Characterization of the proteins encoded by a recently emerged cotton-infecting Polerovirus.

The cotton leafroll dwarf virus (CLDV), an important viral pathogen responsible for substantial losses in cotton crops, has recently emerged in the United States (US). Although CLDV shares similarities with other members of the genus Polerovirus in terms of encoded proteins, their functional characteristics remain largely unexplored. In this study, we expressed and analyzed each protein encoded by CLDV to determine its intracellular localization using fluorescence protein fusion. We also evaluated their potential to induce plant responses, such as the induction of hypersensitive response-like necrosis and the generation of reactive oxygen species. Our findings show that the proteins encoded by CLDV exhibit comparable localization patterns and elicit similar robust plant responses as observed with cognate proteins from other viruses within the genus Polerovirus. This study contributes to our understanding of the functional repertoire of genes carried by Polerovirus members, particularly to CLDV that has recently emerged as a widespread viral pathogen infecting cotton in the US.

The Carboxyl Terminal Regions of P0 Protein Are Required for Systemic Infections of Poleroviruses.

P0 proteins encoded by poleroviruses Brassica yellows virus (BrYV) and Potato leafroll virus (PLRV) are viral suppressors of RNA silencing (VSR) involved in abolishing host RNA silencing to assist viral infection. However, other roles that P0 proteins play in virus infection remain unclear. Here, we found that C-terminal truncation of P0 resulted in compromised systemic infection of BrYV and PLRV. C-terminal truncation affected systemic but not local VSR activities of P0 proteins, but neither transient nor ectopic stably expressed VSR proteins could rescue the systemic infection of BrYV and PLRV mutants. Moreover, BrYV mutant failed to establish systemic infection in DCL2/4 RNAi or RDR6 RNAi plants, indicating that systemic infection might be independent of the VSR activity of P0. Partially rescued infection of BrYV mutant by the co-infected PLRV implied the functional conservation of P0 proteins within genus. However, although C-terminal truncation mutant of BrYV P0 showed weaker interaction with its movement protein (MP) when compared to wild-type P0, wild-type and mutant PLRV P0 showed similar interaction with its MP. In sum, our findings revealed the role of P0 in virus systemic infection and the requirement of P0 carboxyl terminal region for the infection.

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.

Molecular characterization of a new recombinant brassica yellows virus infecting tobacco in China.

Brassica yellows virus (BrYV), prevalently distributed throughout mainland China and South Korea while triggering serious diseases in cruciferous crops, is proposed to be a new species in the genus Polerovirus within the family Luteoviridae. There are three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) reported in cabbage and radish. Here, we describe a new BrYV isolate infecting tobacco plants in the field, which was named BrYV-NtabQJ. The complete genome sequence of BrYV-NtabQJ is 5741 nt in length, and 89% of the sequence shares higher sequence identities (about 90%) with different BrYV isolates. However, it possesses a quite divergent region within ORF5, which is more close to Beet western yellows virus (BWYV), Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV). A significant recombination event was then detected among BrYV-NtabQJ, BrYV-B Beijng isolate (BrYV-BBJ) and BWYV Leonurus sibiricus isolate (BWYV-LS). It is proposed that BrYV-NtabQJ might be an interspecific recombinant between BrYV-BBJ and BWYV-LS, and the recombination might result in the successful aphid transmission of BrYV from cruciferous crops to tobacco. And it also poses new challenges for BrYV diagnosis and the vegetable production.

Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya.

BACKGROUND: Maize lethal necrosis is caused by a synergistic co-infection of Maize chlorotic mottle virus (MCMV) and a specific member of the Potyviridae, such as Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Johnson grass mosaic virus (JGMV). Typical maize lethal necrosis symptoms include severe yellowing and leaf drying from the edges. In Kenya, we detected plants showing typical and atypical symptoms. Both groups of plants often tested negative for SCMV by ELISA. METHODS: We used next-generation sequencing to identify viruses associated to maize lethal necrosis in Kenya through a metagenomics analysis. Symptomatic and asymptomatic leaf samples were collected from maize and sorghum representing sixteen counties. RESULTS: Complete and partial genomes were assembled for MCMV, SCMV, Maize streak virus (MSV) and Maize yellow dwarf virus-RMV (MYDV-RMV). These four viruses (MCMV, SCMV, MSV and MYDV-RMV) were found together in 30 of 68 samples. A geographic analysis showed that these viruses are widely distributed in Kenya. Phylogenetic analyses of nucleotide sequences showed that MCMV, MYDV-RMV and MSV are similar to isolates from East Africa and other parts of the world. Single nucleotide polymorphism, nucleotide and polyprotein sequence alignments identified three genetically distinct groups of SCMV in Kenya. Variation mapped to sequences at the border of NIb and the coat protein. Partial genome sequences were obtained for other four potyviruses and one polerovirus. CONCLUSION: Our results uncover the complexity of the maize lethal necrosis epidemic in Kenya. MCMV, SCMV, MSV and MYDV-RMV are widely distributed and infect both maize and sorghum. SCMV population in Kenya is diverse and consists of numerous strains that are genetically different to isolates from other parts of the world. Several potyviruses, and possibly poleroviruses, are also involved.

Biodiversity and full genome sequence of potato viruses Alfalfa mosaic virus and potato leaf roll virus in Egypt.

Solanum tuberosum (potato) is the second most important vegetable crop in Egypt. It is locally consumed, manufactured or supplied for export to Europe and other Arab countries. Potato is subject to infection by a number of plant viruses, which affect its yield and quality. Potato virus Y (PVY), potato leaf roll virus (PLRV), and Alfalfa mosaic virus (AMV) were detected in major potato-growing areas surveyed. Multiplex-RT-PCR assay was used for the detection of these three viruses in one reaction using three specific primer pairs designed to amplify genomic parts of each virus (1594 bp for PLRV, 795 bp for AMV, 801 bp for PVY). All three viruses were detected in a single reaction mixture in naturally infected field-grown potatoes. Multiplex RT-PCR improved sensitivity necessary for the early detection of infection. Incidence of single, double, or triple infection has been recorded in some locations. Full-length sequencing has been performed for an Egyptian FER isolate of PLRV. Through phylogenetic analysis, it was shown to occupy the same clade with isolate JokerMV10 from Germany. Complete nucleotide sequence of an Egyptian FER isolate of AMV and phylogenetic analysis was also performed; we propose that it is a new distinct strain of AMV belonging to a new subgroup IIC. This is the first complete nucleotide sequence of an Egyptian isolate of AMV. Genetic biodiversity of devastating potato viruses necessitates continuous monitoring of new genetic variants of such viruses.

Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi.

An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB.

Closely related poleroviruses depend on distinct translation initiation factors to infect Arabidopsis thaliana.

In addition to being essential for translation of eukaryotic mRNA, translation initiation factors are also key components of plant-virus interactions. In order to address the involvement of these factors in the infectious cycle of poleroviruses (aphid-transmitted, phloem-limited viruses), the accumulation of three poleroviruses was followed in Arabidopsis thaliana mutant lines impaired in the synthesis of translation initiation factors in the eIF4E and eIF4G families. We found that efficient accumulation of Turnip yellows virus (TuYV) in A. thaliana relies on the presence of eIF (iso)4G1, whereas Beet mild yellowing virus (BMYV) and Beet western yellows virus-USA (BWYV-USA) rely, instead, on eIF4E1. A role for these factors in the infectious processes of TuYV and BMYV was confirmed by direct interaction in yeast between these specific factors and the 5' viral genome-linked protein of the related virus. Although the underlying molecular mechanism is still unknown, this study reveals a totally unforeseen situation in which closely related viruses belonging to the same genus use different translation initiation factors for efficient infection of A. thaliana.

Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions.

Protein interactions are critical determinants of insect transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus.

Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection.

Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.

Genetic analysis of Iranian population of Potato leafroll virus based on ORF0.

Potato leafroll virus (PLRV) is a destructive virus of potatoes and responsible for high yield losses wherever potatoes are grown. In this study, DNA fragments containing ORF0 from each of nine PLRV isolates was sequenced. Sequence analysis data using 36 isolates from 12 different countries including 14 Iranian isolates showed that the identities of ORF0 at both nucleotide and amino acid levels between the Iranian isolates were 96-100 % and these isolates were more similar to the European PLRV isolates than to the other isolates. Furthermore, phylogenetic and population genetic analysis were carried out on the basis of full-length ORF0 and overlapping and non-overlapping regions of ORF0 and ORF1 (ORF0/1) which revealed that PLRV isolates were not geographically resolved. Also, we identified negative selection with different ratios for each of the mentioned genomic regions suggesting effects of F-box motif and -1 frameshift on ORF0 non-overlapping region and ORF0/1 in the selection pressure, respectively. Five recombination events were detected in the Iranian, Australian, and European isolates suggesting an important role for this phenomenon in influencing genetic diversity within this virus population.

Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus.

BACKGROUND: In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae. RESULTS: Deep sequencing of small RNAs (sRNAs) from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt) long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton Dcl transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated. CONCLUSIONS: This is the first report on the profile of sRNAs in a plant infected with a virus from the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases.

Molecular characterization of two genotypes of a new polerovirus infecting brassicas in China.

The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.

Comparative genomics reveals insights into genetic variability and molecular evolution among sugarcane yellow leaf virus populations.

Yellow leaf disease caused by sugarcane yellow leaf virus (SCYLV) is one of the most prevalent diseases worldwide. In this study, six near-complete genome sequences of SCYLV were determined to be 5775-5881 bp in length. Phylogenetic analysis revealed that the two SCYLV isolates from Réunion Island, France, and four from China were clustered into REU and CUB genotypes, respectively, based on 50 genomic sequences (this study = 6, GenBank = 44). Meanwhile, all 50 isolates were clustered into three phylogroups (G1-G3). Twelve significant recombinant events occurred in intra- and inter-phylogroups between geographical origins and host crops. Most recombinant hotspots were distributed in coat protein read-through protein (RTD), followed by ORF0 (P0) and ORF1 (P1). High genetic divergences of 12.4% for genomic sequences and 6.0-24.9% for individual genes were determined at nucleotide levels. The highest nucleotide diversity (π) was found in P0, followed by P1 and RdRP. In addition, purifying selection was a main factor restricting variability in SCYLV populations. Infrequent gene flow between Africa and the two subpopulations (Asia and America) were found, whereas frequent gene flow between Asia and America subpopulations was observed. Taken together, our findings facilitate understanding of genetic diversity and evolutionary dynamics of SCYLV.

A genomic analysis of transcytosis in the pea aphid, Acyrthosiphon pisum, a mechanism involved in virus transmission.

Aphids are the primary vectors of plant viruses. Transmission can occur via attachment to the cuticle lining of the insect (non-circulative transmission) or after internalization in the insect cells with or without replication (circulative transmission). In this paper, we have focused on the circulative and non-propagative mode during which virions enter the cell following receptor-mediated endocytosis, are transported across the cell in vesicles and released by exocytosis without replicating. The correct uptake, transport and delivery of the vesicles cargo relies on the participation of proteins from different families which have been identified in the Acyrthosiphon pisum genome. Assemblage of this annotated dataset provides a useful basis to improve our understanding of the molecules and mechanisms involved in virus transmission by A. pisum and other aphid species.

Identification and characterization of Miscanthus yellow fleck virus, a new polerovirus infecting Miscanthus sinensis.

Miscanthus sinensis is a grass used for sugarcane breeding and bioenergy production. Using high throughput sequencing technologies, we identified a new viral genome in infected M. sinensis leaf tissue displaying yellow fleck symptoms. This virus is most related to members of the genus Polerovirus in the family Luteoviridae. The canonical ORFs were computationally identified, the P3 coat protein was expressed, and virus-like particles were purified and found to conform to icosahedral shapes, characteristic of the family Luteoviridae. We propose the name Miscanthus yellow fleck virus for this new virus.

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.

Isolation of biotic stress resistance genes from cotton (Gossypium arboreum) and their analysis in model plant tobacco (Nicotiana tabacum) for resistance against cotton leaf curl disease complex.

Cotton production is widely effected by Cotton Leaf Curl Virus (CLCuV) in world posing serious losses to cotton yield.The CRT genes from CLCuV resistant G. arboreum and CLCuV susceptible G. hirsutum were cloned and sequenced to know the differences of protein composition in both species. Molecular techniques were used to isolate full length putative biotic stress resistance genes from G. arboreum besides the analysis of identified novel genes in model plant tobacco (Nicotiana tabacum) for resistance to cotton leaf curl disease complex. It was found that transgenic plants over expressing Hydroperoxidelyase (HPL) genes exhibited higher enzyme activity than wild type. In addition the genome sequence information was used for the purpose of gene isolation. Even for the enhanced expression of Calreticulin (CRT), AOS and HPL in G. hirsutum, it still showed susceptibility against CLCuV suggesting alternative genes and pathways involved for the expression of resistance.

Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

Viruses cause many severe plant diseases, resulting in immense losses of crop yield worldwide. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population. Recently, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein (MP). We used the RNAi approach to suppress MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with MP siRNA constructs exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of MP gene expression is an efficient method for generating PLRV-resistant potato plants. Further, we identified novel ATPase motifs in MP that may be involved in DNA binding and translocation through plasmodesmata. We also showed that the ATPase activity of MP was stimulated in the presence of DNA/RNA. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.

Molecular, serological and biological variation among chickpea chlorotic stunt virus isolates from five countries of North Africa and West Asia.

Chickpea chlorotic stunt virus (CpCSV), a proposed new member of the genus Polerovirus (family Luteoviridae), has been reported only from Ethiopia. In attempts to determine the geographical distribution and variability of CpCSV, a pair of degenerate primers derived from conserved domains of the luteovirus coat protein (CP) gene was used for RT-PCR analysis of various legume samples originating from five countries and containing unidentified luteoviruses. Sequencing of the amplicons provided evidence for the occurrence of CpCSV also in Egypt, Morocco, Sudan, and Syria. Phylogenetic analysis of the CP nucleotide sequences of 18 samples from the five countries revealed the existence of two geographic groups of CpCSV isolates differing in CP sequences by 8-10%. Group I included isolates from Ethiopia and Sudan, while group II comprised those from Egypt, Morocco and Syria. For distinguishing these two groups, a simple RFLP test using HindIII and/or PvuII for cleavage of CP-gene-derived PCR products was developed. In ELISA and immunoelectron microscopy, however, isolates from these two groups could not be distinguished with rabbit antisera raised against a group-I isolate from Ethiopia (CpCSV-Eth) and a group-II isolate from Syria (CpCSV-Sy). Since none of the ten monoclonal antibodies (MAbs) that had been produced earlier against CpCSV-Eth reacted with group-II isolates, further MAbs were produced. Of the seven MAbs raised against CpCSV-Sy, two reacted only with CpCSV-Sy and two others with both CpCSV-Sy and -Eth. This indicated that there are group I- and II-specific and common (species-specific) epitopes on the CpCSV CP and that the corresponding MAbs are suitable for specific detection and discrimination of CpCSV isolates. Moreover, CpCSV-Sy (group II) caused more severe stunting and yellowing in faba bean than CpCSV-Eth (group I). In conclusion, our data indicate the existence of a geographically associated variation in the molecular, serological and presumably biological properties of CpCSV.