiveram seu duplo direcionamento demonstrado, a maioria para mitocôndrias e cloroplastos, portanto um fenômeno não tão raro como imaginado. Estudos recentes procuram esclarecer os mecanismos que permitem esse duplo direcionamento, mesmo existindo fatores celulares que garantem a especificidade do transporte para cada organela. O presente trabalho está dividido em três partes. Na primeira, foram investigados aspectos evolutivos do duplo direcionamento. Na análise comparativa de famílias gênicas com membros cujos produtos protéicos apresentam duplo direcionamento, em Arabidopsis thaliana e Oryza sativa, foi demonstrada a conservação do duplo direcionamento de monodeidroascorbato redutase, metionina aminopeptidase e, provavelmente, de THI1 (enzima da biossíntese de tiazol), entre as duas espécies. Os dados sugeriram um mesmo padrão de evolução em famílias incluindo membros com duplo direcionamento. Na segunda parte, o foco foi a seqüência de duplo direcionamento. Documentado o duplo direcionamento, para mitocôndrias e cloroplastos, de proteínas de ligação ao RNA, RBP1a, RBP1b e RPS19, mutações sítiodirigidas foram introduzidas na seqüência de direcionamento de RBP1b. A importância de aminoácidos positivos para o direcionamento de proteínas para mitocôndrias foi confirmada. Demonstrou-se que a mutação da alanina na posição 2, conservada em seqüências de direcionamento ambíguas para mitocôdrias e cloroplastos, não afeta o duplo direcionamento de RBP1b. A informação para o duplo direcionamento foi localizada entre os 17 primeiros aminoácidos da região amino-terminal. Os sinais de direcionamento para cloroplastos se distribuíram ao longo da seqüência. Enquanto a metade amino-terminal da seqüência foi suficiente para determinar o duplo direcionamento, a seqüência compreendendo os 13 aminoácidos seguintes afetaram a eficiência do transporte. Nessas análises, mostrou-se a adequação de método quantitativo na medida dos sinais de fluorescência da GFP, para ser aplicado em estudos quantitativos do direcionamento de proteínas para mitocôndrias e cloroplastos, in vivo. Na terceira parte, o foco foi o mecanismo traducional do duplo direcionamento de THI1, em A. thaliana. Entretanto, não foi possível testar a hipótese da presença de um sítio interno de entrada do ribossomo (IRES) no mRNA de thi1, pois não se encontrou um sistema de expressão transiente capaz de reproduzir dados da literatura que mostraram o duplo direcionamento de THI1. Nos sistemas testados, a proteína foi encontrada somente em cloroplastos, o que inviabilizou o prosseguimento da investigação. / Compartimentalization of the metabolic processes in organelles, each one having a characteristic protein pool and distinct functions, is a property of eukaryote cells. A highly specific cellular system directs proteins, which are synthesized in the cytosol, to the proper organelles. However, due to functional overlaps between organelles, a given protein may be needed in different compartments. This is the case of dual-targeted proteins, which are the product of single nuclear genes, but are somehow directed to different organelles. About 60 plant proteins have had their dual targeting demonstrated, most of them to mitochondria and cloroplasts, the phenomenon being not so rare as previously supposed. Investigations have focused on the mechanisms, which enable protein dual targeting, even in the presence of other cell mechanisms that guarantee the specific protein transport to each organelle. The present work on the subject can be divided in three parts. In the first part, evolutionary aspects of dual targeting were investigated. A comparative analysis of gene families that included members encoding dual-targeted proteins in Arabidopsis thaliana and Oryza sativa demonstrated that the dual targeting of monodehidro-ascorbate reductase, methyonine aminopeptidase and, problably, of the thiazole biosynthetic enzyme THI1 was evolutionary conserved between the two species. In addition, the data suggested the same pattern of evolution for families with members presenting dual targeting. The focus of the second part was the ambiguous sequence for dual targeting. After showing that the RNA-binding proteins RBP1a, RBP1b and RPS19 were dual-targeted to mitochondria and cloroplasts, sitedirected mutations were introduced in the targeting sequence of RBP1b. The importance of positive-charged amino acids for directing the protein to mitochondria was confirmed. Mutation of alanine at position 2, which is conserved in ambiguous sequences, was shown not to affect RBP1b dual targeting. Information for dual targeting was localized among the 17 first amino acids in the amino-terminal region. The signals for directing the protein to cloroplasts appeared distributed along the targeting sequence. While the amino-terminal half of the sequence was sufficient for RBP1b dual targeting, the sequence comprising the next 13 amino acids appeared to affect the efficiency of the transport. In these analyses, a quantitative method to measure the intensity of fluorescent signals of GFP had its efficacy demonstrated to be adopted for in vivo quantitative analysis of dual targeting to mitochondria and cloroplasts. In the third part, the focus was the translational mechanism enabling THI1 dual targeting to mitochondria and cloroplasts, in A. thaliana. It was hypothesized that an internal ribosomal entry site (IRES) was present in thi1 mRNA. However, a transient expression system could not be found that reproduced the literature data demonstrating THI1 dual targeting. In the tested systems the protein was addressed solely to cloroplasts, thus preventing the objective to be pursued.

A targeting peptide at the N-terminus of a precursor protein usually directs the protein synthesized in the cytosol to a specific organelle in the cell. Interestingly, some targeting peptides, so-called dual targeting peptides (dTPs) can target their protein to both mitochondria and chloroplasts. In order to understand the mechanism of dual targeting, a dTP from threonyl tRNA synthetase (ThrRS-dTP) was investigated as a model dTP in this thesis work. The results suggest that ThrRS-dTP is intrinsically disordered in solution but has an α-helical propensity at the N-terminal part. Tom20 and Toc34 are the two primary receptors on the outer membranes of mitochondria and chloroplasts, respectively. We found that the N-terminal half of the ThrRS-dTP sequence, including an amphiphilic helix, is important for the interaction with Tom20. This part also contains a φχχφφ motif, where φ represents a hydrophobic/aromatic residue and χ represents any amino acid residue. In contrast, neither the amphiphilic helix nor φχχφφ motif in ThrRS-dTP has any special role for its interaction with Toc34. Instead, the entire sequence of ThrRS-dTP is important for Toc34 interaction, including the C-terminal part which is barely affected by Tom20 interaction. In addition, the role of lipids in the organelle membrane for the recognition of dual targeting peptides during protein import is also the focus of this thesis. The tendency to form α-helix in ThrRS-dTP, which is not observable in solution by CD, becomes obvious in the presence of lipids and DPC micelles. To be able to study such interactions, DMPC/DHPC isotropic bicelles under different conditions have also been characterized. These results demonstrate that bicelles with a long-chained/short-chained lipid ratio q = 0.5 and a concentration larger than 75 mM should be used to ensure that the classic bicelle morphology persists. Moreover, we developed a novel membrane mimetic system containing the galactolipids, MGDG or DGDG, which have been proposed to be important for protein import into chloroplasts. Up to 30% MGDG or DGDG lipids were able to be integrated into bicelles. The local dynamics of the galactolipids in bicelles displays two types of behavior: the sugar head-group and the glycerol part are rigid, and the acyl chains are flexible. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: In press.</p>

Na célula eucariota, os processos metabólicos estão compartimentalizados em organelas e proteínas sintetizadas no citosol são endereçadas para elas por meio de sistema celular específico. Devido a sobreposições funcionais entre organelas, uma dada proteína pode ser requerida em mais de um compartimento. É o caso de proteínas com duplo direcionamento, em que um gene nuclear é capaz de gerar produtos protéicos direcionados para mais de uma organela. Cerca de 60 proteínas de plantas já tiveram seu duplo direcionamento demonstrado, a maioria para mitocôndrias e cloroplastos, portanto um fenômeno não tão raro como imaginado. Estudos recentes procuram esclarecer os mecanismos que permitem esse duplo direcionamento, mesmo existindo fatores celulares que garantem a especificidade do transporte para cada organela. O presente trabalho está dividido em três partes. Na primeira, foram investigados aspectos evolutivos do duplo direcionamento. Na análise comparativa de famílias gênicas com membros cujos produtos protéicos apresentam duplo direcionamento, em Arabidopsis thaliana e Oryza sativa, foi demonstrada a conservação do duplo direcionamento de monodeidroascorbato redutase, metionina aminopeptidase e, provavelmente, de THI1 (enzima da biossíntese de tiazol), entre as duas espécies. Os dados sugeriram um mesmo padrão de evolução em famílias incluindo membros com duplo direcionamento. Na segunda parte, o foco foi a seqüência de duplo direcionamento. Documentado o duplo direcionamento, para mitocôndrias e cloroplastos, de proteínas de ligação ao RNA, RBP1a, RBP1b e RPS19, mutações sítiodirigidas foram introduzidas na seqüência de direcionamento de RBP1b. A importância de aminoácidos positivos para o direcionamento de proteínas para mitocôndrias foi confirmada. Demonstrou-se que a mutação da alanina na posição 2, conservada em seqüências de direcionamento ambíguas para mitocôdrias e cloroplastos, não afeta o duplo direcionamento de RBP1b. A informação para o duplo direcionamento foi localizada entre os 17 primeiros aminoácidos da região amino-terminal. Os sinais de direcionamento para cloroplastos se distribuíram ao longo da seqüência. Enquanto a metade amino-terminal da seqüência foi suficiente para determinar o duplo direcionamento, a seqüência compreendendo os 13 aminoácidos seguintes afetaram a eficiência do transporte. Nessas análises, mostrou-se a adequação de método quantitativo na medida dos sinais de fluorescência da GFP, para ser aplicado em estudos quantitativos do direcionamento de proteínas para mitocôndrias e cloroplastos, in vivo. Na terceira parte, o foco foi o mecanismo traducional do duplo direcionamento de THI1, em A. thaliana. Entretanto, não foi possível testar a hipótese da presença de um sítio interno de entrada do ribossomo (IRES) no mRNA de thi1, pois não se encontrou um sistema de expressão transiente capaz de reproduzir dados da literatura que mostraram o duplo direcionamento de THI1. Nos sistemas testados, a proteína foi encontrada somente em cloroplastos, o que inviabilizou o prosseguimento da investigação. / Compartimentalization of the metabolic processes in organelles, each one having a characteristic protein pool and distinct functions, is a property of eukaryote cells. A highly specific cellular system directs proteins, which are synthesized in the cytosol, to the proper organelles. However, due to functional overlaps between organelles, a given protein may be needed in different compartments. This is the case of dual-targeted proteins, which are the product of single nuclear genes, but are somehow directed to different organelles. About 60 plant proteins have had their dual targeting demonstrated, most of them to mitochondria and cloroplasts, the phenomenon being not so rare as previously supposed. Investigations have focused on the mechanisms, which enable protein dual targeting, even in the presence of other cell mechanisms that guarantee the specific protein transport to each organelle. The present work on the subject can be divided in three parts. In the first part, evolutionary aspects of dual targeting were investigated. A comparative analysis of gene families that included members encoding dual-targeted proteins in Arabidopsis thaliana and Oryza sativa demonstrated that the dual targeting of monodehidro-ascorbate reductase, methyonine aminopeptidase and, problably, of the thiazole biosynthetic enzyme THI1 was evolutionary conserved between the two species. In addition, the data suggested the same pattern of evolution for families with members presenting dual targeting. The focus of the second part was the ambiguous sequence for dual targeting. After showing that the RNA-binding proteins RBP1a, RBP1b and RPS19 were dual-targeted to mitochondria and cloroplasts, sitedirected mutations were introduced in the targeting sequence of RBP1b. The importance of positive-charged amino acids for directing the protein to mitochondria was confirmed. Mutation of alanine at position 2, which is conserved in ambiguous sequences, was shown not to affect RBP1b dual targeting. Information for dual targeting was localized among the 17 first amino acids in the amino-terminal region. The signals for directing the protein to cloroplasts appeared distributed along the targeting sequence. While the amino-terminal half of the sequence was sufficient for RBP1b dual targeting, the sequence comprising the next 13 amino acids appeared to affect the efficiency of the transport. In these analyses, a quantitative method to measure the intensity of fluorescent signals of GFP had its efficacy demonstrated to be adopted for in vivo quantitative analysis of dual targeting to mitochondria and cloroplasts. In the third part, the focus was the translational mechanism enabling THI1 dual targeting to mitochondria and cloroplasts, in A. thaliana. It was hypothesized that an internal ribosomal entry site (IRES) was present in thi1 mRNA. However, a transient expression system could not be found that reproduced the literature data demonstrating THI1 dual targeting. In the tested systems the protein was addressed solely to cloroplasts, thus preventing the objective to be pursued.

Nos eucariotos, a evolução dos sistemas de transporte molecular foi essencial pois seu alto grau de compartimentalização requer mecanismos com maior especificidade para a localização de proteínas. Com o estabelecimento das mitocôndrias e plastídeos como organelas da célula eucariota, grande parte dos genes específicos para sua atividade e manutenção foram transferidos ao núcleo. Após a transferência gênica, a maioria das proteínas passaram a ser codificadas pelo núcleo, sintetizadas no citosol e direcionadas às organelas por uma maquinaria complexa que envolve receptores nas membranas das organelas, sequências de direcionamento nas proteínas e proteínas citossólicas que auxiliam o transporte. A importação depende em grande parte de uma sequência na região N-terminal das proteínas que contém sinais reconhecidos pelas membranas organelares. No entanto, muito ainda não é compreendido sobre o transporte de proteínas organelares e fatores ainda desconhecidos podem influenciar o direcionamento sub-celular. O objetivo deste trabalho foi a caracterização da General Regulatory Factor 9 (GRF9), uma proteína da família 14-3-3 de Arabidopsis thaliana potencialmente envolvida no direcionamento de proteínas organelares, e a geração de um genótipo para ser utilizado na obtenção de uma população mutante para genes que afetam o direcionamento da proteína Tiamina Monofosfato Sintetase (TH-1). Após experimentos in vivo e in planta, foi observado que GRF9 interage com as proteínas duplo-direcionadas Mercaptopyruvate Sulfurtransferase1 (MST1) e a Thiazole Biosynthetic Enzyme (THI1), e com a proteína direcionada aos cloroplastos TH-1. Experimentos de deleção e interação in vivo mostraram que a região Box1 de GRF9 é essencial para a interação com THI1 e MST1. Com a finalidade de dar continuidade a caracterização da GRF9 e para realização de testes com relação a sua função no direcionamento de proteínas organelares foi gerada uma linhagem homozigota que superexpressa GRF9. Plantas expressando o transgene TH-1 fusionado a Green Fluorescent Protein (GFP) em genótipo deficiente na TH-1 (CS3469/TH-1-GFP) foram obtidas para a geração de população mutante que possibilitará a descoberta de componentes genéticos ainda desconhecidos e responsáveis pelo direcionamento de proteínas aos cloroplastos. / In Eukaryotes, the evolution of molecular transport in the cell was essential due to their increase in compartmentalization, which requires more specific mechanisms for the correct localization of proteins. With the establishment of mitochondria and plastids as organelles, a great number of their genes, either specific for their metabolic functions or maintenance of their own transcription/translation processes, were transferred to the nucleus of the cell. These transfers caused most of the organellar proteins to be coded by the nucleus, then synthesized in the cytosol and targeted to the organelles by a complex machinery which involves membrane receptors in the organelles, targeting sequences in the proteins, and cytosolic proteins which assist them with the transport. Protein import depends greatly on an N-terminal sequence in proteins which has recognizable signals for the organellar membrane receptors. However, much is still not understood about the transport of organellar proteins, and unknown factors may still influence subcellular targeting. The goal of this work was the characterization of General Regulatory Factor 9 (GRF9), a protein of the 14-3-3 family in Arabidopsis thaliana potentially involved in the targeting of organellar proteins, and generating a genotype to be used in obtaining a mutant population for genes affecting the targeting of the protein Thiamine Requiring 1 (TH-1). After in vivo and in planta experiments it was observed that GRF9 interacts with the dual-targeted proteins Mercaptopyruvate Sulfurtransferase1 (MST1) and Thiazole Biosynthetic Enzyme (THI1), and with the chloroplast targeted protein TH-1. Deletion experiments followed by in vivo interaction assays showed that Box 1 region of GRF9 is essential for the interaction with THI1 and MST1. For the continuing characterization of GRF9 and for following tests of its function in the targeting of organellar proteins, a homozygous line was generated overexpressing GRF9. Plants expressing the transgene TH-1 fused to the Green Fluorescent Protein (GFP) in a TH-1 deficient genotype (CS3469/TH-1-GFP) were obtained for the generation of a mutant population which will allow the discovery of genetic components still unknown responsible for targeting proteins to the chloroplasts.

Nos eucariotos, a evolução dos sistemas de transporte molecular foi essencial pois seu alto grau de compartimentalização requer mecanismos com maior especificidade para a localização de proteínas. Com o estabelecimento das mitocôndrias e plastídeos como organelas da célula eucariota, grande parte dos genes específicos para sua atividade e manutenção foram transferidos ao núcleo. Após a transferência gênica, a maioria das proteínas passaram a ser codificadas pelo núcleo, sintetizadas no citosol e direcionadas às organelas por uma maquinaria complexa que envolve receptores nas membranas das organelas, sequências de direcionamento nas proteínas e proteínas citossólicas que auxiliam o transporte. A importação depende em grande parte de uma sequência na região N-terminal das proteínas que contém sinais reconhecidos pelas membranas organelares. No entanto, muito ainda não é compreendido sobre o transporte de proteínas organelares e fatores ainda desconhecidos podem influenciar o direcionamento sub-celular. O objetivo deste trabalho foi a caracterização da General Regulatory Factor 9 (GRF9), uma proteína da família 14-3-3 de Arabidopsis thaliana potencialmente envolvida no direcionamento de proteínas organelares, e a geração de um genótipo para ser utilizado na obtenção de uma população mutante para genes que afetam o direcionamento da proteína Tiamina Monofosfato Sintetase (TH-1). Após experimentos in vivo e in planta, foi observado que GRF9 interage com as proteínas duplo-direcionadas Mercaptopyruvate Sulfurtransferase1 (MST1) e a Thiazole Biosynthetic Enzyme (THI1), e com a proteína direcionada aos cloroplastos TH-1. Experimentos de deleção e interação in vivo mostraram que a região Box1 de GRF9 é essencial para a interação com THI1 e MST1. Com a finalidade de dar continuidade a caracterização da GRF9 e para realização de testes com relação a sua função no direcionamento de proteínas organelares foi gerada uma linhagem homozigota que superexpressa GRF9. Plantas expressando o transgene TH-1 fusionado a Green Fluorescent Protein (GFP) em genótipo deficiente na TH-1 (CS3469/TH-1-GFP) foram obtidas para a geração de população mutante que possibilitará a descoberta de componentes genéticos ainda desconhecidos e responsáveis pelo direcionamento de proteínas aos cloroplastos. / In Eukaryotes, the evolution of molecular transport in the cell was essential due to their increase in compartmentalization, which requires more specific mechanisms for the correct localization of proteins. With the establishment of mitochondria and plastids as organelles, a great number of their genes, either specific for their metabolic functions or maintenance of their own transcription/translation processes, were transferred to the nucleus of the cell. These transfers caused most of the organellar proteins to be coded by the nucleus, then synthesized in the cytosol and targeted to the organelles by a complex machinery which involves membrane receptors in the organelles, targeting sequences in the proteins, and cytosolic proteins which assist them with the transport. Protein import depends greatly on an N-terminal sequence in proteins which has recognizable signals for the organellar membrane receptors. However, much is still not understood about the transport of organellar proteins, and unknown factors may still influence subcellular targeting. The goal of this work was the characterization of General Regulatory Factor 9 (GRF9), a protein of the 14-3-3 family in Arabidopsis thaliana potentially involved in the targeting of organellar proteins, and generating a genotype to be used in obtaining a mutant population for genes affecting the targeting of the protein Thiamine Requiring 1 (TH-1). After in vivo and in planta experiments it was observed that GRF9 interacts with the dual-targeted proteins Mercaptopyruvate Sulfurtransferase1 (MST1) and Thiazole Biosynthetic Enzyme (THI1), and with the chloroplast targeted protein TH-1. Deletion experiments followed by in vivo interaction assays showed that Box 1 region of GRF9 is essential for the interaction with THI1 and MST1. For the continuing characterization of GRF9 and for following tests of its function in the targeting of organellar proteins, a homozygous line was generated overexpressing GRF9. Plants expressing the transgene TH-1 fused to the Green Fluorescent Protein (GFP) in a TH-1 deficient genotype (CS3469/TH-1-GFP) were obtained for the generation of a mutant population which will allow the discovery of genetic components still unknown responsible for targeting proteins to the chloroplasts.

[ES] Los virus de plantas son los agentes causales de un gran número de enfermedades en plantas que ocasionan grandes pérdidas económicas. El virus de las manchas necróticas del melón (MNSV), es un pequeño virus de RNA monocatenario de polaridad positiva, perteneciente al género Gammacarmovirus, cuyo genoma codifica cinco proteínas. La proteína de cubierta (CP), está formada por tres dominios distintos. El descubrimiento de un péptido de transito dual en la región amino-terminal de la CP fue el punto de partida de esta tesis. Al inicio de una infección por MNSV, la CP nuevamente sintetizada es transportada al interior de cloroplastos y mitocondrias mientras que, una parte mucho menor se mantiene en el citoplasma aumentando a medida que avanza la infección. La inhibición de este transporte dual conlleva un aumento de la actividad supresora del silenciamiento del RNA de la CP. Sin embargo, la infección sistémica se ve particularmente afectada. Por tanto, la acumulación de la CP en el citoplasma puede provocar un aumento de la replicación viral pero a su vez una sobreexpresión de la p29, puede provocar una explosión oxidativa y una necrosis que restringe el movimiento viral. De este modo, el transporte de la CP a los orgánulos podría evitar una replicación viral excesiva mediante la modulación de la actividad supresora para gestionar el equilibrio entre la defensa de la planta y la contradefensa viral favoreciendo una interacción compatible entre ambos. Desafortunadamente, Arabidopsis thaliana no es huésped para el MNSV. Por tanto, para entender mejor el transporte de la CP a estos orgánulos, se identificaron los receptores y los poros de los translocones de las membranas externas de las mitocondrias y los cloroplastos en Nicotiana benthamiana. Esta caracterización funcional se realizó principalmente mediante VIGS y RT-qPCR, que mostró una redundancia funcional mayor que la observada entre los homólogos de Arabidopsis. Además, esta herramienta también se utilizó para evaluar la relevancia de cada componente bajo la infección por MNSV, y junto con los estudios de interacción CP-receptor realizados mediante BiFC y Y2H, nos permitió identificar NbToc159A para cloroplastos y NbOm64 para mitocondrias, como principales receptores. A su vez, el silenciamiento de NbToc34, NbToc75 o NbTom40 resultó en una resistencia generalizada no solo a MNSV sino también al virus del arrugamiento del nabo (TCV) y al virus del moteado del clavel (CarMV), lo que respalda la idea actualmente aceptada y que involucra el estado fisiológico del cloroplasto y la mitocondria en la señalización temprana de la respuesta defensiva. Finalmente, se realizó una búsqueda de factores del huésped que interaccionasen con la CP mediante con TurboID, una ligasa de biotina, que permite la detección de interacciones tanto directas e indirectas como transitorias y estables. Así, se obtuvo un gran número de proteínas candidatas utilizando la CP de MNSV y su mutante de localización citoplásmica, ∆NtCP. Tres de ellas, NbSIK1, NbSMU2 y NbMAP3K mostraron un efecto perjudicial constante y repetitivo sobre la acumulación del RNA viral. Después de la validación de las interacciones mediante otro método, y el análisis de la localización subcelular de la CP bajo el silenciamiento del interactor correspondiente, se establecieron dos hipótesis principales. En primer lugar, dado que la función principal de NbSMU2 está relacionada con el procesamiento y regulación del RNA mensajero, esta proteína podría ser secuestrada por la CP provocando la expresión de genes provirales. Por otro lado, NbSIK1 y NbMAP3K, actúan como reguladores positivo y negativo de la respuesta PTI a la infección, respectivamente. Además, ambas proteínas interaccionan entre sí y forman parte de la cascada de MAP quinasas, por lo que en nuestra segunda hipótesis, la CP interaccionaría con este complejo, promoviendo una regulación negativa de la PTI que facilitaría el desarrollo de la infección. / [CA] Els virus de plantes són els principals causants de la major part de malalties en plantes i les consegüents pèrdues econòmiques. El virus de les taques necròtiques del meló (MNSV) és un virus menut d'RNA monocatenari de polaritat positiva, pertanyent al gènere Gammacarmovirus, el genoma del qual codifica cinc proteïnes. La proteïna de coberta (CP) està formada per tres dominis diferents. El descobriment d'un pèptid de trànsit dual a la part aminoterminal de la CP va ser el punt de partida d'aquesta tesi. A l'inici d'una infecció per MNSV, la CP novament sintetitzada és transportada a l'interior dels cloroplasts i mitocondris mentre que, una part molt menor es manté al citoplasma augmentant a mesura que avança la infecció. La inhibició d'aquest transport dual comporta un augment de l'activitat supressora del silenciament de l'RNA de la CP. No obstant això, la infecció sistèmica es va frenar. Per tant, l'acumulació de la CP al citoplasma pot provocar un augment de la replicació viral però alhora una sobreexpressió de la p29, una replicasa auxiliar que ocasiona alteracions morfològiques als mitocondris, una explosió oxidativa i necrosi que restringeix el moviment viral. D'aquesta manera, el transport de la CP als orgànuls podria evitar una replicació viral excessiva mitjançant la modulació de l'activitat supressora per gestionar l'equilibri entre defensa de la planta i contradefensa viral que condueixen a una interacció compatible entre tots dos. Per entendre millor el mecanisme molecular que regeix el transport de la CP a aquests orgànuls, es van identificar els receptors i els porus dels translocon de les membranes externes dels mitocondris i els cloroplasts a Nicotiana benthamiana. Aquesta caracterització funcional es va realitzar principalment mitjançant VIGS i RT-qPCR, que va mostrar una redundància funcional més gran que l'observada entre els homòlegs d'Arabidopsis. A més, aquesta eina també es va utilitzar per avaluar la rellevància de cada component sota la infecció per MNSV, i juntament amb els estudis d'interacció CP-receptor realitzats mitjançant BiFC i Y2H, ens va permetre identificar a NbToc159A per a cloroplasts i NbOm64 per a mitocondris, com els principals receptors implicats en el transport de la CP a aquests orgànuls. Alhora, el silenciament de NbToc34, NbToc75 o NbTom40 va resultar en una resistència generalitzada a MNSV, TCV i CarMV, la qual cosa recolza la idea que circula actualment i que involucra l'estat fisiològic del cloroplast i el mitocondri en la senyalització primerenca de la resposta defensiva. Finalment, es va fer una cerca de factors de l'hoste que interaccionessin amb la CP mitjançant la innovadora tècnica de marcatge de proximitat amb TurboID, una lligasa de biotina, que permet la detecció d'interaccions tant directes i indirectes com transitòries i estables. Així, es va obtenir un gran nombre de proteïnes candidates utilitzant la CP de MNSV i el seu mutant de localització citoplàsmica, ∆NtCP. Tres de elles, NbSIK1, NbSMU2 i NbMAP3K van mostrar un efecte perjudicial constant i repetitiu sobre l'acumulació de l'RNA viral. Després de la validació de les interaccions mitjançant un altre mètode, i l'examen de la localització subcel·lular de la CP sota el silenciament de cada interactor, es van establir dues hipòtesis principals. En primer lloc, atès que la funció principal de NbSMU2 està relacionada amb el processament i la regulació de l'RNA missatger, aquesta proteïna podria ser segrestada per la CP provocant l'expressió de gens provirals. D'altra banda, NbSIK1 i NbMAP3K actuen com a reguladors positiu i negatiu de la resposta PTI a la infecció, respectivament. A més, les dos proteïnes interaccionen entre si i formen part de la cascada de MAP quinases, per la qual cosa en la nostra segona hipòtesi, la CP interaccionaria amb aquest complex, promovent una regulació negativa de la PTI que facilitaria el desenvolupament de la infecció. / [EN] Plant viruses are the causal agents of many plant diseases and the subsequent economic losses, estimated to be US$60 billion worldwide each year. The melon necrotic spot virus (MNSV) is a small, single-stranded, positive-sense RNA virus that belongs to the genus Gammacarmovirus and encodes five proteins. The coat protein (CP) is composed of three distinct domains. The discovery of a dual transit peptide in the amino-terminal part of the CP was the starting point of this thesis. Early in MNSV infection, the new synthesized CP is imported into chloroplasts and mitochondria, while the cytoplasmic pool increases as the infection progresses. Inhibiting this dual transport leads to an increase in the RNA silencing suppressor activity of the CP. However, far from resulting in an enhanced infection development, systemic spread was impaired. Therefore, the accumulation of cytoplasmic CP may cause an increase in viral replication and overexpression of p29, an auxiliary replicase that causes morphological alterations, ROS, and necrosis that may restrict viral movement. Thus, a new role for CP targeting would be to avoid excessive viral replication by modulating the suppressor activity to manage the balance between plant defense and viral counter-defense, leading to a compatible interaction. Unfortunately, Arabidopsis thaliana is not a host for MNSV. Thus, to better understand the molecular mechanism behind the CP dual targeting, the receptors and pores of the Nicotiana benthamiana mitochondrial and chloroplast outer membrane translocons were genome identified, and some functional characterization was carried out. We assigned the following names NbToc75-III, NbToc34, NbToc90, NbToc120, NbToc159A, NbToc159B, NbTic22-III for chloroplast translocon components, and NbTom40, NbTom20-1, NbTom20-2, NbOm64 for mitochondrion translocon components. The functional characterization was mainly carried out by virus-induced gene silencing (VIGS) and RT-qPCR, revealing a functional redundancy higher than that reported for Arabidopsis homologs. Additionally, VIGS was also used to evaluate the relevance of each translocon component in MNSV infection, and together with CP-receptor interaction studies performed by BiFC and Y2H, allowed us to identify NbToc159A for chloroplasts and NbOm64 for mitochondria as the main receptors involved in the CP organelle import. Moreover, silencing of NbToc34, NbToc75, or NbTom40 resulted in a generalized resistance not only to MNSV but also to turnip crinkle virus (TCV), and carnation mottle virus (CarMV), supporting the current idea that involves the chloroplast and mitochondrion physiological state in early defense response signaling. Finally, a search for host factors interacting with the CP was performed by the innovative TurboID proximity labeling tool, which allows the detection of both direct/indirect and transient/stable interactions. Thus, a large number of candidate proteins were obtained that interacted either with the MNSV CP or with ∆NtCP, a cytoplasm-localized mutant. Three of them, NbSIK1, NbSMU2, and NbMAP3K, showed a consistent and repetitive detrimental effect on MNSV RNA accumulation. After the validation of the interactions using another method and the analysis of the subcellular localization of the MNSV CP under each interactor silencing, two main hypotheses were proposed. Firstly, since the main function of NbSMU2 is related to messenger RNA regulation by splicing, this protein could be sequestered by the CP, causing the expression of proviral genes. On the other hand, NbSIK1 and NbMAP3K act as positive and negative regulators of the PTI response to infection, respectively. Moreover, both proteins interact with each other and are part of the MAP kinase cascade, so in our second hypothesis, CP would interact with this complex, promoting a negative regulation of PTI that would facilitate viral infection. / La autora ha disfrutado de un contrato predoctoral de formación de personal investigador (FPI) (PRE-2018-84130) otorgado por el Ministerio de Ciencia e Innovación asociado al proyecto BIO2017-88321-R. Este trabajo de tesis doctoral ha sido realizado con el apoyo económico de los proyectos de investigación del Ministerio de Ciencia e Innovación, BIO2017-88321-R y PID2020-115571RB- I00. / Sáiz Bonilla, M. (2023). Deciphering the intracellular dual targeting of the melon necrotic spot virus coat protein, its interaction with host factors and their roles in plant defense [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/195836