Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor.

Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species.

Genetic Diversity of Rice stripe necrosis virus and New Insights into Evolution of the Genus Benyvirus.

The rice stripe necrosis virus (RSNV) has been reported to infect rice in several countries in Africa and South America, but limited genomic data are currently publicly available. Here, eleven RSNV genomes were entirely sequenced, including the first corpus of RSNV genomes of African isolates. The genetic variability was differently distributed along the two genomic segments. The segment RNA1, within which clusters of polymorphisms were identified, showed a higher nucleotidic variability than did the beet necrotic yellow vein virus (BNYVV) RNA1 segment. The diversity patterns of both viruses were similar in the RNA2 segment, except for an in-frame insertion of 243 nucleotides located in the RSNV tgbp1 gene. Recombination events were detected into RNA1 and RNA2 segments, in particular in the two most divergent RSNV isolates from Colombia and Sierra Leone. In contrast to BNYVV, the RSNV molecular diversity had a geographical structure with two main RSNV lineages distributed in America and in Africa. Our data on the genetic diversity of RSNV revealed unexpected differences with BNYVV suggesting a complex evolutionary history of the genus Benyvirus.

Muntingia yellow spot virus: a novel New World begomovirus infecting Muntingia calabura L.

The whole genome sequence of a begomovirus (family Geminiviridae) infecting Muntingia calabura L. (family Muntingiaceae) from the province of Guayas in Ecuador was determined in this work. The major symptom observed on this plant species was yellow spots on leaves. The nucleotide sequences of three DNA-A clones and one DNA-B clone were compared to those of other begomoviruses. The DNA-A clones displayed the highest similarity to isolates of pepper leafroll virus (PepLRV), with 87.4 to 88.1% sequence identity. Likewise, the DNA-B clone showed the highest similarity (79.3-79.6% sequence identity) to PepLRV isolates. According to the demarcation criteria for begomovirus species, the begomovirus described in this work, for which we propose the name "muntingia yellow spot virus", represents a novel species. To our best knowledge, this is the first report of a begomovirus infecting a plant of the family Muntingiaceae.

RNA silencing machinery contributes to inability of BSBV to establish infection in Nicotiana benthamiana: evidence from characterization of agroinfectious clones of Beet soil-borne virus.

Beet soil-borne virus (BSBV) is a sugar beet pomovirus frequently associated with Beet necrotic yellow veins virus, the causal agent of the rhizomania disease. BSBV has been detected in most of the major beet-growing regions worldwide, yet its impact on this crop remains unclear. With the aim to understand the life cycle of this virus and clarify its putative pathogenicity, agroinfectious clones have been engineered for each segment of its tripartite genome. The biological properties of these clones were then studied on different plant species. Local infection was obtained on agroinfiltrated leaves of Beta macrocarpa. On leaves of Nicotiana benthamiana, similar results were obtained, but only when heterologous viral suppressors of RNA silencing were co-expressed or in a transgenic line down regulated for both dicer-like protein 2 and 4. On sugar beet, local infection following agroinoculation was obtained on cotyledons, but not on other tested plant parts. Nevertheless, leaf symptoms were observed on this host via sap inoculation. Likewise, roots were efficiently mechanically infected, highlighting low frequency of root necrosis and constriction, and enabling the demonstration of transmission by the vector Polymyxa betae. Altogether, the entire viral cycle was reproduced, validating the constructed agroclones as efficient inoculation tools, paving the way for further studies on BSBV and its related pathosystem.

Family Level Phylogenies Reveal Relationships of Plant Viruses within the Order Bunyavirales.

Bunyavirales are negative-sense segmented RNA viruses infecting arthropods, protozoans, plants, and animals. This study examines the phylogenetic relationships of plant viruses within this order, many of which are recently classified species. Comprehensive phylogenetic analyses of the viral RNA dependent RNA polymerase (RdRp), precursor glycoprotein (preGP), the nucleocapsid (N) proteins point toward common progenitor viruses. The RdRp of Fimoviridae and Tospoviridae show a close evolutional relationship while the preGP of Fimoviridae and Phenuiviridae show a closed relationship. The N proteins of Fimoviridae were closer to the Phasmaviridae, the Tospoviridae were close to some Phenuiviridae members and the Peribunyaviridae. The plant viral movement proteins of species within the Tospoviridae and Phenuiviridae were more closely related to each other than to members of the Fimoviridae. Interestingly, distal ends of 3' and 5' untranslated regions of species within the Fimoviridae shared similarity to arthropod and vertebrate infecting members of the Cruliviridae and Peribunyaviridae compared to other plant virus families. Co-phylogeny analysis of the plant infecting viruses indicates that duplication and host switching were more common than co-divergence with a host species.

Description of a Novel Mycovirus in the Phytopathogen Fusarium culmorum and a Related EVE in the Yeast Lipomyces starkeyi.

A new mycovirus was found in the Fusarium culmorum strain A104-1 originally sampled on wheat in Belgium. This novel virus, for which the name Fusarium culmorum virus 1 (FcV1) is suggested, is phylogenetically related to members of the previously proposed family ''Unirnaviridae''. FcV1 has a monopartite dsRNA genome of 2898 bp that harbors two large non-overlapping ORFs. A typical -1 slippery motif is found at the end of ORF1, advocating that ORF2 is translated by programmed ribosomal frameshifting. While ORF2 exhibits a conserved replicase domain, ORF1 encodes for an undetermined protein. Interestingly, a hypothetically transcribed gene similar to unirnaviruses ORF1 was found in the genome of Lipomyces starkeyi, presumably resulting from a viral endogenization in this yeast. Conidial isolation and chemical treatment were unsuccessful to obtain a virus-free isogenic line of the fungal host, highlighting a high retention rate for FcV1 but hindering its biological characterization. In parallel, attempt to horizontally transfer FcV1 to another strain of F. culmorum by dual culture failed. Eventually, a screening of other strains of the same fungal species suggests the presence of FcV1 in two other strains from Europe.

Resistance Against Melon Chlorotic Mosaic Virus and Tomato Leaf Curl New Delhi Virus in Melon.

Thirty-one melon accessions were screened for resistance to the begomoviruses Melon chlorotic mosaic virus (MeCMV) and Tomato leaf curl New Delhi virus (ToLCNDV). Five accessions presented nearly complete resistance to both viruses. Accession IC-274014, showing the highest level of resistance to both viruses, was crossed with the susceptible cultivar Védrantais. The F1, F2, F3/F4, and both backcross progenies were mechanically inoculated with MeCMV. Plants without symptoms or virus detection by enzyme-linked immunosorbent assay and/or PCR were considered as resistant. The segregations were compatible with two recessive and one dominant independent genes simultaneously required for resistance. Inheritance of resistance to ToLCNDV in the F2 was best explained by one recessive gene and two independent dominant genes simultaneously required. Some F3 and F4 families selected for resistance to MeCMV also were resistant to ToLCNDV, suggesting that common or tightly linked genes were involved in resistance to both viruses. We propose the names begomovirus resistance-1 and Begomovirus resistance-2 for these genes (symbols bgm-1 and Bgm-2). Resistance to MeCMV in IC-274014 was controlled by bgm-1, Bgm-2, and the recessive gene melon chlorotic mosaic virus resistance (mecmv); resistance to ToLCNDV was controlled by bgm-1, Bgm-2, and the dominant gene Tomato leaf curl New Delhi virus resistance (Tolcndv).

Tomato Twisted Leaf Virus: A Novel Indigenous New World Monopartite Begomovirus Infecting Tomato in Venezuela.

Begomoviruses are one of the major groups of plant viruses with an important economic impact on crop production in tropical and subtropical regions. The global spread of its polyphagous vector, the whitefly Bemisia tabaci, has contributed to the emergence and diversification of species within this genus. In this study, we found a putative novel begomovirus infecting tomato plants in Venezuela without a cognate DNA-B component. This begomovirus was genetically characterized and compared with related species. Furthermore, its infectivity was demonstrated by agroinoculation of infectious clones in tomato (Solanum lycopersicum) and Nicotiana benthamiana plants. The name Tomato twisted leaf virus (ToTLV) is proposed. ToTLV showed the typical genome organization of the DNA-A component of New World bipartite begomoviruses. However, the single DNA component of ToTLV was able to develop systemic infection in tomato and N. benthamiana plants, suggesting a monopartite nature of its genome. Interestingly, an additional open reading frame ORF was observed in ToTLV encompassing the intergenic region and the coat protein gene, which is not present in other closely related begomoviruses. A putative transcript from this region was amplified by strand-specific reverse transcription-PCR. Along with recent studies, our results showed that the diversity of monopartite begomoviruses from the New World is greater than previously thought.

Complete genome sequence of two tomato-infecting begomoviruses in Venezuela: evidence of a putative novel species and a novel recombinant strain.

At least six begomovirus species have been reported infecting tomato in Venezuela. In this study the complete genomes of two tomato-infecting begomovirus isolates (referred to as Trujillo-427 and Zulia-1084) were cloned and sequenced. Both isolates showed the typical genome organization of New World bipartite begomoviruses, with DNA-A genomic components displaying 88.8% and 90.3% similarity with established begomoviruses, for isolates Trujillo-427 and Zulia-1084, respectively. In accordance to the guidelines for begomovirus species demarcation, the Trujillo-427 isolate represents a putative new species and the name "Tomato wrinkled mosaic virus" is proposed. Meanwhile, Zulia-1084 represents a putative new strain classifiable within species Tomato chlorotic leaf distortion virus, for which a recombinant origin is suggested.

Molecular and biological characterization of a new Tomato mild yellow leaf curl Aragua virus strain producing severe symptoms in tomato.

Tomato mild yellow leaf curl Aragua virus (ToMYLCV) is a begomovirus first reported infecting tomato (Solanum lycopersicum) and milkweed (Euphorbia heterophylla) in Venezuela. In this study, a ToMYLCV isolate (Zulia-219) was completely sequenced and its host range was evaluated. The DNA-A and DNA-B components of isolate Zulia-219 showed 93 and 85% nucleotide sequence identity with the respective counterparts of the ToMYLCV type strain. According to current demarcation criteria for begomovirus species, Zulia-219 is a new strain of ToMYLCV. Interestingly, tomato plants inoculated with ToMYLCV Zulia-219 displayed severe symptoms, including severe chlorotic leaf curling, in contrast to mild symptoms associated with the type strain of this begomovirus. These results indicate potential risks associated with this new ToMYLCV strain for tomato production in Venezuela.

Antiviral Defenses in Plants through Genome Editing.

Plant-virus interactions based-studies have contributed to increase our understanding on plant resistance mechanisms, providing new tools for crop improvement. In the last two decades, RNA interference, a post-transcriptional gene silencing approach, has been used to induce antiviral defenses in plants with the help of genetic engineering technologies. More recently, the new genome editing systems (GES) are revolutionizing the scope of tools available to confer virus resistance in plants. The most explored GES are zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats/Cas9 endonuclease. GES are engineered to target and introduce mutations, which can be deleterious, via double-strand breaks at specific DNA sequences by the error-prone non-homologous recombination end-joining pathway. Although GES have been engineered to target DNA, recent discoveries of GES targeting ssRNA molecules, including virus genomes, pave the way for further studies programming plant defense against RNA viruses. Most of plant virus species have an RNA genome and at least 784 species have positive ssRNA. Here, we provide a summary of the latest progress in plant antiviral defenses mediated by GES. In addition, we also discuss briefly the GES perspectives in light of the rebooted debate on genetic modified organisms (GMOs) and the current regulatory frame for agricultural products involving the use of such engineering technologies.

Tomato chlorotic leaf distortion virus, a new bipartite begomovirus infecting Solanum lycopersicum and Capsicum chinense in Venezuela.

Virus isolate T217L was obtained from a diseased tomato (Solanum lycopersicum) plant showing leaf deformation and chlorotic mottle symptoms near Maracaibo in the state of Zulia, Venezuela. Full-length DNA-A and DNA-B molecules of T217L were cloned and sequenced. The genome organization of T217L was identical to the bipartite genomes of other begomoviruses described from the Americas. Characteristic disease symptoms were reproduced in S. lycopersicum and Capsicum annum plants inoculated using the cloned viral DNA-A and DNA-B components, confirming disease aetiology. A sequence analysis of DNA-A showed that the T217L isolate has the highest sequence identity (84%) with sida yellow mosaic Yucatan virus (SiYMYuV), sida golden mosaic Honduras virus (SiGMHV) and bean dwarf mosaic virus (BDMV) isolates. This is less than the 89% identity in the DNA-A component that has been defined as the threshold value for the demarcation of species in the genus Begomovirus. The molecular data show that isolate T217L belongs to a novel tentative begomovirus species, for which the name tomato chlorotic leaf distortion virus is proposed. TCLDV was also detected in symptomatic C. chinense plants growing near the T217L-infected plant.

[Transmission of tomato Venezuela virus by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), in Maracaibo, Venezuela]. / Transmisión del tomato Venezuela virus por Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), en Maracaibo, Venezuela.

The biological transmission of Tomato Venezuela virus (ToVEV) by biotype B of the whitefly species Bemisia tabaci (Gennadius) increased (21.7-95.0%), and the time for symptom expression decreased (16-12.6 days) as the number of viruliferous whiteflies allowed access for inoculation to susceptible tomato plants increased from 1 to 20 adults/plant. When acquired only as a nymph, adults of this biotype transmitted the virus to 88.3% of susceptible tomato plants, using 15 viruliferous individuals per test plant, corroborating the circulative nature of the transmission. Disease incidence further increased (up to 100%) when the individuals were allowed to feed again on a virus-infected plant as adults. Leaf area, plant height and dry matter were significantly affected in ToVEV infected tomato plants.

Transmisión del tomato Venezuela virus por Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), en Maracaibo, Venezuela / Transmission of tomato Venezuela virus by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), in Maracaibo, Venezuela

The biological transmission of Tomato Venezuela virus (ToVEV) by biotype B of the whitefly species Bemisia tabaci (Gennadius) increased (21.7-95.0 percent), and the time for symptom expression decreased (16-12.6 days) as the number of viruliferous whiteflies allowed access for inoculation to susceptible tomato plants increased from 1 to 20 adults/plant. When acquired only as a nymph, adults of this biotype transmitted the virus to 88.3 percent of susceptible tomato plants, using 15 viruliferous individuals per test plant, corroborating the circulative nature of the transmission. Disease incidence further increased (up to 100 percent) when the individuals were allowed to feed again on a virusinfected plant as adults. Leaf area, plant height and dry matter were significantly affected in ToVEV infected tomato plants.

Almidón modificado de yuca como sustituto económico del agente solidificante para medios de cultivo de tejidos vegetales / Modified cassava starch a cheap gelling agent for plant tissue cultive

Con una historia de más de cinco décadas, los beneficios del cultivo de tejidos vegetales (CTV) no han llegado a los agricultores de las regiones más necesitadas. El problema radica en la complejidad y costo de aplicación de la técnica. En Venezuela, la yuca (Manihot esculenta Crantz) es cultivada en suelos marginales por agricultores de escasos recursos. Las limitaciones del cultivo son la carencia de semilla sana y los bajos niveles de rendimiento. Esta situación puede superarse mediante el uso del CTV para la producción de semilla vegetativa de alta calidad. Un primer paso para lograr la transferencia tecnológica es la sustitución de los componentes de los medios de cultivo convencionales por otros más económicos y accesibles. En el presente trabajo se evaluó el crecimiento de plantas de yuca in vitro, al sustituir el agente solidificante del medio con diferentes almidones modificados de yuca producidos en el país. Para ello fueron sembrados microesquejes en medio Murashige y Skoog (MS) y diferentes agentes solidificantes (Phytagel, AIM TF-351, AIM TF-352 y AIM TP-212). El crecimiento de las plantas fue comparado de acuerdo al peso seco, peso fresco, número de nudos, raíces y altura. Los resultados no mostraron diferencias significativas en el crecimiento y la tasa de multiplicación de plantas crecidas en Phytagel® y AIM TF-351. El almidón AIM TF-351 puede ser utilizado en medios de micropropagación clonal de yuca como una alternativa económica, de fácil manejo y disponibilidad en programas nacionales de producción de semilla

Efecto de tres herbicidas pre-emergentes en el establecimiento en campo de plantas in vitro de yuca (Manihot esculenta crantz) / Effect of three pre-emergent herbicides on field adaptation of cassava (Manihot esculenta crantz) in vitro plants

Entre los principales problemas para el desarrollo del cultivo de la yuca (Manihot esculenta Crantz) en Venezuela esta la carencia de buen material de siembra. Tradicionalmente, la yuca se propaga por estacas, siendo esto una fuente importante en la diseminación de plagas y enfermedades. El uso de plantas in vitro permite obtener material de siembra sano y vigoroso; sin embargo, su adaptación en campo requiere mayor atención en el control de malezas o arvenses. El presente trabajo evaluó el efecto de 3 herbicidas, fluometurón, S-metolacloro, pendimetalín, en comparación con el desyerbe manual, durante el establecimiento en campo de plantas in vitro de yuca. Los ensayos se realizaron en San Pablo, Edo. Yaracuy. Se usó un diseño de bloques al azar con cuatro repeticiones. Aunque los resultados no mostraron diferencias significativas en el porcentaje de supervivencia de las plántulas, su crecimiento fue significativamente superior (p <0,05) en parcelas tratadas con fluometurón y S-metolacloro con respecto al control manual. No se encontró diferencias entre tratamientos en el control general de arvenses, pero el S-metolacloro mostró mejor control de especies de hoja ancha. Se recomienda usar S-metolacloro y fluometurón el mismo día del transplante para mejorar la competitividad de las plántulas.