Estudio del desarrollo de la infección viroidal en mutantes never ripe de tomate

[ES] En respuesta a los ataques por parte de patógenos, así como a las diferentes agresiones ambientales a los que se ven sometidas las plantas, éstas sintetizan multitud de compuestos, que incluyen proteínas de defensa y una gran variedad de metabolitos secundarios que ayudan a la planta a adaptarse y superar la situación de estrés. Esta respuesta viene mediada por un número reducido de moléculas señalizadoras, entre las que destacan el ácido salicílico, el ácido jasmónico y el etileno. El grupo de trabajo dirigido por el Dr. J. Mª Bellés, donde se realizará este trabajo, está realizando estudios dirigidos a elucidar tanto la participación de estas moléculas señal como la implicación de compuestos del metabolismo secundario en la interacción de plantas de tomate con distintos patógenos. Para investigar el papel del etileno en la respuesta de plantas infectadas por el viroide de la exocortis de los cítricos (CEVd), se utilizará como material biológico mutantes de tomate Never ripe (Nr), que presentan insensibilidad a etileno, por lo que muestran fenotipos anómalos en la maduración de sus frutos y en su respuesta a patógenos. Disponemos de plantas Never ripe de los cultivares Rutgers y Pearson , así como sus correspondientes líneas parentales (accesiones LA3001, LA0162, LA1090 y LA0012, https://tgrc.ucdavis.edu). Se caracterizará fenotípicamente los mutantes de etileno Nr frente a la infección por CEVd, mediante el estudio de la sintomatología y la acumulación del patógeno. Se caracterizará molecularmente mediante el análisis de la expresión de genes implicados en las rutas de señalización defensiva y en la biosíntesis de etileno, así como el estudio del estrés ribosomal causado por este patógeno. Por último, se llevará a cabo una caracterización química, mediante el análisis de los niveles de etileno que refleje el estado bioquímico de estos mutantes como consecuencia de la infección viroidal. Este proyecto contribuirá al conocimiento del sistema defensivo de las plantas frente a patógenos, tanto en sus aspectos de señalización como en los que se refieren a los componentes de la respuesta final de la planta, con vistas a su posible aplicación a la obtención de plantas más resistentes mediante técnicas de biología molecular.[EN] To study the role of ethylene in the defensive response of tomato plants to the citrus exocortis viroid (CEVd), tomato plants from Rutgers and Pearson cultivars and their ethylene insensitive mutants Never rie (Nr) were infected with CEVd. Both cultivars and their corresponding mutants developed several symptoms including bunchiness and epinasty of the leaves, marked growth retardation, stunting and necrosis at 3 weeks post inoculation. Symptoms were more severe in Nr mutants than their corresponding control. As the infection developed, an accumulation of pathogen-related proteins and viroid transcript was observed. The induction of genes related to defensive signalling like PR1 was quantified. The expression of genes related to ethylene biosynthesis (1- aminocyclopropane-1-carboxylic acid oxidase (ACO) and 1-aminocy- clopropane-1- carboxylate synthase (ACS)) was also quantified, as well as ethylene levels emitted by infected and control plants. Results show an induction of these genes caused by an infection with CEVd. However, the development of symptoms correlates with ethylene levels emitted and not with the accumulation of viroid transcript. As a final assay, alterations in the translation machinery caused by CEVd was studied as a new aspect of viroid pathogenesis. A higher expression level of the ribosomal stress mediator SlNAC082 and an accumulation of non-mature rRNA was detected in CEVd-infected tomato leaves. These results indicate the induction of ribosomal stress in an infection with CEVd.[CA] Per a investigar el paper de l'etilé en la resposta de les plantes de tomaca infectades pel viroid de l’exocortis dels citrics (CEVd), plantes de tomaca de les varietats Rutgers i Pearson i mutants insensibles a etilé Never ripe (Nr) van ser infectats amb el CEVd. Les dues varietats i també els mutants van desenvolupar diversos símptomes incloent la reducció i arrugamiento de les fulles, nanisme, epinastia pronunciada i necrosi a les 3 setmanes post-inoculació, encara que els símptomes no van ser tan severs en plantes Rutgers i Pearson com en els mutants Nr. A mesura que va avançar la malaltia es va produir l'acumulació de proteïnes relacionades amb la patogénesis denominaes PR i transcrit de CEVd. A més, es va quantificar la inducció de gens implicats en les rutes de senyalització defensiva com PR1, així com, l'expressió del gen que codifica l'enzim 1- aminociclopropano-1-carboxilato sintasa (ACS) i la 1-aminociclopropano-1-carboxilato oxidasa (ACO), enzims implicats en la ruta de biosíntesi de l'etilé. D'altra banda, es van analitzar els nivells d'etilé emesos per les fulles de les plantes infectades i dels corresponents controls. Els resultats obtinguts suggereixen que la infecció pel viroid indueix l'expressió d'aquestos gens. No obstant això, els símptomes produïts per la infecció amb CEVd semblen dependre dels nivells d'etilé emesos i no de l'acumulació del viroid. Finalment, es van estudiar les alteracions causades pel CEVd en la maquinària de traducció en plantes de tomaca en un nou aspecte de la patogénesi dels viroids. Es va detectar un major nivell d'expressió del mediador d'estrés ribosòmic NAC082 en les fulles de tomaca infectades amb CEVd. Estos resultats podrán permetre dilucidar els factors implicats en l'augment de l'estrés ribosomal per una infecció amb el CEVd. Els resultats obtinguts en este projecte aumenten el coneixement del sistema defensiu de les plantes enfront de patògens, tant en els aspectes de senyalització com en els components de la resposta final de la planta, tot aixó amb l’objetiu últim d’obtenir plantes més resistents a differents patógenes.Vázquez Prol, F. (2020). Estudio del desarrollo de la infección viroidal en mutantes never ripe de tomate. http://hdl.handle.net/10251/136734TFG

Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection

[EN] Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. 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Symptom severity, infection progression and plant responses in Solanum plants caused by three Pospiviroids vary with the inoculation procedure

Trabajo presentado a la XV Reunión de Biología Molecular y Celular de Plantas, celebrada on-line del 26 al 27 de noviembre de 2020

Symptom Severity, Infection Progression and Plant Responses in Solanum Plants Caused by Three Pospiviroids Vary with the Inoculation Procedure

[EN] Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid¿host interactionsThis work was supported by the Spanish Ministry of Economy and Competitiveness (co-supported by FEDER) Grants BIO2017-88321-R (V.P.) and PID2019-104126RB-I00 (G.G.). The funders had no role in the experiment design, data analysis, decision to publish, or preparation of the manuscriptVázquez Prol, F.; Márquez-Molins, J.; Rodrigo Bravo, I.; López-Gresa, MP.; Belles Albert, JM.; Gomez, GG.; Pallás Benet, V.... (2021). Symptom Severity, Infection Progression and Plant Responses in Solanum Plants Caused by Three Pospiviroids Vary with the Inoculation Procedure. International Journal of Molecular Sciences. 22(12):1-16. https://doi.org/10.3390/ijms22126189S116221