High-throughput RNA sequencing analysis of Mallotus japonicus revealed novel polerovirus and amalgavirus.

High-throughput RNA sequencing (RNA-seq) analysis of samples from Mallotus japonicus, a traditional medicinal plant, yielded two novel RNA viruses tentatively named Mallotus japonicus virus A (MjVA) and Mallotus japonicus virus B (MjVB). The MjVA and MjVB genomes encode proteins showing amino acid sequence similarities to those of poleroviruses (the genus Polerovirus, the family Solemoviridae) and amalgaviruses (the genus Amalgavirus, the family Amalgaviridae), respectively. The MjVA genome contains seven highly overlapping open reading frames, which are translated to seven proteins through various translational mechanisms, including -1 programmed ribosomal frameshifting (PRF) at the slippery motif GGGAAAC, non-AUG translational initiation, and stop codon readthrough. The MjVB genome encodes two proteins; one of which is translated by +1 PRF mechanism at the slippery motif UUUCGN. The abundance analysis of virus-derived RNA fragments revealed that MjVA is highly concentrated in plant parts with well-developed phloem tissues as previously demonstrated in other poleroviruses, which are transmitted by phloem feeders, such as aphids. MjVB, an amalgavirus generally transmitted by seeds, is distributed in all samples at low concentrations. Thus, this study demonstrates the effectiveness and usefulness of RNA-seq analysis of plant samples for the identification of novel RNA viruses and analysis of their tissue distribution. Keywords: Polerovirus; Amalgavirus; Mallotus japonicus; RNA virus; viral genome; programmed ribosomal frameshifting.

Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato.

Potatoes are developed vegetatively from tubers, and therefore potato virus transmission is always a possibility. The potato leafroll virus (PLRV) is a highly devastating virus of the genus Polerovirus and family Luteoviridae and is regarded as the second-most destructive virus after Potato virus Y. Multiple species of aphids are responsible for the persistent and non-propagating transmission of PLRV. Due to intrinsic tuber damage (net necrosis), the yield and quality are drastically diminished. PLRV is mostly found in phloem cells and in extremely low amounts. Therefore, we have attempted to detect PLRV in both potato tuber and leaves using a highly sensitive, reliable and cheap method of one-step reverse transcription-recombinase polymerase amplification (RT-RPA). In this study, an isothermal amplification and detection approach was used for efficient results. Out of the three tested primer sets, one efficiently amplified a 153-bp product based on the coat protein gene. In the present study, there was no cross-reactivity with other potato viruses and the optimal amplification reaction time was thirty minutes. The products of RT-RPA were amplified at a temperature between 38 and 42 °C using a simple heating block/water bath. The present developed protocol of one-step RT-RPA was reported to be highly sensitive for both leaves and tuber tissues equally in comparison to the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. By using template RNA extracted employing a cellular disc paper-based extraction procedure, the method was not only simplified but it detected the virus as effectively as purified total RNA. The simplified one-step RT-RPA test was proven to be successful by detecting PLRV in 129 samples of various potato cultivars (each consisting of leaves and tubers). According to our knowledge, this is the first report of a one-step RT-RPA performed using simple RNA extracted from cellular disc paper that is equally sensitive and specific for detecting PLRV in potatoes. In terms of versatility, durability and the freedom of a highly purified RNA template, the one-step RT-RPA assay exceeds the RT-PCR assay, making it an effective alternative for the certification of planting materials, breeding for virus resistance and disease monitoring.

A new simple and effective method for PLRV infection to screen for virus resistance in potato.

Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.

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.

Affinity Purification-Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency.

The vast majority of plant viruses are transmitted by insect vectors, with many crucial aspects of the transmission process being mediated by key protein-protein interactions. Still, very few vector proteins interacting with viruses have been identified and functionally characterized. Potato leafroll virus (PLRV) is transmitted most efficiently by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using affinity purification coupled to high-resolution mass spectrometry (AP-MS), we identified 11 proteins from M. persicaedisplaying a high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Three of these aphid proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV in cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Artificial diet delivery to aphids of a chemical inhibitor of C1QBP leads to increased PLRV acquisition by aphids and subsequently increased titer in inoculated plants, supporting a role for C1QBP in the acquisition and transmission efficiency of PLRV by M. persicae. This study presents the first use of AP-MS for the in vivo isolation of a functionally relevant insect vector-virus protein complex. MS data are available from ProteomeXchange.org using the project identifier PXD022167.

Complete genome sequence of artemisia virus B, a new polerovirus infecting Artemisia princeps in South Korea.

The complete genome sequence of a new polerovirus found naturally infecting Artemisia princeps, artemisia virus B (ArtVB), was determined using high-throughput sequencing. The ArtVB genome comprises 6,141 nucleotides and contains six putative open reading frames (ORF0 to ORF5) with a genome structure typical of poleroviruses. A multiple sequence alignment showed that the complete ArtVB genome shares 50.98% nucleotide sequence identity with ixeridium yellow mottle virus 1 (IxYMaV-1, GenBank accession no. KT868949). ArtVB shares the highest amino acid sequence identity in P0 and P3-P5 (21.54%-51.69%) with other known poleroviruses. Phylogenetic analysis indicated that ArtVB should be considered a member of a new species within the genus Polerovirus, family Luteoviridae.

A polerovirus, Potato leafroll virus, alters plant-vector interactions using three viral proteins.

Potato leafroll virus (PLRV), genus Polerovirus, family Luteoviridae, is a major pathogen of potato worldwide. PLRV is transmitted among host plants by aphids in a circulative-nonpropagative manner. Previous studies have demonstrated that PLRV infection increases aphid fecundity on, and attraction to, infected plants as compared to controls. However, the molecular mechanisms mediating this relationship are still poorly understood. In this study, we measured the impact of PLRV infection on plant-aphid interactions and plant chemistry in two hosts: Solanum tuberosum and Nicotiana benthamiana. Our study demonstrates that PLRV infection attenuates the induction of aphid-induced jasmonic acid and ethylene in S. tuberosum and N. benthamiana. Using transient expression experiments, insect bioassays and chemical analysis, we show that expression of three PLRV proteins (P0, P1, and P7) mediate changes in plant-aphid interactions and inhibition of aphid-induced jasmonic acid and ethylene in N. benthamiana. This study enhances our understanding of the plant-vector-pathogen interface by elucidating new mechanisms by which plant viruses transmitted in a circulative manner can manipulate plant hosts.

Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences.

Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.

The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection.

Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection.

Long-term preservation of potato leafroll virus, potato virus S, and potato spindle tuber viroid in cryopreserved shoot tips.

Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips (58-60%) than in 0.5-mm-ones (30-38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRV-preserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation, similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.

Biodiversity and full genome sequence of potato viruses Alfalfa mosaic virus and potato leaf roll virus in Egypt.

Solanum tuberosum (potato) is the second most important vegetable crop in Egypt. It is locally consumed, manufactured or supplied for export to Europe and other Arab countries. Potato is subject to infection by a number of plant viruses, which affect its yield and quality. Potato virus Y (PVY), potato leaf roll virus (PLRV), and Alfalfa mosaic virus (AMV) were detected in major potato-growing areas surveyed. Multiplex-RT-PCR assay was used for the detection of these three viruses in one reaction using three specific primer pairs designed to amplify genomic parts of each virus (1594 bp for PLRV, 795 bp for AMV, 801 bp for PVY). All three viruses were detected in a single reaction mixture in naturally infected field-grown potatoes. Multiplex RT-PCR improved sensitivity necessary for the early detection of infection. Incidence of single, double, or triple infection has been recorded in some locations. Full-length sequencing has been performed for an Egyptian FER isolate of PLRV. Through phylogenetic analysis, it was shown to occupy the same clade with isolate JokerMV10 from Germany. Complete nucleotide sequence of an Egyptian FER isolate of AMV and phylogenetic analysis was also performed; we propose that it is a new distinct strain of AMV belonging to a new subgroup IIC. This is the first complete nucleotide sequence of an Egyptian isolate of AMV. Genetic biodiversity of devastating potato viruses necessitates continuous monitoring of new genetic variants of such viruses.

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen.

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

Insights in luteovirid structural biology guided by chemical cross-linking and high resolution mass spectrometry.

Interactions among plant pathogenic viruses in the family Luteoviridae and their plant hosts and insect vectors are governed by the topology of the viral capsid, which is the sole vehicle for long distance movement of the viral genome. Previous application of a mass spectrometry-compatible cross-linker to preparations of the luteovirid Potato leafroll virus (PLRV; Luteoviridae: Polerovirus) revealed a detailed network of interactions between viral structural proteins and enabled generation of the first cross-linking guided coat protein models. In this study, we extended application of chemical cross-linking technology to the related Turnip yellows virus (TuYV; Luteoviridae: Polerovirus). Remarkably, all cross-links found between sites in the viral coat protein found for TuYV were also found in PLRV. Guided by these data, we present two models for the TuYV coat protein trimer, the basic structural unit of luteovirid virions. Additional cross-links found between the TuYV coat protein and a site in the viral protease domain suggest a possible role for the luteovirid protease in regulating the structural biology of these viruses.

Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi.

An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB.

Beet western yellows virus infects the carnivorous plant Nepenthes mirabilis.

Although poleroviruses are known to infect a broad range of higher plants, carnivorous plants have not yet been reported as hosts. Here, we describe the first polerovirus naturally infecting the pitcher plant Nepenthes mirabilis. The virus was identified through bioinformatic analysis of NGS transcriptome data. The complete viral genome sequence was assembled from overlapping PCR fragments and shown to share 91.1 % nucleotide sequence identity with the US isolate of beet western yellows virus (BWYV). Further analysis of other N. mirabilis plants revealed the presence of additional BWYV isolates differing by several insertion/deletion mutations in ORF5.

Phylogenetic relationships and the occurrence of interspecific recombination between beet chlorosis virus (BChV) and Beet mild yellowing virus (BMYV).

Samples containing two viruses belonging to the genus Polerovirus, beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV), were collected from French and Polish sugar beet fields. The molecular properties of 24 isolates of BChV and BMYV were investigated, and their genetic diversity was examined in the coat protein (CP)- and P0-encoding genes. For the first time, we have demonstrated that beet polerovirus populations include recombinants between BChV and BMYV containing breakpoints within the CP gene. Moreover, a partial correlation between geographic origin and phylogenetic clustering was observed for BMYV isolates.

Amino acid sequence motifs essential for P0-mediated suppression of RNA silencing in an isolate of potato leafroll virus from Inner Mongolia.

Polerovirus P0 suppressors of host gene silencing contain a consensus F-box-like motif with Leu/Pro (L/P) requirements for suppressor activity. The Inner Mongolian Potato leafroll virus (PLRV) P0 protein (P0(PL-IM)) has an unusual F-box-like motif that contains a Trp/Gly (W/G) sequence and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other P0 proteins. We used Agrobacterium infiltration-mediated RNA silencing assays to establish that P0(PL-IM) has a strong suppressor activity. Mutagenesis experiments demonstrated that the P0(PL-IM) F-box-like motif encompasses amino acids 76-LPRHLHYECLEWGLLCG THP-95, and that the suppressor activity is abolished by L76A, W87A, or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141 region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of P0(PL-IM). As has been observed with other P0 proteins, P0(PL-IM) suppression is correlated with reduced accumulation of the host AGO1-silencing complex protein. However, P0(PL-IM) fails to bind SKP1, which functions in a proteasome pathway that may be involved in AGO1 degradation. These results suggest that P0(PL-IM) may suppress RNA silencing by using an alternative pathway to target AGO1 for degradation. Our results help improve our understanding of the molecular mechanisms involved in PLRV infection.

Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye.

The most common virus affecting potatoes in the field worldwide is Potato Leafroll virus (PLRV), belonging to the family Luteoviridae, genius Plerovirus. There are several molecular methods to detect PLRV including polymerase chain reaction (PCR), Multiplex AmpliDet RNA and double antibody sandwich ELISA (DAS-ELISA). But these techniques take a long time for 3h to two days, requiring sophisticated tools. The aim of this study was to reduce the time required to detect PLRV, using a newly designed loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock. PLRV RNA was extracted from overall 80 infected naturally potato leaves. A set of six novel primers for the LAMP reaction was designed according to the highly conserved sequence of the viral coat protein (CP) gene. LAMP was carried out under isothermal conditions, applying the Bst DNA polymerase enzyme; the LAMP products were detected visually using the GeneFinder™ florescence dye. A positive result using the GeneFinder™ dye was a color change from the original orange to green. Results confirmed LAMP with GeneFinder™ provides a rapid and safe assay for detection of PLRV. Since with other molecular methods, equipping laboratories with a thermocycler or expensive detector systems is unavoidable, this assay was found to be a simple, cost-effective molecular method that has the potential to replace other diagnostic methods in primary laboratories without the need for expensive equipment or specialized techniques. It can also be considered as a reliable alternative viral detection system in further investigations.

Engineered resistance in potato against potato leafroll virus, potato virus A and potato virus Y.

Transgenic potato plants of Solanum tuberosum cultivar Vales Sovereign were generated that expressed fused, tandem, 200 bp segments derived from the capsid protein coding sequences of potato virus Y (PVY strain O) and potato leafroll virus (PLRV), as well as the cylindrical inclusion body coding sequences of potato virus A (PVA), as inverted repeat double-stranded RNAs, separated by an intron. The orientation of the expressed double-stranded RNAs was either sense-intron-antisense or antisense-intron-sense RNAs, and the double-stranded RNAs were processed into small RNAs. Four lines of such transgenic potato plants were assessed for resistance to infection by PVY-O, PLRV, or PVA, all transmitted by a natural vector, the green-peach aphid, Myzus persicae. Resistance was assessed by the absence of detectable virus accumulation in the foliage. All four transgenic potato lines tested showed 100% resistance to infection by either PVY-O or PVA, but variable resistance to infection by PLRV, ranging from 72 to 96% in different lines. This was regardless of the orientation of the viral inserts in the construct used to generate the transgenic plants and the gene copy number of the transgene. This demonstrates the potential for using tandem, fused viral segments and the inverted-repeat expression system to achieve multiple virus resistance to viruses transmitted by aphids in potato.