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.

Production of Double-Stranded RNA in Planta by a Potato Mop-Top Virus (PMTV)-Based Vector for Inducing Gene Silencing.

RNA silencing (also known as gene silencing) is an evolutionary conserved mechanism that is involved in regulating gene expression, suppressing mobile elements, and defensing virus infection. RNA silencing is triggered by double-stranded RNA via Dicer or Dicer-like riboendonucleases. DsRNAs are also the replication intermediates of all RNA viruses; as a result, plant RNA viruses are ideal candidates to induce RNA silencing. A large body of plant viruses have been modified into vectors for RNA silencing in varied plant species. Here, we described a simple, time-saving, and operable system for gene function and genetic breeding study of potato and Nicotiana benthamiana using a potato mop-top (MPTV)-based vector.

Potato Virus Y Strain N-Wi Offers Cross-Protection in Potato Against Strain NTN-NW by Superior Competition.

Potato virus Y (PVY) is one of the most economically important pathogens of potato. PVY exhibits different phenotypes in dissimilar potato cultivars. Previously, we observed that two recombinant isolates, PVYN-Wi-HLJ-BDH-2 (BDH) and PVYNTN-NW(SYR-II)-INM-W-369-12 (369), exhibited different virulence levels in potato cultivar Kexin 13 despite high genome sequence identity. Indeed, 369 induced severe necrosis and plant death in severe cases in Kexin 13 and severe mosaic in cultivar Yanshu 8, whereas BDH caused mainly mosaic symptoms on the plants of both cultivars. We hypothesized that preinfection of plants with BDH could cross-protect them from 369 infection, and not vice versa. Challenge inoculation, either by mechanical wounding or through grafting, with 369 on plants that were preinfected with BDH did not augment the symptom expression in both cultivars. Reverse transcription quantitative PCR analysis showed that, after challenge inoculation with 369, the titer of the isolate on BDH-preinfected plants remained at a low level (about 3 × 104 copy/µl) during the tested time course (0 h to 30 days). In contrast, in plants that were preinoculated with buffer (mock) and challenge inoculated with 369, the titer of 369 increased continuously until reaching its highest level of about 2 × 107 (Yanshu 8) and about 4 × 108 (Kexin 13) during the time course. Surprisingly, in plants that were preinfected with 369 and challenge inoculated with BDH, the accumulation of BDH reached nearly the same level as that in plants that were preinoculated with buffer and challenge inoculated with BDH. Taken together, these results suggest that PVYN-Wi mediated cross-protection against PVYNTN-NW(SYR-II) by superior competition and better fitness.

Evolutionary history and global spatiotemporal pattern of alfalfa mosaic virus.

Alfalfa mosaic virus (AMV) is an important plant virus causing considerable economic loss to alfalfa production. Knowledge of the evolutionary and demographic history of the pathogen is limited but essential to the development of effective and sustainable pathogen management schemes. In this study, we performed worldwide phylodynamic analyses of AMV based on 154 nucleotide sequences of the coat protein gene, sampled from 1985 to 2020, to understand the epidemiology of this pathogen. Bayesian phylogenetic reconstruction estimates that the crown group of AMV dates back to 1840 (95% credibility interval, 1687-1955). We revealed that AMV continuously evolves at a rate of 4.14 × 10-4 substitutions/site/year (95% credibility interval, 1.04 × 10-4 - 6.68 × 10-4). Our phylogeographic analyses identified multiple migration links between Europe and other regions, implying that Europe played a key role in spreading the virus worldwide. Further analyses showed that the clustering pattern of AMV isolates is significantly correlated to geographic regions, indicating that geography-driven adaptation may be a factor that affects the evolution of AMV. Our findings may be potentially used in the development of effective control strategies for AMV.

Geography-Driven Evolution of Potato Virus A Revealed by Genetic Diversity Analysis of the Complete Genome.

Potato virus A (PVA), a member of the genus Potyvirus, is an important potato pathogen that causes 30%-40% yield reduction to global potato production. Knowledge on the genetic structure and the evolutionary forces shaping the structure of this pathogen is limited but vital in developing effective management strategies. In this study, we investigated the population structure and molecular evolution of PVA by analyzing novel complete genomic sequences from Chinese isolates combined with available sequences from Europe, South America, Oceania, and North America. High nucleotide diversity was discovered among the populations studied. Pairwise F ST values between geographical populations of PVA ranged from 0.22 to 0.46, indicating a significant spatial structure for this pathogen. Although purifying selection was detected at the majority of polymorphic sites, significant positive selection was identified in the P1, NIa, and NIb proteins, pointing to adaptive evolution of PVA. Further phylogeny-trait association analysis showed that the clustering of PVA isolates was significantly correlated with geographic regions, suggesting that geography-driven adaptation may be an important determinant of PVA diversification.

Potato Tuber Necrosis Induced by Alfalfa Mosaic Virus Depends on Potato Cultivar Rather Than on Virus Strain.

Alfalfa mosaic virus (AMV) was identified as the causal agent of internal tuber necrosis in the potato cultivar Innovator in New Brunswick, Canada. Further pathological characterization of the isolate (designated as isolate CaM) was performed on six potato cultivars and one breeding clone. Upon mechanical inoculation, four cultivars (Innovator, Yukon Gold, Rochdale Gold-Dorée, and Shepody) showed needle-sized necrotic spots and increasing calico symptoms on new leaves, whereas the remaining cultivars only developed calico symptoms on new leaves. All tubers of CaM-infected Innovator and Shepody plants developed sporadic internal necrotic spots, as did ca. 23 and 8% tubers of CaM-infected Yukon Gold and Rochdale Gold-Dorée, respectively. Sequence analysis of the CP gene of CaM with AMV isolates from potato, all presumed belonging to the "non-necrotic" strain and retrieved from GenBank, indicated that CaM shared >97.1% sequence identity with all but four Egyptian isolates. At the complete genome level, phylogenetic analysis of all available sequences demonstrated that RNA 1 and RNA 3 can be grouped into three major clades each, whereas RNA 2 can be clustered into two clades. CaM and Ca175-1, an AMV isolate that was deemed non-necrotic in a previous study, had different phylogenetic clade patterns, indicating different RNA 1-RNA 2-RNA 3 haplotypes: IA-I-IB (CaM) versus Ca175-1 (IB-II-IA). Despite the difference in haplotype composition, CaM and Ca175-1 induced similar levels of internal necrosis in tubers of Innovator and its parent Shepody. The results suggest that the internal necrosis in AMV-infected tubers depends on potato cultivar rather than on AMV strain/haplotype, and CaM is just a "regular" isolate of AMV.

Molecular Evolutionary Analysis of Potato Virus Y Infecting Potato Based on the VPg Gene.

Potato virus Y (PVY) is an important plant pathogen infecting solanaceous crops, causing significant losses to global potato and tobacco production. Some aspects of the plant pathology and molecular biology of PVY have been studied intensively, but the evolutionary dynamics of this virus are poorly understood. Here, we performed a comprehensive set of rigorous evolutionary analyses using 177 nucleotide sequences of the viral genome linked protein (VPg) gene, which interacts with the plant eukaryotic translation initiation factor 4E (eIF4E). Our Bayesian analysis reveals that the VPg gene of PVY has been evolving at a rate of 5.60 × 10-4 subs/site/year (95% credibility interval 3.35 × 10-4-8.17 × 10-4), which is equivalent to those of other plant-infecting RNA viruses. We identified different evolutionary constraints on the two clades of PVY, clade N and clade O, whose diverge time were estimated at the year 1861 CE (95% credibility interval 1750-1948 CE). We also found that genetic variations were correlated with geographic regions, suggesting that the evolution of this pathogen is strongly affected by geographical associated factors. Taken together, the results of our study have potential implications for the control strategies of PVY.

Genetic Diversity of Potato virus Y in Potato Production Areas in Northeast China.

In 2011-2014, ELISA or nucleic acid spot hybridization (NASH) testing for common potato viruses or Potato spindle tuber viroid (PSTVd) was performed on 500 leaf samples collected in potato fields in the northeast provinces Heilongjiang and Inner Mongolia, China. The results revealed that 38.4% (Heilongjiang) and 27.7% (Inner Mongolia) were positive for Potato virus Y (PVY). To unveil the strain composition and population structure of PVY in the region, the multiplex RT-PCR described by Chikh-Ali et al. was performed on all of the ELISA-PVY-positive samples. Of the 158 samples whose PVY strain scenarios could be determined, PVYNTN-NW-SYR-II and PVYN-Wi were the most abundant strains, occurring in 58.9 and 47.5% samples, followed by PVYNTN-NW-SYR-I (31.0%), PVYN:O (19.6%), Eu-PVYNTN (7.6%), NA-PVYN (1.3%), and PVYO (0.6%). In the 84 single-strain-infected samples, PVYN-Wi accounted for 41.7%, PVYNTN-NW-SYR-II for 40.5%, PVYNTN-NW-SYR-I for 14.3%, and PVYN:O and Eu-PVYNTN for 3.6% each. Seven isolates representing PVYNTN-NW-SYR-I (HLJ-6-1 and HLJ-9-4), PVYNTN-NW-SYR-II (INM-W-369-12 and SC-1-1-2), PVYN:O (HLJ-30-2), and PVYN-Wi (HLJ-BDH-2 and HLJ-C-429) were sequenced and analyzed molecularly. Whereas the sequence identities for isolates belonging to the same strain group were >98.5%, they fell for isolates belonging to different strain groups to 92.7-98.1% at the genome level and 96.1-98.4% at the polyprotein level. Interestingly, the exact location of the recombination events varied among isolates within a strain group. Phylogenetic analysis of all 42 full length PVY sequences from China indicated that most clustered to various recombinant groups, despite the fact that the PVY isolates were isolated from at least five host species. Pathological analysis of four isolates representing PVYN:O, PVYN-Wi, PVYNTN-NW-SYR-I, and PVYNTN-NW-SYR-II revealed that the PVYNTN-NW-SYR-II isolate incited the most severe symptoms on potato cultivar Kexin 13, followed by PVYNTN-NW-SYR-I, PVYN:O and PVYN-Wi. The PVYNTN-NW-SYR-I and PVYNTN-NW-SYR-II isolates also caused necrotic ringspots on the tubers of Kexin 13, indicating their ability to induce the potato tuber necrotic ringspot disease in potato.

Synthesis and biological evaluation of nannocystin analogues toward understanding the binding role of the (2R,3S)-Epoxide in nannocystin A.

Nannocystin A is a potent antiproliferative cyclodepsipeptide targeting eukaryotic translation elongation factor 1α. To elucidate the binding role of its (2R,3S)-epoxide, we designed and synthesized a focused library of 10 nannocystin analogues. Variable temperature NMR experiments demonstrated the importance of the (2R,3S)-epoxide in controlling the macrocyclic conformation. Biological evaluation of these compounds against three typical cancer cell lines established a clear structure-activity relationship at the epoxide region, which was rationalized by comparing the superimposed conformations of different nannocystin analogues and in silico docking analysis. Our results showed that the (2R,3S)-epoxide of nannocystin A is mainly responsible for controlling the macrocyclic conformation, rather than binding directly to the target.

A Novel Recombined Potato virus Y Isolate in China.

This study reports the findings of a distinct Potato virus Y (PVY) isolate found in Northeast China. One hundred and ten samples (leaves and tubers) were collected from potato plants showing mosaic symptoms around the city of Harbin in Heilongjiang province of China. The collected tubers were planted and let to grow in a greenhouse. New potato plants generated from these tubers showed similar symptoms, except for one plant. Subsequent serological analyses revealed PVY as the causing agent of the disease. A novel PVY isolate (referred to as HLJ-C-44 in this study) was isolated from this sample showing unique mild mosaic and crisped leaf margin symptoms. The complete genome of this isolate was analyzed and determined. The results showed that HLJ-C-44 is a typical PVY isolate. Phylogenetic analysis indicated that this isolate belongs to the N-Wi strain group of PVY recombinants (PVYN-Wi) and also shared the highest overall sequence identity (nucleotide and amino acid) with other members of this strain group. However, recombination analysis of isolate HLJ-C-44 revealed a recombination pattern that differed from that of other PVYN-Wi isolates. Moreover, biological assays in four different potato cultivars and in Nicotiana tabacum also revealed a different phenotypic response than that of a typical PVYN-Wi isolate. This data, combined, suggest that HLJ-C-44 is a novel PVY recombinant with distinct biological properties.

[Characteristics of P1 gene of potato virus Y in Heilongjiang].

Objective: Based on different potato virus Y isolates gene sequencing, we studied the diversity of potato virus Y strains, to provide information for molecular detection, prevention and control of the virus. Methods: P1 gene of 15 samples of potato virus Y of Heilongjiang Province was cloned and then the sequences of genes were analyzed by using phylogenetic tree. Results: Samples were divided into two groups. According to a comparative analysis, 10 samples have highly conservative and homologous genes. They are the dominant population in the research area and have certain genetic distance to other domestic samples and foreign samples. In another group, 5 samples differ significantly with local dominant population in term of P1 gene. These 5 samples also have some differences and their P1 genes are close to those of other domestic samples and foreign samples. By comparing PVY strain data provided by uploaded sequences in GenBank, it found that P1 gene of test samples is similar with PVYNTN-NW strains. These 15 samples as well as other domestic samples are evolved from PVYN strains. Conclusions: The P1 gene analysis demonstrated that PVY is influenced by environment significantly and PVY of 10 samples in Heilongjiang develops local characteristics in the long-term evolution. The later 5 samples reflect that most PVY in China may be introduced by foreign cultivars. At the same time, PVY spreads through regional resource exchange and tuber transportation in China.

Development of universal primers for detection of potato carlaviruses by RT-PCR.

To facilitate efficient and accurate detection of potato-infecting carlaviruses, degenerated universal primers were designed based on conserved amino acid and nucleotide sequences. Two sense primers, Car-F1 and Car-F2, were based on the amino acid sequences "SNNMA" and "GLGVPTE", respectively, in the coat protein. The reverse primer, Car-R, which was located at the border of the nucleic acid binding protein gene and the 3' untranslated region, and dT-B, which was derived from the oligo-dT targeting the poly(A) tail, were selected. Successful application of fragments within the predicted size range of carlaviruses was obtained using Car-F1 paired with either Car-R or dT-B from tested carlaviruses (Potato virus S, M and latent) by RT-PCR. The Car-F2 failed to yield clear-cut fragments within the predicted size range when paired with either Car-R or dT-B in RT-PCR. However, a less degenerated version of the primer, Car-F2b, resulted in amplicons within the predicted size range when paired with either Car-R or dT-B. Sequencing of the tentative carlavirus-fragments resulting from Car-F1/Car-R and Car-F2b/dT-B proved their carlavirus-origin, thus indicating the high specificity of these primers. The sensitivity of Car-F1/Car-R or Car-F2b/Car-R mediated RT-PCR for the detection of carlavirus-infected potato tubers were assessed using composite samples containing one carlavirus-infected-potato-tuber RNA sample with up to 49 virus-free-potato-tuber RNA samples under the optimal annealing temperature. The target carlaviruses were detected readily from all composites, demonstrating a high sensitivity. The method was further evaluated using presumed virus-free or carlavirus-infected potatoes of several cultivars, and reliable results were obtained.