Revisiting a pollen-transmitted ilarvirus previously associated with angular mosaic of grapevine.

We report the characterization of a novel tri-segmented RNA virus infecting Mercurialis annua, a common crop weed and model species in plant science. The virus, named "Mercurialis latent virus" (MeLaV) was first identified in a mixed infection with the recently described Mercurialis orthotospovirus 1 (MerV1) on symptomatic plants grown in glasshouses in Lausanne (Switzerland). Both viruses were found to be transmitted by Thrips tabaci, which presumably help the inoculation of infected pollen in the case of MeLaV. Complete genome sequencing of the latter revealed a typical ilarviral architecture and close phylogenetic relationship with members of the Ilarvirus subgroup 1. Surprisingly, a short portion of MeLaV replicase was found to be identical to the partial sequence of grapevine angular mosaic virus (GAMV) reported in Greece in the early 1990s. However, we have compiled data that challenge the involvement of GAMV in angular mosaic of grapevine, and we propose alternative causal agents for this disorder. In parallel, three highly-conserved MeLaV isolates were identified in symptomatic leaf samples in The Netherlands, including a herbarium sample collected in 1991. The virus was also traced in diverse RNA sequencing datasets from 2013 to 2020, corresponding to transcriptomic analyses of M. annua and other plant species from five European countries, as well as metaviromics analyses of bees in Belgium. Additional hosts are thus expected for MeLaV, yet we argue that infected pollen grains have likely contaminated several sequencing datasets and may have caused the initial characterization of MeLaV as GAMV.

Editing melon eIF4E associates with virus resistance and male sterility.

The cap-binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap-dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single-nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non-transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non-mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild-type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild-type. An RNA-Seq analysis identified critical genes in pollen development that were down-regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E-specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

Autoxidation Products of the Methanolic Extract of the Leaves of Combretum micranthum Exert Antiviral Activity against Tomato Brown Rugose Fruit Virus (ToBRFV).

Tomato brown rugose fruit virus (ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of C. micranthum, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Phylogenetics and Evolution of Potato Virus V: Another Potyvirus that Originated in the Andes.

Potato virus V (PVV) causes a disease of potato (Solanum tubersosum) in South and Central America, Europe, and the Middle East. We report here the complete genomic sequences of 42 new PVV isolates from the potato's Andean domestication center in Peru and of eight historical or recent isolates from Europe. When the principal open reading frames of these genomic sequences together with those of nine previously published genomic sequences were analyzed, only two from Peru and one from Iran were found to be recombinant. The phylogeny of the 56 nonrecombinant open reading frame sequences showed that the PVV population had two major phylogroups, one of which formed three minor phylogroups (A1 to A3) of isolates, all of which are found only in the Andean region of South America (Peru and Colombia), and the other formed two minor phylogroups, a basal one of Andean isolates (A4) that is paraphyletic to a crown cluster containing all the isolates found outside South America (World). This suggests that PVV originated in the Andean region, with only one minor phylogroup spreading elsewhere in the world. In minor phylogroups A1 and A3, there were two subclades on long branches containing isolates from S. phureja evolving more rapidly than the others, and these interfered with dating calculations. Although no temporal signal was directly detected among the dated nonrecombinant sequences, PVV and potato virus Y (PVY) are from the same potyvirus lineage and are ecologically similar, so "subtree dating" was done via a single maximum likelihood phylogeny of PVV and PVY sequences, and PVY's well-supported 157 ce "time to most common recent ancestor" was extrapolated to date that of PVV as 29 bce. Thus the independent historical coincidences supporting the datings of the PVV and PVY phylogenies are the same; PVV arose ≥2,000 years ago in the Andes and was taken to Europe during the Columbian Exchange, where it diversified around 1853 ce, soon after the European potato late blight pandemic. PVV is likely to be more widespread than currently realized and is of biosecurity relevance for world regions that have not yet recorded its presence.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of a Novel Geminivirus in Fraxinus rhynchophylla in Korea.

Fraxinus rhynchophylla, common name ash, belongs to the family Oleaceae and is found in China, Korea, North America, the Indian subcontinent, and eastern Russia. It has been used as a traditional herbal medicine in Korea and various parts of the world due to its chemical constituents. During a field survey in March 2019, mild vein thickening (almost negligible) was observed in a few ash trees. High-throughput sequencing of libraries of total DNA from ash trees, rolling-circle amplification (RCA), and polymerase chain reaction (PCR) allowed the identification of a Fraxinus symptomless virus. This virus has five confirmed open reading frames along with a possible sixth open reading frame that encodes the movement protein and is almost 2.7 kb in size, with a nonanucleotide and stem loop structure identical to begomoviruses. In terms of its size and structure, this virus strongly resembles begomoviruses, but does not show any significant sequence identity with them. To confirm movement of the virus within the trees, different parts of infected trees were examined, and viral movement was successfully observed. No satellite molecules or DNA B were identified. Two-step PCR confirmed the virion and complementary strands during replication in both freshly collected infected samples of ash tree and Nicotiana benthamiana samples agro-inoculated with infectious clones. This taxon is so distantly grouped from other known geminiviruses that it likely represents a new geminivirus genus.

Potato leafroll virus reduces Buchnera aphidocola titer and alters vector transcriptome responses.

Viruses in the Luteoviridae family, such as Potato leafroll virus (PLRV), are transmitted by aphids in a circulative and nonpropagative mode. This means the virions enter the aphid body through the gut when they feed from infected plants and then the virions circulate through the hemolymph to enter the salivary glands before being released into the saliva. Although these viruses do not replicate in their insect vectors, previous studies have demonstrated viruliferous aphid behavior is altered and the obligate symbiont of aphids, Buchnera aphidocola, may be involved in transmission. Here we provide the transcriptome of green peach aphids (Myzus persicae) carrying PLRV and virus-free control aphids using Illumina sequencing. Over 150 million paired-end reads were obtained through Illumina sequencing, with an average of 19 million reads per library. The comparative analysis identified 134 differentially expressed genes (DEGs) between the M. persicae transcriptomes, including 64 and 70 genes that were up- and down-regulated in aphids carrying PLRV, respectively. Using functional classification in the GO databases, 80 of the DEGs were assigned to 391 functional subcategories at category level 2. The most highly up-regulated genes in aphids carrying PLRV were cytochrome p450s, genes related to cuticle production, and genes related to development, while genes related to heat shock proteins, histones, and histone modification were the most down-regulated. PLRV aphids had reduced Buchnera titer and lower abundance of several Buchnera transcripts related to stress responses and metabolism. These results suggest carrying PLRV may reduce both aphid and Buchnera genes in response to stress. This work provides valuable basis for further investigation into the complicated mechanisms of circulative and nonpropagative transmission.

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe.

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen-alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.

Characterization of the Mycovirome from the Plant-Pathogenic Fungus Cercospora beticola.

Cercospora leaf spot (CLS) caused by Cercospora beticola is a devastating foliar disease of sugar beet (Beta vulgaris), resulting in high yield losses worldwide. Mycoviruses are widespread fungi viruses and can be used as a potential biocontrol agent for fugal disease management. To determine the presence of mycoviruses in C. beticola, high-throughput sequencing analysis was used to determine the diversity of mycoviruses in 139 C. beticola isolates collected from major sugar beet production areas in China. The high-throughput sequencing reads were assembled and searched against the NCBI database using BLASTn and BLASTx. The results showed that the obtained 93 contigs were derived from eight novel mycoviruses, which were grouped into 3 distinct lineages, belonging to the families Hypoviridae, Narnaviridae and Botourmiaviridae, as well as some unclassified (-)ssRNA viruses in the order Bunyavirales and Mononegavirales. To the best of our knowledge, this is the first identification of highly diverse mycoviruses in C. beticola. The novel mycoviruses explored in this study will provide new viral materials to biocontrol Cercospora diseases. Future studies of these mycoviruses will aim to assess the roles of each mycovirus in biological function of C. beticola in the future.

Pepper Mottle Virus and Its Host Interactions: Current State of Knowledge.

Pepper mottle virus (PepMoV) is a destructive pathogen that infects various solanaceous plants, including pepper, bell pepper, potato, and tomato. In this review, we summarize what is known about the molecular characteristics of PepMoV and its interactions with host plants. Comparisons of symptom variations caused by PepMoV isolates in plant hosts indicates a possible relationship between symptom development and genetic variation. Researchers have investigated the PepMoV-plant pathosystem to identify effective and durable genes that confer resistance to the pathogen. As a result, several recessive pvr or dominant Pvr resistance genes that confer resistance to PepMoV in pepper have been characterized. On the other hand, the molecular mechanisms underlying the interaction between these resistance genes and PepMoV-encoded genes remain largely unknown. Our understanding of the molecular interactions between PepMoV and host plants should be increased by reverse genetic approaches and comprehensive transcriptomic analyses of both the virus and the host genes.

Ten new nortriterpenes from Euphorbia resinifera and their anti-tomato yellow leaf curl virus activities.

Ten new nortriterpenes, euphorbiumrins A-J (1-10), together with three known analogues (11-13) were isolated from the latex of Euphorbia resinifera. Their structures were established on the basis of extensive spectroscopic analyses (IR, UV, HRESIMS, 1D and 2D NMR). Their inhibitions on tomato yellow leaf curl virus (TYLCV) were evaluated and compound 5 exhibited significant anti-TYLCV activity with an inhibition rate of 71.7% at concentration of 40 µg/mL.

One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y.

BACKGROUND: Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine. RESULTS: We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay. CONCLUSION: This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

The Resistance Responses of Potato Plants to Potato Virus Y Are Associated with an Increased Cellular Methionine Content and an Altered SAM:SAH Methylation Index.

Plant-virus interactions are frequently influenced by elevated temperature, which often increases susceptibility to a virus, a scenario described for potato cultivar Chicago infected with potato virus Y (PVY). In contrast, other potato cultivars such as Gala may have similar resistances to PVY at both normal (22 °C) and high (28 °C) temperatures. To elucidate the mechanisms of temperature-independent antivirus resistance in potato, we analysed responses of Gala plants to PVY at different temperatures using proteomic, transcriptional and metabolic approaches. Here we show that in Gala, PVY infection generally upregulates the accumulation of major enzymes associated with the methionine cycle (MTC) independently of temperature, but that temperature (22 °C or 28 °C) may finely regulate what classes accumulate. The different sets of MTC-related enzymes that are up-regulated at 22 °C or 28 °C likely account for the significantly increased accumulation of S-adenosyl methionine (SAM), a key component of MTC which acts as a universal methyl donor in methylation reactions. In contrast to this, we found that in cultivar Chicago, SAM levels were significantly reduced which correlated with the enhanced susceptibility to PVY at high temperature. Collectively, these data suggest that MTC and its major transmethylation function determines resistance or susceptibility to PVY.

Virus and helper component interactions favour the transmission of recombinant potato virus Y strains.

In recent years, several recombinant strains of potato virus Y, notably PVYNTN and PVYN:O have displaced the ordinary strain, PVYO, and emerged as the predominant strains affecting the USA potato crop. Previously we reported that recombinant strains were transmitted more efficiently than PVYO when they were acquired sequentially, regardless of acquisition order. In another recent study, we showed that PVYNTN binds preferentially to the aphid stylet over PVYO when aphids feed on a mixture of PVYO and PVYNTN. To understand the mechanism of this transmission bias as well as preferential virus binding, we separated virus and active helper component proteins (HC), mixed them in homologous and heterologous combinations, and then fed them to aphids using Parafilm sachets. Mixtures of PVYO HC with either PVYN:O or PVYNTN resulted in efficient transmission. PVYN:O HC also facilitated the transmission of PVYO and PVYNTN, albeit with reduced efficiency. PVYNTN HC failed to facilitate transmission of either PVYO or PVYN:O. When PVYO HC or PVYN:O HC was mixed with equal amounts of the two viruses, both viruses in all combinations were transmitted at high efficiencies. In contrast, no transmission occurred when combinations of viruses were mixed with PVYNTN HC. Further study evaluated transmission using serial dilutions of purified virus mixed with HCs. While PVYNTN HC only facilitated the transmission of the homologous virus, the HCs of PVYO and PVYN:O facilitated the transmission of all strains tested. This phenomenon has likely contributed to the increase in the recombinant strains affecting the USA potato crop.

Molecular insights on potato yellow vein crinivirus infections in the highlands of Colombia.

Potato yellow vein virus (PYVV) was detected in potatoes grown in the Central highlands, north of Bogotá (~3000 m altitude), Colombia. At this altitude viral whitefly vectors are largely absent, but infection persists because of the use of uncertified tubers. Plants with typical PYVV-induced yellowing symptoms, as well as with atypical yellowing or non-symptomatic symptoms were sampled at three separate geographical locations. PYVV presence was assessed by RT-PCR, and several plants were subjected to high-throughput sequencing (HTS) of their small RNA (sRNA) populations. Complete or almost complete sequences of four PYVV isolates were thus reconstructed, all from symptomatic plants. Three viral isolates infected plants singly, while the fourth co-infected the plant together with a potyvirus. Relative proportions of sRNAs to each of the three crinivirus genomic RNAs were found to remain comparable among the four infections. Genomic regions were identified as hotspots of sRNA formation, or as regions that poorly induced sRNAs. Furthermore, PYVV titres in the mixed versus single infections remained comparable, indicating an absence of synergistic/antagonistic effects of the potyvirus on the accumulation of PYVV. Daughter plants raised in the greenhouse from tubers of the infected, field-sampled plants displayed mild PYVV infection symptoms that disappeared with time, demonstrating the occurrence of recovery and asymptomatic infection phenotypes in this pathosystem.

A new Streptomyces scabies-infecting bacteriophage from Egypt with promising biocontrol traits.

Potato common scab caused by Streptomyces scabies is one of the most economically important diseases infecting potato. It reduces the quality of potato tubers, which subsequently decreases the tuber prices and causes significant economic losses for potato growers. Biological control using bacteriophages is a promising strategy for controlling this disease. In this study, a novel bacteriophage with high lytic efficacy against S. scabies was isolated from a potato field at El-Minya, Egypt, and was designated SscP1EGY. The phage has an icosahedral head of 55 nm and a short tail of 7.5 nm, typical of a podovirus. Its infection cycle was 90 min, including 50 min of latent time and 40 min of rise period with a burst size of approximately 200 PFU per infected cell. The genome of SscP1EGY consists 51,751 nucleotides with 76 predicted genes. SscP1EGY infected and completely lysed seven tested S. scabies strains but showed no lytic activity against three beneficial Streptomyces species, other beneficial bacterial species, and non-target plant pathogenic bacteria. In greenhouse experiments, treatment of S. scabies-inoculated potato tubers with phage SscP1EGY resulted in reductions of (1) the severity of scab, (2) the number of lesions, and (3) the percentage of lesion surface, as compared to the inoculated tubers without phage treatment. Also, scab lesions appeared superficial in phage-treated tubers but pitted in non-phage-treated tubers. Our results suggest that SscP1EGY has a potential as a biological control agent for S. scabies. Based on our knowledge, SscP1EGY is the first sequenced S. scabies-infecting phage in Egypt.

The Beta vulgaris-derived resistance gene Rz2 confers broad-spectrum resistance against soilborne sugar beet-infecting viruses from different families by recognizing triple gene block protein 1.

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.

The Phylogeography of Potato Virus X Shows the Fingerprints of Its Human Vector.

Potato virus X (PVX) occurs worldwide and causes an important potato disease. Complete PVX genomes were obtained from 326 new isolates from Peru, which is within the potato crop's main domestication center, 10 from historical PVX isolates from the Andes (Bolivia, Peru) or Europe (UK), and three from Africa (Burundi). Concatenated open reading frames (ORFs) from these genomes plus 49 published genomic sequences were analyzed. Only 18 of them were recombinants, 17 of them Peruvian. A phylogeny of the non-recombinant sequences found two major (I, II) and five minor (I-1, I-2, II-1, II-2, II-3) phylogroups, which included 12 statistically supported clusters. Analysis of 488 coat protein (CP) gene sequences, including 128 published previously, gave a completely congruent phylogeny. Among the minor phylogroups, I-2 and II-3 only contained Andean isolates, I-1 and II-2 were of both Andean and other isolates, but all of the three II-1 isolates were European. I-1, I-2, II-1 and II-2 all contained biologically typed isolates. Population genetic and dating analyses indicated that PVX emerged after potato's domestication 9000 years ago and was transported to Europe after the 15th century. Major clusters A-D probably resulted from expansions that occurred soon after the potato late-blight pandemic of the mid-19th century. Genetic comparisons of the PVX populations of different Peruvian Departments found similarities between those linked by local transport of seed potato tubers for summer rain-watered highland crops, and those linked to winter-irrigated crops in nearby coastal Departments. Comparisons also showed that, although the Andean PVX population was diverse and evolving neutrally, its spread to Europe and then elsewhere involved population expansion. PVX forms a basal Potexvirus genus lineage but its immediate progenitor is unknown. Establishing whether PVX's entirely Andean phylogroups I-2 and II-3 and its Andean recombinants threaten potato production elsewhere requires future biological studies.

Transcriptional Regulatory Networks Associate with Early Stages of Potato Virus X Infection of Solanum tuberosum.

Potato virus X (PVX) belongs to genus Potexvirus. This study characterizes the cellular transcriptome responses to PVX infection in Russet potato at 2 and 3 days post infection (dpi). Among the 1242 differentially expressed genes (DEGs), 268 genes were upregulated, and 37 genes were downregulated at 2 dpi while 677 genes were upregulated, and 265 genes were downregulated at 3 dpi. DEGs related to signal transduction, stress response, and redox processes. Key stress related transcription factors were identified. Twenty-five pathogen resistance gene analogs linked to effector triggered immunity or pathogen-associated molecular pattern (PAMP)-triggered immunity were identified. Comparative analysis with Arabidopsis unfolded protein response (UPR) induced DEGs revealed genes associated with UPR and plasmodesmata transport that are likely needed to establish infection. In conclusion, this study provides an insight on major transcriptional regulatory networked involved in early response to PVX infection and establishment.

In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus RNA Silencing Suppressor, a Viral RNase III.

Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.