Tomato spotted wilt virus (TSWV) is an economically important viral pathogen of a wide range of field and horticultural crops. We developed an artificial microRNA (amiRNA) strategy against TSWV, targeting the nucleoprotein (N) and silencing suppressor (NSs) genes. The amiRNA constructs replaced the natural miRNA in a shortened Arabidopsis 173-nucleotide (nt) miR159a precursor backbone (athmiR159a) without the stem base extending beyond the miR/miR* duplex. Further, each amiRNA was modified to contain a mismatch (wobble) sequence at nucleotide position 12 and 13 on the complementary strand amiRNA*, mimicking the endogenous miR159a sequence structure. Transient expression in Nicotiana benthamiana demonstrated that the introduction of a wobble sequence did not alter amiRNA expression levels. Following challenge inoculation with TSWV, plants expressing N-specific amiRNAs with or without the wobble remained asymptomatic and were negative for TSWV by ELISA. In contrast, plants expressing the NSs-specific amiRNAs were symptomatic and accumulated high levels of TSWV. Similar findings were obtained in stably transformed Nicotiana tabacum plants. Our results show that a shortened 173-nt athmiR159a backbone is sufficient to express amiRNAs and that the presence of mismatch at position 12-13 does not influence amiRNA expression or conferring of resistance. We also show that selection of target gene and positional effect are critical in amiRNA-based approach for introducing resistance. These findings open the possibility of employing the amiRNA approach for broad-spectrum resistance to tospoviruses as well as other viruses. (C) 2015 Elsevier B.V. All rights reserved.

The dahlia (Dahlia variabilis) genome contains an endogenous pararetrovirus sequence (EPRS) tentatively designated as DvEPRS. The DvEPRS shares genome structure and organization that is typical of members of the Caulimovirus genus. Studies were carried out to better understand the nature of this integration and to determine the gene expression of this DvEPRS. Genomic Southern hybridization showed multiple and random integration events of the DvEPRS in the dahlia genome. To investigate the presence of DvEPRS transcripts, RT-PCR was done on DNase-treated total RNA from DvEPRS-infected dahlia plants. Results showed the expression of open reading frames I, V, and VI. Direct PCR from sap extracts produced more intense DNA amplicons of Dahlia mosaic virus and Dahlia common mosaic virus which are believed to exist as typical episomal caulimoviruses, whereas significantly less intense amplicon was seen in case of DvEPRS in comparison with internal transcribed spacer region of dahlias amplicon. The DvEPRS in wild and cultivated species of Dahlia offer a model system to study the molecular events underlying the ecology, evolution and spread of DvEPRS within natural and managed ecosystems and the factors affecting integration of these EPRS in the plant genome.

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus (DCMV), were found to be associated with dahlia mosaic disease. Additionally, an endogenous caulimovirus, Dahlia variabilis endogenous plant pararetroviral sequence (DvEPRS), was found to be present in many cultivated and wild Dahlia spp. Biological properties including mechanical and aphid transmission were investigated for DMV, DCMV and DvEPRS using Verbesina encelioides (crownbeard) as an experimental host. DMV and DCMV were transmitted by Myzus persicae at the rate of 50 and 73%, respectively. DMV and DCMV were mechanically transmissible to V. encelioides and the two effective buffers for mechanical transmission were phosphate buffer (pH 7.0) with nicotinic acid (1%) and sodium bisulfite (5%). Mechanical transmission rates of 58% and 36% were obtained for DMV and DCMV, respectively. DvEPRS was not transmissible by either aphids or mechanical inoculation. Availability of aphid and mechanical inoculation protocols for DMV and DCMV would facilitate further biological studies on caulimoviruses associated with Dahlia. ResumeDeux distincts caulimovirus ont ete trouves associes avec la maladie mosaique de dahlia, Dahlia mosaic virus (DMV) et Dahlia common mosaic virus (DCMV). En plus, un endogene caulimovirus, Dahlia variabilis plante pararetroviral endogene sequence (DvEPRS), a ete detecte dans plusieurs cultivars de dahlias et meme dans l'habitat naturel de dahlias. Les proprietes biologiques concernant la transmission mecanique et transmission par les pucerons ont ete etudiees pour DMV, DCMV, et DvEPRS en utilisant Verbesina encelioides comme un hote experimental. DMV et DCMV ont ete transmis par les pucerons Myzus pesicae aux taux de 50 et 73% respectivement. DMV et DCMV ont ete transmis mecaniquement a V. encelioides en utilisant deux efficace solutions le tampon de phosphate avec 1% acide nicotinique ou 5% bisulfite de sodium. Taux de transmission mecanique etait 58% et 36% pour DMV et DCM respectivement. DvEPRS n'etait pas transmis mecaniquement ni par les pucerons. La disponibilite de la transmission mecanique et la transmission par les pucerons protocoles pour DMC and DCMV facilite encore plus les etudes biologiques des caulimovirus associes a Dahlia.

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus, and an endogenous plant pararetroviral sequence (DvEPRS) were reported in Dahlia spp. DvEPRS, previously referred to as DMV-D10, was originally identified in the US from the cultivated Dahlia variabilis, and has also been found in New Zealand, Lithuania and Egypt, as well as in wild dahlia species growing in their natural habitats in Mexico. Sequence analysis of three new EPRSs from cultivated dahlias from Lithuania [D10-LT; 7,159 nucleotide level (nt)], New Zealand (D10-NZ, 7,156 nt), and the wild species, Dahlia rupicola, from Mexico (D10-DR, 7,133 nt) is reported in this study. The three EPRSs have the structure and organization typical of a caulimovirus species and showed identities among various open reading frames (ORFs) ranging between 71 and 97 % at the nt when compared to those or the known DvEPRS from the US. Examination of a dataset of seven full-length EPRSs obtained to date from cultivated and wild Dahlia spp. provided clues into genetic diversity of these EPRSs from diverse sources of dahlia. Phylogenetic analyses, mutation frequencies, potential recombination events, selection, and fitness were evaluated as evolutionary evidences for genetic variation. Assessment of all ORFs using phylogenomic and population genetics approaches suggests a wide genetic diversity of EPRSs occurring in dahlias. Phylogenetic analyses show that the EPRSs from various sources form one clade indicating a lack of clustering by geographical origin. Grouping of various EPRSs into two host taxa (cultivated vs. wild) shows little divergence with respect to their origin. Population genetic parameters demonstrate negative selection for all ORFs, with the reverse transcriptase region more variable than other ORFs. Recombination events were found which provide evolutionary evidence for genetic diversity among dahlia-associated EPRSs. This study contributes to an increased understanding of molecular population genetics and evolutionary pathways of these reverse transcribing viral elements.

Selected wild Dahlia species in their natural habitats from west‐central Mexico were tested for the presence of three caulimoviruses known to be associated with cultivated dahlia (Dahlia variabilis), viz. Dahlia mosaic virus (DMV), DMV‐D10 and Dahlia common mosaic virus. Virus species‐specific primers and PCR were used followed by cloning and sequencing of the amplicons. Results showed that the wild dahlia species in their natural habitat contained DMV‐D10, which is an endogenous plant pararetrovirus. Viral sequences were found in 91% of the samples (n = 56) representing four different wild species. The gene coding for the movement protein of DMV‐D10 from Dahlia coccinea and all other species was cloned and sequenced. Sequence comparisons showed divergence of this gene when compared to that of DMV‐D10 from cultivated dahlias. The discovery of plant pararetroviruses in wild dahlia species in their natural habitats suggests a possible emergence, co‐existence and co‐evolution of pararetroviruses and their host plants.

The genome structure and organization of endogenous caulimovirus sequences from dahlia (Dahlia spp), dahlia mosaic virus (DMV)-D10 from three wild species, D. coccinea (D10-DC), D. sherffii (D10-DS) and D. tenuicaulis (D10-DT), were determined and compared to those from cultivated species of dahlia, D. variabilis (DvEPRS). The complete ca. 7-kb dsDNA genomes of D10-DC, D10-DS, and D10-DT had a structure and organization typical of a caulimovirus and shared 89.3 to 96.6% amino acid sequence identity in various open reading frames (ORF) when compared to DvEPRS. The absence of the aphid transmission factor and the truncated coat protein fused with the reverse transcriptase ORF were common among these DMV-D10 isolates from wild Dahlia species.

The presence of Dahlia mosaic virus D10 (DMV-D10) was confirmed for the first time in dahlia (Dahlia variabilis) in Egypt. DMV-D10 was recently described as a caulimovirus that exists as an endogenous pararetroviral sequence (EPRS). DMV-D10 was confirmed by amplification of the ORF1 (encoding for the movement protein) using species specific primers (D10F1/R1). The expected size (900 bp) was amplified from 36 samples with no evidence of infection with either DMV or DCMV. The same dahlia plants were tested for the presence of CMV, INSV, TSV, and TSWV and they were all negative. Sequence comparisons of the Egyptian DMV-D10 ORF1, GenBank accession HM007162, amplified from these samples revealed that the amplicon had the highest sequence identity (96%) with that of DMV-D10 (isolated from US dahlia cultivars). Cluster dendogram based on the amino acid sequences of movement protein of all known caulimoviruses placed D10-US (isolated from US dahlia cultivars), D10-NZ (isolated from New Zealand dahlia cultivars), D10-DC (isolated from D. coccinea) and D10-Egypt (isolated from Egyptian dahlia cultivars) in one clade.

Dahlia mosaic is a serious disease affecting dahlias. In addition to the Dahlia mosaic virus (DMV) reported previously, we characterized two putative new caulimoviruses, tentatively designated as DMV-D10 and Dahlia common mosaic virus (DCMV), from dahlia. To better understand their relative incidence in dahlia, a total of 213 samples were collected during 2007 and 2008 from several varieties of cultivated dahlia (D. variabilis) in the United States. Samples were tested for the three caulimoviruses using virus-specific primers in a polymerase chain reaction. Amplicons were cloned and sequenced to confirm the infection of dahlia with these viruses. Results showed that DMV-D10 was the most prevalent (94%) followed by DCMV (48.5%) and DMV (23%). Mixed infections were common and viruses were detected irrespective of symptom expression at the time of sampling. Two percent of the samples were not infected by any of the three tested caulimoviruses. Results suggest that caulimovirus infections are widespread in dahlia and highlight the need for testing and production of virus-free material to reduce their spread.