Detección y caracterización del virus meridional del tomate (STV)

[ES] El virus meridional del tomate (Southern tomato virus, STV) es un virus persistente (género Amalgavirus, familia Amalgaviridae) que se ha detectado en diversos países como España e Italia. Inicialmente, fue asociado a síntomas de decoloración y falta de maduración en el fruto de tomate. Sin embargo, la presencia frecuente de virus agudos en las plantas infectadas con el STV y la detección de éste en plantas asintomáticas, ponen en duda su patogenicidad y el impacto que puede tener en el cultivo. En esta tesis doctoral se puso a punto la RT-LAMP y la RT-qPCR para la detección específica y sensible del STV. La RT-LAMP permitió reducir costes y simplificar el procedimiento, siendo útil para la detección en campo. La RT-qPCR nos permitió detectar y cuantificar el STV en distintos tipos de tejidos de tomate, incluyendo semillas individuales. El virus se acumulaba principalmente en las raíces y hojas, y en las semillas se encontraba tanto en cubierta como en embrión, lo que dificulta las tareas de desinfección. La tasa de transmisión por semilla del virus, la incidencia en campo y en viveros era muy elevada, afectando más a variedades comerciales que locales. Los análisis filogenéticos mostraron que el STV tenía una baja diversidad genética con una fuerte presión de selección negativa. Además, no había una correlación entre distancia genética del virus y origen geográfico debido a una rápida dispersión de semillas infectadas y/o una fuerte presión de selección negativa. Se comprobó que el STV en condiciones de infección simple no inducía síntomas, no alteraba la producción, ni afectaba a parámetros fisiológicos como conductividad estomática, fotosíntesis y peso, en condiciones de estrés salino. Tampoco se observaron cambios a nivel tisular ni celular, ni se encontraron partículas virales. Sin embargo, el virus modificaba la expresión de algunos miRNAs con importantes funciones. Se detectaron muy poca cantidad de vsiRNAs derivados del STV, lo cual podría deberse a la supresión del silenciamiento génico de la planta por acción de un supresor codificado por el virus. Los ensayos de expresión transitoria en plantas de N. benthamiana 16C determinaron que la p42 del STV no tenía actividad supresora de silenciamiento génico. Finalmente, estudiamos el efecto del STV en infecciones múltiples con otros virus agudos como el CMV y el PepMV. Se observaron complejas interacciones entre los virus que implicaban variaciones en la severidad de síntomas, en los niveles de acumulación viral y en las poblaciones de siRNAs. El STV y el CMV establecían una interacción sinérgica que producía el adelanto y aumento de la severidad de los síntomas, y de la acumulación del CMV en las primeras fases de la infección. La presencia del STV en plantas infectadas con el PepMV también producía un adelanto de los síntomas sin cambios en la acumulación viral del PepMV. En las plantas coinfectadas con el CMV y PepMV se observó un efecto antagónico que disminuía la concentración del CMV y alteraba los síntomas. El STV era capaz de romper este efecto antagónico restableciendo la concentración del CMV y modificando los síntomas. Los análisis de siRNAs permitieron identificar un total de 78 miRNAs, 47 noveles, que se expresaban diferencialmente en los grupos de plantas infectadas con los diferentes virus respecto a las plantas sin infectar. Estos miRNAs estaban implicados en la regulación de importantes funciones y tanto su número como su nivel de expresión variaba dependiendo de la combinación viral. También se identificaron vsiRNAs de origen viral y se vio que su proporción variaba dependiendo de la combinación viral. La cantidad de vsiRNAs del STV se incrementaba notablemente con la presencia de otros virus. La frecuencia de acumulación de vsiRNAs en los genomas virales no era uniforme y no se veía influenciada por las combinaciones de virus.[CA] El virus meridional de la tomaca (Southern tomato virus, STV) és un virus persistent (gènere Amalgavirus, família Amalgaviridae) que s'ha detectat en diversos països com Espanya i Itàlia. Inicialment, STV va ser associat a distints símptomes de decoloració i anomalies en la maduració del fruit. Però la presència freqüent de virus aguts en les plantes infectades amb STV i la detecció d'aqueste en plantes asimptomàtiques, posen en dubte la seua patogenicitat i l'impacte que pot tindre en el cultiu. En aquesta tesi doctoral es va realitzar la posada al punt de la RT-LAMP i la RT- qPCR per a la detecció específica i sensible de STV. La RT-LAMP va permetre reduir costos i simplificar el procediment, sent útil per a la detecció del virus en camp. La RT-qPCR és una tècnica molt sensible que ens va permetre detectar i quantificar STV en distints tipus de teixits, incloent-hi llavors individuals. El virus s'acumulava principalment en arrels i fulles, i en les llavors es trobava tant en la coberta com en l'embrió. Es va comprovar que les taxes de transmissió per llavor, la incidència en camp i en vivers era molt elevada, major en les varietats comercials que en les locals. Els estudis filogenètics realitzats van mostrar que el virus tenia una baixa diversitat genètica amb una forta pressió de selecció negativa. No hi havia una correlació entre distància genètica del virus i origen geogràfic, degut per una ràpida dispersió a traves de llavors infectades i/o a la forta pressió de selecció negativa. En aquest treball es van obtindre evidències de què STV en condicions d'infecció simple no induïa símptomes en la planta, no alterava la producció, ni afectava paràmetres fisiològics com a conductivitat estomacal, fotosíntesi i pes en condicions d'estrés salí. Tampoc, es van observar cap presència de partícules virals ni canvis a nivell tissular ni cel·lular. No obstant això, STV era capaç de modificar l'expressió d'alguns miRNAs amb importants funcions. Es van detectar molt poca quantitat de vsiRNAs derivats del STV, podria deure's a la supressió del mecanisme de silenciamient gènic per acció d'un supressor. Els assajos d'expressió transitòria en plantes de N. benthamiana 16C va determinar que la p42 de STV no va tindre capacitat supressora de silenciamient gènic. Per finalitzar, vam estudiar l'efecte que podia tindre STV en infeccions mixtes amb altres virus aguts com CMV i PepMV. Els resultats obtinguts d'un assaig amb diferents combinacions d'infeccions van mostrar interaccions complexes entre els virus que implicaven variacions en la severitat de símptomes, en els nivells d'acumulació viral i en les poblacions de siRNA. STV i CMV establien una interacció sinèrgica que produïa l'avanç i l'increment dels símptomes, i un augment de l'acumulació de CMV. D'altra banda, la presència de STV en plantes infectades amb PepMV també produïa un avanç dels símptomes, però no hi havia variacions en l'acumulació de PepMV. En el grup de plantes co-infectades amb CMV i PepMV es va observar un efecte antagònic que dificultava la replicació de CMV, alterant-se els símptomes de la planta. STV era capaç de trencar aquest efecte antagònic restaurant la concentració de CMV i modificant els símptomes. Els anàlisis de siRNAs van permetre identificar un total de 78 miRNAs, 47 corresponien a miRNAs novells, que s'expressaven de forma diferent als grups de plantes infectades amb els diversos virus, respecte a les plantes control sense infectar. Aquests miRNAs estaven implicats en la regulació d'importants funcions i tant el seu nombre com el seu nivell d'expressió variaven. També es van identificar vsiRNAs d'origen viral i es va observar que la seua proporció variava depenent de la combinació viral. La quantitat de vsiRNAs de STV s'incrementava notablement amb la presència d'altres virus. Les freqüències de vsiRNA en els genomes virals no eren uniformes, no obstant això, els pat[EN] Southern tomato virus (STV) is a persistent virus (genus Amalgavirus, family Amalgaviridae) which was detected in several countries such Spain and Italy. STV was associated with symptoms of discoloration and maturation of tomato fruit. However, STV was frequently detected in mixed infections with acute viruses and in some asymptomatic tomato plants. For these reasons, it is not clear the STV pathogenic role and its real impact on tomato crops. In this PhD we improved the specific and sensitive detection of the virus by using the RT-LAMP and RT-qPCR. RT-LAMP is very useful for field STV detection since it is a simple and cheap technique. The high RT-qPCR sensitivity enabled this technique to detect and quantify STV from different plant tissues even individual sees. The highest STV concentrations were found in tomato leaves and roots. In the seeds, STV could be detected in both coat and embryo. The virus transmission by seed and the STV incidence in fields and seedlings was very high, being higher in commercial tomato varieties than in local ones. Phylogenetic analysis from different STV isolates showed a low genetic diversity with a high negative selection pressure. Moreover, there was no correlation between genetic diversity and geographic region. This could be explained by a quick dispersion of infected seeds and/or by the high negative selection pressure. It was shown that STV did not induce any apparent plant symptom and did not affect the plant production in single infection conditions. Also, physiological parameters related to stomatic conductivity, photosynthesis, and plant weight were no affected by STV infection in saline stress conditions. Optic and transmission electron microscopy did not reveal viral particles or structural changes in STV tomato tissues. However, the population analysis of miRNAs showed that STV was able to modify the expression of some miRNAs which modulated important plant functions. Low vsiRNAs were detected in STV tomato infected plants. It could be produced by the action of a suppressor which could suppress the gene silencing pathway in the plants. A transient expression assay of p42 in N. benthamiana 16C plants did not show suppressor activity of this STV protein. Finally, we studied the effect of STV in mixed infections with other acute viruses such as CMV and PepMV. The virus mixes infection assay in tomato plants showed complex interactions between viruses that modify the symptoms severity, the viral accumulation and the siRNA population. STV and CMV established a synergistic interaction in co-infected tomato plants producing the advancement of the symptoms and an increase in its severity. STV and CMV co-infection increased the CMV accumulation in the early stages of infection. On the other hand, the presence of STV in plants co-infected with PepMV also produced an advance of symptoms, but with no variation in the PepMV accumulation. In the group of plants co-infected with CMV and PepMV, it was observed an antagonistic effect that delayed the CMV accumulation, altering the symptoms of the plant with respect to the simple infections. STV was able to break this antagonistic effect by increasing the CMV viral concentration and changing the symptomatology. The siRNAs analysis allowed to identify a total of 78 miRNAs, 47 corresponding to novel miRNAs, that were expressed differentially in the plants infected respect to no infected plants. These miRNAs were involved in the regulation of important functions and their number and their level of expression varied depending on the virus combination. vsiRNAs of the different viruses were also identified and it was observed that rates varied depending on the virus combination. The number of vsiRNAs in STV single infected tomato plants was very small, but it increased with the presence of the other viruses. The frequencies of vsiRNAs in the viral genomes were not uniform and these frequencies were not influenced by other viruses in mixed infections.Elvira González, L. (2021). Detección y caracterización del virus meridional del tomate (STV) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/165207TESI

Persistent Southern Tomato Virus (STV) Interacts with Cucumber Mosaic and/or Pepino Mosaic Virus in Mixed- Infections Modifying Plant Symptoms, Viral Titer and Small RNA Accumulation

Southern tomato virus (STV) is a persistent virus that was, at the beginning, associated with some tomato fruit disorders. Subsequent studies showed that the virus did not induce apparent symptoms in single infections. Accordingly, the reported symptoms could be induced by the interaction of STV with other viruses, which frequently infect tomato. Here, we studied the effect of STV in co- and triple-infections with Cucumber mosaic virus (CMV) and Pepino mosaic virus (PepMV). Our results showed complex interactions among these viruses. Co-infections leaded to a synergism between STV and CMV or PepMV: STV increased CMV titer and plant symptoms at early infection stages, whereas PepMV only exacerbated the plant symptoms. CMV and PepMV coinfection showed an antagonistic interaction with a strong decrease of CMV titer and a modification of the plant symptoms with respect to the single infections. However, the presence of STV in a triple-infection abolished this antagonism, restoring the CMV titer and plant symptoms. The siRNAs analysis showed a total of 78 miRNAs, with 47 corresponding to novel miRNAs in tomato, which were expressed differentially in the plants that were infected with these viruses with respect to the control mock-inoculated plants. These miRNAs were involved in the regulation of important functions and their number and expression level varied, depending on the virus combination. The number of vsiRNAs in STV single-infected tomato plants was very small, but STV vsiRNAs increased with the presence of CMV and PepMV. Additionally, the rates of CMV and PepMV vsiRNAs varied depending on the virus combination. The frequencies of vsiRNAs in the viral genomes were not uniform, but they were not influenced by other viruses

Proteomic insights into the effect of Broad bean wilt virus-1 infection in Nicotiana benthamiana plants

Broad bean wilt virus 1 (BBWV-1, genus Fabavirus, family Secoviridae) infects many plants species, including important horticultural crops. Since some tobacco plants such as Nicotiana benthamiana, are susceptible to many viruses, they are used as experimental hosts and therefore abundantinformation about these species is available in databases. Here, the protein differential expression in N. benthamiana plants infected with BBWV-1 wasstudied using Liquid Chromatography coupled with Mass Spectrometry in tandem analysis (LC–MS/MS). Also, we studied the role of viral VP37 protein which is a BBWV-1 determinant of pathogenicity in the accumulation of the host proteins. For this purpose, we agroinfiltrated N. benthamiana plants with two BBWV-1 cDNA infectious clones: pBBWV1-Wt wilt type and the pBBWV1-G492C mutant knocking out for the viral VP37 protein. Virus infection induced the differential expression of 44 host proteins: 22 were overexpressed and the other 22 were underexpressed. These proteins were involved in important plant processes and located in different cell organelles, mainly in chloroplasts. Finally, 24 of these proteins were expressed differentially according to the presence of VP37 protein. Relation among host proteins that were differentially expressed, plant symptoms, and subcellular alterations are discussed

Current ozone levels threaten gross primary production and yield of Mediterranean annual pastures and nitrogen modulates the response

Pastures are among the most important ecosystems in Europe considering their biodiversity and dis- tribution area. However, their response to increasing tropospheric ozone (O 3 ) and nitrogen (N) deposi- tion, two of the main drivers of global change, is still uncertain. A new Open-Top Chamber (OTC) experiment was performed in central Spain, aiming to study annual pasture response to O 3 and N in close to natural growing conditions. A mixture of six species of three representative families was sowed in the fi eld. Plants were exposed for 40 days to four O 3 treatments: fi ltered air, non- fi ltered air (NFA) repro- ducing ambient levels and NFA supplemented with 20 and 40 nl l � 1 O 3 . Three N treatments were considered to reach the N integrated doses of “ background ” , þ 20 or þ 40 kg N ha � 1 . Ozone signi fi cantly reduced green and total aboveground biomass (maximum reduction 25%) and increased the senescent biomass (maximum increase 40%). Accordingly, O 3 decreased community Gross Primary Production due to both a global reduction of ecosystem CO 2 exchange and an increase of ecosystem respiration. Nitrogen could partially counterbalance O 3 effects on aboveground biomass when the levels of O 3 were moderate, but at the same time O 3 exposure reduced the fertilization effect of higher N availability. Therefore, O 3 must be considered as a stress factor for annual pastures in the Mediterranean areas

Urinary transferrin pre-emptively identifies the risk of renal damage posed by subclinical tubular alterations

Nephrotoxicity is an important limitation to the clinical use of many drugs and contrast media. Drug nephrotoxicity occurs in acute, subacute and chronic manifestations ranging from glomerular, tubular, vascular and immunological phenotypes to acute kidney injury. Pre-emptive risk assessment of drug nephrotoxicity poses an urgent need of precision medicine to optimize pharmacological therapies and interventional procedures involving nephrotoxic products in a preventive and personalized manner. Biomarkers of risk have been identified in animal models, and risk scores have been proposed, whose clinical use is abated by their reduced applicability to specific etiological models or clinical circumstances. However, our present data suggest that the urinary level of transferrin may be indicative of risk of renal damage, where risk is induced by subclinical tubular alterations regardless of etiology. In fact, urinary transferrin pre-emptively correlates with the subsequent renal damage in animal models in which risk has been induced by drugs and toxins affecting the renal tubules (i.e. cisplatin, gentamicin and uranyl nitrate); whereas transferrin shows no relation with the risk posed by a drug affecting renal hemodynamics (i.e. cyclosporine A). Our experiments also show that transferrin increases in the urine in the risk state (i.e. prior to the damage) precisely as a consequence of reduced tubular reabsorption. Finally, urinary transferrin pre-emptively identifies subpopulations of oncological and cardiac patients at risk of nephrotoxicity. In perspective, urinary transferrin might be further explored as a wider biomarker of an important mechanism of predisposition to renal damage induced by insults causing subclinical tubular alterations.Research from the authors’ laboratory supporting part of the information incorporated into this article was funded by grants from Instituto de Salud Carlos III (PI14/01776, DT15S/00166, PI15/01055 and PI17/01979, and Retic RD016/0009/0025, REDINREN), Ministerio de Economía y Competitividad (IPT-2012-0779-010000), Junta de Castilla y León (Consejería de Sanidad, BIO/SA20/14, BIO/SA66/15; and Consejería de Educación, SA359U14), and FEDER funds

Urinary transferrin pre-emptively identifies the risk of renal damage posed by subclinical tubular alterations

Nephrotoxicity is an important limitation to the clinical use of many drugs and contrast media. Drug nephrotoxicity occurs in acute, subacute and chronic manifestations ranging from glomerular, tubular, vascular and immunological phenotypes to acute kidney injury. Pre-emptive risk assessment of drug nephrotoxicity posesman urgent need of precision medicine to optimize pharmacological therapies and interventional procedures involving nephrotoxic products in a preventive and personalized manner. Biomarkers of risk have been identified in animal models, and risk scores have been proposed, whose clinical use is abated by their reduced applicability to specific etiological models or clinical circumstances. However, our present data suggest that the urinary level of transferrin may be indicative of risk of renal damage, where risk is induced by subclinical tubular alterations regardless of etiology. In fact, urinary transferrin pre-emptively correlates with the subsequent renal damage in animal models in which risk has been induced by drugs and toxins affecting the renal tubules (i.e. cisplatin, gentamicin and uranyl nitrate); whereas transferrin shows no relation with the risk posed by a drug affecting renal hemodynamics (i.e. cyclosporine A). Our experiments also show that transferrin increases in the urine in the risk state (i.e. prior to the damage) precisely as a consequence of reduced tubular reabsorption. Finally, urinary transferrin pre-emptively identifies subpopulations of oncological and cardiac patients at risk of nephrotoxicity. In perspective, urinary transferrin might be further explored as a wider biomarker of an important mechanism of predisposition to renal damage induced by insults causing subclinical tubular alterations

Use of a monitoring system for heat stress to check and optimize cooling strategies

Heat stress in dairy cows can have detrimental effects on milk production and reproduction. Traditional indicators like the temperature-humidity index (THI) provide an estimate of potential stress but fail to measure the actual impact on the animals. To address this, the study utilized the SenseHub® monitoring system, which records panting, a direct indicator of heat stress, along with intake and rumination data. The objectives of this work were to evaluate the impact of heat stress on cows in different production phases and assess the effectiveness of cooling strategies such as showers. The study monitored 77 cows from various production cycles, including lactating, dry, and postpartum cows, using the SenseHub® collar. Information on panting, intake, and rumination was collected over 24 hours, as well as daily and monthly averages from June 2022 to September 2022 in a commercial farm located in Bétera (Valencia, Spain). THI values were recorded during the whole experimental period. For prepartum cows, two cooling strategies were compared: 2 hours per day in three showers vs. 4 hours per day in six showers. Dry and postpartum cows received 4 hours of cooling per day in six showers. The results showed that lactating cows experienced the highest impact of heat stress, while dry cows were less affected. The monitoring revealed a significant difference between day and night impacts, with the highest panting percentage occurring during the day for lactating cows. In addition, the number of showers did not affect to dry cows as expected. Panting duration correlated inversely with intake and rumination activity, indicating the severity of heat stress. Descriptional analysis as well as T-test with paired data was performed with SAS System®. The beneficial effect of cooling on heat stress was clear during the day, but some doubts appear during the night. The average percentage of panting animals varied depending on whether they were in the cooling period or not. Comparing the two cooling strategies for lactating cows, no significant biological differences were observed. Despite the positive impact of cooling, heat stress remained high, with THI values above 72 resulting in reduced production and conception rates. In conclusion, the monitoring system used in this work proved to be a valuable tool for evaluating heat stress impacts and optimizing cooling strategies. However, under conditions of high ambient humidity, further evaluation of alternative cooling strategies is necessary to effectively mitigate the negative impacts of heat stress while optimizing available resource