ABSTRACT Cassava is currently the second most important source of carbohydrates on the African continent. In the last two decades, cassava crops have been severely affected by outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus (SACMV) has been associated with CMD outbreaks in the Mpumalanga province. Advances in post-transcriptional gene silencing (PTGS) technology have provided promising new strategies for the engineering of virus resistance in plants. Inverted repeat (IR) constructs are currently the most potent inducers of PTGS, however, these constructs are inherently unstable. The purpose of this study was to develop IR constructs with an improved stability for the efficient induction of PTGS in plants. Two mismatched inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize the linear conformation of the IR constructs, as they were efficiently propagated by E.coli DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found that the number of mismatches on the BC1 construct (17.5%) was ideal, as the subsequent stability of the predicted RNA hairpin was not affected. Due to the higher number of mismatches on the AC1 construct (23.5%), it was found that the loop region of the RNA hairpin was marginally destabilized. Despite this, long stretches of stable dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. Interestingly, it was observed that the mismatched IR constructs, although still replicated in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that the stability of a mismatched IR construct may be influenced by the particular intracellular environment of an organism. Due to the recalcitrance of cassava to transformation, a model plant system, Nicotiana benthamiana, was used to screen constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated transformation and regeneration of N. benthamiana was optimized, and 86% transformation efficiency was achieved when using leaf disk explants. It was found that the addition of an ethylene scrubber, potassium permanganate, substantially increased the rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene iv integration was confirmed by PCR amplification of the hptII gene in the T-DNA region. Transgene expression was confirmed by screening for GUS and GFP reporter genes. No toxic responses to the transgene have been observed thus far. Studies are currently underway to confirm the stability of the mismatched IR constructs in N. benthamiana. PAGE Northern blotting is being done, as the detection of siRNAs derived from the transgene will confirm that constructs are functional. In addition, infectivity assays are underway to determine the efficacy of BC1 knockdown by a stably integrated construct. Due to the enhanced stability of mismatched IR constructs, they may be an appealing alternative to currently available intron-spliced, or exact matched hairpin systems.

Plant microRNAs play important roles in plant growth and development. Here we investigated the roles of miRNAs in the plant abiotic stress, development and viral infection. MicroRNA membrane array analysis using five different abiotic stress treatments resulted in the identification of 8 novel stress inducible miRNA-families. Functional studies on novel stress inducible miR168 revealed its functional relation with abiotic stress. Over expression of miR168 in Arabidopsis showed upregulation of four stress related miRNAs (miR163, miR167, miR398 and miR408). Analysis of 9 independent transgenic lines showed induction of miR398, an oxidative stress responsive miRNA with a corresponding down regulation of its target genes. Heavy metal oxidative stress tolerance bioassays confirmed the susceptibility of transgenics compared to the wild types indicating the fact that the miR168 is indirectly involved in plant abiotic stress by inducing other stress responsive miRNAs. MicroRNAs are highly conserved across the plant kingdom. A miRNA atlas was drafted for different tomato organs and fruit stages using the known miRNA sequences from different plants species. A large variation in both number and level of miRNA expression pattern was observed among different organs as well as among fruit stages. In the present investigation, we have found a window of expression for different miRNAs during the fruit development. A gradual decrease in the expression levels of miR160h, miR167a and miR399d and a gradual increase in miR164a have been noticed towards the fruit maturation while miR398b showed dual peaks during fruit development indicating a potential role of various miRNAs in fruit development and maturation. Sonchus yellow net virus (SYNV) infected Nicotinana benthamiana leaves showed severe disease symptoms at two weeks post infection (WPI) and gradually recovered from the SYNV infection after 4-5 WPI correlating with the overall miRNA levels. The miRNA array and northern analysis showed an overall reduction of miRNA biogenesis during 2WPI followed by restoration to normal levels supporting the idea that the SYNV indeed interfered with the host miRNA levels which caused the symptoms and recovery phenotypes. Overall studies on plant abiotic stress, development and viral infection showed important roles of miRNAs in different aspects of plant life.

Le silencing transcriptionnel limite la transcription des gènes et des éléments transposables dont l’expression pourrait être délétère à la cellule. Il dépend d’une diversité de modifications de la chromatine comme la méthylation ADN ou les marques répressives des histones. De façon à mieux comprendre les mécanismes moléculaires à l’origine du silencing transcriptionnel, nous avons mené une approche de génétique directe à l’aide d’un transgène soumis au silencing dans la plante modèle Arabidopsis thaliana. Cette stratégie nous a permis d'isoler à la fois des mutants déficients pour le maintien du silencing transcriptionnel et des mutations qui empêchent la réactivation transcriptionnelle des éléments transposables en réponse à un stress thermique. Nous avons caractérisé les défauts provoqués par ces mutations en combinant des approches de biologie moléculaire, de cytologie et de génomique.Nous montrons ainsi que MED14, la sous-unité centrale du complexe Mediator, et UVH6, composant du complexe TFIIH, sont requis pour la transcription de l'hétérochromatine en stress thermique. MED14 stimule aussi la transcription de l'hétérochromatine en l'absence de stress, mais ne semble fonctionner qu'en présence de la méthylation ADN. En plus de cette fonction originale, nous identifions un nouveau rôle de MED14 dans le maintien de la méthylation ADN, possiblement via la voie de méthylation ADN dirigée par les petits ARN.Par ailleurs, nos résultats nous ont permis d’identifier le rôle des protéines MAIN et MAIL1, qui définissent une voie de silencing transcriptionnelle indépendante des voies connues jusqu'alors. De façon intéressante, MAIN et MAIL1 possèdent un domaine protéique partagé avec les éléments transposables, qui aurait successivement été capturé par les éléments transposables et leur hôte au cours de l’histoire évolutive des plantes à fleurs.Enfin, en isolant une nouvelle mutation du gène POL2A, nous confirmons le rôle de l’ADN polymérase epsilon dans le silencing transcriptionnel et caractérisons les propriétés chromatiniennes qui dépendent de POL2A. Nous montrons que les défauts de silencing des mutants pol2a corrèlent avec une désorganisation importante de l’hétérochromatine sans diminution drastique des marques qui y sont associées. Au contraire, nous détectons une hyperméthylation ADN prononcée dans le mutant, et explorons différentes hypothèses pour expliquer ce phénotype particulier. Nos données suggèrent que plusieurs mécanismes moléculaires sont à l’origine des défauts des mutants pol2a. Elles confirment le rôle prépondérant de la chromométhylase CMT3 dans la régulation de la méthylation ADN, et suggèrent qu’un stress réplicatif pourrait causer une hyperméthylation de l’ADN.Dans l’ensemble, ces travaux de thèse proposent des pistes de travail dont l’exploration pourrait permettre d’expliquer les effets des déficiences réplicatives dans le maintien du silencing transcriptionnel et de l’homéostasie de la méthylation ADN. Ils suggèrent en outre que MED14 a une fonction dédiée à la transcription de l’hétérochromatine qui pourrait stimuler le maintien de la méthylation ADN. / Transcriptional gene silencing hinders deleterious transcription of some genes and transposable elements. Silencing is maintained by numerous chromatin modifications such as DNA methylation and repressive histone marks. To better understand the molecular mechanisms of silencing, we conducted a forward genetic screen using a transgene reporter system targeted by transcriptional gene silencing in the model plant Arabidopsis thaliana. We isolated a first type of mutants with diminished maintenance of silencing and a second category that displayed deficient release of transgene silencing upon heat stress. We then combined molecular, cytological and genomic methods to characterize the defects associated with these mutations.First, we show that the Mediator subunit MED14 and the TFIIH complex subunit UVH6 are required for heat-stress-induced release of silencing. We further show that MED14, but not UVH6, promotes transcriptional activation of transposable elements in mutant contexts where silencing is defective. Importantly, MED14 is only required when DNA methylation is not affected, suggesting that MED14 has a specialized function to promote transcription of heterochromatin. Furthermore, we show that MED14 promote DNA methylation at targets regulated by RNA-directed DNA methylation.Characterizing mutants from the first category, we unveil the contribution of the MAIN and MAIL1 proteins into transcriptional gene silencing, and show that they likely act through a pathway independent of known silencing factors. Interestingly, MAIN and MAIL1 bear a protein domain that is shared with transposable elements, and that has been captured by transposable elements and genes throughout the evolutionary history of flower plants.Additionally, we confirm the involvement of the DNA polymerase epsilon in transcriptional gene silencing by isolating a new mutation of the POL2A gene among mutants of the first category. We characterize the effects of the pol2a mutation on several heterochromatin properties, and show that the pol2a mutant retains high levels of heterochromatin marks despite having highly disorganized heterochromatin. We actually detect a strong elevation of DNA methylation in the pol2a mutant and explore different hypothesis to explain this unusual phenotype. We show that increased expression of the CMT3 chromomethylase is a likely cause, but that additional molecular mechanisms are probably involved. Further exploration suggests that constitutive replicative stress occurring in pol2a mutants could be an additional cause of DNA hypermethylation.To summarize, this work provide putative causes for DNA hypermethylation and silencing defects in a situation of replicative deficiency. Further investigation will be required to identify the molecular components involved in the mechanism. Our data further suggest that MED14 has a function dedicated to heterochromatin transcription that could promote DNA methylation maintenance.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13017号 / 論農博第2827号 / 新制||農||1042(附属図書館) / 学位論文||H28||N4964(農学部図書室) / 32945 / 京都大学大学院農学研究科農学専攻 / (主査)教授 土井 元章, 教授 裏出 令子, 教授 奥本 裕 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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No / Eczema is widely considered to be an exacerbation of alkaline stress to the skin. Epidermal barrier dysfunction is a feature of eczema pathology, which predisposes affected individuals to distressing morbid symptoms. At least two serine proteases, stratum corneum chymotryptic enzyme (kallikrein 7 [KLK7]) and stratum corneum tryptic enzyme (kallikrien 5 [KLK5]), have increased activity levels in eczematous lesions and both have been implicated in the destruction of corneodesomosomes, which are crucial to epidermal integrity. The present in vitro study investigated whether transcriptional gene silencing after siRNA transfection could influence the activity of these signature enzymes in an in vitro model of eczema induced by alkaline shock. HaCaT epithelial cells were subjected to alkaline stress by the addition of 1,1,3,3-tetramethyl guanidine “superbase” (TMG) to the culture media. The culture media were subsequently tested for chymotryspin, trypsin, plasmin, and urokinase activity using colorimetric peptide assays and for reactive oxygen species using WST1 cell viability reagent. Cells that had been transfected with small interfering ribonucleic acid (siRNA) against KLK5 and KLK7 for 24 h before alkaline shock did not exhibit the increase in serine protease levels observed in untreated controls. Moreover, an endpoint MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) confirmed that detachment of cells from the culture substrate observed in alkaline-stressed cells did not occur in siRNA-treated cells. This in vitro study has established the proof-of-principle that siRNA therapy appears to mitigate the consequences of alkaline shock to the serine protease-associated fragility of epithelial cells that is characteristic of eczema.

ARGONAUTE proteins (AGOs) are generally accepted as key components of the post transcriptional gene silencing mechanism, also involved in plant antiviral defense. Except for reports on the antiviral roles of AGO1, AGO2 and AGO7 in Arabidopsis, the exact roles played by the individual AGOs in other plant species are largely unknown. This research focused on the identification and characterization of AGOs involved in antiviral RNAi response to various viruses in N. benthamiana. Based on the temporal and spatial distribution of AGO transcripts in 3 and 8-week old plant root, stem and leaf tissues, expressions of NbAGO mRNAs were found to vary with age and tissue specificity. Plant endogenous AGO mRNAs were knocked down through virus induced gene silencing techniques using the Tobacco rattle virus vector system and posteriorly challenged with a GFP-chimeric virus construct deficient of a silencing suppressor. Unlike in control non-silenced plants, the Tomato bushy stunt virus construct deficient of its P19 silencing suppressor was consistently seen to exhibit a strong fluorescence on N. benthamiana plants silenced for NbAGOs 2 and X. Similar results were also obtained upon silencing of NbAGO2 using hairpin vector techniques. Comparable observations were also made when Tobacco mosaic virus GFP constructs were agroinfiltrated on NbAGO2 silenced plants further hinting the antiviral defense roles played by these AGOs. Agroinfiltration of Foxtailmosaic virus, Sunnhemp mosaic virus, and Turnip crinkle virus GFP chimeric constructs on NbAGO2 silenced N. benthamiana plants, however did not result in accumulation of GFP indicating the AGO antiviral defense specificity to TBSV and TMV. The results also hinted at a role for AGO7. Collectively my findings suggest that the expression of AGOs in N. benthamiana is tissue and age dependent, and that unlike in the model plant Arabidopsis where the main antiviral AGO is thought to be AtAGO1; in N. benthamiana, NbAGOs 2 and X seem to be involved in an antiviral defense role against TBSV and TMV with other AGOs perhaps contributing.

The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

Le virus des nervures jaunes et nécrotiques de la betterave (Beet necrotic yellow vein virus, BNYVV) est l’agent infectieux responsable de la rhizomanie de la betterave sucrière, une maladie caractérisée par une prolifération anarchique du chevelu racinaire. Le Beet soil-borne mosaic virus (BSBMV) appartient également au genre Benyvirus mais n’est retrouvé qu’en Amérique du Nord. Ce virus, identifié pour la première fois au Texas, est morphologiquement et génétiquement semblable au BNYVV mais sérologiquement éloigné. Compte tenu des différences moléculaires existant, le BSBMV et BNYVV correspondent à deux espèces virales distinctes. Mon projet de thèse a consisté à étudier les interactions moléculaires entre le BNYVV et le BSBMV et rechercher les mécanismes impliqués dans la pathogénicité de ces deux virus. Des clones complets cDNA infectieux du BNYVV étaient disponibles, tout comme ceux de BSBMV. Compte tenu de l’aspect versatile de l’obtention de transcrits infectieux de ces différents clones, j’ai entrepris de produire des clones cDNA de chacun des ARN viraux sous contrôle d’un promoteur constitutive végétal pour initier l’infection par agroinfiltration. Les plantes hôtes Chenopodium quinoa et Nicotiana benthamiana ont été inoculées par des transcrits et agroinfiltrées pour initier l’infection virale et étudier l’interaction entre les ARN génomiques 1 et 2 des deux virus et étudier les propriétés de constructions chimères. En parallèle à ce travail, j’ai réalisé la caractérisation du suppresseur de RNA silencing du BSBMV en le comparant à celui du BNYVV. / The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

La tristeza es la enfermedad viral más grave del cultivo de los cítricos y su agente causal es el virus de la tristeza de los cí­tricos (CTV). CTV induce uno o más de los siguientes sí­ndromes: I) decaimiento y muerte de los naranjos dulces (ND), pomelos y mandarinos injertados sobre el patrón naranjo amargo (NA), sí­ndrome conocido como "tristeza", II) enanismo, acanaladuras en la madera y fruta de pequeño calibre (stem pitting, SP), y III) enanismo y amarillamiento de plantas de semilla de limonero, pomelo y NA (seedling yellows, SY). La gama de huéspedes de CTV es muy restringida y hasta hace poco no se conocí­a ningún huésped herbáceo experimental. Actualmente, se sabe que la agroinfiltración de Nicotiana benthamiana, con clones de DNA complemantario (cDNA) del aislado T36 de CTV produce la infección sistémica de la planta, acompañada de sí­ntomas similares a los inducidos en cí­tricos, si bien la infección no queda limitada al floema. El aislado T36 induce SY y SP de lima Mejicana (LM), pero no en otros huéspedes como pomelo o ND. El estudio de los determinantes genéticos responsables de la inducción del sí­ndrome de SP requerí­a desarrollar un sistema genético basado en clones agroinfecciosos de un aislado inductor de estos sí­ntomas, como el aislado español T318A. Para ello, se partió de clones de cDNA de longitud completa de T318A previamente desarrollados en el laboratorio, capaces de replicarse en hojas de N. benthamiana pero incapaces de inducir infección sistémica y que presentaban varias mutaciones en su proteína de cápsida minoritaria p27. La corrección de dichas mutaciones y la construcción de nuevos clones de longitud completa de T318A marcados con el gen gfp, mostraron una correcta replicación en hojas agroinfiltradas de N. benthamiana, pero resultaron incapaces de inducir infección sistémica en este huésped experimental. La respuesta diferencial de N. benthamiana frente a distintas cepas de CTV permite estudiar los factores implicados en la interacción virus-huésped. Se analizó la interacción de las proteí­nas virales p20 y p25 de los aislados T36 y T318A con proteí­nas de N. benthamiana utilizando un abordaje consistente en: i) la expresión transitoria de p20/p25 marcadas con una etiqueta Strep-Tag en hojas de N. benthamiana, ii) purificación de los complejos proteí­na CTV-proteína huésped y análisis interactómico de los datos, y iii) estudio de la interacción directa entre p20/p25 y proteínas seleccionadas del huésped mediante análisis del doble hibrido en levadura y complementación bimolecular de fluorescencia (BIFC). Este abordaje proteómico mostró claras diferencias entre aislados que pueden explicar, en parte, el comportamiento diferencial de los aislados T36 y T318A en dicho huésped experimental. La inducción el síndrome de decaimiento por parte de CTV ha obligado a utilizar patrones tolerantes al decaimiento. Dichos patrones son menos adecuados. Las plantas de cí­tricos propagadas sobre NA e infectadas por CTV muestran necrosis en los tubos cribosos y disminución del floema funcional. Éstos desórdenes podrí­an ser consecuencia de la activación de los mecanismos de defensa como la reacción de hipersensibilidad desencadenada por la ruta del ácido salicí­lico o el silenciamiento génico mediado por RNA (post-transcriptional gene silencing, PTGS). Con el objetivo de avanzar en el mecanismo molecular de la resistencia del NA a la infección por CTV, se estudió el papel de diferentes genes de la planta implicados en las rutas mediante el uso de un vector viral basado en el genoma del virus del manchado foliar de los cítricos (citrus leaf blotch virus, CLBV). El silenciamiento génico de las rutas del AS o del PTGS en plantas NA y la inoculación de tres aislados de CTV patogénicamente diferentes mostró la implicación de ambas rutas en la defensa del NA frente a CTV. / Tristeza is the most important viral disease affecting citrus plants and Citrus tristeza virus (CTV) is the causal agent of this disease. CTV induces at least one of this syndromes: I) decline and death of sweet orange (SwO), grapefruits and mandarin trees grafted on sour orange (SO) rootstock, this syndromes is known as "tristeza", II) stunting, stem pitting (SP) and small fruits, and III) stunting and leaf chlorosis of lemon, grapefruit and SO seedlings (seedling yellows, SY). The host range of CTV is restricted and until recently no experimental herbaceous host was known. The agroinoculation Nicotiana benthamiana with clones of complementary DNA (cDNA) from the CTV isolate T36 cause the systemic infection of the plant and similar symptoms to those observed in citrus, although the infection is not limited to the phloem. T36 isolate induces SY and SP of Mexican lime (ML), but not in other hosts such as grapefruit and SwO. Therefore, to study the genetic determinants responsible of the SP syndrome induction was necessary to develop a genetic system based on agroinoculated clones from an isolate able to induce these symptoms, such as the Spanish isolate T318A. To do this, full length cDNA clones from T318A were obtained. They are able to replicate in N. benthamiana leaves but unable to induce systemic infection and showed several mutations in their protein of the minor coat, p27. The correction of these mutations and the construction of new clones of complete length from T318A labeled with the gfp gene, showed a proper replication in agroinoculated leaves of N. benthamiana, but they were still unable to induce systemic infection in this experimental host. The differential response of N. benthamiana to different CTV strains allows the study of the potential factors involved in the virus-host interaction. The aim of this work was study the interaction between the viral proteins p20 and p25 from the isolates T36 and T318A with N. benthamiana proteins with an analysis consisted in: I) the transitory expression of p20/p25 fused to Strep-Tag in N. benthamiana leaves, II) purification of the CTV protein-host protein complex and interatomic analysis of the data, and III) the study of the direct interaction between p20/p25 and selected plant proteins by the analysis of the double hybrid in yeast and bimolecular complementation of fluorescence (BIFC). The proteomic analysis showed strong differences between isolates that may partially explain the differential behavior of the T36 and T318A isolates in this experimental host. The induction of decline syndrome by CTV in citrus has leaded the use of tolerant rootstocks to decline. However, the use of such rootstocks is less suitable. Citrus plants propagated on SO rootstock and infected by CTV show phloem necrosis below the bud union that reduces the flow of carbohydrates to the roots. These symptoms may be a consequence of the activation of defense pathways in the plant, such as the hypersensitive reaction, hormone salicylic acid (SA) pathways or the RNA mediated post-transcriptional gene silencing (PTGS). Their relation is essential to know their implication in the decline. Therefore, the role of different genes involved in SA and PTGS has been studied by the silencing of plant genes using a viral vector (VIGS) based in the genome of the citrus leaf blotch virus (CLBV). The gene silencing of the SA and PTGS in SO and the inoculation of three different pathogenicity CTV isolates showed that both pathways are involved in the SO defense against CTV. The analysis of the proteins p20, p23 and p25 as possible suppressors of the AS indicating that the more virulent CTV isolates possess the more powerful suppressors. / La Tristesa és la malaltia viral més greu del cultiu dels cítrics. CTV induïx un o més de les sí­ndromes següents: I) decaïment i mort de taronger dolç§ (ND), pomelo i mandariner empeltats sobre el patró taronger amarg (NA), sí­ndrome conegut com "Tristesa", II) nanisme, estries en la fusta i fruita de xicotet calibre (SP) i III) nanisme i tonalitat groguenta de plantes de llavor de llimera, pomelo i taronger amarg (SY). El rang d'hostes de CTV és molt restringit i fins fa poc no es coneixia cap hoste herbaci experimental. Actualment es sap que la infecciò sistèmica en Nicotiana benthamiana amb clons de DNA complementari (cDNA) de l`aïllat de T36 provoca la infecció sistemàtica de la planta, acompanyada de síntomes similars als induïts en cí­trics, si be la infecció no queda llimitada al floema. L' aïllat T36 induïx SY i estries en la fusta de Llima Mexicana (LM), però no en altres hostes com a pomelo, ND o NA, l'estudi dels determinants genètics responsables de la inducció de la síndrome de SP requeria desenvolupar un sistema genètic basat en clons agroinfecciosos d'un aïllat inductor d'estos sí­mptomes, com l'aïllat espanyol T318A. Per a això, es va partir de clons de cDNA longitud completa de T318A prèviament desenvolupats al laboratori, capaços de replicar-se en fulls de N. benthamiana però incapaços d'induir infecció sistèmica i que presentaven varies mutacions en la seua proteïna de càpsida minoritatia p27. La correcció d`aquestes mutacions i la construcció de nous clons T318A de longitud completa marcats amb el gen gfp, van mostrar una correcta replicació en fulls agroinfiltradas de N. benthamiana però van resultar incapaços d'induir infecció sistèmica en aquest hoste experimental. La resposta diferencial dependent d'aïllat en N. benthamiana front CTV permet estudiar els possibles factors de la interacció virus- hoste. Es va dur a terme l'estudi de la funció de les proteínes virals p20 i p25 dels aïllats T36 i T318A amb proteïnes de N. benthamiana utilitzant un abordatge consistent en: i) l' expressió transitòria de les dues proteïnes p20/p25 marcades amb una etiqueta Strep-Tag en fulls de N. benthamiana, ii) purificació dels complexos proteïna CTV-proteïna hoste i anàlisi interactómic de les dades, i iii) estudi de la interacció directa per mitjà  de doble híbrid en llevat i complementació bimolecular de fluorescència (BIFC) de les proteïnes virals i determinades proteïnes de N. benthamiana. Aquest abordatge proteòmic va mostrar clares diferències entre aïllats que poden explicar el comportament diferencial dels aïllats T36 i T318A en aquest hoste experimental. La inducció de la sí­ndrome de decaïment per part de CTV en cí­trics ha obligat la utilització de patrons tolerants al decaïment. No obstant, aquestos patrons són agronòmicament menys adequats. Les plantes de cítrics propagades sobre NA i infectades por CTV mostren necrosi als tubs cribosos i disminució del floema funcional. Aquestos sí­mptomes poden ser conseqüència de l'activació de les rutes de defensa de la planta com la reacció d'hipersensibilitat, desencadenada per la ruta de l'àcid salicí­lic o el silenciamient gènic mediat per RNA (PTGS). Amb l'objectiu d'analitzar la implicació d¿aquestes rutes en la defensa, es va estudiar el paper de diferents gens implicats en la ruta de l'AS i del PTGS per mitjà  del silenciamient gènic induït per virus basat en el genoma del tacat foliar dels cítrics (CLBV). El silenciamient gènic de les rutes AS o PTGS en plantes NA i la inoculació de tres aïllats de CTV patogènicament diferents va mostrar la implicació de les dues rutes en la defensa del NA front CTV. L'analisis de les proteïnes p20, p23 i p25 com a possibles supressors de la ruta de l'AS va indicar que els aïllats més virulents de CTV posseïxen supressors més potents. / Gómez Muñoz, N. (2017). ESTUDIO DE LA INTERACCIÓN DIFERENCIAL ENTRE AISLADOS DEL VIRUS DE LA TRISTEZA DE LOS CÍTRICOS (CTV) Y SUS HUÉSPEDES [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/94624 / TESIS