Beet Soil-borne Virus Research Articles

Meta-transcriptomic analysis reveals the geographical expansion of known sugarbeet-infecting viruses and the occurrence of a novel virus in sugarbeet in the United States.

In this study, meta-transcriptome sequencing was conducted on a total of 18 sugarbeet (Beta vulgaris L. subsp. vulgaris) sample libraries to profile the virome of field-grown sugarbeet to identify the occurrence and distribution of known and potentially new viruses from five different states in the United States. Sugarbeet roots with symptoms resembling rhizomania caused by beet necrotic yellow vein virus (BNYVV), or leaves exhibiting leaf-curling, yellowing to browning, or green mosaic were collected from the sugarbeet growing areas of California, Colorado, Idaho, Minnesota, and North Dakota. In silico analysis of de novo assembled contigs revealed the presence of nearly full-length genomes of BNYVV, beet soil-borne virus (BSBV), and beet soil-borne mosaic virus (BSBMV), which represent known sugarbeet-infecting viruses. Among those, BNYVV was widespread across the locations, whereas BSBV was prevalent in Minnesota and Idaho, and BSBMV was only detected in Minnesota. In addition, two recently reported Beta vulgaris satellite virus isoforms (BvSatV-1A and BvSatV-1B) were detected in new locations, indicating the geographical expansion of this known virus. Besides these known sugarbeet-infecting viruses, the bioinformatic analysis identified the widespread occurrence of a new uncharacterized Erysiphe necator-associated abispo virus (En_abispoV), a fungus-related virus that was identified in all 14 libraries. En_abispoV contains two RNA components, and nearly complete sequences of both RNA1 and RNA2 were obtained from RNASeq and were further confirmed by primer-walking RT-PCR and Sanger sequencing. Phylogenetic comparison of En_abispoV isolates obtained in this study showed varying levels of genetic diversity within RNA1 and RNA2 compared to previously reported isolates. The undertaken meta-transcriptomic approach revealed the widespread nature of coexisting viruses associated with field-grown sugarbeet exhibiting virus disease-like symptoms in the United States.

Beet Soil-Borne Virus Is a Helper Virus for the Novel Beta vulgaris Satellite Virus 1A.

Sugar beet (Beta vulgaris) is grown in temperate regions around the world as a source of sucrose used for natural sweetening. Sugar beet is susceptible to a number of viral diseases, but identification of the causal agent(s) under field conditions is often difficult due to mixtures of viruses that may be responsible for disease symptoms. In this study, the application of RNAseq to RNA extracted from diseased sugar beet roots obtained from the field and from greenhouse-reared plants grown in soil infested with the virus disease rhizomania (causal agent beet necrotic yellow vein virus; BNYVV) yielded genome-length sequences from BNYVV, as well as beet soil-borne virus (BSBV). The nucleotide identities of the derived consensus sequence of BSBV RNAs ranged from 99.4 to 96.7% (RNA1), 99.3 to 95.3% (RNA2), and 98.3 to 95.9% (RNA3) compared with published BSBV sequences. Based on the BSBV genome consensus sequence, clones of the genomic RNAs 1, 2, and 3 were obtained to produce RNA copies of the genome through in vitro transcription. Capped RNA produced from the clones was infectious when inoculated into leaves of Chenopodium quinoa and B. vulgaris, and extracts from transcript-infected C. quinoa leaves could infect sugar beet seedling roots through a vortex inoculation method. Subsequent exposure of these infected sugar beet seedling roots to aviruliferous Polymyxa betae, the protist vector of both BNYVV and BSBV, confirmed that BSBV derived from the infectious clones could be transmitted by the vector. Co-inoculation of BSBV synthetic transcripts with transcripts of a cloned putative satellite virus designated Beta vulgaris satellite virus 1A (BvSat1A) resulted in the production of lesions on leaves of C. quinoa similar to those produced by inoculation with BSBV alone. Nevertheless, accumulation of genomic RNA and the encoded protein of the satellite virus in co-inoculated leaves was readily detected on Northern and Western blots, respectively, whereas no accumulation of satellite virus products occurred when satellite virus RNA was inoculated alone. The predicted sequence of the detected protein encoded by BvSat1A bears hallmarks of coat proteins of other satellite viruses, and virions of a size consistent with a satellite virus were observed in samples testing positive for the virus. The results demonstrate that BSBV is a helper virus for the novel satellite virus BvSat1A.

Molecular characterization based on coat protein gene of Beet soil-borne virus isolates obtained from the soils of sugar beet growing areas in Turkey

Şeker pancarı (Beta vulgaris L.), Türkiye'de şeker endüstrisinin hammaddesi olup, yaygın olarak yetiştirilmektedir. Pancar bitkisi, plazmodiophorid vektör Polymyxa betae tarafından taşınan bazı toprak kaynaklı virüsler tarafından enfekte edilmektedir. Taksonomik olarak Virgaviridae familyası içinde yer alan ve Pomovirus cinsine ait Beet soil-borne virus (BSBV) ve Beet virus Q (BVQ) şeker pancarında enfeksiyon oluşturan toprak kökenli virüs türleridir. Bu virüslerden BSBV, Türkiye'de ilk olarak 2003 yılında kayıt edilmiş ve daha sonra şeker pancarı üretim alanlarında oldukça yaygın olduğu (% 49.8) belirlenmiştir. Bu çalışmada, BSBV ile bulaşık 10 toprak örneği coğrafik orjinlerine göre rastgele seçilmiş ve bu toprak örneklerinden tuzak bitki testi yöntemiyle elde edilen izolatlar, moleküler yönden analiz edilmiştir. BSBV kılıf protein (CP) genine spesifik primerler ile uygulanan RT-PCR yöntemi ve elde edilen DNA ürünlerinin baz dizi analizi sonrasında, tüm BSBV izolatlarının CP bölgelerinin 495 nükleotit ve 164 amino asit (aa) içerdiği tespit edilmiştir. İlginç bir şekilde, farklı lokasyonlardan elde edilen 9 BSBV izolatı birbirleriyle karşılaştırıldığında, bu izolatların aa düzeyinde % 100 benzer oldukları saptanmıştır. Bununla birlikte, Yozgat ilinden elde edilen YZT-355S izolatının, diğer izolatlarla aa benzerlik oranının % 98.17 olduğu ve 92., 121. ve 122. pozisyonlarda yer alan aa’lerin diğer Türkiye izotları ve daha önce tanımlanan BSBV dünya izolatlarından farklılık sergilediği tespit edilmiştir. Bunun yanı sıra, 24 BSBV izolatının (bu çalışmadan 10 ve GenBank'tan 14) filogenetik analizi, tüm izolatların I, II, III ve IV olmak üzere dört ana grupta toplandığını göstermiştir. Grup I'e ait Türkiye izolatları iki alt gruba (alt grup a ve c) ayrılırken, diğer Türkiye izolatları grup III ve grup IV içerisinde yer almışlardır.

Beet soil-borne mosaic virus

This datasheet on Beet soil-borne mosaic virus covers Identity, Distribution, Hosts/Species Affected, Further Information.

The Beta vulgaris-derived resistance gene Rz2 confers broad-spectrum resistance against soilborne sugar beet-infecting viruses from different families by recognizing triple gene block protein 1.

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled‐coil and leucine‐rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV‐encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)‐like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.

Complete Genomic Characterization of Two Beet Soil-Borne Virus Isolates from Turkey: Implications of Comparative Analysis of Genome Sequences.

Sugar beet (Beta vulgaris L.) is known as a key product for agriculture in several countries across the world. Beet soil-borne virus (BSBV) triggers substantial economic damages to sugar beet by reducing the quantity of the yield and quality of the beet sugars. We conducted the present study to report the complete genome sequences of two BSBV isolates in Turkey for the first time. The genome organization was identical to those previously established BSBV isolates. The tripartite genome of BSBV-TR1 and -TR3 comprised a 5,835-nucleotide (nt) RNA1, a 3,454-nt RNA2, and a 3,005-nt RNA3 segment. According to sequence identity analyses, Turkish isolates were most closely related to the BSBV isolate reported from Iran (97.83–98.77% nt identity). The BSBV isolates worldwide (n = 9) were phylogenetically classified into five (RNA-coat protein read through gene [CPRT], TGB1, and TGB2 segments), four (RNA-rep), or three (TGB3) lineages. In genetic analysis, the TGB3 revealed more genetic variability (Pi = 0.034) compared with other regions. Population selection analysis revealed that most of the codons were generally under negative selection or neutral evolution in the BSBV isolates studied. However, positive selection was detected at codon 135 in the TGB1, which could be an adaptation in order to facilitate the movement and overcome the host plant resistance genes. We expect that the information on genome properties and genetic variability of BSBV, particularly in TGB3, TGB1, and CPRT genes, assist in developing effective control measures in order to prevent severe losses and make amendments in management strategies.

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.

RNAseq Analysis of Rhizomania-Infected Sugar Beet Provides the First Genome Sequence of Beet Necrotic Yellow Vein Virus from the USA and Identifies a Novel Alphanecrovirus and Putative Satellite Viruses.

“Rhizomania” of sugar beet is a soilborne disease complex comprised of beet necrotic yellow vein virus (BNYVV) and its plasmodiophorid vector, Polymyxa betae. Although BNYVV is considered the causal agent of rhizomania, additional viruses frequently accompany BNYVV in diseased roots. In an effort to better understand the virus cohort present in sugar beet roots exhibiting rhizomania disease symptoms, five independent RNA samples prepared from diseased beet seedlings reared in a greenhouse or from field-grown adult sugar beet plants and enriched for virus particles were subjected to RNAseq. In all but a healthy control sample, the technique was successful at identifying BNYVV and provided sequence reads of sufficient quantity and overlap to assemble > 98% of the published genome of the virus. Utilizing the derived consensus sequence of BNYVV, infectious RNA was produced from cDNA clones of RNAs 1 and 2. The approach also enabled the detection of beet soilborne mosaic virus (BSBMV), beet soilborne virus (BSBV), beet black scorch virus (BBSV), and beet virus Q (BVQ), with near-complete genome assembly afforded to BSBMV and BBSV. In one field sample, a novel virus sequence of 3682 nt was assembled with significant sequence similarity and open reading frame (ORF) organization to members within the subgenus Alphanecrovirus (genus Necrovirus; family Tombusviridae). Construction of a DNA clone based on this sequence led to the production of the novel RNA genome in vitro that was capable of inducing local lesion formation on leaves of Chenopodium quinoa. Additionally, two previously unreported satellite viruses were revealed in the study; one possessing weak similarity to satellite maize white line mosaic virus and a second possessing moderate similarity to satellite tobacco necrosis virus C. Taken together, the approach provides an efficient pipeline to characterize variation in the BNYVV genome and to document the presence of other viruses potentially associated with disease severity or the ability to overcome resistance genes used for sugar beet rhizomania disease management.

First Report of Beet Soil-Borne Virus on Red Table Beet in Brazil

First Report of Beet Soil-Borne Virus on Red Table Beet in Brazil

Beet soil-borne mosaic virus: development of virus-specific detection tools

To have an opportunity for reliable detection of benyvirus Beet soil-borne mosaic virus (BSBMV) and its discrimination from Beet necrotic yellow vein virus (BNYVV), an attempt to generate a specific immunoassay was made. Using a purified recombinant polypeptide corresponding to the 55 C-terminal amino acid residues of viral coat protein as antigen, a specific polyclonal antiserum against BSBMV was raised. The obtained immunoglobulin G fraction reacted with high specificity in western blot analysis with both the recombinant polypeptide expressed in Escherichia coli and total protein extract from BSBMV-infected plants. In parallel, to improve the identification of benyviruses, a duplex RT-PCR method for simultaneous detection of BNYVV and BSBMV was developed. This method is based on the combination of multiple primer sets to different pathogens into single multiplexed amplification reaction, which share single standard amplification conditions.

Detection and prevalence of viruses associated with sugarbeet in the Tadla region of Morocco

Fifty-five soil samples were collected from sugarbeet fields with rhizomania occurrence in the region of Tadla. BNYVV was ascertained by RT-PCR in 40 out of 55 fields surveyed and the positive samples were subsequently examined for the presence of the different pathotype of BNYVV. Using the same technique, a survey was simultaneously undertaken to identify the occurrence of other soil-borne viruses, which might frequently associate with BNYVV such as beet soilborne virus (BSBV), beet soilborne mosaic virus (BSBMV) and beet virus Q (BVQ). Results indicated that most samples were infected with A-type BNYVV, but only 9% of the samples were positive for B-type BNYVV. BNYVV (72.7%) was the most frequently detected, followed respectively by BSBV (36.36%) and BVQ (30.9%). However, P-type BNYVV was not detected at all. Our results also indicated the likely absence of BSBMV in the Tadla region. Surprisingly, among the 40 BNYVV-infected samples, 17 were also infected with BVQ and 20 with BSBV. In conclusion, BSBV occurred in triple infections with BNYVV and BVQ. In addition, there was only one sample revealed positive for both viruses BNYVV-B and BVQ and four samples for BSBV infection. Therefore, this study is the first investigation of BSBV and BVQ in fields-grown sugarbeet of Morocco.

Fluorescent labelling of Beet necrotic yellow vein virus and Beet soil-borne mosaic virus for co- and superinfection experiments in Nicotiana benthamiana.

Infectious full-length clones of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV), both genus Benyvirus, were used for fluorescent labelling with the objective to study their interaction in coinfection and superinfection experiments. Fluorescent labelling was achieved by replacing a part of the RNA2 encoded coat protein read-through domain with either GFP or mRFP fluorescent marker proteins. This resulted in a translational fusion comprising the coat and the fluorescent protein. The labelled viruses were infectious and moved systemically in Nicotiana benthamiana, producing wild-type-like symptoms. Virus particles could be observed by electron microscopy, demonstrating that the viral read-through domain is dispensable for particle formation. Coinfection experiments revealed a spatial separation of differentially labelled populations of both identical and different Benyvirus species after N. benthamiana agro-inoculation. Identical observations were obtained when Tobacco rattle virus (TRV) was differentially labelled and used for coinfection. In contrast, coinfections of BSBMV with Potato virus X (PVX) or TRV resulted in many co-infected cells lacking spatial separation. Micro-projectile co-bombardment of N. benthamiana leaves revealed that two differently labelled populations of the same virus co-infected only a few cells before starting to separate. In superinfection experiments with N. benthamiana, BSBMV and BNYVV were unable to establish a secondary infection in plants that were previously infected with BNYVV or BSBMV. Taken together, this is the first work to describe the interaction between two economically important Benyviruses using fluorescence-labelled full-length clones.

Long Term Management of Rhizomania Disease-Insight Into the Changes of the Beet necrotic yellow vein virus RNA-3 Observed Under Resistant and Non-resistant Sugar Beet Fields.

Rhizomania disease, caused by the Beet necrotic yellow vein virus (BNYVV), is considered as one of the major constraints for sugar beet production, worldwide. As a result of the introgression of major resistance genes (Holly, Rz2) in commercially available sugar beet varieties, the virus has endured strong selection pressure since the 90s'. Understanding the virus response and diversity to sugar beet resistance is a key factor for a sustainable management of only few resistance genes. Here we report rhizomania surveys conducted in a rhizomania hot spot, the Pithiviers area (France) during a 4-year period and complementary to the study of Schirmer et al. (2005). The study aimed at evaluating the intra- and inter-field BNYVV diversity in response to different sources of resistance and over the growing season. To follow rhizomania development over the sugar beet growing season, extensive field samplings combined with field assays were performed in this study. The evolution of the BNYVV diversity was assessed at intra- and inter-field levels, with sugar beet cultivars containing different resistance genes (Rz1, Rz1 + Heterodera schachtii resistance and Rz1Rz2). Intra-field diversity was analyzed at the beginning and the end of the growing season of each field. From more than one thousand field samples, the simultaneous presence of the different A, B and P types of BNYVV was confirmed, with 21 variants identified at positions 67–70 of the p25 tetrad. The first variant, AYHR, was found most commonly followed by SYHG. Numerous mixed infections (9.93% of the samples), mostly of B-type with P-type, have also been evidenced. Different tetrads associated with the A- or B-type were also found with a fifth RNA-genome component known to allow more aggressiveness to BNYVV on sugar beet roots. Cultivars with Rz1+Rz2 resistant genes showed few root symptoms even if the BNYVV titre was quite high according to the BNYVV type present. The virus infectious potential in the soil at the end of the growing season with such cultivars was also lower despite a wider diversity at the BNYVV RNA3 sequence level. Rz1+Rz2 cultivars also exhibited a lower presence of Beet soil-borne virus (BSBV), a P. betae-transmitted Pomovirus. Cultivars with Rz1 and nematode (N) resistance genes cultivated in field infected with nematodes showed lower BNYVV titre than those with Rz1 or Rz1+Rz2 cultivars. Overall, the population structure of BNYVV in France is shown to be different from that previously evidenced in different world areas. Implications for long-term management of the resistance to rhizomania is discussed.

Distribution of resistance-breaking isolates of beet necrotic yellow vein virus differing in virulence in sugar beet fields in Turkey

ABSTRACTRhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is one of the most devastating diseases on sugar beet throughout the world. A total of 235 BNYVV-infested soil samples taken from 29 provinces during the growing seasons of 2004–2005 and 2010–2011 were investigated for resistance-breaking properties of BNYVV isolates. BNYVV resistant (Rz1, Rz1 + R2, Rz1 + C48 + minor genes) and susceptible (rz1) sugar beet cultivars were grown in the BNYVV-infested soils by the bait plant technique, and virus infection was evaluated based on disease symptom and DAS-ELISA. Of the soil samples tested, 57.4% produced infection in the roots of sugar beet cultivar (cv.) containing the Rz1 gene, 51.9% in cv. carrying the Rz1 + Rz2 and 18.9% in cv. carrying the Rz1 + C48 + minor genes. Also, the presence of Beet soil-borne virus (BSBV) was evaluated by TAS-ELISA. Additionally, the presence of BNYVV RNA-5 component was investigated by RT–PCR using the primers specific for p26 coding region. Our results revealed that BNYVV isolates compromising resistance genes are highly common and widespread in sugar beet production areas in Turkey, and it seems unlikely that BSBV and/or RNA-5 were directly involved in RB-event.

Biological properties of Beet soil-borne mosaic virus and Beet necrotic yellow vein virus cDNA clones produced by isothermal in vitro recombination: Insights for reassortant appearance

Two members of the Benyviridae family and genus Benyvirus, Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV), possess identical genome organization, host range and high sequence similarity; they infect Beta vulgaris with variable symptom expression. In the US, mixed infections are described with limited information about viral interactions. Vectors suitable for agroinoculation of all genome components of both viruses were constructed by isothermal in vitro recombination. All 35S promoter-driven cDNA clones allowed production of recombinant viruses competent for Nicotiana benthamiana and Beta macrocarpa systemic infection and Polymyxa betae transmission and were compared to available BNYVV B-type clone. BNYVV and BSBMV RNA1 + 2 reassortants were viable and spread long-distance in N. benthamiana with symptoms dependent on the BNYVV type. Small genomic RNAs were exchangeable and systemically infected B. macrocarpa. These infectious clones represent a powerful tool for the identification of specific molecular host-pathogen determinants.

P25 pathogenicity factor deletion mutants of beet necrotic yellow vein virus occurring in sugar beet fields in Turkey

Beet necrotic yellow vein virus (BNYVV) causes rhizomania which is one of the most devastating diseases in sugar beet fields worldwide. BNYVV symptoms were observed in Rz1-resistant sugar beet plants in a number of fields in Aksaray and Konya Provinces during surveys in 2011. Seven soil samples, taken from problematic areas in these provinces and known to be infested with populations of Polymyxa betae Keskin carrying BNYVV, were used in bait plant and molecular studies. Initially, the presence of other soil-borne viruses was investigated by using ELISA and RT-PCR. It was determined that BNYVV was in co-infections with pomoviruses, namely beet soil-borne virus and/or beet virus Q. The partial RNA-3 segment of BNYVV was amplified by RT-PCR, and the nucleotide sequences involving the p25 coding region were obtained. The deduced amino acid (aa) sequences of the p25 protein indicated that four BNYVV populations had ‘ACHG’ or ‘VHHG’ residues at positions 67–70. In addition, three populations containing three nucleotide deletions, not shifting the reading frame but affecting the aa at position 67 (-DHG) or 68 (D-HG), were identified. A deleted form of the p25 was identified in both the A- and J-type BNYVV populations. The pathogenicities of these BNYVV populations in the sugar beet genotypes rz1 and Rz1 were investigated under controlled conditions. All seven BNYVV isolates were capable of overcoming Rz1 resistance. These results seem to indicate that mutants of BNYVV with a deletion are aggressive, as are the isolates without deletion. Resistant beet cultivars will most likely be more productive in the future if these isolates become widespread in Turkey.

The widespread occurrences of Beet soil borne virus and RNA-5 containing Beet necrotic yellow vein virus isolates in sugar beet production areas in Turkey

A total of 632 soil samples were collected from different sugar beet fields in 32 provinces during 2005, 2010 and 2011 growing seasons. Incidences of Beet necrotic yellow vein virus (BNYVV), Beet soil-borne virus (BSBV) and their vector Polymyxa betae were determined by bait plant tests using ELISA and root staining techniques. The percentages of BNYVV and BSBV infested soil samples were 38 and 49.8 %, respectively. The study showed that P. betae was very common (87.7 %) in the sampled sugar beet fields. Additionally, the presence of BNYVV RNA-5 component was investigated by RT-PCR using the primers specific for p26 coding region. Out of 240 BNYVV-infected samples, 85 were selected according to their geographic origin, and 62.4 % of them belonging to 29 provinces were found to contain the RNA-5 segment. The partial nucleotide sequences of RNA-5 of BNYVV isolates from Igdir, Edirne, Ankara and Konya provinces of Turkey were obtained. Nucleotide sequencing and phylogenetic analyses showed that RNA-5 of Turkish BNYVV isolates is closer to the J type BNYVV isolates than the P type isolates. These results also indicated that BNYVV populations containing RNA-5 is highly widespread in sugar beet production areas in Turkey.

Molecular mechanisms involved in the pathogenesis of beet soil-borne viruses.

The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

Characterization of burdock mottle virus, a novel member of the genus Benyvirus, and the identification of benyvirus-related sequences in the plant and insect genomes

The complete nucleotide sequence of the burdock mottle virus (BdMoV) isolated from an edible burdock plant (Arctium lappa) in Japan has been determined. BdMoV has a bipartite genome, whose organization is similar to RNA1 and RNA2 of benyviruses, beet necrotic yellow vein virus (BNYVV), beet soil-borne mosaic virus (BSBMV), and rice stripe necrosis virus (RSNV). BdMoV RNA1 (7038 nt) contains a single open reading frame (ORF) encoding a 249-kDa polypeptide that consists of methyl-transferase, helicase, papain-like protease, AlkB-like, and RNA-dependent RNA polymerase domains. The AlkB-like domain sequence is not present in the proteins encoded by other known benyviruses, but is found in replication-associated proteins of viruses mainly belonging to the families Alfaflexiviridae and Betaflexiviridae. BdMoV RNA2 (4315 nt) contains six ORFs that are similar to those of benyviruses: these are coat protein (CP), CP readthrough, triple gene block movement and cysteine-rich proteins. Phylogenetic analyses showed that BdMoV is more closely related to BNYVV and BSBMV than to RSNV. Database searches showed that benyvirus replicase-related sequences are present in the chromosomes of a chickpea plant (Cicer arietinum) and a blood-sucking insect (Rhodnius prolixus). Some other benyvirus-related sequences are found in the transcriptome shotgun libraries of a few species of plants and a bark beetle. Our results show that BdMoV is a distinct species of the genus Benyvirus and that ancestral and extant benyviruses may have infected or currently infect a wide range of hosts, including plants and insects.

The Benyvirus RNA Silencing Suppressor Is Essential for Long-Distance Movement, Requires Both Zinc-Finger and NoLS Basic Residues but Not a Nucleolar Localization for Its Silencing-Suppression Activity

The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.