Tryptanthrin Derivative B1 Binds Viral Genome-Linked Protein (VPg) of Potato Virus Y.

Potato virus Y (PVY) is a plant virus that is known to be responsible for substantial economic losses in agriculture. Within the PVY genome, viral genome-linked protein (VPg) plays a pivotal role in the viral translation process. In this study, VPg was used as a potential target for analyzing the antiviral activity of tryptanthrin derivatives. In vitro, the dissociation constants of B1 with PVY VPg were 0.69 µmol/L (measured by microscale thermophoresis) and 4.01 µmol/L (measured via isothermal titration calorimetry). B1 also strongly bound to VPg proteins from three other Potyviruses. Moreover, in vivo experiments demonstrated that B1 effectively suppressed the expression of the PVY gene. Molecular docking experiments revealed that B1 formed a hydrogen bond with N121 and that no specific binding occurred between B1 and the PVY VPgN121A mutant. Therefore, N121 is a key amino acid residue in PVY VPg involved in B1 binding. These results highlight the potential of PVY VPg as a potential target for the development of antiviral agents.

Transcriptome analysis of the synergistic mechanisms between two strains of potato virus Y in Solanum tuberosum L.

Many viruses employ a process known as superinfection exclusion (SIE) to block subsequent entry or replication of the same or closely related viruses in the cells they occupy. SIE is also referred to as Cross-protection refers to the situation where a host plant infected by a mild strain of a virus or viroid gains immunity against a more severe strain closely related to the initial infectant. The mechanisms underlying cross-protection are not fully understood. In this study, we performed a comparative transcriptomic analysis of potato (Solanum tuberosum L.) leaves. The strains PVYN-Wi-HLJ-BDH-2 and PVYNTN-NW-INM-W-369-12 are henceforth designated as BDH and 369, respectively. In total, 806 differentially expressed genes (DEGs) were detected between the Control and JZ (preinfected with BDH and challenge with 369) treatment. Gene Ontology (GO) analysis showed that the response to external biological stimulation, signal transduction, kinase, immunity, redox pathways were significantly enriched. Among these pathways, we identified numerous differentially expressed metabolites related to virus infection. Moreover, our data also identified a small set of genes that likely play important roles in the establishment of cross-protection. Specifically, we observed significant differential expression of the A1-II gamma-like gene, elongation factor 1-alpha-like gene, and subtilisin-like protease StSBT1.7 gene, with StSBT1.7 being the most significant in our transcriptome data. These genes can stimulate the expression of defense plant genes, induce plant chemical defense, and participate in the induction of trauma and pathogenic bacteria. Our findings provided insights into the mechanisms underlying the ability of mild viruses to protect host plants against subsequent closely related virus infection in Solanum tuberosum L.

Complete genome sequence of polygonatum kingianum mottle virus infecting Polygonatum kingianum Coll. et Hemsl in Yunnan, China.

The complete genome sequence of a putative novel potyvirus, tentatively named "polygonatum kingianum mottle virus" (PKgMV; GenBank accession no. ON428226), infecting Polygonatum kingianum in China, was obtained by next-generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), and rapid amplification of cDNA ends (RACE). PKgMV exhibits the typical genome organization and characteristics of members of the genus Potyvirus, with a length of 10,002 nucleotides (nt) and a large open reading frame (nt 108 to 9,746) encoding a polyprotein of 3,212 amino acids (aa) (363.68 kDa). Pairwise comparisons revealed that the PKgMV polyprotein shares 50.5-68.6% nt and 43.1-72.2% aa sequence identity with reported members of the genus Potyvirus. Moreover, phylogenetic analysis indicated that PKgMV is closely related to polygonatum kingianum virus 1 (PKgV1; accession no. MK427056). These results suggest that the PKgMV is a novel member of the genus Potyvirus of the family Potyviridae.

Turnip mosaic virus NIb weakens the function of eukaryotic translation initiation factor 6 facilitating viral infection in Nicotiana benthamiana.

Viruses rely completely on host translational machinery to produce the proteins encoded by their genes. Controlling translation initiation is important for gaining translational advantage in conflicts between the host and virus. The eukaryotic translation initiation factor 4E (eIF4E) has been reported to be hijacked by potyviruses for virus multiplication. The role of translation regulation in defence and anti-defence between plants and viruses is not well understood. We report that the transcript level of eIF6 was markedly increased in turnip mosaic virus (TuMV)-infected Nicotiana benthamiana. TuMV infection was impaired by overexpression of N. benthamiana eIF6 (NbeIF6) either transiently expressed in leaves or stably expressed in transgenic plants. Polysome profile assays showed that overexpression of NbeIF6 caused the accumulation of 40S and 60S ribosomal subunits, the reduction of polysomes, and also compromised TuMV UTR-mediated translation, indicating a defence role for upregulated NbeIF6 during TuMV infection. However, the polysome profile in TuMV-infected leaves was not identical to that in leaves overexpressing NbeIF6. Further analysis showed that TuMV NIb protein, the RNA-dependent RNA polymerase, interacted with NbeIF6 and interfered with its effect on the ribosomal subunits, suggesting that NIb might have a counterdefence role. The results propose a possible regulatory mechanism at the translation level during plant-virus interaction.

Genetic basis of Arabidopsis thaliana responses to infection by naïve and adapted isolates of turnip mosaic virus.

Plant viruses account for enormous agricultural losses worldwide, and the most effective way to combat them is to identify genetic material conferring plant resistance to these pathogens. Aiming to identify genetic associations with responses to infection, we screened a large panel of Arabidopsis thaliana natural inbred lines for four disease-related traits caused by infection by A. thaliana-naïve and -adapted isolates of the natural pathogen turnip mosaic virus (TuMV). We detected a strong, replicable association in a 1.5 Mb region on chromosome 2 with a 10-fold increase in relative risk of systemic necrosis. The region contains several plausible causal genes as well as abundant structural variation, including an insertion of a Copia transposon into a Toll/interleukin receptor (TIR-NBS-LRR) coding for a gene involved in defense, that could be either a driver or a consequence of the disease-resistance locus. When inoculated with TuMV, loss-of-function mutant plants of this gene exhibited different symptoms than wild-type plants. The direction and severity of symptom differences depended on the adaptation history of the virus. This increase in symptom severity was specific for infections with the adapted isolate. Necrosis-associated alleles are found worldwide, and their distribution is consistent with a trade-off between resistance during viral outbreaks and a cost of resistance otherwise, leading to negative frequency-dependent selection.

Molecular Characteristics of Bean Common Mosaic Virus Occurring in Inner Mongolia, China.

Bean common mosaic virus (BCMV) was detected on common bean (Phaseolus vulgaris) plants showing wrinkled and/or narrow leaves, curling, shrinking and chlorosis of leaves, dwarfing of plants, and mottled pods in Inner Mongolia and named BCMV-22Huhe. Its genome has a size of 10,062 bp and was deposited in GenBank under the accession number OR778613. It is closely related to BCMV-Az (GenBank accession no. KP903372, in China) in the lineage of AzBMV. A recombination event was detected for BCMV-22Huhe among the 99 BCMV isolates published in the NCBI GenBank database, showing that BCMV-CJ25 (MK069986, found in Mexico) was a potential major parent, and the minor parent is unknown. This work is the first description of the occurrence of BCMV in Inner Mongolia, China.

A natural substitution of a conserved amino acid in eIF4E confers resistance against multiple potyviruses.

Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D71 located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.

Phenotypic and genomic changes during Turnip mosaic virus adaptation to Arabidopsis thaliana mutants lacking epigenetic regulatory factors.

In this study, we investigated how an emerging RNA virus evolves, interacts, and adapts to populations of a novel host species with defects in epigenetically controlled plant defense mechanisms. Mutations in epigenetic regulatory pathways would exert different effects on defense-response genes but also induce large-scale alterations in cellular physiology and homeostasis. To test whether these effects condition the emergence and subsequent adaptation of a viral pathogen, we have evolved five independent lineages of a naive turnip mosaic virus (TuMV) strain in a set of Arabidopsis thaliana genotypes carrying mutations that influence important elements of two main epigenetic pathways and compare the results with those obtained for viral lineages evolved in wild-type plants. All evolved lineages showed adaptation to the lack of epigenetically regulated responses through significant increases in infectivity, virulence, and viral load although the magnitude of the improvements strongly depended on the plant genotype. In early passages, these traits evolved more rapidly, but the rate of evolution flattened out in later ones. Viral load was positively correlated with different measures of virulence, though the strength of the associations changed from the ancestral to the evolved viruses. High-throughput sequencing was used to evaluate the viral diversity of each lineage, as well as characterizing the nature of fixed mutations, evolutionary convergences, and potential targets of TuMV adaptation. Within each lineage, we observed a net increase in genome-wide genetic diversity, with some instances where nonsynonymous alleles experienced a transient rise in abundance before being displaced by the ancestral allele. In agreement with previous studies, viral VPg protein has been shown as a key player in the adaptation process, even though no obvious association between fixed alleles and host genotype was found.

High-throughput characterization and phenotyping of resistance and tolerance to virus infection in sweetpotato.

Breeders have made important efforts to develop genotypes able to resist virus attacks in sweetpotato, a major crop providing food security and poverty alleviation to smallholder farmers in many regions of Sub-Saharan Africa, Asia and Latin America. However, a lack of accurate objective quantitative methods for this selection target in sweetpotato prevents a consistent and extensive assessment of large breeding populations. In this study, an approach to characterize and classify resistance in sweetpotato was established by assessing total yield loss and virus load after the infection of the three most common viruses (SPFMV, SPCSV, SPLCV). Twelve sweetpotato genotypes with contrasting reactions to virus infection were grown in the field under three different treatments: pre-infected by the three viruses, un-infected and protected from re-infection, and un-infected but exposed to natural infection. Virus loads were assessed using ELISA, (RT-)qPCR, and loop-mediated isothermal amplification (LAMP) methods, and also through multispectral reflectance and canopy temperature collected using an unmanned aerial vehicle. Total yield reduction compared to control and the arithmetic sum of (RT-)qPCR relative expression ratios were used to classify genotypes into four categories: resistant, tolerant, susceptible, and sensitives. Using 14 remote sensing predictors, machine learning algorithms were trained to classify all plots under the said categories. The study found that remotely sensed predictors were effective in discriminating the different virus response categories. The results suggest that using machine learning and remotely sensed data, further complemented by fast and sensitive LAMP assays to confirm results of predicted classifications could be used as a high throughput approach to support virus resistance phenotyping in sweetpotato breeding.

Alfalfa vein mottling virus, a novel potyvirid infecting Medicago sativa L.

BACKGROUND: We have recently identified a novel virus detected in alfalfa seed material. The virus was tentatively named alfalfa-associated potyvirus 1, as its genomic fragments bore similarities with potyvirids. In this study, we continued investigating this novel species, expanding information on its genomic features and biological characteristics. METHODS: This research used a wide range of methodology to achieve end results: high throughput sequencing, bioinformatics tools, reverse transcription-polymerase chain reactions, differential diagnostics using indicator plants, virus purification, transmission electron microscopy, and others. RESULTS: In this study, we obtained a complete genome sequence of the virus and classified it as a tentative species in the new genus, most closely related to the members of the genus Ipomovirus in the family Potyviridae. This assumption is based on the genome sequence and structure, phylogenetic relationships, and transmission electron microscopy investigations. We also demonstrated its mechanical transmission to the indicator plant Nicotiana benthamiana and to the natural host Medicago sativa, both of which developed characteristic symptoms therefore suggesting a pathogenic nature of the disease. CONCLUSIONS: Consistent with symptomatology, the virus was renamed to alfalfa vein mottling virus. A name Alvemovirus was proposed for the new genus in the family Potyviridae, of which alfalfa vein mottling virus is a tentative member.

Soybean 40S Ribosomal Protein S8 (GmRPS8) Interacts with 6K1 Protein and Contributes to Soybean Susceptibility to Soybean Mosaic Virus.

Soybean mosaic virus (SMV), a member of Potyvirus, is the most destructive and widespread viral disease in soybean production. Our earlier studies identified a soybean 40S ribosomal protein S8 (GmRPS8) using the 6K1 protein of SMV as the bait to screen a soybean cDNA library. The present study aims to identify the interactions between GmRPS8 and SMV and characterize the role of GmRPS8 in SMV infection in soybean. Expression analysis showed higher SMV-induced GmRPS8 expression levels in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting that GmRPS8 was involved in the response to SMV in soybean. Subcellular localization showed that GmRPS8 was localized in the nucleus. Moreover, the yeast two-hybrid (Y2H) experiments showed that GmRPS8 only interacted with 6K1 among the eleven proteins encoded by SMV. The interaction between GmRPS8 and 6K1 was further verified by a bimolecular fluorescence complementation (BiFC) assay, and the interaction was localized in the nucleus. Furthermore, knockdown of GmRPS8 by a virus-induced gene silencing (VIGS) system retarded the growth and development of soybeans and inhibited the accumulation of SMV in soybeans. Together, these results showed that GmRPS8 interacts with 6K1 and contributes to soybean susceptibility to SMV. Our findings provide new insights for understanding the role of GmRPS8 in the SMV infection cycle, which could help reveal potyviral replication mechanisms.

A Non-Canonical Pathway Induced by Externally Applied Virus-Specific dsRNA in Potato Plants.

The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18-30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection.

Characterization of a Putative New Member of the Genus Potyvirus from Kudzu (Pueraria montana var. lobata) in Mississippi.

Kudzu (Pueraria montana var. lobata), a plant native to Southeastern Asia, has become a major noxious weed covering millions of hectares in the Southern United States. A kudzu patch displaying virus-like symptoms located in Ackerman, northeastern Mississippi (MS), was used as a source for virus isolation and characterization involving mechanical and vector transmission, ultrastructural observation, surveys, Sanger and high-throughput genome sequencing, and sequence analyses. The results revealed the presence of a new potyvirus in infected kudzu, closely related to wisteria vein mosaic virus (WVMV) and provisionally named kudzu chlorotic ring blotch virus (KudCRBV). Genome features and pairwise comparison with six WVMV genomes currently available in GenBank and three additional isolates from MS sequenced in this work suggest that KudCRBV is likely a member of a new species in the genus Potyvirus. Furthermore, under experimental conditions, KudCRBV was successfully transmitted by cotton and potato aphids and mechanically to soybean and beans. A state-wide survey revealed several kudzu patches infected by the virus in northern MS.

Plant and animal positive-sense single-stranded RNA viruses encode small proteins important for viral infection in their negative-sense strand.

Positive-sense single-stranded RNA (+ssRNA) viruses, the most abundant viruses of eukaryotes in nature, require the synthesis of negative-sense RNA (-RNA) using their genomic (positive-sense) RNA (+RNA) as a template for replication. Based on current evidence, viral proteins are translated via viral +RNAs, whereas -RNA is considered to be a viral replication intermediate without coding capacity. Here, we report that plant and animal +ssRNA viruses contain small open reading frames (ORFs) in their -RNA (reverse ORFs [rORFs]). Using turnip mosaic virus (TuMV) as a model for plant +ssRNA viruses, we demonstrate that small proteins encoded by rORFs display specific subcellular localizations, and confirm the presence of rORF2 in infected cells through mass spectrometry analysis. The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes. The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase, and mutation of rORF2 completely abolishes virus infection, whereas ectopic expression of rORF2 rescues the mutant virus. Furthermore, we show that several rORFs in the -RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have the ability to suppress type I interferon production and facilitate the infection of vesicular stomatitis virus. In addition, we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs. Taken together, these findings indicate that the -RNA of +ssRNA viruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection, revealing a viral proteome larger than previously thought.

Quantitative Analysis of RNA Polymerase Slippages for Production of P3N-PIPO Trans-frame Fusion Proteins in Potyvirids.

Potyvirids, members of the family Potyviridae, produce the P3N-PIPO protein, which is crucial for the cell-to-cell transport of viral genomic RNAs. The production of P3N-PIPO requires an adenine (A) insertion caused by RNA polymerase slippage at a conserved GAAAAAA (GA6) sequence preceding the PIPO open reading frame. Presently, the slippage rate of RNA polymerase has been estimated in only a few potyvirids, ranging from 0.8 to 2.1%. In this study, we analyzed publicly available plant RNA-seq data and identified 19 genome contigs from 13 distinct potyvirids. We further investigated the RNA polymerase slippage rates at the GA6 motif. Our analysis revealed that the frequency of the A insertion variant ranges from 0.53 to 4.07% in 11 potyviruses (genus Potyvirus). For the two macluraviruses (genus Macluravirus), the frequency of the A insertion variant was found to be 0.72% and 10.96% respectively. Notably, the estimated RNA polymerase slippage rates for 12 out of the 13 investigated potyvirids were reported for the first time in this study. Our findings underscore the value of plant RNA-seq data for quantitative analysis of potyvirid genome variants, specifically at the GA6 slippage site, and contribute to a more comprehensive understanding of the RNA polymerase slippage phenomenon in potyvirids.

The Temporal and Geographical Dynamics of Potato Virus Y Diversity in Russia.

Potato virus Y, an important viral pathogen of potato, has several genetic variants and geographic distributions which could be affected by environmental factors, aphid vectors, and reservoir plants. PVY is transmitted to virus-free potato plants by aphids and passed on to the next vegetative generations through tubers, but the effects of tuber transmission in PVY is largely unknown. By using high-throughput sequencing, we investigated PVY populations transmitted to potato plants by aphids in different climate zones of Russia, namely the Moscow and Astrakhan regions. We analyzed sprouts from the tubers produced by field-infected plants to investigate the impact of tuber transmission on PVY genetics. We found a significantly higher diversity of PVY isolates in the Astrakhan region, where winters are shorter and milder and summers are warmer compared to the Moscow region. While five PVY types, NTNa, NTNb, N:O, N-Wi, and SYR-I, were present in both regions, SYRI-II, SYRI-III, and 261-4 were only found in the Astrakhan region. All these recombinants were composed of the genome sections derived from PVY types O and N, but no full-length sequences of such types were present. The composition of the PVY variants in the tuber sprouts was not always the same as in their parental plants, suggesting that tuber transmission impacts PVY genetics.

Sword Bean (Canavalia gladiata): a new host of Bean common mosaic virus.

BACKGROUND: Sword bean (Canavalia gladiata) is an underutilized legume that has the potential to become an important food source owing to its wide range of nutritional and medicinal properties. In May 2023, symptoms induced by a possible virus infection such as mosaic, mottling and vein banding were observed on the leaves of about 20% of the Sword bean plants growing at the experimental research farm of the Indian Agricultural Research Institute in Pune, Maharashtra, India. METHODS AND RESULTS: Symptomatic and asymptomatic samples were screened by ELISA for the presence of Potyvirus, Cucumber mosaic virus and Tobacco mosaic virus. All symptomatic samples tested positive for Potyvirus in ELISA as well as in RT-PCR assay using the universal potyvirus primer pair (CPUP /P9502) which amplify c. 700 bp of the partial coat protein region and 3'UTR. Asymptomatic samples tested negative for all tested viruses in both serological and molecular assays. BLASTn sequence analysis of the amplicons revealed that the sequence shares more than 98% identity with an Indian isolate of Bean common mosaic virus (BCMV). Sequence analysis enabled the identification of the Potyvirus as BCMV. Furthermore, the present Sword bean isolate clustered with other BCMV isolates in the phylogenetic analysis. CONCLUSION: In the present study, BCMV was found to be naturally infecting Sword bean for the first time in the world. This is of epidemiological importance, as BCMV is known to cause significant yield losses in legumes and could severely hamper Sword bean production.

Cas13a-based multiplex RNA targeting for potato virus Y.

MAIN CONCLUSION: The Cas13a-based multiplex RNA targeting system can be engineered to confer resistance to RNA viruses, whereas the number and expression levels of gRNAs have no significant effect on viral interference. The CRISPR-Cas systems provide adaptive immunity to bacterial and archaeal species against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However, whether the number and expression levels of guide RNAs (gRNAs) have effects on the efficiency of RNA virus inhibition is unknown. Here, we repurpose CRISPR/Cas13a in combination with an endogenous tRNA-processing system (polycistronic tRNA-gRNA) to target four genes of potato virus Y (PVY) with varying expression levels. We expressed Cas13a and four different gRNAs in potato lines, and the transgenic plants expressing multiple gRNAs displayed similar suppression of PVY accumulation and reduced disease symptoms as those expressing a single gRNA. Moreover, PTG/Cas13a-transformed plants with different expression levels of multiple gRNAs displayed similar resistance to PVY strains. Collectively, this study suggests that the Cas13a-based multiplex RNA targeting system can be utilized to engineer resistance to RNA viruses in plants, whereas the number and expression levels of gRNAs have no significant effect on CRISPR/Cas13a-mediated viral interference in plants.

The mystery remains: How do potyviruses move within and between cells?

The genus Potyvirus is considered as the largest among plant single-stranded (positive-sense) RNA viruses, causing considerable economic damage to vegetable and fruit crops worldwide. Through the coordinated action of four viral proteins and a few identified host factors, potyviruses exploit the endomembrane system of infected cells for their replication and for their intra- and intercellular movement to and through plasmodesmata (PDs). Although a significant amount of data concerning potyvirus movement has been published, no synthetic review compiling and integrating all information relevant to our current understanding of potyvirus transport is available. In this review, we highlight the complexity of potyvirus movement pathways and present three potential nonexclusive mechanisms based on (1) the use of the host endomembrane system to produce membranous replication vesicles that are targeted to PDs and move from cell to cell, (2) the movement of extracellular viral vesicles in the apoplasm, and (3) the transport of virion particles or ribonucleoprotein complexes through PDs. We also present and discuss experimental data supporting these different models as well as the aspects that still remain mostly speculative.