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.

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.

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.

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.

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.

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.

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.

The 3'-proximal part of the Plum pox virus P1 gene determinates the symptom expression in two herbaceous host plants.

Three major strains of the Plum pox virus (PPV) are the most important in Europe: PPV-D, PPV-M, and PPV-Rec. By combining the genomes of two different strains of PPV (PPV-D with PPV-Rec; PPV-D with PPV-M), 20 inter-strain chimeric infectious clones (CICPPV) were constructed. Biological properties of CICPPV were tested by inoculating them on different herbaceous host species susceptible to PPV. Four of the seven species tested, exhibited visible symptoms. In Nicotiana benthamiana all CICPPV induced systemic mosaic and leaf malformation. Pisum sativum showed a broad range of symptom severity (systemic chlorotic and necrotic lesions) but neither qualitative nor quantitative aspects of symptomatology were related to a single PPV genome locus. Nicotiana occidentalis and Nicandra physaloides proved to be suitable for symptom-based differentiation. Depending on the virus strain/chimera, N. occidentalis showed two types of symptoms: mild systemic chlorotic spots or local necrotic lesions/systemic vein necroses. N. physaloides reacted to the PPV infection either symptomless or by local necrotic lesions. Our results demonstrated that the P1/HC-pro region of the PPV genome appears to be the determinant of the symptom manifestation in these host plants. In silico analysis mapped it to the 3'-proximal part of the P1 gene.

The Cucumber vein yellowing virus silencing suppressor P1b can functionally replace HCPro in Plum pox virus infection in a host-specific manner.

Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silencing suppressors (RSS) to counteract antiviral RNA silencing responses. HCPro is the RSS of Potyvirus spp., and its activity is enhanced by the upstream P1 protein. Distinctively, the ipomovirus Cucumber vein yellowing virus (CVYV) lacks HCPro but contains two P1 copies in tandem (P1aP1b), the second of which functions as RSS. Using chimeras based on the potyvirus Plum pox virus (PPV), we found that P1b can functionally replace HCPro in potyviral infections of Nicotiana plants. Interestingly, P1a, the CVYV protein homologous to potyviral P1, disrupted the silencing suppression activity of P1b and reduced the infection efficiency of PPV in Nicotiana benthamiana. Testing the influence of RSS in host specificity, we found that a P1b-expressing chimera poorly infected PPV's natural host, Prunus persica. Conversely, P1b conferred on PPV chimeras the ability to replicate locally in cucumber, CVYV's natural host. The deleterious effect of P1a on PPV infection is host dependent, because the P1aP1b-expressing PPV chimera accumulated in cucumber to higher levels than PPV expressing P1b alone. These results demonstrate that a potyvirus can use different RSS, and that particular RSS and upstream P1-like proteins contribute to defining the virus host range.

The determinant of potyvirus ability to overcome the RTM resistance of Arabidopsis thaliana maps to the N-terminal region of the coat protein.

In Arabidopsis thaliana Columbia (Col-0) plants, the restriction of Tobacco etch virus (TEV) long-distance movement involves at least three dominant RTM (restricted TEV movement) genes named RTM1, RTM2, and RTM3. Previous work has established that, while the RTM-mediated resistance is also effective against other potyviruses, such as Plum pox virus (PPV) and Lettuce mosaic virus (LMV), some isolates of these viruses are able to overcome the RTM mechanism. In order to identify the viral determinant of this RTM-resistance breaking, the biological properties of recombinants between PPV-R, which systemically infects Col-0, and PPV-PSes, restricted by the RTM resistance, were evaluated. Recombinants that contain the PPV-R coat protein (CP) sequence in an RTM-restricted background are able to systemically infect Col-0. The use of recombinants carrying chimeric CP genes indicated that one or more PPV resistance-breaking determinants map to the 5' half of the CP gene. In the case of LMV, sequencing of independent RTM-breaking variants recovered after serial passages of the LMV AF199 isolate on Col-0 plants revealed, in each case, amino acid changes in the CP N-terminal region, close to the DAG motif. Taken together, these findings demonstrate that the potyvirus CP N-terminal region determines the outcome of the interaction with the RTM-mediated resistance.

Host-specific effect of P1 exchange between two potyviruses.

The potyviruses Plum pox virus (PPV) and Tobacco vein mottling virus (TVMV) have distinct host ranges and induce different symptoms in their common herbaceous hosts. To test the relevance of the P1 protein in host compatibility and pathogenicity, hybrid viruses were constructed in which the P1 coding sequence of PPV was completely or partially replaced by the corresponding sequences from TVMV. Infections induced by these chimeric viruses revealed that the TVMV P1 and a PPV/TVMV hybrid P1 proteins are functionally equivalent in herbaceous plants to the P1 protein of a PPV isolate adapted to these hosts, in spite of having high sequence divergence. Moreover, the presence of TVMV P1 sequences enhanced the competence of a low-infectivity PPV-D-derived chimera in Nicotiana clevelandii. Conversely, all PPV/TVMV hybrids were unable to infect Prunus persicae, a specific host for PPV, suggesting that TVMV P1 is not functionally competent in this plant. Together, these data highlight the importance of the P1 protein in defining the virus host range.

Identification of Plum pox virus pathogenicity determinants in herbaceous and woody hosts.

Plum pox virus (PPV) is a member of the genus Potyvirus that is able to infect a large variety of plant species, including trees of the genus Prunus, its natural host. When some PPV isolates are propagated for an extended time in herbaceous plants, their ability to infect trees is reduced. The molecular basis of this change in host infectivity is poorly understood. We report the construction of hybrid viruses from cDNA clones of two D-strain isolates of PPV, PPV-D and PPV-R, which differ in their host range. PPV-D can infect GF305 peach seedlings efficiently, however, it is unable to infect Nicotiana clevelandii plants. Conversely, PPV-R infects N. clevelandii, but not GF305 peach seedlings. The analyses of the hybrid viruses showed that, although determinants of PPV pathogenicity are extensively spread throughout the PPV genome, the 3' terminal region of the PPV-R genome, including the 3' noncoding region and the coding regions for the coat protein (CP), NIb, and part of NIa protein, is sufficient to confer infectivity of N. clevelandii in a PPV-D background. Our data demonstrate a high concentration of amino acid substitutions in the CP and a host-specific effect of a deletion at the N terminus of this protein in PPV pathogenicity in peach and N. clevelandii infectivity experiments. These results suggest that relevant host specificity determinants are located in the N-terminal region of the CP. The analyses of the PPV-R and PPV-D chimeras also showed that key host-specific pathogenicity determinants lie in the 5' terminal third of the PPV genome, a region that spans proteins P1, HCPro, and P3. The selection of mutations in only a few specific residues in proteins P1, P3, and 6K1 after partial adaptation of a chimeric virus (BD-GFP) to N. clevelandii further suggests a relevant role for these proteins in host adaptation.

Electrophoretic mobility of the capsid protein of the Plum pox virus strain PPV-Rec indicates its partial phosphorylation.

A double-band SDS-PAGE profile was found reproducible for capsid protein (CP) of Plum pox virus (PPV) isolates belonging to the strain PPV-Rec. The double-band was also present in the virus population multiplied in various plants. A single-lesion passage in a hypersensitive host Chenopodium foetidum showed that its presence was not a result of a mixed infection. We found that the two electrophoretic forms of CP shared identical N-terminus. Therefore, they did not originate from an alternative proteolytic processing, but were different in their posttranslational modification. The slower band of CP could be converted to the faster one by the phosphatase treatment. We assumed that CP protein was present in both phosphorylated and dephosphorylated forms in the infected plants.

A chimeric plum pox virus shows reduced spread and cannot compete with its parental wild-type viruses in a mixed infection.

The effect of a recombination event in the genomic 3' end on the biological properties and competitiveness of plum pox virus (PPV) was investigated. Therefore, a fragment spanning the coat protein (CP) coding region and a part of the 3' non-translated region of a non-aphid-transmissible strain of PPV (PPV-NAT) was replaced by the corresponding region of a PPV sour cherry isolate (PPV-SoC). The resulting chimera (PPV-NAT/SoC) caused severe symptoms in Nicotiana benthamiana, resembling those of PPV-NAT. In mixed infections with either of the parental viruses, the chimera PPV-NAT/SoC was less competitive. Labelling experiments with DsRed showed that PPV-NAT/SoC (PPV-NAT/SoC-red) moved more slowly from cell to cell than PPV-NAT (PPV-NAT-red). In mixed infections of PPV-NAT/SoC-red with a green fluorescent protein-expressing PPV-NAT (PPV-NAT-AgfpS), spatial separation of the viruses was observed. These data suggest that, in PPV infections, symptom severity and competitiveness are independent aspects and that spatial separation may contribute to the displacement of a recombinant virus.

Production of plum pox virus HC-Pro functionally active for aphid transmission in a transient-expression system.

Potyviruses are non-persistently transmitted by aphid vectors with the assistance of a viral accessory factor known as helper component (HC-Pro), a multifunctional protein that is also involved in many other essential processes during the virus infection cycle. A transient Agrobacterium-mediated expression system was used to produce Plum pox virus (PPV) HC-Pro in Nicotiana benthamiana leaves from constructs that incorporated the 5' region of the genome, yielding high levels of HC-Pro in agroinfiltrated leaves. The expressed PPV HC-Pro was able to assist aphid transmission of purified virus particles in a sequential feeding assay, and to complement transmission-defective variants of the virus. Also, HC-Pro of a second potyvirus, Tobacco etch virus (TEV), was expressed and found to be functional for aphid transmission. These results show that this transient system can be useful for production of functionally active HC-Pro in potyviruses, and the possible uses of this approach to study the mechanism of transmission are discussed.

Mapping of two O-GlcNAc modification sites in the capsid protein of the potyvirus Plum pox virus.

A large number of O-linked N-acetylglucosamine (O-GlcNAc) residues have been mapped in vertebrate proteins, however targets of O-GlcNAcylation in plants still have not been characterized. We show here that O-GlcNAcylation of the N-terminal region of the capsid protein of Plum pox virus resembles that of animal proteins in introducing O-GlcNAc monomers. Thr-19 and Thr-24 were specifically O-GlcNAcylated. These residues are surrounded by amino acids typical of animal O-GlcNAc acceptor sites, suggesting that the specificity of O-GlcNAc transferases is conserved among plants and animals. In laboratory conditions, mutations preventing O-GlcNAcylation of Thr-19 and Thr-24 did not have noticeable effects on PPV competence to infect Prunus persicae or Nicotiana clevelandii. However, the fact that Thr-19 and Thr-24 are highly conserved among different PPV strains suggests that their O-GlcNAc modification could be relevant for efficient competitiveness in natural conditions.