Potyvirus Infection Research Articles

First report of lily mottle virus naturally infecting lily (Lilium asiatica) in Texas, USA.

Lily (Lilium asiatica) is an important plant grown for its range of flower colors and heavy scent. In March 2024, potyvirus-like symptoms consisting of light-yellow mottling and mosaic were noticed on 12/20 lily plants in a private property in Weslaco, Hidalgo County, Texas. Two symptomatic plants (WTX1 and WTX2) were sampled randomly for virus diagnosis. The leaf extracts of both samples were negative for the potyvirus group using Agdia's Poty ImmunoStrip® (Agdia, Inc., Elkhart, IN, USA). However, rub-inoculation of the extracts onto healthy Nicotiana benthamiana and Vigna unguiculata plants (n=4, each) induced mild mottle symptoms on the systemic leaves of both herbaceous test plants 20 to 28 days post inoculation, indicating the presence of a mechanically transmissible agent in the samples. No virus-like symptoms were observed on the mock-inoculated plants (n=1, each) of both species. To test for suspected potyvirus infection, 2-µg of total nucleic acid extracts (Dellaporta et al. 1983) from WTX1 and WTX2 were used for complimentary DNA (cDNA) synthesis with Oligo(dT) primer and the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, USA). One microliter aliquot of each cDNA served as template in 12.5-µl conventional PCR reaction volumes with 5 U Taq polymerase (Roche Diagnostics, Indianapolis, IN), and two pairs of degenerate primers targeting the partial cylindrical inclusion (CI) gene and the helper component protease (HC-Pro) of potyviruses (Ha et al. 2008). The expected ~700-bp DNA product of each gene target was amplified from both samples. The amplicons were excised, gel eluted (Zymoclean™ Gel DNA Recovery kit) and cloned individually into the pJET1.2/Blunt vector (Life Technologies). Recombinant plasmids from two transformed Escherichia coli cells per cloned DNA insert were Sanger sequenced. BLASTn analysis of each gene-specific nucleotide (nt) sequence fragment revealed their identities (83 to 92 % nt) as lily mottle virus (LMoV; genus Potyvirus) at 93 to 98% query coverage. In pairwise comparison, the obtained sequences of LMoV isolates from TX specific to the CI (GenBank accession nos. PQ037260-61) and the HC-Pro (PQ037262-63) shared ~99% nt and 100% amino acid (aa) identities with each other and ~91-92% nt and 98-100% aa identities with the closest isolate, Bate5 (JN127341) from Australia. Based on these results, a pair of LMoV-specific primers (LM_v109-F: 5'-GGCCAGTAATGTGCACAAGC-3' and LM_c527-R: 5'-TCGCTGTAGCTAGCGACGTAC-3') was newly designed to target the partial CI gene, internal to the 700-bp fragment obtained above with the generic primers. The primer pair was used to screen each of the mechanically inoculated test plants by RT-PCR as previously described above. The expected 438-bp fragment of the LMoV CI gene was amplified from all the inoculated plants of both N. benthamiana and V. unguiculata; the results were confirmed by Sanger sequencing as described above. The mock-inoculated plant of each experimental host plant tested negative for LMoV. LMoV is a quarantine pest and was previously reported in the U.S. (Brierley and Smith, 1944) and other lily-producing parts of the world, such as the Netherlands, China, Korea, and Brazil. To our knowledge, this is the first confirmed report of the virus in Texas, thus expanding its geographical range. Testing of lily foundation stocks before propagation is essential to prevent further spread of LMoV via planting material.

Adaptation of turnip mosaic virus to Arabidopsis thaliana involves rewiring of VPg-host proteome interactions.

The outcome of a viral infection depends on a complex interplay between the host physiology and the virus, mediated through numerous protein-protein interactions. In a previous study, we used high-throughput yeast two-hybrid (HT-Y2H) to identify proteins in Arabidopsis thaliana that bind to the proteins encoded by the turnip mosaic virus (TuMV) genome. Furthermore, after experimental evolution of TuMV lineages in plants with mutations in defense-related or proviral genes, most mutations observed in the evolved viruses affected the VPg cistron. Among these mutations, D113G was a convergent mutation selected in many lineages across different plant genotypes, including cpr5-2 with constitutive expression of systemic acquired resistance. In contrast, mutation R118H specifically emerged in the jin1 mutant with affected jasmonate signaling. Using the HT-Y2H system, we analyzed the impact of these two mutations on VPg's interaction with plant proteins. Interestingly, both mutations severely compromised the interaction of VPg with the translation initiation factor eIF(iso)4E, a crucial interactor for potyvirus infection. Moreover, mutation D113G, but not R118H, adversely affected the interaction with RHD1, a zinc-finger homeodomain transcription factor involved in regulating DNA demethylation. Our results suggest that RHD1 enhances plant tolerance to TuMV infection. We also discuss our findings in a broad virus evolution context.

First report of saffron latent virus in Crocus sativus from Hungary.

In early April of 2018 we sampled asymptomatic autumn flowering Crocus plants (Fig. S1.) in a private collection in Hajdú-Bihar county, Hungary. From each species (Cr. kotschyanus subsp. kotschyanus, Cr. sativus, Cr. speciosus) 200 mg leaf sample was collected from 5 neighboring shoot, which were treated as one sample. ELISA tests were carried out in duplicates using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) on the samples (Agdia, Elkhart, IN, USA). A sample was considered positive if the absorbance was at least three times greater than that of the negative control. Only one sample tested positive; the absorbance values of Cr. sativus leaves were 0.013 and 0.014, while the negative controls were 0.002 and 0.003, respectively. The samples were further tested by RT-PCR for potyviruses (Salamon and Palkovics 2005), tomato spotted wilt virus (TSWV) (Nemes and Salánki 2020) and nepovirus subgroup A (Digiaro et al. 2007). Total nucleic acid was extracted with the phenol-chloroform method of White and Kaper (1989), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) using random hexamer primer. The samples were negative for TSWV and nepovirus subgroup A, but a single PCR product of ~ 1700 nucleotide (nt) was amplified with potyvirus specific primers and cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA). The 1726 nt long insert sequence, including the complete coat protein region was determined and deposited in the NCBI GenBank database (Accession No: OR425160). Digestion of the original PCR products with restriction enzyme SacI yielded only the predicted restriction fragments (364 / 1362 bp), indicating the presence of only a single potyvirus in the infected sample. BLASTn analysis of the CP cistron revealed the highest nt identities to saffron latent virus (SaLV) Iranian isolates (GenBank AccNo.: MN990394 - 85.44%, MN990415 - 85.39% and RefSeq: NC_036802 - 84.05%). For phylogenetic analyses MEGA11 (Tamura et al. 2021) was used. The resulting Maximum Likelihood tree (Fig. S2) showed that all Iranian SaLV isolates grouped together, while the Hungarian isolate is on an adjacent branch, separate from other virus species, and supported with 100% bootstrap values. From these results, it appears that the Hungarian isolate has been separated from the Iranian clade, and has evolved separately as a distinct lineage. We were unable to fulfill Koch's postulates as all available Crocus sativus plants were infected with SaLV. Latent potyvirus infection of Crocus species, by bean yellow mosaic virus (BYMV), iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV) and turnip mosaic virus (TuMV) has been reported by Grilli Caiola and Faoro (2011). SaLV was first reported from Iran (Parizad et al. 2017), but to our knowledge has never been reported from Europe or from any current EPPO member state. Since Crocus species can be asymptomatic virus reservoirs, it is important that any certification scheme for production should require laboratory tests to prove the health of the plants; or advise growers to keep possible high value susceptible crops such as breeding material and nuclear stocks at a distance from crocuses to mitigate virus transmission between stocks. It is also advisable to grow infected lots far from healthy stocks and protected wild hosts. To our knowledge, this is the first report of SaLV from Hungary and from Europe.

First Report of Natural Infection of Watermelon mosaic virus (WMV) Infecting Bottle Gourd and Snake Melon

Cucurbitaceous crops, one of the main crops of agriculture, are sensitive to many plant viruses. In August 2019, virus-like symptoms were observed on some cucurbit plants grown in private home gardens in Antalya and Denizli provinces (Turkey). A total of 53 leaf samples were sampled from plants with the most symptoms (melon (Cucumis melo L.), watermelon (Citrullus lanatus L.), bottle gourd (Lagenaria siceraria (Molina) Standl.), and snake melon (Cucumis melo var. flexuosus) and tested by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) against possible watermelon mosaic potyvirus (WMV) infection. The coat protein gene (CP) specific primer sets amplified a gene product of nearly 820 bp fragment from symptomatic plants. WMV infections were detected in 31 individual cucurbit plants, including 11 melons, 8 watermelons, 7 snake melons and 5 bottle gourds. The presence of viral infection was found only in ornamental squash plants in Antalya province and in all cucurbits sampled in Denizli province. To better comprehend the molecular characteristics of virus isolates, the amplified viral DNA fragments were cloned in a proper prokaryotic plasmid, sequenced by Next Generation Sequencing (NGS) and recorded to GenBank. Bioinformatic analyses using the Basic Local Alignment Search Tool (BLAST) showed that the identified CP gene sequences exhibited significant nucleotide homogeneity, supported by a high nucleotide similarity index with that of other isolates around the world. In addition, Turkish isolates isolated from Antalya and Denizli regions showed approximately 94% nucleotide similarity among themselves. For phylogenetic inference, WMV sequences were subjected to multiple alignments with isolates from different geographic origins of the same viruses. Molecular phylogeny showed that all WMV isolates are closely related to other world WMV isolates at variable rates. WMV is wide host range viruses in cucurbit crops, however, this work is the first scientific report of WMV isolates detected in bottle gourd and snake melon from the South and West Regions of Turkey all over the world.WMV are broad spectrum viruses in cucurbit crops. This work is the first scientific report of Watermelon mosaic potyvirus isolates detected in bottle gourd and snake melon from the South-West Region of Turkey in all over the world.

Monitoring the Intracellular Trafficking of Virus-Induced Structures and Intercellular Spread of Viral Infection in Plants Using Endomembrane Trafficking Pathway-Specific Chemical Inhibitor and Organelle-Selective Fluorescence Dye.

Infection by positive-strand RNA viruses induces extensive remodeling of the host endomembrane system in favor of viral replication and movement. The integral membrane protein 6K2 of potyviruses induces the formation of membranous virus replication vesicles at the endoplasmic reticulum exit site (ERES). The intracellular trafficking of 6K2-induced vesicles along with microfilaments requires the vesicular transport pathway, actomyosin motility system, and possibly post-Golgi compartments such as endosomes as well. Recent studies have shown that endocytosis is essential for the intracellular movement of potyviruses from the site of viral genome replication/assembly site to plasmodesmata (PD) to enter neighboring cells. In this chapter, we describe a detailed protocol of how to use endomembrane trafficking pathway-specific chemical inhibitors and organelle-selective fluorescence dye to study the trafficking of potyviral proteins and potyvirus-induced vesicles and to unravel the role of endocytosis and the endocytic pathway in potyvirus infection in Nicotiana benthamiana plants.

Arabidopsis eIF4E1 protects the translational machinery during TuMV infection and restricts virus accumulation.

Successful subversion of translation initiation factors eIF4E determines the infection success of potyviruses, the largest group of viruses affecting plants. In the natural variability of many plant species, resistance to potyvirus infection is provided by polymorphisms at eIF4E that renders them inadequate for virus hijacking but still functional in translation initiation. In crops where such natural resistance alleles are limited, the genetic inactivation of eIF4E has been proposed for the engineering of potyvirus resistance. However, recent findings indicate that knockout eIF4E alleles may be deleterious for plant health and could jeopardize resistance efficiency in comparison to functional resistance proteins. Here, we explored the cause of these adverse effects by studying the role of the Arabidopsis eIF4E1, whose inactivation was previously reported as conferring resistance to the potyvirus clover yellow vein virus (ClYVV) while also promoting susceptibility to another potyvirus turnip mosaic virus (TuMV). We report that eIF4E1 is required to maintain global plant translation and to restrict TuMV accumulation during infection, and its absence is associated with a favoured virus multiplication over host translation. Furthermore, our findings show that, in the absence of eIF4E1, infection with TuMV results in the production of a truncated eIFiso4G1 protein. Finally, we demonstrate a role for eIFiso4G1 in TuMV accumulation and in supporting plant fitness during infection. These findings suggest that eIF4E1 counteracts the hijacking of the plant translational apparatus during TuMV infection and underscore the importance of preserving the functionality of translation initiation factors eIF4E when implementing potyvirus resistance strategies.

Biological and molecular characterization of bidens mosaic virus infecting patchouli (Pogostemon cablin) in Brazil

AbstractSix patchouli (Pogostemon cablin Benth.) plants showing symptoms of viral infection were found in Brazil. Transmission electron microscopy revealed flexuous particles and characteristic pinwheel‐shaped cytoplasmic inclusions of potyvirus in sap and cells of symptomatic leaves. Potyvirus infection was confirmed through RT‐PCR, using universal primers followed by nucleotide sequencing. The obtained sequences, comprising part of the CP gene and the 3′ non‐translated region, showed 97.71%–97.87% identity with an isolate of bidens mosaic virus (BiMV). The nucleotide sequences of the CP, HC‐Pro and CI genes of BiMV isolates from patchouli plants were obtained through RT‐PCR with specific primers followed by nucleotide sequencing. The obtained sequences showed 95.78%–97.1% identities with BiMV isolates from Brazil. One BiMV isolate from a patchouli infected plant was sap‐transmitted to different plant species. Chenopodium amaranticolor exhibited local lesions, Bidens pilosa developed leaf mosaic, and Nicotiana benthamiana exhibited chlorosis and stunting. The BiMV isolate was transmitted from infected patchouli plant to B. pilosa using Myzus persicae aphids. This is the first confirmation of patchouli infected with BiMV in Brazil. Further studies are necessary to assess the incidence of this potyvirus in commercial patchouli crops, as well as the damage caused to plant development and the quantity and quality of essential oils produced.

Characterization of sugarcane mosaic virus from Cymbopogon spp. plants in Brazil

AbstractIn April 2022, virus‐like symptoms were observed in a Cymbopogon spp. plant grown in the backyard of a house and in Java citronella (C. winterianus) plants in a flower shop in Piracicaba, São Paulo State, Brazil. In April 2023, ca. 700,000 Ceylon citronella (C. nardus) plants showing symptoms of mosaic were observed in a field crop in Dois Corrégos (SP). Evidence for a possible potyvirus infection was revealed by electron microscopy and confirmed by molecular assays. Nucleotide sequencing of amplicons from RT‐PCR identified the potyvirus infecting a Cymbopogon spp. plant, Java and Ceylon citronella as sugarcane mosaic virus (SCMV, genus Potyvirus). Phylogenetic analysis of the coat protein gene indicated that the SCMV isolates from Cymbopogon (C1), Java (JC1), and Ceylon citronella (CC) belong to the clade of SCMV from sugarcane. The C1 isolate, and an isolate from sugarcane (SCMV‐SU) (control) were mechanically transmitted to Java citronella, sorghum, sugarcane and maize plants. Aphis gossypii transmitted the C1 and SU isolates to Java citronella plants, while Melanaphis sacchari transmitted only the SU isolate to Java citronella plants. This is the first molecular characterization of SCMV from Cymbopogon spp. plants.

First report of meadow saffron breaking virus on wild Colchicum autumnale from a stricly protected Natura2000 site at a Hungarian National Park.

In mid-April of 2018 light green to greenish yellow linear stripes (Fig. S1.) were observed on the foliage of meadow saffron (Colchicum autumnale) plants - which are native to Hungary - at a strictly protected Natura2000 site maintained by the Duna-Ipoly National Park (DNPI). By autumn, during the flowering season, flower breaking symptoms (Fig. S2.) were noticed, which indicated possible viral infection. With the permit of the Government Office of Pest County and the DNPI, 200 mg leaf sample was collected from one symptomatic plant in spring 2021 and stored at -70 °C until further processing. At the time of the sampling about 2.5 % of the ~ 5000 meadow saffron were symptomatic. Multiplex RT-PCR testing of the sample and an asymptomatic C. autumnale plant for cucumber mosaic virus, tomato spotted wilt virus (Nemes and Salánki 2020) and Nepovirus subgroup-A (Digiaro et al. 2007) gave negative results. The asymptomatic plant also tested negative for potyviruses (Salamon and Palkovics 2005). The asymptomatic (healthy) C. autumnale plant was inoculated with leaf sap of the sample (0.02M Sörensen's phosphate buffer pH 7.2 + 2 % PVP-40 (m/v)) resulting in symptoms of flower breaking in autumn of 2021, and linear stripes on the foliage in spring 2022, identical to symptoms on the originally infected plant. ELISA tests were carried out on the source plants in duplicate using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) (Agdia, Elkhart, IN, USA). Absorbance values were 1.519 and 1.530, while the negative controls were 0.003 and 0.007, respectively indicating potyvirus infection of the sample. Molecular tests were carried out on the source and inoculated plant samples in 2022. Total nucleic acid was extracted with the modified CTAB protocol of Xu et al. (2004), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) with poly T2 (5'-CGGGGATCCTCGAGAAGCTTTTTTTTTTTTTTTTT-3') primer (Salamon and Palkovics 2005). PCR amplification was carried out with poty7941 (5'-GGAATTCCCGCGGNAAYAAYAGYGGNCARCC-3') and poly T2 primers as described earlier (Salamon and Palkovics 2005). A PCR product of ~ 1.6 kb was obtained in each case (Fig. S3.), cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA) and transformed into E. coli DH5α strain. The obtained 1642 nucleotide (nt) sequence encompassing the complete coat protein (CP) was determined (Accession No: OP057214). The virus sequence present in the source and inoculated plants shared 100% nt identity. EcoRV digestion of the PCR products yielded two restriction fragments (369/1273 bp), indicating the presence of a single potyvirus in the infected plant tissue (Fig. S3.). BLASTN analysis of the CP cistron revealed highest nt identity (93.91 %) to meadow saffron breaking virus (MSBV) isolate FR GenBank Acc. No.: AY388995. MSBV was first reported in the Alsace region of France at an INRA research station in cultivated meadow saffron plants showing similar symptoms and the disease reached 100% incidence within a year (Poutaraud et al. 2004). Potyviruses are transmitted mechanically and by aphids (Inoue-Nagata et al. 2022). The spread of MSBV could lead to the infection and decline of the population of Colchicum in protected ecosystems. To our knowledge, this is the first report of MSBV on wild meadow saffron plant from a strictly protected Natura2000 site at a Hungarian National Park.

Mixed infection of phytoplasmas and potyvirus in Phlox drummondii in India

Mixed infection of phytoplasmas and potyvirus in Phlox drummondii in India

An overview of Potyviruses infecting daffodil and their disease management

AbstractDaffodil (Narcissus tazetta L.) is a bulbous ornamental plant which belongs to the family Amaryllidaceae. It is popular for sweet-smelling beautiful flowers and is used for the fragrant oil and perfumes. It is also a source of alkaloids used in traditional medicines. Daffodil is commercially important ornamental plant, therefore, it is important to maintain its quality and production. Various type of viruses affect daffodil cultivation and among them, potyviruses are of great concern as they cause more economic losses to its commercial cultivation in term of reduction in bulb size, number of bulbs and the quality of daffodil flowers. In this review, a general overview of daffodils, genus Potyvirus, disease symptoms in daffodils linked to potyvirus infection, potyviruses described in daffodils, Potyvirus identification methods and possible management strategies of potyviruses in daffodil cultivation are described in detail. The study will be helpful to daffodil growers for improvement of the production/yield and quality of daffodil crop.

Binding immunoglobulin 2 functions as a proviral factor for potyvirus infections in Nicotiana benthamiana.

Infection of viruses from the genera Bromovirus, Potyvirus, and Potexvirus in Nicotiana benthamiana induces significant up-regulation of the genes that encode the HSP70 family, including binding immunoglobulin protein 2 (BiP2). Three up-regulated genes were knocked down and infection assays with these knockdown lines demonstrated the importance of the BiP2 gene for potyvirus infection but not for infection by the other tested viruses. Distinct symptoms of cucumber mosaic virus (CMV) and potato virus X (PVX) were observed in the BiP2 knockdown line at 10 days postagroinfiltration. Interestingly, following inoculation with either soybean mosaic virus (SMV) or pepper mottle virus (PepMoV) co-expressing green fluorescent protein (GFP), neither crinkle symptoms nor GFP signals were observed in the BiP2 knockdown line. Subsequent reverse transcription-quantitative PCR analysis demonstrated that knockdown of BiP2 resulted in a significant decrease of SMV and PepMoV RNA accumulation but not PVX or CMV RNA accumulation. Further yeast two-hybrid and co-immunoprecipitation analyses validated the interaction between BiP2 and nuclear inclusion protein b (NIb) of SMV. Together, our findings suggest the crucial role of BiP2 as a proviral host factor necessary for potyvirus infection. The interaction between BiP2 and NIb may be the critical factor determining susceptibility in N.benthamiana, but further studies are needed to elucidate the underlying mechanism.

Analysis of the RNA-Dependent RNA Polymerase 1 (RDR1) Gene Family in Melon.

RNA-dependent RNA polymerase 1 (RDR1) plays a crucial defense role against plant viruses by secondary amplification of viral double-stranded RNA in the gene-silencing pathway. In this study, it was found that melon (Cucumis melo) encodes four RDR1 genes (CmRDR1a, b, c1 and c2) similar to the CsRDR1 gene family of cucumber (C. sativus). However, in contrast to cucumber, melon harbors a truncated CmRDR1b gene. In healthy plants, CmRDR1a was expressed, whereas the expression of CmRDR1c1/c2 was not detected. CmRDR1a expression level increased 20-fold upon cucumber mosaic virus (CMV) infection and was not increased in melon plants infected with zucchini yellow mosaic virus (ZYMV), cucumber vein yellowing virus (CVYV) and cucumber green mottle mosaic virus (CGMMV). The expression of CmRDR1c1/c2 genes was induced differentially by infection with viruses from different families: high levels of ~340-, 172- and 115-fold increases were induced by CMV, CVYV and CGMMV, respectively, and relatively low-level increases by potyvirus infection (4- to 6-fold). CMV mutants lacking the viral silencing suppressor 2b protein did not cause increased CmRDR1c/c2 expression; knockout of CmRDR1c1/c2 by CRISPR/Cas9 increased susceptibility to CMV but not to ZYMV. Therefore, it is suggested that the sensitivity of melon to viruses from different families is a result of the loss of function of CmRDR1b.

A new strain of Papaya ring spot virus infecting ivy gourd (Coccinia grandis L.) in India

Ivy gourd (Coccinia grandis L.) plants showing virus likesymptoms such as mosaic mottling and thickening of leaflamina were collected from Tindivanam, Tamil Nadu. ThroughReverse transcription polymerase chain reaction, infectionof potyvirus was confirmed with the yield of desiredamplicon (~1.0 kb) using universal primer pair. Sequenceanalysis of amplified product showed the infection of Papayaring spot virus (PRSV). Further association of PRSV wasconfirmed with PRSV specific primer pair (GK PRSV F/R).The nucleotide sequence analysis showed identity of coatprotein region shared only 84% with PRSV isolate fromThailand in BLAST analysis. In phylogenetic analysis, PRSVinfecting ivy gourd formed a separate cluster from otherPRSV isolates. This confirms the association of distinct strainof PRSV on Ivy gourd in India.

Defects in plant immunity modulate the rates and patterns of RNA virus evolution.

It is assumed that host genetic variability for susceptibility to infection conditions virus evolution. Differences in host susceptibility can drive a virus to diversify into strains that track different defense alleles (e.g. antigenic diversity) or to infect only the most susceptible genotypes. Here, we have studied how variability in host defenses determines the evolutionary fate of a plant RNA virus. We performed evolution experiments with Turnip mosaic potyvirus in Arabidopsis thaliana mutants that had disruptions in infection-response signaling pathways or in genes whose products are essential for potyvirus infection. Plant genotypes were classified into five phenogroups according to their response to infection. We found that evolution proceeded faster in more restrictive hosts than in more permissive ones. Most of the phenotypic differences shown by the ancestral virus across host genotypes were removed after evolution, suggesting the combined action of selection and chance. When all evolved viral lineages were tested in all plant genotypes used in the experiments, we found compelling evidences that the most restrictive plant genotypes selected for more generalist viruses, while more permissive genotypes selected for more specialist viruses. Sequencing the genomes of the evolved viral lineages, we found that selection targeted the multifunctional genome-linked protein VPg in most host genotypes. Overall, this work illustrates how different host defenses modulate the rates and extent of virus evolution.

AlkB RNA demethylase homologues and N6 -methyladenosine are involved in Potyvirus infection.

Proteins of the alkylation B (AlkB) superfamily show RNA demethylase activity removing methyl adducts from N 6‐methyladenosine (m6A). m6A is a reversible epigenetic mark of RNA that regulates human virus replication but has unclear roles in plant virus infection. We focused on Potyvirus—the largest genus of plant RNA viruses—and report here the identification of AlkB domains within P1 of endive necrotic mosaic virus (ENMV) and an additional virus of a putative novel species within Potyvirus. We show that Nicotiana benthamiana m6A levels are reduced by infection of plum pox virus (PPV) and potato virus Y (PVY). The two potyviruses lack AlkB and the results suggest a general involvement of RNA methylation in potyvirus infection and evolution. Methylated RNA immunoprecipitation sequencing of virus‐infected samples showed that m6A peaks are enriched in plant transcript 3′ untranslated regions and in discrete internal and 3′ terminal regions of PPV and PVY genomes. Down‐regulation of N. benthamiana AlkB homologues of the plant‐specific ALKBH9 clade caused a significant decrease in PPV and PVY accumulation. In summary, our study provides evolutionary and experimental evidence that supports the m6A implication and the proviral roles of AlkB homologues in Potyvirus infection.

Targeted Knockout of Eukaryotic Translation Initiation Factor 4E Confers Bymovirus Resistance in Winter Barley.

The Eukaryotic Translation Initiation Factor 4E (EIF4E) is a well-known susceptibility factor for potyvirus infections in many plant species. The barley yellow mosaic virus disease, caused by the bymoviruses Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), can lead to yield losses of up to 50% in winter barley. In autumn, the roots of young barley plants are infected by the soil-borne plasmodiophoraceous parasite Polymyxa graminis L. that serves as viral vector. Upon viral establishment and systemic spreading into the upper parts of the plants, yellow mosaics occur as first symptoms on leaves. In the further course of plant development, the disease entails leaf necrosis and increased susceptibility to frost damage. Thanks to the rym4 and rym5 allelic variants of the HvEIF4E gene, more than two thirds of current European winter barley cultivars are resistant to BaYMV and BaMMV. However, several strains of BaYMV and BaMMV have already overcome rym4- and rym5-mediated resistance. Accordingly, new resistance-conferring alleles are needed for barley breeding. Therefore, we performed targeted mutagenesis of the EIF4E gene by Cas9 endonuclease in BaMMV/BaYMV-susceptible winter barley cv. “Igri”. Small insertions were generated, resulting in a shift of the translational reading frame, thereby causing the loss-of-function of EIF4E. The mutations occurred in the homozygous state already in the primary mutants. Their progeny proved invariably homozygous and fully resistant to mechanical inoculation with BaMMV. EIF4E knockout plants showed normal growth habit and produced grains, yet exhibited a yield penalty.

Sternbergia lutea, a new host of Narcissus late season yellows virus

In autumn 2017, autumn daffodil plants with yellow-green stripes on the leaves were observed at a botanical garden in Budapest, Hungary. Indicator plants were inoculated, but symptoms did not develop. RT-PCR tests of the indicator plants were also negative for the viruses. Potyvirus specific ACP-ELISA and RT-PCR were carried out on the symptomatic S. lutea leaf samples. RT-PCR with universal potyvirus primers resulted in one, approx. 1700 base pair PCR product. Phylogenetic analysis of the nucleotide sequence of the coat protein demonstrated 98.78-99.51% identity with three Japanese isolates of Narcissus late season yellows virus. While unidentified potyvirus infection of autumn daffodil has been previously reported, sequence data have not been published. Therefore, this is the first report of Sternbergia lutea as a host of Narcissus late season yellows virus.

Molecular characterization and identification of economically important Potyviruses in Cucurbitaceae family from Gujranwala division of Punjab, Pakistan

Cucurbits belongs to Cucurbitaceae family and they are main source of energy, vitamins and minerals. The plant viruses in general and potyviruses in particular are considered to hamper the successful production of cucurbits in Punjab province of Pakistan. The present study was undertaken to determine the current status of major potyviruses infecting cucurbits in Gujranwala division of Punjab. Overall, 528 samples of cucurbit species were collected from 46 fields of six districts (Narowal, Sialkot, Gujranwala, Gujrat, Mandi Baha Uddin and Hafizabad) in Gujranwala division. All disease samples were screened for the presence of potyvirus infection by indirect plate-trapped antigen (PTA)-ELISA using “Poty-group test” kit (Bioreba AG, Switzerland). These samples were further tested for Papaya ring spot virus (PRSV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV) (Bioreba AG) using virus specific DAS-ELISA. Zucchini yellow mosaic virus (ZYMV) was also further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). ZYMV was found as the most predominant virus of cucurbits in Gujranwala division with an average incidence of 20.8%, followed by PRSV (7.2%) and WMV (1.7%). Maximum mixed double infection was observed in combination of ZYMV+PRSV (1.3%) followed by ZYMV+WMV (0.4%) and WMV+PRSV (0.8%). Overall highest disease incidence of potyviruses was in Gujranwala (40.86%) followed by Narowal (34.65%), Gujrat (28.6%), Mandi Baha Uddin (26.56%), Hafizabad (25%) and Sialkot (23.21%). Likewise, the maximum ZYMV incidence was recorded in Angel Gourd whereas no viral infection was observed in snap melon and ash gourd in Gujranwala division. All cucurbits vegetables were found to be highly prone to ZYMV followed by PRSV and WMV. The sequence of CI region of ZYMV AABRM-CI ridge gourd isolate was deposited in GenBank with an accession no. MN897101, which showed nucleotide identities from 91.501-98.401% with other isolates of ZYMV available at database. Highest nucleotide identity 98.401% was found with the sequence of ZYMV South Korean isolate BR1 (MH042024) followed by 98.301% with ZYMV isolate Y23 from Turkey (KP828425). The results of the current study would be useful in devising management strategies to mitigate potyvirus losses in cucurbits from Pakistan.

Nuclear exportin 1 facilitates turnip mosaic virus infection by exporting the sumoylated viral replicase and by repressing plant immunity.

Exportin 1/XPO1 is an important nuclear export receptor that binds directly to cargo proteins and translocates the cargo proteins to the cytoplasm. To understand XPO1 protein functions during potyvirus infections, we investigated the nuclear export of the NIb protein encoding the RNA-dependent RNA polymerase (RdRp) of turnip mosaic virus (TuMV). Previously, we found that NIb is transported to the nucleus after translation and sumoylated by the sumoylation (small ubiquitin-like modifier) pathway to support viral infection. Here, we report that XPO1 interacts with NIb to facilitate translocation from the nucleus to the viral replication complexes (VRCs) that accumulate in the perinuclear regions of TuMV-infected cells. XPO1 contains two NIb-binding domains that recognize and interact with NIb in the nucleus and in the perinuclear regions, respectively, which facilitates TuMV replication. Moreover, XPO1 is involved in nuclear export of the sumoylated NIb and host factors tagged with SUMO3 that is essential for suppression of plant immunity in the nucleus. Deficiencies of XPO1 in Arabidopsis and Nicotiana benthamiana plants inhibit TuMV replication and infection. These data demonstrate that XPO1 functions as a host factor in TuMV infection by regulating NIb nucleocytoplasmic transport and plant immunity.