Sweet potato chlorotic stunt virus : Studies on viral synergism and suppression of RNA silencing

The studies presented in this thesis aimed to a better understanding of the molecular biology of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus, Closteroviridae) and its role in the development of synergistic viral diseases. The emphasis was on the severe sweet potato virus disease (SPVD) that results from a synergistic interaction of SPCSV and Sweet potato feathery mottle virus (SPFMV, Potyvirus, Potyviridae). SPVD is the most important disease affecting sweetpotato. It is manifested as a significant increase in symptom severity and SPFMV titres. This is accompanied by a dramatic sweetpotato yield reduction. SPCSV titres remain little affected in the diseased plants. Viral synergistic interactions have been associated with the suppression of an adaptive general defence mechanism discovered in plants and known as RNA silencing. In the studies of this thesis two novel proteins (RNase3 and p22) identified in the genome of a Ugandan SPCSV isolate were shown to be involved in suppression of RNA silencing. RNase3 displayed a dsRNA-specific endonuclease activity that enhanced the RNA-silencing suppression activity of p22. Comparative analyses of criniviral genomes revealed variability in the gene content at the 3´end of the genomic RNA1. Molecular analyses of different isolates of SPCSV indicated a marked intraspecific heterogeneity in this region where the p22 and RNase3 genes are located. Isolates of the East African strain of SPCSV from Tanzania and Peru and an isolate from Israel were missing a 767-nt fragment that included the p22 gene. However, regardless of the absence of p22, all SPCSV isolates acted synergistically with SPFMV in co-infected sweetpotato, enhanced SPFMV titres and caused SPVD. These results showed that p22 is dispensable for development of SPVD. The role of RNase3 in SPVD was then studied by generating transgenic plants expressing the RNase3 protein. These plants had increased titres of SPFMV (ca. 600-fold higher in comparison with nontransgenic plants) 2-3 weeks after graft inoculation and displayed the characteristic SPVD symptoms. RNA silencing suppression (RSS) activity of RNase3 was detected in agroinfiltrated leaves of Nicotiana bethamiana. In vitro studies showed that RNase3 was able to cleave small interferring RNAs (siRNA) to products of ~14-nt. The data thus identified RNase3 as a suppressor of RNA silencing able to cleave siRNAs. RNase3 expression alone was sufficient for breaking down resistance to SPFMV in sweetpotato and for the development of SPVD. Similar RNase III-like genes exist in animal viruses which points out a novel and possibly more general mechanism of RSS by viruses. A reproducible method of sweetpotato transformation was used to target RNA silencing against the SPCSV polymerase region (RdRp) with an intron-spliced hairpin construct. Hence, engineered resistance to SPCSV was obtained. Ten out of 20 transgenic events challenged with SPCSV alone showed significantly reduced virus titres. This was however not sufficient to prevent SPVD upon coinfection with SPFMV. Immunity to SPCSV seems to be required to control SPVD and targeting of different SPCSV regions need to be assessed in further studies. Based on the identified key role of RNase3 in SPVD the possibility to design constructs that target this gene might prove more efficient in future studies.Ravintokasvien ryhmässä bataatin vuotuinen tuotantomäärä on viidenneksi suurin kehitysmaissa. Bataatti on kasvuoloiltaan vaatimaton mutta satoisa, joten sillä on erityisen keskeinen rooli omavaraistuotannossa kuivuina kausina ja nälänhädän uhatessa. Kiinan jälkeen merkittävin tuotantoalue on Itä-Afrikka, erityisesti Victoria-järven ympäristö, jossa Uganda on suurin tuottaja. Bataatin viljelyn merkittävin tautiongelma ovat virukset, joiden leviämistä käytössä olevat viljelymenetelmät ja virusalttiit lajikkeet edistävät. Bataatin kitukasvuviruksen (SPCSV) tartunta altistaa sinänsä melko viruskestävät itäafrikkalaiset bataattilajikkeet muiden virusten tartunnoille. Lisäksi sekainfektio aiheuttaa erityisen ankaria oireita, joiden takia sato voidaan menettää. Tämän tutkimuksen tarkoituksena on selvittää, kuinka bataatin kitukasvuvirus estää viruskestävyyden ilmentymisen ja edistää muiden virusten tartuntoja. Lisäksi tutkitaan bataattien kestävyysmekanismeja, joilla sekainfektioiden aiheuttama ankara tauti voitaisiin estää

¿Método tradicional o alternativo? Hacia la consolidación de una estrategia didáctica para el aprendizaje del tema segunda guerra mundial acorde a las necesidades educativas actuales

La Segunda Guerra Mundial, un acontecimiento de discusiones políticas y dominio social que parte la historia en dos y finaliza con la creación de nuevas instituciones que establecen un orden mundial. En el ámbito educativo actualmente predomina el método de enseñanza tradicional el cual fue pensado para responder en las necesidades de un contexto, por ello es pertinente y necesario se dé un proceso de transición en los métodos.   Para alcanzar el objetivo propuesto, se desarrollan los referentes teóricos y se selecciona la metodología cuantitativa de tipo experimental con la cual se usan como instrumentos para la recolección de información el cuestionario, la observación y una guía didáctica la cual integra varias estrategias didácticas. Cabe mencionar que como muestra se selecciona los dos grados octavos del colegio Cooperativo la Presentación integrado por 43 estudiantes, un grupo llamado control realiza su proceso de aprendizaje mediante el método tradicional y el otro grupo experimental mediante las estrategias didácticas de las TIC, la metodología por proyectos y el trabajo colaborativo-cooperativo.   Luego de desarrollar las actividades por medio de las estrategias didácticas y recolectar la información encontramos que el grupo experimental tiene motivación, autonomía, interés, inquietud por desarrollar su proceso de aprendizaje cuando se emplea esta metodología a diferencia del grupo control que, aunque con resultados académicos buenos los factores mencionados no son evidentes durante el proceso de enseñanza

Nanopore-based complete genome sequence of a Sri Lankan cassava mosaic virus (Geminivirus) strain from Thailand

Sri Lankan cassava mosaic virus is an emerging pathogen in Southeast Asia. Here, we report the complete genome of a Thai isolate obtained using Nanopore technology. The isolate was collected in 2019 from the northeastern province of Surin, soon after disease eradication was reported in the country

Challenge of virus disease threats to ensuring sustained uptake of vitamin-A-rich sweetpotato in Africa

Orange-fleshed sweetpotatoes (OFSP) are a rich source of pro-vitamin A and can alleviate vitamin A deficiency in the developing world. In Africa, traditional varieties have been almost exclusively white-fleshed and introduction and breeding of orange-fleshed varieties into Africa has been severely hampered by virus diseases to which many varieties are susceptible. Breeding progress to generate resistant varieties has been slow due to rare and recessive occurrence of resistance in breeding populations. Production of virus-free seed is complicated by the fact that most sweetpotato viruses show no or only limited symptoms and very low virus concentrations when infected by individual viruses, making them difficult to detect. Even single infections can lead to significant yield losses, but when they combine severe disease complexes are generated, which can lead to total crop failure. Significant efforts have been made in characterizing and understanding virus interactions in sweetpotato over the last two decades to address this challenge; they are reviewed in this chapter. We also review the state of the art in detection of viruses in support of seed systems and breeding. We conclude with recommendations for the most urgent future research directions needed to address virus problems in sweetpotatoes

Genome sequence of SARS-CoV-2 isolate Cali-01, from Colombia, obtained using Oxford Nanopore MinION Sequencing

We report the genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate obtained from a patient with symptoms of coronavirus disease 2019 (COVID-19) who was infected in Cali, Colombia. The patient had no recent travel record and did not require hospitalization. The virus genome was obtained using Oxford Nanopore MinION sequencing

Cassava torrado-like virus encodes a gene that facilitates the mechanical transmission to Nicotiana benthamiana of Cassava virus X

Cassava-torrado-like virus (CsTLV) is a bipartite single-stranded RNA virus belonging to the family Secoviridae. The virus has been reported in Brazil and Colombia, and is usually found in mixed infections, and in plants affected by Cassava Frogskin Disease (CFSD), an endemic cassava disease in the Americas. Genome analysis of CsTLV identifies a gene in RNA2 implicated in pathogen movement in other torradoviruses. This gene (RNA2-ORF1) and another one no related to virus movement (Maf/Ham1) were amplified by PCR and cloned into constructs under the 35S promoter of cauliflower mosaic virus (CaMV), which then were transfered to agrobacterium binary vectors. When agro-infiltrated in Nicotiana benthamiana plants, only RNA2-ORF1 had a positive effect on the mechanical inoculation of cassava virus X (CsVX), a potexvirus that has a low rate of mechanical infection in N. benthamiana. Efficiency of CsVX mechanical transmission was measured by the number of infected plants, presence of symptoms, and titers of CsVX as measured by ELISA, two weeks after infection. On average, CsVX could infect 2.3 times more plants when these were previously agro-infiltrated with A. tumefaciens binary vector encoding CsTLV RNA2-ORF1. We conclude that the novel secovirid CsTLV associated with leaf spot symptoms in cassava, encodes a gene that could enhance other viral infections in N. benthamiana. Further studies are required to elucidate this effect and its role in mixed infections, often observed in cassava plants affected by CFSD

High throughput sequencing for the detection and characterization of new virus found in arracacha (Arracacia xanthorrhiza)

Arracacha is a root crop cultivated in several countries of South America. The commercial product, which is the storage root, has multiple nutritional properties: its fine starch makes it easily digestible, and it contains high levels of calcium and vitamin A. High throughput sequencing (HTS) technology has been applied successfully for virus discovery in different agricultural crops, and it has been proposed to apply it in routine pathogen detection. Using HTS, novel sequences related to crinivirus and vitivirus were identified in apparently symptomeless arracacha, which were assembled into contigs located in different positions of the genome. Based on those sequences’ primers were designed to amplify corresponding sequences from further arracacha accessions and potato plant samples collected from farmers’ fields in Colombia and Peru. It was possible to determine the near complete genomes of these viruses. In addition, sequences related to an enamovirus and ST9-like RNA were also identified in arracacha plants using HTS

Natural co-infection of solanum tuberosum crops by the potato yellow vein virus and potyvirus in colombia

The Potato yellow vein virus (PYVV), a Crinivirus with an RNA tripartite genome, is the causal agent of the potato yellow vein disease, reported in Colombian since 1950, with yield reductions of up to 50%. Co-infection of two or more viruses is common in nature and can be associated with differences in virus accumulation and symptom expression. No evidence of mixed infection between PYVV and other viruses has been reported. In this study, eight plants showing yellowing PYVV symptoms: four Solanum tuberosum Group Phureja (P) and four Group Andigena (A), were collected in Cundinamarca, Colombia to detect mixed infection in the isolates using next generation sequencing (NGS). The Potato virus Y (PVY) complete genome (similar to N strain) and the Potato virus V (PVV) partial genomes were detected using NGS and re-confirmed by RT-PCR. Preliminary field screening in a large sample showed that PYVV and PVY co-infect potato plants with a prevalence of 21% within the P group and 23% within the A group. This is the first report of co-infection of PYVV and potyvirus in Colombia and with the use of NGS. Considering that potyviruses enhance symptom severity and/or yield reductions in mixed infections, our results may be relevant for disease diagnosis, breeding programs and tuber certification