Rosana Blawid

Construction of an infectious clone of a plant RNA virus in a binary vector using one-step Gibson Assembly

The construction of full-length infectious clones of RNA viruses is often laborious due to the many cloning steps required and the DNA exclusion within the plasmid during Escherichia coli transformation. We demonstrate single-step cloning procedure of an infectious cDNA of the tomato blistering mosaic virus (ToBMV) using Gibson Assembly (GA), which drastically reduces the number of cloning steps. By agro-inoculation with the construct obtained by this procedure, ToBMV was recovered six days post-inoculation in Nicotiana benthamiana plants. The symptoms induced by the recovered virus were indistinguishable from those caused by the wild-type virus. We conclude that the GA is very useful method particularly to construct a full-length cDNA clone of a plant RNA virus in a binary vector.

A novel vitivirus-like sequence found in Arracacia xanthorrhiza plants by high throughput sequencing

High throughput sequencing (HTS) is a very powerful tool for detecting and discovering novel viral-like sequences without prior knowledge of the sequence. Here we describe the complete genome of a new vitivirus-like sequence that was found in arracacha (Arracacia xanthorrhiza) plants using HTS technology. The complete genome sequence was validated by Sanger sequencing. The genomic organization of the new putative vitivirus resembles that of grapevine virus B (GVB) and grapevine virus D (GVD). The putative coat protein showed 41 to 49% identity with similar proteins of known vitiviruses, while the RNA-dependent RNA polymerase shared 52 to 55% identity with those encoded by grapevine vitiviruses. Based on the demarcation criteria for the genus Vitivirus, the virus described in this work, provisionally named as “Arracacha virus V”, represents a novel species in this taxon.

Construction of an agroinfectious clone of bean rugose mosaic virus using Gibson Assembly

Construction of agroinfectious viral clones usually requires many steps of cloning and sub-cloning and also a binary vector, which makes the process laborious, time-consuming, and frequently susceptible to some degree of plasmid instability. Nowadays, novel methods have been applied to the assembly of infectious viral clones, and here we have applied isothermal, single-step Gibson Assembly (GA) to construct an agroinfectious clone of Bean rugose mosaic virus (BRMV) using a small binary vector. The procedure has drastically reduced the cloning steps, and BRMV could be recovered from agroinfiltrated common bean twenty days after inoculation, indicating that the infectious clone could spread in the plant tissues and efficiently generate a systemic infection. The virus was also recovered from leaves of common bean and soybean cultivars mechanically inoculated with infectious clone two weeks after inoculation, confirming the efficiency of GA cloning procedure to produce the first BRMV agroinfectious clone to bean and soybean.

Discovery and molecular characterization of a novel enamovirus, Grapevine enamovirus-1

In this study, we describe a novel putative Enamovirus member, Grapevine enamovirus-1 (GEV-1), discovered by high-throughput sequencing (HTS). A limited survey using HTS of 17 grapevines (Vitis spp.) from the south, southeast, and northeast regions of Brazil led to the detection of GEV-1 exclusively on southern plants, infecting four grapevine cultivars (Cabernet Sauvignon, Semillon, CG 90450, and Cabernet franc) with a remarkable identity of around 99% at the nucleotide level. This novel virus was only detected in multiple-virus infected plants exhibiting viral-like symptoms. GEV-1 was also detected on a cv. Malvasia Longa by RT-PCR. We performed graft-transmissibility assays on GEV-1. The organization, products, and cis-acting regulatory elements of GEV-1 genome are also discussed here. The near complete genome sequence of GEV-1 was obtained during the course of this study, lacking only part of the 3′ untranslated terminal region. This is the first report of a virus in the family Luteoviridae infecting grapevines. Based on its genomic properties and phylogenetic analyses, GEV-1 should be classified as a new member of the genus Enamovirus.

Complete genome sequence of melon yellowing-associated virus from melon plants with the severe yellowing disease in Brazil

Here, we describe the complete genome sequence of melon yellowing-associated virus (MYaV), found in melon plants with severe yellowing disease, determined by high-throughput and Sanger sequencing. MYaV has an RNA genome of 9073 nucleotides plus a poly(A) tail. At least six open reading frames were predicted, with a typical carlavirus genomic organisation. Phylogenetic analysis of the complete genome sequence and the amino acid sequences of the RNA-dependent RNA polymerase confirmed that MYaV belongs to the genus Carlavirus, with the highest genome-wide nucleotide sequence identity of 59.8% to sweet potato yellow mottle virus.

Fluorescence in situ hybridization analysis of endosymbiont genera reveals novel infection patterns in a tomato-infesting Bemisia tabaci population from Brazil

The Bemisia tabaci cryptic species complex harbors a diversified flora of primary and secondary endosymbionts, which plays crucial roles in many aspects of the insect biology. The endosymbiont infection pattern is dependent upon many factors, including host plant and geographic origin. In Brazil, the invasion of B. tabaci Middle East-Asia Minor-1 (MEAM 1) populations was observed in tomato (Solanum lycopersicum L.) fields in the 1990s, which was followed by severe begomovirus epidemics. Here we confirmed the vertical transmission of “Candidatus Portiera” and the localization of secondary endosymbionts in distinct B. tabaci developmental stages. Hamiltonella defensa was detected in bacteriocytes but also scattered in the leg muscles as well as in the male heads. Wolbachia-specific signals were found in eggs, nymphs, male adults, and female gonads in predominant association with bacteriocytes. These results were somewhat surprising since Wolbachia infection is rarely found in MEAM 1 populations from the Old World. Our results reinforce the notion that endosymbiont infection pattern may vary according to the insect population, gender, developmental stage, and geographic origin. This characterization will provide tools to study the endosymbiont function in the transmission ability/efficiency of a complex of tomato-infecting bipartite begomoviruses by B. tabaci under Neotropical conditions.

High-throughput sequencing reveals a novel closterovirus in arracacha (Arracacia xanthorrhiza)

High-throughput sequencing analysis detected a clostero-like virus from arracacha plants (Arracacia xanthorrhiza) in Brazil. The complete genome sequence, confirmed by RACE and Sanger sequencing, consists of 15,763 nucleotides with nine predicted open reading frames (ORFs) in a typical closterovirus genome organisation. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (Hsp70h), and coat protein showed 55-65, 38-44, and 20-36% amino acid sequence identity, respectively, to the homologous proteins of known closteroviruses. Phylogenetic analysis of Hsp70h showed that this putative novel arracacha plant virus was related to members of the genus Closterovirus in the family Closteroviridae. These results suggest that this virus, tentatively named “arracacha virus 1” (AV-1), is a novel member of the genus Closterovirus. This is the first closterovirus identified in arracacha plants.

Subcellular localization of p29, a putative movement protein of pepper ringspot virus

Pepper ringspot virus (PepRSV) is a member of the genus Tobravirus. It possesses a bipartite single-strand RNA genome in a positive-sense polarity. The p29 protein is encoded by RNA 1 and is presumed to be the movement protein (MP) of this virus. In this study, the intracellular distribution of the p29 protein was analyzed by confocal microscopy. Transient expression of the PepRSV p29 protein fused to green fluorescent protein was observed as punctate spots localized next to the cell wall. This protein partially co-localized with the eCFP-tagged tobacco mosaic virus 30K MP, which is known to associate with plasmodesmata. This result suggests that the p29 protein is most probably the movement protein for PepRSV.