Turnip yellows virus variants differ in host range, transmissibility, and virulence.

Turnip yellows virus (TuYV; family Solemoviridae, genus Polerovirus, species Turnip yellows virus) is a genetically diverse virus that infects a broad range of plant species across the world. Due to its global economic significance, most attention has been given to the impact of TuYV on canola (syn. oilseed rape; Brassica napus). In Australia, a major canola-exporting country, TuYV isolates are highly diverse, with the most variation concentrated in open reading frame 5 (ORF 5), which encodes the readthrough domain (P5) component of the readthrough protein (P3P5), which plays an important role in host adaptation and aphid transmission. When analysing ORF 5, Australian TuYV isolates form three phylogenetic groups with just 45 to 49% amino acid sequence identity: variants P5-I, P5-II, and P5-III. Despite the possible implications for TuYV epidemiology and management, research examining phenotypic differences between TuYV variants is scarce. This study was designed to test the hypothesis that three TuYV isolates, representing each of the Australian P5 variants, differ phenotypically. In particular, the host range, vector species, transmissibility, and virulence of isolates 5414 (P5-I5414), 5509 (P5-II5509), and 5594 (P5-III5594) were examined in a series of glasshouse experiments. Only P5-I5414 readily infected faba bean (Vicia faba), only P5-II5509 infected chickpea (Cicer arietinum), and only P5-I5414 and P5-III5594 infected lettuce (Lactuca sativa). Myzus persicae transmitted each isolate, but Brevicoryne brassicae and Lipaphis pseudobrassicae did not. When using individual M. persicae to inoculate canola seedlings, P5-I5414 had significantly higher transmission rates (82%) than P5-II5509 (62%) and P5-III5594 (59%). As indicated by enzyme-linked immunosorbent assay absorbance values, P5-I5414 reached higher virus titers in canola than P5-II5509, which, in turn, reached higher titers than P5-III5594. P5-I5414 was also more virulent in canola than P5-II5509 and P5-III5594, inducing more severe foliar symptoms, stunting, and, in one of two experiments, seed yield loss. Results from this study compared to those of previous studies suggest that analysis of ORF 5 alone is insufficient to assign isolates to coherent strain categories, and further sequencing and phenotyping of field isolates is required.

Integrating High throughput Sequencing into Survey Design Reveals Turnip Yellows Virus and Soybean Dwarf Virus in Pea (Pisum Sativum) in the United Kingdom.

There is only limited knowledge of the presence and incidence of viruses in peas within the United Kingdom, therefore high-throughput sequencing (HTS) in combination with a bulk sampling strategy and targeted testing was used to determine the virome in cultivated pea crops. Bulks of 120 leaves collected from twenty fields from around the UK were initially tested by HTS, and presence and incidence of virus was then determined using specific real-time reverse-transcription PCR assays by testing smaller mixed-bulk size samples. This study presents the first finding of turnip yellows virus (TuYV) in peas in the UK and the first finding of soybean dwarf virus (SbDV) in the UK. While TuYV was not previously known to be present in UK peas, it was found in 13 of the 20 sites tested and was present at incidences up to 100%. Pea enation mosaic virus-1, pea enation mosaic virus-2, pea seed-borne mosaic virus, bean yellow mosaic virus, pea enation mosaic virus satellite RNA and turnip yellows virus associated RNA were also identified by HTS. Additionally, a subset of bulked samples were re-sequenced at greater depth to ascertain whether the relatively low depth of sequencing had missed any infections. In each case the same viruses were identified as had been identified using the lower sequencing depth. Sequencing of an isolate of pea seed-borne mosaic virus from 2007 also revealed the presence of TuYV and SbDV, showing that both viruses have been present in the UK for at least a decade, and represents the earliest whole genome of SbDV from Europe. This study demonstrates the potential of HTS to be used as a surveillance tool, or for crop-specific field survey, using a bulk sampling strategy combined with HTS and targeted diagnostics to indicate both presence and incidence of viruses in a crop.

Nine viruses from eight lineages exhibiting new evolutionary modes that co-infect a hypovirulent phytopathogenic fungus.

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation.

Sugarcane mosaic virus remodels multiple intracellular organelles to form genomic RNA replication sites.

Positive-stranded RNA viruses usually remodel the host endomembrane system to form virus-induced intracellular vesicles for replication during infections. The genus Potyvirus of the family Potyviridae represents the largest number of positive single-stranded RNA viruses, and its members cause great damage to crop production worldwide. Although potyviruses have a wide host range, each potyvirus infects a relatively limited number of host species. Phylogenesis and host range analysis can divide potyviruses into monocot-infecting and dicot-infecting groups, suggesting that they differ in their infection mechanisms, probably during replication. Comprehensive studies on the model dicot-infecting turnip mosaic virus have shown that the 6K2-induced replication vesicles are derived from the endoplasmic reticulum (ER) and subsequently target chloroplasts for viral genome replication. However, the replication site of monocot-infecting potyviruses is unknown. In this study, we show that the precursor 6K2-VPg-Pro polyproteins of dicot-infecting potyviruses and monocot-infecting potyviruses cluster phylogenetically in two separate groups. With a typical gramineae-infecting potyvirus-sugarcane mosaic virus (SCMV)-we found that replicative double-stranded RNA (dsRNA) forms aggregates in the cytoplasm but does not associate with chloroplasts. SCMV 6K2-VPg-Pro-induced vesicles colocalize with replicative dsRNA. Moreover, SCMV 6K2-VPg-Pro-induced structures target multiple intracellular organelles, including the ER, Golgi apparatus, mitochondria, and peroxisomes, and have no evident association with chloroplasts.

Introducing SELEX via a semester-long course-based undergraduate research experience (CURE).

With the growing importance of the field of RNA biology, undergraduates need to perform RNA-related research. Systematic evolution of ligands by exponential enrichment (SELEX) has become an important method in RNA biology. The principles of SELEX were applied to a semester-long course-based undergraduate research experience (CURE) in which two rounds of in vivo functional selection of regions of a viral RNA were performed. As the labwork had an unknown outcome, students indicated that they were excited by the work and became invested in the experience. By completing two rounds of SELEX, the students repeated molecular methods (e.g., RNA extraction, RT-PCR, agarose gel electrophoresis, DNA purification, cloning, and sequence analysis) and reported that repetition reinforced their learning and helped them build confidence in their lab abilities. Students also appreciated that they did not learn a "technique-per-week" without context, but rather they understood why certain methods were used for certain molecular tasks. Results from a 19-question multiple-choice assessment indicated increased comprehension of theory underlying methods performed. Details regarding experimental methods and timeline, and assessment and attitudinal results from three student cohorts, are described herein.

Molecular characterization of poleroviruses isolated from oilseed rape in Greece.

In 2018 virus-like symptoms, typical of polerovirus infection were observed in several oilseed rape crops in northern Greece. In order to identify the etiological agent of these symptoms a polerovirus-generic RT-PCR assay was applied. Sequencing of the amplicons revealed the presence of virus isolates genetically close to turnip yellows virus (TuYV). Further molecular characterization of the near complete genome of '1-2', 'Geo1', 'Geo7' and 'Geo15' isolates revealed that they share > 96% nt identity with various TuYV sequences. On the other hand, the fifth, characterized isolate from oilseed rape, termed '1-1', showed higher sequence similarity to brassica yellows virus (BrYV) regarding the 5' part of the complete coding sequence, whereas the 3' part was closely related to TuYV isolates. A recombination analysis using RDP indicated the presence of a putative breakpoint (nucleotide position 2964) in '1-1' genome and it is proposed that the virus isolate '1-1' might be an interspecies recombinant between BrYV and TuYV. To our knowledge, this is the first time that the complete coding sequences of Greek TuYV isolates have been determined and the first detection of a BrYV/TuYV recombinant isolate infecting oilseed rape in Greece.

Plant virus evolution under strong drought conditions results in a transition from parasitism to mutualism.

Environmental conditions are an important factor driving pathogens' evolution. Here, we explore the effects of drought stress in plant virus evolution. We evolved turnip mosaic potyvirus in well-watered and drought conditions in Arabidopsis thaliana accessions that differ in their response to virus infection. Virus adaptation occurred in all accessions independently of watering status. Drought-evolved viruses conferred a significantly higher drought tolerance to infected plants. By contrast, nonsignificant increases in tolerance were observed in plants infected with viruses evolved under standard watering. The magnitude of this effect was dependent on the plant accessions. Differences in tolerance were correlated to alterations in the expression of host genes, some involved in regulation of the circadian clock, as well as in deep changes in the balance of phytohormones regulating defense and growth signaling pathways. Our results show that viruses can promote host survival in situations of abiotic stress, with the magnitude of such benefit being a selectable trait.

Comparison of two Turnip mosaic virus P1 proteins in their ability to co-localize with the Arabidopsis thaliana G3BP-2 protein.

Turnip mosaic virus (TuMV), belonging to the genus Potyvirus (family Potyviridae), has a large host range and consists of a single-stranded positive sense RNA genome encoding 12 proteins, including the P1 protease. This protein which is separated from the polyprotein by cis cleavage at its respective C-terminus, has been attributed with different functions during potyviral infection of plants. P1 of Turnip mosaic virus (P1-TuMV) harbors an FGSF-motif and FGSL-motif at its N-terminus. This motif is predicted to be a binding site for the host Ras GTPase-activating protein-binding protein (G3BP), which is a key factor for stress granule (SG) formation in mammalian systems and often targeted by viruses to inhibit SG formation. We therefore hypothesized that P1-TuMV might interact with G3BP to control and regulate plant SGs to optimize cellular conditions for the production of viral proteins. Here, we analyzed the co-localization of the Arabidopsis thaliana G3BP-2 with the P1 of two TuMV isolates, namely UK 1 and DEU 2. Surprisingly, P1-TuMV-DEU 2 co-localized with AtG3BP-2 under abiotic stress conditions, whereas P1-TuMV-UK 1 did not. AtG3BP-2::RFP showed strong SGs formation after stress, while P1-UK 1::eGFP maintained a chloroplastic signal under stress conditions, the signal of P1-DEU 2::eGFP co-localized with that of AtG3BP-2::RFP. This indicates a specific interaction between P1-DEU 2 and the AtG3BP family which is not solely based on the canonical interaction motifs.

Genetic diversity and recombination between turnip yellows virus strains in Australia.

Disease outbreaks caused by turnip yellows virus (TuYV), a member of the genus Polerovirus, family Luteoviridae, regularly occur in canola and pulse crops throughout Australia. To understand the genetic diversity of TuYV for resistance breeding and management, genome sequences of 28 TuYV isolates from different hosts and locations were determined using high-throughput sequencing (HTS). We aimed to identify the parts of the genome that were most variable and clarify the taxonomy of viruses related to TuYV. Poleroviruses contain seven open reading frames (ORFs): ORF 0-2, 3a, and 3-5. Phylogenetic analysis based on the genome sequences, including isolates of TuYV and brassica yellows virus (BrYV) from the GenBank database, showed that most genetic variation among isolates occurred in ORF 5, followed by ORF 0 and ORF 3a. Phylogenetic analysis of ORF 5 revealed three TuYV groups; P5 group 1 and group 3 shared 45-49% amino acid sequence identity, and group 2 is a recombinant between the other two. Phylogenomic analysis of the concatenated ORFs showed that TuYV is paraphyletic with respect to BrYV, and together these taxa form a well-supported monophyletic group. Our results support the hypothesis that TuYV and BrYV belong to the same species and that the phylogenetic topologies of ORF 0, 3a and 5 are incongruent and may not be informative for species demarcation. A number of beet western yellow virus (BWYV)- and TuYV-associated RNAs (aRNA) were also identified by HTS for the first time in Australia.

Frontiers in Dissecting and Managing Brassica Diseases: From Reference-Based RGA Candidate Identification to Building Pan-RGAomes.

The Brassica genus contains abundant economically important vegetable and oilseed crops, which are under threat of diseases caused by fungal, bacterial and viral pathogens. Resistance gene analogues (RGAs) are associated with quantitative and qualitative disease resistance and the identification of candidate RGAs associated with disease resistance is crucial for understanding the mechanism and management of diseases through breeding. The availability of Brassica genome assemblies has greatly facilitated reference-based quantitative trait loci (QTL) mapping for disease resistance. In addition, pangenomes, which characterise both core and variable genes, have been constructed for B. rapa, B. oleracea and B. napus. Genome-wide characterisation of RGAs using conserved domains and motifs in reference genomes and pangenomes reveals their clustered arrangements and presence of structural variations. Here, we comprehensively review RGA identification in important Brassica genome and pangenome assemblies. Comparison of the RGAs in QTL between resistant and susceptible individuals allows for efficient identification of candidate disease resistance genes. However, the reference-based QTL mapping and RGA candidate identification approach is restricted by the under-represented RGA diversity characterised in the limited number of Brassica assemblies. The species-wide repertoire of RGAs make up the pan-resistance gene analogue genome (pan-RGAome). Building a pan-RGAome, through either whole genome resequencing or resistance gene enrichment sequencing, would effectively capture RGA diversity, greatly expanding breeding resources that can be utilised for crop improvement.

Turnip mosaic virus in oilseed rape activates networks of sRNA-mediated interactions between viral and host genomes.

Virus-induced plant diseases in cultivated plants cause important damages in yield. Although the mechanisms of virus infection are intensely studied at the cell biology level, only little is known about the molecular dialog between the invading virus and the host genome. Here we describe a combinatorial genome-wide approach to identify networks of sRNAs-guided post-transcriptional regulation within local Turnip mosaic virus (TuMV) infection sites in Brassica napus leaves. We show that the induction of host-encoded, virus-activated small interfering RNAs (vasiRNAs) observed in virus-infected tissues is accompanied by site-specific cleavage events on both viral and host RNAs that recalls the activity of small RNA-induced silencing complexes (RISC). Cleavage events also involve virus-derived siRNA (vsiRNA)-directed cleavage of target host transcripts as well as cleavage of viral RNA by both host vasiRNAs and vsiRNAs. Furthermore, certain coding genes act as virus-activated regulatory hubs to produce vasiRNAs for the targeting of other host genes. The observations draw an advanced model of plant-virus interactions and provide insights into the complex regulatory networking at the plant-virus interface within cells undergoing early stages of infection.

Gene presence-absence variation associates with quantitative Verticillium longisporum disease resistance in Brassica napus.

Although copy number variation (CNV) and presence-absence variation (PAV) have been discovered in selected gene families in most crop species, the global prevalence of these polymorphisms in most complex genomes is still unclear and their influence on quantitatively inherited agronomic traits is still largely unknown. Here we analyze the association of gene PAV with resistance of oilseed rape (Brassica napus) against the important fungal pathogen Verticillium longisporum, as an example for a complex, quantitative disease resistance in the strongly rearranged genome of a recent allopolyploid crop species. Using Single Nucleotide absence Polymorphism (SNaP) markers to efficiently trace PAV in breeding populations, we significantly increased the resolution of loci influencing V. longisporum resistance in biparental and multi-parental mapping populations. Gene PAV, assayed by resequencing mapping parents, was observed in 23-51% of the genes within confidence intervals of quantitative trait loci (QTL) for V. longisporum resistance, and high-priority candidate genes identified within QTL were all affected by PAV. The results demonstrate the prominent role of gene PAV in determining agronomic traits, suggesting that this important class of polymorphism should be exploited more systematically in future plant breeding.

Mycovirus-Induced Hypervirulence of Leptosphaeria biglobosa Enhances Systemic Acquired Resistance to Leptosphaeria maculans in Brassica napus.

Phoma stem canker (blackleg) is one of the most important diseases of winter oilseed rape (Brassica napus) worldwide and is caused by a complex that comprises at least two species: Leptosphaeria maculans and L. biglobosa. Screening a panel of field Leptosphaeria isolates from B. napus for the presence of mycoviruses revealed the presence of a novel double-stranded RNA quadrivirus in L. biglobosa and no viruses in L. maculans. Following elimination of the mycovirus, virus-infected and virus-free isogenic lines of L. biglobosa were created. A direct comparison of the growth and virulence of these isogenic lines illustrated that virus infection caused hypervirulence and resulted in induced systemic resistance toward L. maculans in B. napus following lower leaf preinoculation with the virus-infected isolate. Analysis of the plant transcriptome suggests that the presence of the virus leads to subtle alterations in metabolism and plant defenses. For instance, transcripts involved in carbohydrate and amino acid metabolism are enriched in plants treated with the virus-infected isolate, while pathogenesis-related proteins, chitinases and WRKY transcription factors are differentially expressed. These results illustrate the potential for deliberate inoculation of plants with hypervirulent L. biglobosa to decrease the severity of Phoma stem canker later in the growing season.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Impact of Turnip yellows virus infection on seed yield of an open-pollinated and hybrid canola cultivar when inoculated at different growth stages.

Turnip yellows virus (TuYV; family Luteoviridae, genus Polerovirus) is the most economically damaging virus infecting canola (Brassica napus) in the south-west Australian grainbelt. However, the impact of TuYV infection at different growth stages on canola seed yield has not been examined. This information is vital for implementing targeted management strategies. Four glasshouse experiments were conducted to examine seed yield losses incurred by an open-pollinated (ATR Bonito) and hybrid (Hyola® 404RR) canola cultivar when aphid-inoculated with TuYV at GS12 (two leaves unfolded), GS17 (seven leaves unfolded), GS30 (beginning of stem elongation) and GS65 (full flowering). When inoculated at GS12 and GS17, cv. Bonito plants incurred 30 % and 36 % seed yield losses, respectively, compared to healthy plants. Similarly, cv. 404RR incurred 41 % and 26 % seed yield losses at GS12 and GS17, respectively. However, when inoculated at GS30, whilst cv. Bonito plants incurred a 26 % seed yield loss, cv. 404RR incurred no significant loss. Neither cultivar incurred seed yield losses from inoculation at GS65. Additional information was collected from these experiments to improve sampling protocols to enhance TuYV detection, with a molecular and serological technique. When canola plants were at pre-flowering growth stages, TuYV was reliably detected 7-14 days after inoculation (DAI) in the youngest leaf. Once flowering had begun, TuYV was consistently detected 7-14 DAI in petals and flower buds. In contrast, regardless of growth stage, testing the oldest leaf regularly resulted in delayed detection or false negatives. Information generated in this study helps to quantify the value of management strategies targeted at preventing TuYV spread in pre-flowering canola crops and ultimately increase the efficiency of resource use.

Identification and QTL mapping of resistance to Turnip yellows virus (TuYV) in oilseed rape, Brassica napus.

KEY MESSAGE: Partially dominant resistance to Turnip yellows virus associated with one major QTL was identified in the natural allotetraploid oilseed rape cultivar Yudal. Turnip yellows virus (TuYV) is transmitted by the peach-potato aphid (Myzus persicae) and causes severe yield losses in commercial oilseed rape crops (Brassica napus). There is currently only one genetic resource for resistance to TuYV available in brassica, which was identified in the re-synthesised B. napus line 'R54'. In our study, 27 mostly homozygous B. napus accessions, either doubled-haploid (DH) or inbred lines, representing a diverse subset of the B. napus genepool, were screened for TuYV resistance/susceptibility. Partial resistance to TuYV was identified in the Korean spring oilseed rape, B. napus variety Yudal, whilst the dwarf French winter oilseed rape line Darmor-bzh was susceptible. QTL mapping using the established Darmor-bzh × Yudal DH mapping population (DYDH) revealed one major QTL explaining 36% and 18% of the phenotypic variation in two independent experiments. A DYDH line was crossed to Yudal, and reciprocal backcross (BC1) populations from the F1 with either the susceptible or resistant parent revealed the dominant inheritance of the TuYV resistance. The QTL on ChrA04 was verified in the segregating BC1 population. A second minor QTL on ChrC05 was identified in one of the two DYDH experiments, and it was not observed in the BC1 population. The TuYV resistance QTL in 'R54' is within the QTL interval on Chr A04 of Yudal; however, the markers co-segregating with the 'R54' resistance are not conserved in Yudal, suggesting an independent origin of the TuYV resistances. This is the first report of the QTL mapping of TuYV resistance in natural B. napus.

A novel Waikavirus (the family Secoviridae) genome sequence identified in rapeseed (Brassica napus).

The genome sequence of a novel species of the genus Waikavirus (the family Secoviridae), which we named Brassica napus RNA virus 1 (BnRV1), was identified in a rapeseed (Brassica napus) transcriptome dataset. The BnRV1 genome was 12,293 nucleotides long followed by a poly(A) tail. Two open reading frames (ORFs), called ORF1 and ORFX, were predicted. The larger ORF, ORF1, encodes a polyprotein of 3,471 amino acids and the smaller ORF, ORFX, overlaps ORF1 and encodes an 87 aa long protein of unknown function. The BnRV1 ORF1 polyprotein was predicted to undergo proteolytic processing to yield seven mature proteins, including an RNA-dependent RNA polymerase and three distinct coat proteins. The ORF1 and ORFX proteins share sequence similarities with the respective proteins of viruses in the genus Waikavirus, including the bellflower vein chlorosis virus, rice tungro spherical virus, and maize chlorotic dwarf virus. A phylogenetic tree inferred from a conserved segment of the polyproteins of several Secoviridae viruses confirmed that BnRV1 is a novel species of the genus Waikavirus. The BnRV1 genome sequence identified in this study may be useful for the study of waikavirus biology and waikavirus-derived diseases. Keywords: Brassica napus RNA virus 1; Waikavirus; Secoviridae; rapeseed.

In-field capable loop-mediated isothermal amplification detection of Turnip yellows virus in plants and its principal aphid vector Myzus persicae.

Widespread Turnip yellows virus (TuYV) infection causes severe seed yield and quality losses in rapeseed (Brassica napus) crops grown in broadacre agricultural systems worldwide. Current TuYV detection protocols are expensive and time consuming, and can have poor specificity and sensitivity. Typically, they are used as a diagnostic tool to test already symptomatic plants, limiting their practical value to reactive disease management. To improve diagnostic services so that they provide earlier, cheaper, faster, more specific and sensitive TuYV detection, novel and innovative protocols that utilise new technology are required. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect TuYV in crude and total RNA extractions of leaf material and its principal aphid vector Myzus persicae. The assay was based on a set of six primers, highly sensitive and specific to TuYV, derived from a TuYV isolate originating from the south-west Australian grainbelt. TuYV was readily detected in 1 in 100 dilutions of (i) infected to uninfected leaf material, and (ii) viruliferous to non-viruliferous M. persicae. Furthermore, detection was successful in a majority of aphids stored for at least 8 weeks in various trapping and storage substances, including 30% ethylene glycol, sticky trap glue and 70% ethanol. This RT-LAMP assay protocol enables quicker and cheaper diagnosis for TuYV than currently adopted laboratory-based diagnostic techniques. Ultimately, it has the potential for earlier in-field TuYV detection in combination with aphid trapping surveillance programs.

Effects of infection by Turnip mosaic virus on the population growth of generalist and specialist aphid vectors on turnip plants.

Recent studies have revealed that relationships between plant pathogens and their vectors differ depending on species, strains and associated host plants. Turnip mosaic virus (TuMV) is one of the most important plant viruses worldwide and is transmitted by at least 89 aphid species in a non-persistent manner. TuMV is fundamentally divided into six phylogenetic groups; among which Asian-BR, basal-BR and world-B groups are known to occur in Japan. In Kyushu Japan, basal-BR has invaded approximately 2000 and immediately replaced the predominant world-B virus group. To clarify the relationships between TuMV and vector aphids, we examined the effects of the TuMV phylogenetic group on the population growth of aphid vectors in turnip plants. The population growth of a generalist aphid, Myzus persicae, was not significantly different between non-infected and TuMV-infected treatments. The population growth of a specialist aphid, Lipaphis erysimi, was higher in TuMV-infected plants than non-infected ones. Similar results were obtained in experiments using world-B and basal-BR groups of TuMV. Therefore, we conclude that L. erysimi is more mutualistic with TuMV than M. persicae, and differences in TuMV phylogenetic groups do not affect the growth of aphid vectors on turnip plants.

Formation of large viroplasms and virulence of Cauliflower mosaic virus in turnip plants depend on the N-terminal EKI sequence of viral protein TAV.

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11-13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.

The Timescale of Emergence and Spread of Turnip Mosaic Potyvirus.

Plant viruses have important global impacts on crops, and identifying their centre and date of emergence is important for planning control measures. Turnip mosaic virus (TuMV) is a member of the genus Potyvirus in the family Potyviridae and is a major worldwide pathogen of brassica crops. For two decades, we have collected TuMV isolates, mostly from brassicas, in Turkey and neighbouring countries. This region is thought to be the centre of emergence of this virus. We determined the genomic sequences of 179 of these isolates and used these to estimate the timescale of the spread of this virus. Our Bayesian coalescent analyses used synonymous sites from a total of 417 novel and published whole-genome sequences. We conclude that TuMV probably originated from a virus of wild orchids in Germany and, while adapting to wild and domestic brassicas, spread via Southern Europe to Asia Minor no more than 700 years ago. The population of basal-B group TuMVs in Asia Minor is older than all other populations of this virus, including a newly discovered population in Iran. The timescale of the spread of TuMV correlates well with the establishment of agriculture in these countries.