TERMINAL FLOWER-1/CENTRORADIALIS inhibits tuberisation via protein interaction with the tuberigen activation complex.

Potato tuber formation is a secondary developmental programme by which cells in the subapical stolon region divide and radially expand to further differentiate into starch-accumulating parenchyma. Although some details of the molecular pathway that signals tuberisation are known, important gaps in our knowledge persist. Here, the role of a member of the TERMINAL FLOWER 1/CENTRORADIALIS gene family (termed StCEN) in the negative control of tuberisation is demonstrated for what is thought to be the first time. It is shown that reduced expression of StCEN accelerates tuber formation whereas transgenic lines overexpressing this gene display delayed tuberisation and reduced tuber yield. Protein-protein interaction studies (yeast two-hybrid and bimolecular fluorescence complementation) demonstrate that StCEN binds components of the recently described tuberigen activation complex. Using transient transactivation assays, we show that the StSP6A tuberisation signal is an activation target of the tuberigen activation complex, and that co-expression of StCEN blocks activation of the StSP6A gene by StFD-Like-1. Transcriptomic analysis of transgenic lines misexpressing StCEN identifies early transcriptional events in tuber formation. These results demonstrate that StCEN suppresses tuberisation by directly antagonising the function of StSP6A in stolons, identifying StCEN as a breeding marker to improve tuber initiation and yield through the selection of genotypes with reduced StCEN expression.

Natural resistance to Potato virus Y in Solanum tuberosum Group Phureja.

KEY MESSAGE: Novel major gene resistance against Potato virus Y in diploid populations of Solanum tuberosum Groups Phureja and Tuberosum was biologically and genetically characterised. Named Ry(o)phu, it mapped to chromosome 9. A new source of genetic resistance derived from Solanum tuberosum Group Phureja against Potato virus Y (PVY) was identified and genetically characterised in three diploid biparental potato populations. Segregation data for two populations (05H1 and 08H1) suggested the presence of a single dominant gene for resistance to PVY which, following DaRT analysis of the 08H1 cross, was mapped to chromosome 9. More detailed genetic analysis of resistance utilised a well-characterised SNP-linkage map for the 06H1 population, together with newly generated marker data. In these plants, which have both S. tuberosum Group Phureja and S. tuberosum Group Tuberosum in their pedigree, the resistance was shown to map to chromosome 9 at a locus not previously associated with PVY resistance, although there is evidence for at least one other genetic factor controlling PVY infection. The resistance factor location on chromosome 9 (named as Ry(o)phu) suggests a potential role of NB-LRR genes in this resistance. Phenotypic analysis using a GUS-tagged virus revealed that a small amount of PVY replication occurred in occasional groups of epidermal cells in inoculated leaves of resistant plants, without inducing any visible hypersensitive response. However, the virus did not enter the vascular system and systemic spread was completely prevented.

Kodoja: A workflow for virus detection in plants using k-mer analysis of RNA-sequencing data.

RNA-sequencing of plant material allows for hypothesis-free detection of multiple viruses simultaneously. This methodology relies on bioinformatics workflows for virus identification. Most workflows are designed for human clinical data, and few go beyond sequence mapping for virus identification. We present a new workflow (Kodoja) for the detection of plant virus sequences in RNA-sequence data. Kodoja uses k-mer profiling at the nucleotide level and sequence mapping at the protein level by integrating two existing tools Kraken and Kaiju. Kodoja was tested on three existing RNA-seq datasets from grapevine, and two new RNA-seq datasets from raspberry. For grapevine, Kodoja was shown to be more sensitive than a method based on contig building and blast alignments (27 viruses detected compared to 19). The application of Kodoja to raspberry, showed that field-grown raspberries were infected by multiple viruses, and that RNA-seq can identify lower amounts of virus material than reverse transcriptase PCR. This work enabled the design of new PCR-primers for detection of Raspberry yellow net virus and Beet ringspot virus. Kodoja is a sensitive method for plant virus discovery in field samples and enables the design of more accurate primers for detection. Kodoja is available to install through Bioconda and as a tool within Galaxy.

Potato Mop-Top Virus Co-Opts the Stress Sensor HIPP26 for Long-Distance Movement.

Virus movement proteins facilitate virus entry into the vascular system to initiate systemic infection. The potato mop-top virus (PMTV) movement protein, TGB1, is involved in long-distance movement of both viral ribonucleoprotein complexes and virions. Here, our analysis of TGB1 interactions with host Nicotiana benthamiana proteins revealed an interaction with a member of the heavy metal-associated isoprenylated plant protein family, HIPP26, which acts as a plasma membrane-to-nucleus signal during abiotic stress. We found that knockdown of NbHIPP26 expression inhibited virus long-distance movement but did not affect cell-to-cell movement. Drought and PMTV infection up-regulated NbHIPP26 gene expression, and PMTV infection protected plants from drought. In addition, NbHIPP26 promoter-reporter fusions revealed vascular tissue-specific expression. Mutational and biochemical analyses indicated that NbHIPP26 subcellular localization at the plasma membrane and plasmodesmata was mediated by lipidation (S-acylation and prenylation), as nonlipidated NbHIPP26 was predominantly in the nucleus. Notably, coexpression of NbHIPP26 with TGB1 resulted in a similar nuclear accumulation of NbHIPP26. TGB1 interacted with the carboxyl-terminal CVVM (prenyl) domain of NbHIPP26, and bimolecular fluorescence complementation revealed that the TGB1-HIPP26 complex localized to microtubules and accumulated in the nucleolus, with little signal at the plasma membrane or plasmodesmata. These data support a mechanism where interaction with TGB1 negates or reverses NbHIPP26 lipidation, thus releasing membrane-associated NbHIPP26 and redirecting it via microtubules to the nucleus, thereby activating the drought stress response and facilitating virus long-distance movement.

Engineering heat tolerance in potato by temperature-dependent expression of a specific allele of HEAT-SHOCK COGNATE 70.

For many commercial potato cultivars, tuber yield is optimal at average daytime temperatures in the range of 14-22 °C. Further rises in ambient temperature can reduce or completely inhibit potato tuber production, with damaging consequences for both producer and consumer. The aim of this study was to use a genetic screen based on a model tuberization assay to identify quantitative trait loci (QTL) associated with enhanced tuber yield. A candidate gene encoding HSc70 was identified within one of the three QTL intervals associated with elevated yield in a Phureja-Tuberosum hybrid diploid potato population (06H1). A particular HSc70 allelic variant was linked to elevated yield in the 06H1 progeny. Expression of this allelic variant was much higher than other alleles, particularly on exposure to moderately elevated temperature. Transient expression of this allele in Nicotiana benthamiana resulted in significantly enhanced tolerance to elevated temperature. An TA repeat element was present in the promoter of this allele, but not in other HSc70 alleles identified in the population. Expression of the HSc70 allelic variant under its native promoter in the potato cultivar Desiree resulted in enhanced HSc70 expression at elevated temperature. This was reflected in greater tolerance to heat stress as determined by improved yield under moderately elevated temperature in a model nodal cutting tuberization system and in plants grown from stem cuttings. Our results identify HSc70 expression level as a significant factor influencing yield stability under moderately elevated temperature and identify specific allelic variants of HSc70 for the induction of thermotolerance via conventional introgression or molecular breeding approaches.

Foundational and Translational Research Opportunities to Improve Plant Health.

Reader Comments | Submit a Comment The white paper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an over-reliance on nonsustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana.

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.

Distinct circular single-stranded DNA viruses exist in different soil types.

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity.

Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisin-like protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates.

Deciphering the mechanism of defective interfering RNA (DI RNA) biogenesis reveals that a viral protein and the DI RNA act antagonistically in virus infection.

Potato mop-top virus (PMTV) produces a defective RNA (D RNA) encompassing the 5'-terminal 479 nucleotides (nt) and 3'-terminal 372 nt of RNA-TGB (where TGB is triple gene block). The mechanism that controls D RNA biogenesis and the role of D RNA in virus accumulation was investigated by introducing deletions, insertions, and point mutations into the sequences of the open reading frames (ORFs) of TGB1 and the 8-kilodalton (8K) protein that were identified as required for efficient production of the D RNA. Transient expression of RNA-TGB in the absence of RNA-Rep (which encodes the replicase) did not result in accumulation of D RNA, indicating that its production is dependent on PMTV replication. The D RNA could be eliminated by disrupting a predicted minus-strand stem-loop structure comprising complementary sequences of the 5' TGB1 ORF and the 3' 8K ORF, suggesting intramolecular template switching during positive-strand synthesis as a mechanism for the D RNA biogenesis. Virus accumulation was reduced when the 8K ORF was disrupted but D RNA was produced. Conversely, the virus accumulated at higher titers when the 8K ORF was intact and D RNA production was blocked. These data demonstrate that the D RNA interferes with virus infection and therefore should be referred to as a defective interfering RNA (DI RNA). The 8K protein was shown to be a weak silencing suppressor. This study provides an example of the interplay between a pathogen and its molecular parasite where virus accumulation was differentially regulated by the 8K protein and DI RNA, indicating that they play antagonistic roles and suggesting a mechanism by which the virus can attenuate replication, decreasing viral load and thereby enhancing its efficiency as a parasite.

A new simple and effective method for PLRV infection to screen for virus resistance in potato.

Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.

Allelic variants of a potato HEAT SHOCK COGNATE 70 gene confer improved tuber yield under a wide range of environmental conditions.

Previously, we developed and applied a glasshouse screen for potato tuber yield under heat stress and identified a candidate gene (HSc70) for heat tolerance by genetic analysis of a diploid potato population. Specific allelic variants were expressed at high levels on exposure to moderately elevated temperature due to variations in gene promoter sequence. In this study, we aimed to confirm the results from the glasshouse screen in field trials conducted over several seasons and locations including those in Kenya, Malawi and the UK. We extend our understanding of the HSc70 gene and demonstrate that expression level of HSc70 correlates with tolerance to heat stress in a wide range of wild potato relatives. The physiological basis of the protective effect of HSc70 was explored and we show that genotypes carrying the highly expressed HSc70 A2 allele are protected against photooxidative damage to PSII induced by abiotic stresses. Overall, we show the potential of HSc70 alleles for breeding resilient potato genotypes for multiple environments.

Climate Change and Defense against Pathogens in Plants.

Most reviews of climate change are epidemiological, focusing on impact assessment and risk mapping. However, there are many reports of the effects of environmental stress factors on defense mechanisms in plants against pathogens. We review those representative of key climate change-related stresses to determine whether there are any patterns or trends in adaptation responses. We recognize the complexity of climate change itself and the multitrophic nature of the complex biological interactions of plants, microbes, soil, and the environment and, therefore, the difficulty of reductionist dissection approaches to resolving the problems. We review host defense genes, germplasm, and environmental interactions in different types of organisms but find no significant group-specific trends. Similarly, we review by host defense mechanism type and by host-pathogen trophic relationship but identify no dominating mechanism for stress response. However, we do identify core stress response mechanisms playing key roles in multiple response pathways whether to biotic or abiotic stress. We suggest that these should be central to mechanistic climate change plant defense research. We also recognize biodiversity, heterogeneity, and the need for understanding stress in a true systems biology approach as being essential components of progressing our understanding of and response to climate change.

In vivo expression and binding activity of scFv-RWAV, which recognizes the coat protein of tomato leaf curl New Delhi virus (family Geminiviridae).

Recombinant antibodies expressed in plants have the potential to interrupt virus infections by blocking essential stages of the infection cycle. Here, we show that the expression of a recombinant single-chain variable fragment (scFv) that recognizes the coat protein of tomato leaf curl New Delhi virus (ToLCNDV) in vitro can also bind to a recombinant coat protein in vivo in the reducing environment of the plant cytosol. The scFv and its target were both expressed as fluorescent protein fusions, one incorporating green fluorescent protein (GFP) and the other DsRed. We found that the incorporation of a nuclear localization signal into the scFv construct resulted in the nuclear import of the antibody-antigen complex, as shown by colocalization of the two fluorescent signals. This demonstrates that recombinant antibodies can be targeted to the nucleus and will bind to geminivirus coat proteins therein, allowing the virus infection cycle to be interrupted during its critical replicative phase.

Phloem connectivity and transport are not involved in mature plant resistance (MPR) to Potato Virus Y in different potato cultivars, and MPR does not protect tubers from recombinant strains of the virus.

The aims of this study were: i) to investigate mature plant resistance (MPR) against four strains of Potato virus Y (PVYO, PVYN, PVYNTN and PVYN-Wi) in potato cultivars that differ in maturity (e.g. early or maincrop) at different developmental stages, and ii) to determine whether phloem translocation of photoassimilates at different stages including the source-sink transition influences MPR. The data showed that MPR was functional by the flowering stage in all cultivars, and that the host-pathogen interaction is highly complex, with all three variables (potato cultivar, virus strain and developmental stage of infection) having a significant effect on the outcome. However, virus strain was the most important factor, and MPR was less effective in protecting tubers from recombinant virus strains (PVYNTN and PVYN-Wi). Development of MPR was unrelated to foliar phloem connectivity, which was observed at all developmental stages, but a switch from symplastic to apoplastic phloem unloading early in tuber development may be involved in the prevention of tuber infections with PVYO. Recombinant virus strains were more infectious than parental strains and PVYNTN has a more effective silencing suppressor than PVYO, another factor that may contribute to the efficiency of MPR. The resistance conferred by MPR against PVYO or PVYN may be associated with or enhanced by the presence of the corresponding strain-specific HR resistance gene in the cultivar.

Varied movement strategies employed by triple gene block-encoding viruses.

Several RNA virus genera belonging to the Virgaviridae and Flexiviridae families encode proteins organized in a triple gene block (TGB) that facilitate cell-to-cell and long-distance movement. The TGB proteins have been traditionally classified as hordei-like or potex-like based on phylogenetic comparisons and differences in movement mechanisms of the Hordeivirus and Potexvirus spp. However, accumulating data from other model viruses suggests that a revised framework is needed to accommodate the profound differences in protein interactions occurring during infection and ancillary capsid protein requirements for movement. The goal of this article is to highlight common features of the TGB proteins and salient differences in movement properties exhibited by individual viruses encoding these proteins. We discuss common and divergent aspects of the TGB transport machinery, describe putative nucleoprotein movement complexes, highlight recent data on TGB protein interactions and topological properties, and review membrane associations occurring during subcellular targeting and cell-to-cell movement. We conclude that the existing models cannot be used to explain all TGB viruses, and we propose provisional Potexvirus, Hordeivirus, and Pomovirus models. We also suggest areas that might profit from future research on viruses harboring this intriguing arrangement of movement proteins.

The N-terminal domain of PMTV TGB1 movement protein is required for nucleolar localization, microtubule association, and long-distance movement.

The triple-gene-block (TGB)1 protein of Potato mop-top virus (PMTV) was fused to fluorescent proteins and expressed in epidermal cells of Nicotiana benthamiana under the control of the 35S promoter. TGB1 fluorescence was observed in the cytoplasm, nucleus, and nucleolus and occasionally associated with microtubules. When expressed from a modified virus (PMTV.YFP-TGB1) which formed local lesions but was not competent for systemic movement, yellow fluorescent protein (YFP)-TGB1 labeled plasmodesmata in cells at the leading edge of the lesion and plasmodesmata, microtubules, nuclei, and nucleoli in cells immediately behind the leading edge. Deletion of 84 amino acids from the N-terminus of unlabeled TGB1 within the PMTV genome abolished movement of viral RNA to noninoculated leaves. When the same deletion was introduced into PMTV.YFP-TGB1, labeling of microtubules and nucleoli was abolished. The N-terminal 84 amino acids of TGB1 were fused to green fluorescent protein (GFP) and expressed in epidermal cells where GFP localized strongly to the nucleolus (not seen with unfused GFP), indicating that these amino acids contain a nucleolar localization signal; the fusion protein did not label microtubules. This is the first report of nucleolar and microtubule association of a TGB movement protein. The results suggest that PMTV TGB1 requires interaction with nuclear components and, possibly, microtubules for long-distance movement of viral RNA.

Generation and characterization of a scFv against recombinant coat protein of the geminivirus tomato leaf curl New Delhi virus.

We report the establishment of a hybridoma cell line secreting the monoclonal antibody (mAb) HAV, which recognizes the coat (AV1) protein of tomato leaf curl New Delhi virus (ToLCNDV), a begomovirus. The cell line was obtained following immunization of mice with purified recombinant AV1 fused to glutathione S-transferase (GST). A single-chain variable fragment (scFv-SAV) was assembled from hybridoma cDNA, but sequence analysis revealed a single nucleotide deletion causing a frame shift that resulted in a 21-residue N-terminal truncation. The missing nucleotide was restored by in vitro site-directed mutagenesis to create scFv-RWAV. The binding properties of mAb HAV and the corresponding scFvs were characterized by western blot, ELISA and surface plasmon resonance spectroscopy. MAb HAV bound to AV1 with nanomolar affinity but reacted neither with the N-terminal region of the protein nor with the GST fusion partner. This suggested that the antibody recognized a linear epitope in a region of the coat protein that is conserved among begomoviruses. Both scFvs retained the antigen specificity of mAb HAV, although the dissociation rate constant of scFv-RWAV was tenfold greater than that of scFv-SAV, showing the importance of restoring the 21 N-terminal amino acids.

Potato Virus Y Emergence and Evolution from the Andes of South America to Become a Major Destructive Pathogen of Potato and Other Solanaceous Crops Worldwide.

The potato was introduced to Europe from the Andes of South America in the 16th century, and today it is grown worldwide; it is a nutritious staple food eaten by millions and underpins food security in many countries. Unknowingly, potato virus Y (PVY) was also introduced through trade in infected potato tubers, and it has become the most important viral pathogen of potato. Phylogenetic analysis has revealed the spread and emergence of strains of PVY, including strains causing economically important diseases in tobacco, tomato and pepper, and that the virus continues to evolve with the relatively recent emergence of new damaging recombinant strains. High-throughput, next-generation sequencing platforms provide powerful tools for detection, identification and surveillance of new PVY strains. Aphid vectors of PVY are expected to increase in incidence and abundance in a warmer climate, which will increase the risk of virus spread. Wider deployment of crop cultivars carrying virus resistance will be an important means of defence against infection. New cutting-edge biotechnological tools such as CRISPR and SIGS offer a means for rapid engineering of resistance in established cultivars. We conclude that in future, human activities and ingenuity should be brought to bear to control PVY and the emergence of new strains in key crops by increased focus on host resistance and factors driving virus evolution and spread.

RNA sequence analysis of diseased groundnut (Arachis hypogaea) reveals the full genome of groundnut rosette assistor virus (GRAV).

The complete genome sequences for two variant isolates of groundnut rosette assistor virus (GRAV) have been determined from symptomatic groundnut plants in western Kenya. The sequences of the two GRAV isolates (sc7.1 and sc7.2) are 84.2% identical at the nucleotide level and 98.5% identical at the coat protein level. The variants sc7.1 and sc7.2 comprise 5850 and 5879 nucleotides respectively, and show similar genome organizations with 7 predicted ORFs (P0, P1, P2, P3a, P3 (coat protein, CP), P4 (movement protein, MP) and P5 (coat protein-readthrough protein, CP-RT). Currently, GRAV is an unassigned virus in the Luteoviridae family, due to the fact that only the sequence of the coat protein was previously obtained. The presence of both ORF0 and ORF 4 within the genome sequence determined in the current work suggest that GRAV should be classified as a member of the genus Polerovirus.