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.

Nanobiotechnological Approaches to Enhance Potato Resistance against Potato Leafroll Virus (PLRV) Using Glycyrrhizic Acid Ammonium Salt and Salicylic Acid Nanoparticles

The present research was aiming to study In-Silico the effect of Glycyrrhizic Acid ammonium salt (GAS) and Salicylic acid (SA) on the coat protein of potato leafroll virus (PLRV). In addition, in-vitro studying the effect of (GAS NPs) and Salicylic acid (SA NPs) nanoparticles at concentrations 0.15, 0.30, 1.25 and 2.5 mM, respectively, to control, decline or reduce the presence of PLRV in potato plants Solanum tuberosum L. selena. (GAS NPs) and (SA NPs) were applied in the MS medium at concentrations 0.15, 0.30, 1.25 and 2.5 mM, respectively. Results revealed that, enhancement or decline the PLRV according to the initiation of specific pathways. The expression level of Kinase 3 gene increased significantly due to the two used concentrations of GAS NPs. While the expression of callose gene was upregulated significantly in response to treatment of PLRV infected plant with (GAS NPs) with concentration (0.30 mM). Treatment with (SA NPs) caused upregulation significance only of callose gene at (2.5 mM) concentration. The molecular modeling results of used compounds (glycyrrhizic acid ammonium salt and salicylic acid) showed highest score of binding and the best rms define value with a very good binding mode and perfect interactions with amino acids of the three subunits (A, B and C) forming the protein coat of leaf roll virus. Glycyrrhizic acid ammonium salt and salicylic acid nanoparticles could be perfect solution to produce potato plant free virus in-vitro. Further larger studies are needed to investigate the role of the studied compounds in vivo.

Crystal structure of the potato leafroll virus coat protein and implications for viral assembly

Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of staple food crops. Previous cryo-electron microscopy studies of virus-like particles indicate that luteovirid viral capsids are built from a structural coat protein that organizes with T=3 icosahedral symmetry. Here we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.57-Å resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation, suggesting that the CP N-terminus and its interactions with RNA serve as a key driver for generating capsid curvature.

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 green peach aphid, Myzus persicae, transcriptome in response to a circulative, nonpropagative polerovirus, Potato leafroll virus.

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 down- and up-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 histone 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 nonpropogative transmission.

Direct and Indirect Effect via Endophytism of Entomopathogenic Fungi on the Fitness of Myzus persicae and Its Ability to Spread PLRV on Tobacco

Aphids are major crop pests that transmit more than half of all insect-vectored plant viruses responsible for high yield losses worldwide. Entomopathogenic fungi (EPF) are biological control agents mainly used by foliar application to control herbivores, including sap-sucking pests such as aphids. Their ability to colonize plant tissues and to interact with diverse plant pathogenic microorganisms have been reported. In our study, we evaluated the effectiveness of Beauveria bassiana ((Balsamo-Crivelli) Vuillemin) directly applied by contact or/and indirectly via endophytism in tobacco plants (Nicotiana tabacum L.) against the virus vector Myzus persicae (Sulzer) carrying the Potato leafroll virus (PLRV) or not. We found that both contact treatment and endophytic colonization of leaves significantly increased aphid mortality and decreased the fecundity rate when compared to control plants. In addition, on fungal-colonized leaves, viruliferous aphids were more negatively impacted than virus-free ones and nymph mortality was significantly higher than on fungal-free plants. Furthermore, we assessed PLRV transmission by M. persicae on tobacco plants inoculated with either B. bassiana or Metarhizium acridum ((Driver and Milner) JF Bischoff, Rehner, and Humber) as source or/and recipient plants. Myzus persicae was found to acquire and transmit PLRV regardless of the treatment. Nevertheless, the infection rate of endophytically colonized plants was lower at a seven-day incubation period and had increased to almost 100% after fifteen days. These results suggest that B. bassiana is effective against aphids, both by contact and via endophytism, and both B. bassiana and M. acridum delayed PLRV infection in tobacco.