Adaptive substitutions at two amino acids of HCPro modify its functional properties to separately increase the virulence of a potyviral chimera.

We had previously reported that a plum pox virus (PPV)-based chimera that had its P1-HCPro bi-cistron replaced by a modified one from potato virus Y (PVY) increased its virulence in some Nicotiana benthamiana plants, after mechanical passages. This correlated with the natural acquisition of amino acid substitutions in several proteins, including in HCPro at either position 352 (Ile→Thr) or 454 (Leu→Arg), or of mutations in non-coding regions. Thr in position 352 is not found among natural potyviruses, while Arg in 454 is a reversion to the native PVY HCPro amino acid. We show here that both mutations separately contributed to the increased virulence observed in the passaged chimeras that acquired them, and that Thr in position 352 is no intragenic suppressor to a Leu in position 454, because their combined effects were cumulative. We demonstrate that Arg in position 454 improved HCPro autocatalytic cleavage, while Thr in position 352 increased its accumulation and the silencing suppression of a reporter in agropatch assays. We assessed infection by four cloned chimera variants expressing HCPro with none of the two substitutions, one of them or both, in wild-type versus DCL2/4-silenced transgenic plants. We found that during infection, the transgenic context of altered small RNAs affected the accumulation of the four HCPro variants differently and hence, also infection virulence.

Viral Reservoir Capacity of Wild Prunus Alternative Hosts of Plum Pox Virus Through Multiple Cycles of Transmission and Dormancy.

Plum pox virus (PPV) is a significant pathogen of Prunus worldwide and is known for having a broad experimental host range. Many of these hosts represent epidemiological risks as potential wild viral reservoirs. A comparative study of the PPV reservoir capacity of three commonly found native North American species, western choke cherry (Prunus virginiana var. demissa), black cherry (Prunus serotina), and American plum (Prunus americana) was conducted. Pennsylvania isolates of PPV-D were transmitted from the original host peach (Prunus persica cv. GF305) to all three species. Viral accumulation and transmission rates to alternative hosts and peach were monitored over the course of five vegetative growth and cold induced dormancy (CID) cycles. The three alternative host species demonstrated differences in their ability to maintain PPV-D and the likelihood of transmission to additional alternative hosts or back transmission to peach. Western choke cherry had low (5.8%) initial infection levels, PPV-D was not transmissible to additional western choke cherry, and transmission of PPV-D from western choke cherry to peach was only possible before the first CID cycle. Black cherry had intermediate initial infection levels (26.6%) but did not maintain high infection levels after repeated CID cycles. Conversely, American plum had a high level (50%) of initial infection that was not significantly different from initial infection in peach (72.2%) and maintained moderate levels (15 to 25%) of infection and PPV-D transmission to both American plum and peach through all five cycles of CID. Our results indicate that American plum has the greatest potential to act as a reservoir host for Pennsylvania isolates of PPV-D.

Robust Response to Plum pox virus Infection via Plant Biotechnology.

Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.

Intra-strain biological and epidemiological characterization of plum pox virus.

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.

Sterol isomerase HYDRA1 interacts with RNA silencing suppressor P1b and restricts potyviral infection.

Plants use RNA silencing as a strong defensive barrier against virus challenges, and viruses counteract this defence by using RNA silencing suppressors (RSSs). With the objective of identifying host factors helping either the plant or the virus in this interaction, we have performed a yeast two-hybrid screen using P1b, the RSS protein of the ipomovirus Cucumber vein yellowing virus (CVYV, family Potyviridae), as a bait. The C-8 sterol isomerase HYDRA1 (HYD1), an enzyme involved in isoprenoid biosynthesis and cell membrane biology, and required for RNA silencing, was isolated in this screen. The interaction between CVYV P1b and HYD1 was confirmed in planta by Bimolecular Fluorescence Complementation assays. We demonstrated that HYD1 negatively impacts the accumulation of CVYV P1b in an agroinfiltration assay. Moreover, expression of HYD1 inhibited the infection of the potyvirus Plum pox virus, especially when antiviral RNA silencing was boosted by high temperature or by coexpression of homologous sequences. Our results reinforce previous evidence highlighting the relevance of particular composition and structure of cellular membranes for RNA silencing and viral infection. We report a new interaction of an RSS protein from the Potyviridae family with a member of the isoprenoid biosynthetic pathway.

Photosynthetic and Stress Responsive Proteins Are Altered More Effectively in Nicotiana benthamiana Infected with Plum pox virus Aggressive PPV-CR versus Mild PPV-C Cherry-Adapted Isolates.

Plum pox virus (PPV, family Potyviridae) is one of the most important viral pathogens of Prunus spp. causing considerable damage to stone-fruit industry worldwide. Among the PPV strains identified so far, only PPV-C, PPV-CR, and PPV-CV are able to infect cherries under natural conditions. Herein, we evaluated the pathogenic potential of two viral isolates in herbaceous host Nicotiana benthamiana. Significantly higher accumulation of PPV capsid protein in tobacco leaves infected with PPV-CR (RU-30sc isolate) was detected in contrast to PPV-C (BY-101 isolate). This result correlated well with the symptoms observed in the infected plants. To further explore the host response upon viral infection at the molecular level, a comprehensive proteomic profiling was performed. Using reverse-phase ultra-high-performance liquid chromatography followed by label-free mass spectrometry quantification, we identified 38 unique plant proteins as significantly altered due to the infection. Notably, the abundances of photosynthesis-related proteins, mainly from the Calvin-Benson cycle, were found more aggressively affected in plants infected with PPV-CR isolate than those of PPV-C. This observation was accompanied by a significant reduction in the amount of photosynthetic pigments extracted from the leaves of PPV-CR infected plants. Shifts in the abundance of proteins that are involved in stimulation of photosynthetic capacity, modification of amino acid, and carbohydrate metabolism may affect plant growth and initiate energy formation via gluconeogenesis in PPV infected N. benthamiana. Furthermore, we suggest that the higher accumulation of H2O2 in PPV-CR infected leaves plays a crucial role in plant defense and development by activating the glutathione synthesis.

Estimation of the dispersal distances of an aphid-borne virus in a patchy landscape.

Characterising the spatio-temporal dynamics of pathogens in natura is key to ensuring their efficient prevention and control. However, it is notoriously difficult to estimate dispersal parameters at scales that are relevant to real epidemics. Epidemiological surveys can provide informative data, but parameter estimation can be hampered when the timing of the epidemiological events is uncertain, and in the presence of interactions between disease spread, surveillance, and control. Further complications arise from imperfect detection of disease and from the huge number of data on individual hosts arising from landscape-level surveys. Here, we present a Bayesian framework that overcomes these barriers by integrating over associated uncertainties in a model explicitly combining the processes of disease dispersal, surveillance and control. Using a novel computationally efficient approach to account for patch geometry, we demonstrate that disease dispersal distances can be estimated accurately in a patchy (i.e. fragmented) landscape when disease control is ongoing. Applying this model to data for an aphid-borne virus (Plum pox virus) surveyed for 15 years in 605 orchards, we obtain the first estimate of the distribution of flight distances of infectious aphids at the landscape scale. About 50% of aphid flights terminate beyond 90 m, which implies that most infectious aphids leaving a tree land outside the bounds of a 1-ha orchard. Moreover, long-distance flights are not rare-10% of flights exceed 1 km. By their impact on our quantitative understanding of winged aphid dispersal, these results can inform the design of management strategies for plant viruses, which are mainly aphid-borne.

Metabolomics and Biochemical Approaches Link Salicylic Acid Biosynthesis to Cyanogenesis in Peach Plants.

Despite the long-established importance of salicylic acid (SA) in plant stress responses and other biological processes, its biosynthetic pathways have not been fully characterized. The proposed synthesis of SA originates from chorismate by two distinct pathways: the isochorismate and phenylalanine (Phe) ammonia-lyase (PAL) pathways. Cyanogenesis is the process related to the release of hydrogen cyanide from endogenous cyanogenic glycosides (CNglcs), and it has been linked to plant plasticity improvement. To date, however, no relationship has been suggested between the two pathways. In this work, by metabolomics and biochemical approaches (including the use of [13C]-labeled compounds), we provide strong evidences showing that CNglcs turnover is involved, at least in part, in SA biosynthesis in peach plants under control and stress conditions. The main CNglcs in peach are prunasin and amygdalin, with mandelonitrile (MD), synthesized from phenylalanine, controlling their turnover. In peach plants MD is the intermediary molecule of the suggested new SA biosynthetic pathway and CNglcs turnover, regulating the biosynthesis of both amygdalin and SA. MD-treated peach plants displayed increased SA levels via benzoic acid (one of the SA precursors within the PAL pathway). MD also provided partial protection against Plum pox virus infection in peach seedlings. Thus, we propose a third pathway, an alternative to the PAL pathway, for SA synthesis in peach plants.

Virulence determines beneficial trade-offs in the response of virus-infected plants to drought via induction of salicylic acid.

It has been hypothesized that plants can get beneficial trade-offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus-induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought-tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus-infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid-independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non-infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.

Plum pox virus capsid protein suppresses plant pathogen-associated molecular pattern (PAMP)-triggered immunity.

The perception of pathogen-associated molecular patterns (PAMPs) by immune receptors launches defence mechanisms referred to as PAMP-triggered immunity (PTI). Successful pathogens must suppress PTI pathways via the action of effectors to efficiently colonize their hosts. So far, plant PTI has been reported to be active against most classes of pathogens, except viruses, although this defence layer has been hypothesized recently as an active part of antiviral immunity which needs to be suppressed by viruses for infection success. Here, we report that Arabidopsis PTI genes are regulated upon infection by viruses and contribute to plant resistance to Plum pox virus (PPV). Our experiments further show that PPV suppresses two early PTI responses, the oxidative burst and marker gene expression, during Arabidopsis infection. In planta expression of PPV capsid protein (CP) was found to strongly impair these responses in Nicotiana benthamiana and Arabidopsis, revealing its PTI suppressor activity. In summary, we provide the first clear evidence that plant viruses acquired the ability to suppress PTI mechanisms via the action of effectors, highlighting a novel strategy employed by viruses to escape plant defences.

An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene.

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees.

Genome-wide association links candidate genes to resistance to Plum Pox Virus in apricot (Prunus armeniaca).

In fruit tree species, many important traits have been characterized genetically by using single-family descent mapping in progenies segregating for the traits. However, most mapped loci have not been sufficiently resolved to the individual genes due to insufficient progeny sizes for high resolution mapping and the previous lack of whole-genome sequence resources of the study species. To address this problem for Plum Pox Virus (PPV) candidate resistance gene identification in Prunus species, we implemented a genome-wide association (GWA) approach in apricot. This study exploited the broad genetic diversity of the apricot (Prunus armeniaca) germplasm containing resistance to PPV, next-generation sequence-based genotyping, and the high-quality peach (Prunus persica) genome reference sequence for single nucleotide polymorphism (SNP) identification. The results of this GWA study validated previously reported PPV resistance quantitative trait loci (QTL) intervals, highlighted other potential resistance loci, and resolved each to a limited set of candidate genes for further study. This work substantiates the association genetics approach for resolution of QTL to candidate genes in apricot and suggests that this approach could simplify identification of other candidate genes for other marked trait intervals in this germplasm.

Gene Expression Analysis of Plum pox virus (Sharka) Susceptibility/Resistance in Apricot (Prunus armeniaca L.).

RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, "Rojo Pasión" and "Z506-7", resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.

Analysis of gene expression changes in peach leaves in response to Plum pox virus infection using RNA-Seq.

Differences in gene expression were studied after Plum pox virus (PPV, sharka disease) infection in peach GF305 leaves with and without sharka symptoms using RNA-Seq. For each sample, more than 80% of 100-nucleotide paired-end (PE) Illumina reads were aligned on the peach reference genome. In the symptomatic sample, a significant proportion of reads were mapped to PPV reference genomes (1.04% compared with 0.00002% in non-symptomatic leaves), allowing for the ultra-deep assembly of the complete genome of the PPV isolate used (9775 nucleotides, missing only 11 nucleotides at the 5' genome end). In addition, significant alternative splicing events were detected in 359 genes and 12 990 single nucleotide polymorphisms (SNPs) were identified, 425 of which could be annotated. Gene ontology annotation revealed that the high-ranking mRNA target genes associated with the expression of sharka symptoms are mainly related to the response to biotic stimuli, to lipid and carbohydrate metabolism and to the negative regulation of catalytic activity. A greater number of differentially expressed genes were observed in the early asymptomatic phase of PPV infection in comparison with the symptomatic phase. These early infection events were associated with the induction of genes related to pathogen resistance, such as jasmonic acid, chitinases, cytokinin glucosyl transferases and Lys-M proteins. Once the virus had accumulated, the overexpression of Dicer protein 2a genes suggested a gene silencing plant response that was suppressed by the virus HCPro and P1 proteins. These results illustrate the dynamic nature of the peach-PPV interaction at the transcriptome level and confirm that sharka symptom expression is a complex process that can be understood on the basis of changes in plant gene expression.

Single amino acid changes in the 6K1-CI region can promote the alternative adaptation of Prunus- and Nicotiana-propagated Plum pox virus C isolates to either host.

Plum pox virus (PPV) C is one of the less common PPV strains and specifically infects cherry trees in nature. Making use of two PPV-C isolates that display different pathogenicity features, i.e., SwCMp, which had been adapted to Nicotiana species, and BY101, which had been isolated from cherry rootstock L2 (Prunus lannesiana) and propagated only in cherry species, we have generated two infective full-length cDNA clones in order to determine which viral factors are involved in the adaptation to each host. According to our results, the C-P3(PIPO)/6K1/N-CI (cylindrical inclusion) region contains overlapping but not coincident viral determinants involved in symptoms development, local viral amplification, and systemic movement capacity. Amino acid changes in this region promoting the adaptation to N. benthamiana or P. avium have trade-off effects in the alternative host. In both cases, adaptation can be achieved through single amino acid changes in the NIapro protease recognition motif between 6K1 and CI or in nearby sequences. Thus, we hypothesize that the potyvirus polyprotein processing could depend on specific host factors and the adaptation of PPV-C isolates to particular hosts relies on a fine regulation of the proteolytic cleavage of the 6K1-CI junction.

Plum pox virus and sharka: a model potyvirus and a major disease.

TAXONOMIC RELATIONSHIPS: Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae. PPV diversity is structured into at least eight monophyletic strains. GEOGRAPHICAL DISTRIBUTION: First discovered in Bulgaria, PPV is nowadays present in most of continental Europe (with an endemic status in many central and southern European countries) and has progressively spread to many countries on other continents. GENOMIC STRUCTURE: Typical of potyviruses, the PPV genome is a positive-sense single-stranded RNA (ssRNA), with a protein linked to its 5' end and a 3'-terminal poly A tail. It is encapsidated by a single type of capsid protein (CP) in flexuous rod particles and is translated into a large polyprotein which is proteolytically processed in at least 10 final products: P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIapro, NIb and CP. In addition, P3N-PIPO is predicted to be produced by a translational frameshift. PATHOGENICITY FEATURES: PPV causes sharka, the most damaging viral disease of stone fruit trees. It also infects wild and ornamental Prunus trees and has a large experimental host range in herbaceous species. PPV spreads over long distances by uncontrolled movement of plant material, and many species of aphid transmit the virus locally in a nonpersistent manner. SOURCES OF RESISTANCE: A few natural sources of resistance to PPV have been found so far in Prunus species, which are being used in classical breeding programmes. Different genetic engineering approaches are being used to generate resistance to PPV, and a transgenic plum, 'HoneySweet', transformed with the viral CP gene, has demonstrated high resistance to PPV in field tests in several countries and has obtained regulatory approval in the USA.

Diverse amino acid changes at specific positions in the N-terminal region of the coat protein allow Plum pox virus to adapt to new hosts.

Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N terminus of the coat protein (CP) includes host-specific pathogenicity determinants. Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3' region of PPV-D (including the region coding for the CP) in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and N. benthamiana when specific amino acids were modified or deleted in a short 30-amino-acid region of the N terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus spp. although others impaired systemic infection in this host. We propose a model in which the N terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanism in Nicotiana spp. Mutations in this short region allow PPV to overcome the defense response in this host but can compromise the efficiency of PPV systemic movement in other hosts such as Prunus spp.

O-GlcNAc modification of the coat protein of the potyvirus Plum pox virus enhances viral infection.

O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus.

Silencing of the host factor eIF(iso)4E gene confers plum pox virus resistance in plum.

Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species.