RNA-binding protein immunoprecipitation as a tool to investigate plant miRNA processing interference by regulatory proteins of diverse origin

Background: Due to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) of viral and host origin for replication, dissemination and evasion of host RNA degradation pathways. Some viruses interfere with the microRNA (miRNA) pathway to generate better fitness. The development of an adjusted, reliable and sensitive ribonucleoprotein immunoprecipitation (RIP) assay is needed to study the interaction between RBP of different origin (including viral origin) and miRNA precursors. The method could be further applied to transiently expressed heterologous proteins in different plant species. Results: Here we describe a modified RIP assay applied to nuclear epitope-tagged proteins of heterologous origin and transiently expressed in Nicotiana benthamiana. The assay includes a combination of optimized steps as well as the careful selection of control samples and rigorous data analysis. It has proven efficient to detect and quantify miRNA processing intermediates associated with regulatory proteins. Conclusions: The RIP method described here provides a reliable tool to study the interaction of RBPs, such as transiently expressed regulatory proteins with lowly represented host RNA, as is the case of miRNA precursors. This modified method was efficiently adjusted to recover nuclear proteins and reduce unspecific background. The purification scheme optimized here for GFP-tagged proteins can be applied to a wide array of RBPs. The subsequent application of next-generation sequencing technologies will permit to sequence and characterize all RNA species bound in vivo by a given RBP.Fil: Marmisollé, Facundo Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Garcia, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Transgenic‐based solutions for citrus disease management in Argentina

Citrus is a major fruit crop with economic importance worldwide, with citriculture historically threatened at times by a diverse array of pathogens. As a leading producer and exporter, Argentina has been dealing with endemic and quarantine diseases of citrus by implementing conventional management strategies. In recent decades, the pursuit of pathogen-resistant transgenic citrus has been explored in the country as part of a long-term and sustainable disease management strategy. Successful genetically modified organisms (GMOs) were created locally, engineered to resist viruses and bacteria for the control of tristeza, psorosis, canker, and huanglongbing diseases of citrus. Although the Argentine regulatory system accommodated these developments, there were also difficulties that demand further recognition and analysis. In the present work, we describe four major diseases affecting Argentine citriculture and a series of GMO-oriented strategies for their management. We explore the methodologies behind these strategies, including transgenic-based approaches, the current state of regulations, and what further actions may be taken to ensure continuing protection for citriculture.Fil: de Francesco, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Sendín, Lorena Noelia. Instituto Nacional de Tecnología Agropecuaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gómez, Rocio Liliana. Instituto Nacional de Tecnología Agropecuaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Ensayos de infección con Xanthomonas citri en plantas réplicas de líneas transgénicas de Citrus sinensis cv "Pineapple"

La producción de cítricos es una de las actividades frutícolas más importantes en Argentina, contribuyendo significativamente a las exportaciones y al desarrollo económico a nivel regional. La cancrosis, causada por Xanthomonas citri subsp citri (Xcc) provoca problemas en la exportación de los cítricos y afecta con diferente severidad a distintas especies y variedades. Las estrategias biotecnológicas ofrecen herramientas prometedoras para el control de diversas bacteriosis en forma conjunta. Anteriormente este grupo de trabajo obtuvo plantas de naranja "Pineapple" transformadas con una construcción genética que favorece la acumulación constitutiva del péptido antimicrobiano (AMP) dermaseptina, cuya expresión mostró niveles de reducción de los síntomas de hasta un 50% en comparación con las plantas no transformadas. Con el objetivo de evaluar la continuidad de la resistencia a la enfermedad, en este trabajo se realizaron ensayos de infección con Xcc en plantas réplicas obtenidas de yemas de las líneas madre transgénicas. Los ensayos de infección se realizaron en cámara de crecimiento en condiciones controladas utilizando tres concentraciones distintas del inóculo. La técnica de inoculación consistió en la infiltración infra-epidérmica de las suspensiones bacterianas utilizando la mitad de la lámina. Se utilizaron siete plantas réplicas y un control no transgénico, incluyendo por cada ensayo de 4 a 9 hojas por planta. A los 21 días post-infección se recolectó y se evaluó la incidencia de cancros típicos de la enfermedad (les/cm2). Análisis preliminares de datos dan como resultado que al menos una de las líneas transgénicas réplica muestra persistencia en la reducción de síntomas de cancrosis respecto de los controles sin transformar. Estos resultados avalan que el uso de AMPs es una estrategia promisoria para el control de la cancrosis y respalda su evaluación para el control de otras enfermedades bacterianas incluido el HuanglongbingFil: Vandecaveye, Melina Antonella. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Bella Vista; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Furman, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Kobayashi, Ken. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Gochez, Alberto Martin. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Bella Vista; ArgentinaFil: Lezcano, Cecilia Carolina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Bella Vista; ArgentinaFil: Hermosis, Fabian. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Bella Vista; ArgentinaFil: Soliz, Jorge Andres. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Bella Vista; ArgentinaFil: Benitez, Rolando José. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Bella Vista; ArgentinaFil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Garcia, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Canteros, Blanca Isabel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Bella Vista; ArgentinaIX Congreso Argentino de CitriculturaSan Miguel de TucumánArgentinaInstituto Nacional de Tecnología Agropecuaria. Estación Experimental Agroindustrial Obispo Colombre

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

RNA-binding protein immunoprecipitation as a tool to investigate plant miRNA processing interference by regulatory proteins of diverse origin

Background: Due to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) of viral and host origin for replication, dissemination and evasion of host RNA degradation pathways. Some viruses interfere with the microRNA (miRNA) pathway to generate better fitness. The development of an adjusted, reliable and sensitive ribonucleoprotein immunoprecipitation (RIP) assay is needed to study the interaction between RBP of different origin (including viral origin) and miRNA precursors. The method could be further applied to transiently expressed heterologous proteins in different plant species. Results: Here we describe a modified RIP assay applied to nuclear epitope-tagged proteins of heterologous origin and transiently expressed in Nicotiana benthamiana. The assay includes a combination of optimized steps as well as the careful selection of control samples and rigorous data analysis. It has proven efficient to detect and quantify miRNA processing intermediates associated with regulatory proteins. Conclusions: The RIP method described here provides a reliable tool to study the interaction of RBPs, such as transiently expressed regulatory proteins with lowly represented host RNA, as is the case of miRNA precursors. This modified method was efficiently adjusted to recover nuclear proteins and reduce unspecific background. The purification scheme optimized here for GFP-tagged proteins can be applied to a wide array of RBPs. The subsequent application of next-generation sequencing technologies will permit to sequence and characterize all RNA species bound in vivo by a given RBP.Instituto de Biotecnologia y Biologia Molecula

Identification of a microRNA encoded by Anticarsia gemmatalis Multiple Nucleopolyhedrovirus

MicroRNAs are regulatory RNAs that are scarcely described in Baculoviruses. In this work we predicted a microRNA in silico, denominated agmnpv-miR-4, encoded in the genome of Anticarsia gemmatalis Nucleopolyhedrovirus (AgMNPV), which is homologous to the already validated bmnpv-miR-4 from Bombyx mori Nucleopolyhedrovirus (BmNPV). Considering information known for bmnpv-miR-4 such as seed sequence, coding location in the genome and putative target binding, we searched for the coding sequence of agmnpv-miR-4 in AgMNPV genome. A precursor sequence of agmnpv-miR-4 was predicted, and we identified a putative 23 nt mature microRNA, agmnpv-miR-4, coded in the complementary strand of AgMNPV-2D between positions 49,450 and 49,472. We validated agmnpv-miR-4 by Northern blot from HighFive cells and A. gemmatalis larve extracts infected with AgMNPV.Fil: Ferrelli, Maria Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Garcia, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Romanowski, Victor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Uncontrolled Citrus psorosis virus infection in Citrus sinensis transgenic plants expressing a viral 24K-derived hairpin that does not trigger RNA silencing

Citrus psorosis virus (CPsV) is the causal agent of psorosis disease of citrus. Pineapple sweet orange plants were transformed with a hairpin construct derived from the viral 24k gene (lines ihp24K). Contrary to expectations, these lines did not trigger efficient RNA silencing, and when infected with CPsV they showed a phenotype of exacerbated symptoms with a persistent and homogeneous infection without the recovery observed in non-transgenic plants. Ihp24K lines did not behave similarly when challenged with Citrus tristeza virus. All these results indicate that hypersusceptibility is likely related to the specific action of 24K-derived hairpin over CPsV multiplication.Fil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: de Francesco, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Ocolotobiche, Eliana Evelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Costa, N.. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Garcia, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Citrus genetic transformation: an overview of the current strategies and insights on the new emerging technologies

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.Instituto de BiotecnologíaFil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Conti, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Conti, Gabriela. Universidad de Buenos Aires. Faculta de Agronomía. Cátedra de Genética; ArgentinaFil: Xoconostle-Cázares, Beatriz. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional. Departamento de Biotecnología y Bioingeniería; MéxicoFil: Marcelino-Pérez, Gabriel. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional. Departamento de Biotecnología y Bioingeniería; MéxicoFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Hopp, Horacio Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hopp, Horacio Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Reyes Martinez, Carina Andrea. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentin

Up-regulation of microRNA targets correlates with symptom severity in Citrus sinensis plants infected with two different isolates of citrus psorosis virus

Main conclusion: miRNA targets from Citrus sinensis are predicted and validated using degradome data. They show an up-regulation upon infection with CPsV, with a positive correlation between target expression and symptom severity. Abstract: Sweet orange (Citrus sinensis) may suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. Infection of sweet orange plants with two isolates of citrus psorosis virus (CPsV), expressing different symptomatologies, alters the accumulation of a set of endogenous microRNAs (miRNAs). Here, we predicted ten putative targets from four down-regulated miRNAs: three belonging to the CCAAT-binding transcription factor family (CBFAs); an Ethylene-responsive transcription factor (RAP2-7); an Integrase-type DNA-binding superfamily protein (AP2B); Transport inhibitor response 1 (TIR1); GRR1-like protein 1-related (GRR1); Argonaute 2-related (AGO2), Argonaute 7 (AGO7), and a long non-coding RNA (ncRNA). We validated six of them through analysis of leaf degradome data. Expressions of the validated targets increase in infected samples compared to healthy tissue, showing a more striking up-regulation those samples with higher symptom severity. This study contributes to the understanding of the miRNA-mediated regulation of important transcripts in Citrus sinensis through target validation and shed light in the manner a virus can alter host regulatory mechanisms leading to symptom expression.Fil: Marmisollé, Facundo Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Arizmendi, Ailín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Ribone, Andrés Ignacio. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Garcia, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin