Transgenerational Tolerance to Salt and Osmotic Stresses Induced by Plant Virus Infection.

Following pathogen infection, plants have developed diverse mechanisms that direct their immune systems towards more robust induction of defense responses against recurrent environmental stresses. The induced resistances could be inherited by the progenies, rendering them more tolerant to stressful events. Although within-generational induction of tolerance to abiotic stress is a well-documented phenomenon in virus-infected plants, the transgenerational inheritance of tolerance to abiotic stresses in their progenies has not been explored. Here, we show that infection of Nicotiana benthamiana plants by Potato virus X (PVX) and by a chimeric Plum pox virus (PPV) expressing the P25 pathogenicity protein of PVX (PPV-P25), but not by PPV, conferred tolerance to both salt and osmotic stresses to the progeny, which correlated with the level of virulence of the pathogen. This transgenerational tolerance to abiotic stresses in the progeny was partially sustained even if the plants experience a virus-free generation. Moreover, progenies from a Dicer-like3 mutant mimicked the enhanced tolerance to abiotic stress observed in progenies of PVX-infected wild-type plants. This phenotype was shown irrespective of whether Dicer-like3 parents were infected, suggesting the involvement of 24-nt small interfering RNAs in the transgenerational tolerance to abiotic stress induced by virus infection. RNAseq analysis supported the upregulation of genes related to protein folding and response to stress in the progeny of PVX-infected plants. From an environmental point of view, the significance of virus-induced transgenerational tolerance to abiotic stress could be questionable, as its induction was offset by major reproductive costs arising from a detrimental effect on seed production.

Targeting of genomic and negative-sense strands of viral RNA contributes to antiviral resistance mediated by artificial miRNAs and promotes the emergence of complex viral populations.

Technology based on artificial small RNAs, including artificial microRNAs (amiRNAs), exploits natural RNA silencing mechanisms to achieve silencing of endogenous genes or pathogens. This technology has been successfully employed to generate resistance against different eukaryotic viruses. However, information about viral RNA molecules effectively targeted by these small RNAs is rather conflicting, and factors contributing to the selection of virus mutants escaping the antiviral activity of virus-specific small RNAs have not been studied in detail. In this work, we transformed Nicotiana benthamiana plants with amiRNA constructs designed against the potyvirus plum pox virus (PPV), a positive-sense RNA virus, and obtained lines highly resistant to PPV infection and others showing partial resistance. These lines have allowed us to verify that amiRNA directed against genomic RNA is more efficient than amiRNA targeting its complementary strand. However, we also provide evidence that the negative-sense RNA strand is cleaved by the amiRNA-guided RNA silencing machinery. Our results show that the selection pressure posed by the amiRNA action on both viral RNA strands causes an evolutionary explosion that results in the emergence of a broad range of virus variants, which can further expand in the presence, and even in the absence, of antiviral challenges.

AlkB RNA demethylase homologues and N6 -methyladenosine are involved in Potyvirus infection.

Proteins of the alkylation B (AlkB) superfamily show RNA demethylase activity removing methyl adducts from N6 -methyladenosine (m6 A). m6 A is a reversible epigenetic mark of RNA that regulates human virus replication but has unclear roles in plant virus infection. We focused on Potyvirus-the largest genus of plant RNA viruses-and report here the identification of AlkB domains within P1 of endive necrotic mosaic virus (ENMV) and an additional virus of a putative novel species within Potyvirus. We show that Nicotiana benthamiana m6 A levels are reduced by infection of plum pox virus (PPV) and potato virus Y (PVY). The two potyviruses lack AlkB and the results suggest a general involvement of RNA methylation in potyvirus infection and evolution. Methylated RNA immunoprecipitation sequencing of virus-infected samples showed that m6 A peaks are enriched in plant transcript 3' untranslated regions and in discrete internal and 3' terminal regions of PPV and PVY genomes. Down-regulation of N. benthamiana AlkB homologues of the plant-specific ALKBH9 clade caused a significant decrease in PPV and PVY accumulation. In summary, our study provides evolutionary and experimental evidence that supports the m6 A implication and the proviral roles of AlkB homologues in Potyvirus infection.

Suitability of different plant species for experimental agroinfection with Plum pox virus-based expression vector for potential production of edible vaccines.

Nine herbaceous plant species were tested for susceptibility to Plum pox virus (PPV) by Agrobacterium-mediated delivery of its infectious cDNA clone. Two of them became infected, namely spinach (local infection) and oilseed poppy (systemic infection). As a control, PPV infection was successfully established in plum seedlings following agroinfiltration, thus providing the first report of agroinfection in Prunus species. According to our results, oilseed poppy can be considered as a candidate host for the production of edible vaccines by a PPV-derived expression vector. Keywords: agroinfiltration; virus host; poppy; spinach.

New Cherry-Adapted Plum Pox Virus Phylogroups Discovered in Russia.

Plum pox virus (PPV) is the most pathogenic virus of stone fruit crops worldwide. Unusual PPV isolates were discovered on sour cherry (Prunus cerasus L.) and steppe cherry (P. fruticosa Pall.) in the Republic of Tatarstan and the Middle Ural region, Russia. They induced typical sharka symptoms and tested positive for PPV by ELISA and RT-PCR, but were not detected by PCR using known strain-specific primers. Their complete genomes were determined using high-throughput sequencing. Phylogenetic analysis allocated new isolates to four clearly distinguished lineages (SC, TAT, Y, Tat-26) within a cluster of PPV cherry-adapted strains. The phylogroups SC and TAT had 84.5 to 86.9% average nucleotide identity to each other and strain CR, with which they comprised a common subcluster. Isolates from the Middle Ural region (group Y) were closer to strain C, sharing 96.9% identity. The fourth lineage is represented by the isolate Tat-26, which was a recombinant of strain CR and C isolates as major and minor parents, respectively. These results show that the genetic diversity of PPV is higher than thought and may contribute to a better understanding of the origin and evolution of cherry-adapted strains of the virus. P. fruticosa was reported as a new natural PPV host for the first time.

sRNA Analysis Evidenced the Involvement of Different Plant Viruses in the Activation of RNA Silencing-Related Genes and the Defensive Response Against Plum pox virus of 'GF305' Peach Grafted with 'Garrigues' Almond.

Plum pox virus (PPV) causes sharka disease in Prunus trees. Peach (P. persica) trees are severely affected by PPV, and no definitive source of genetic resistance has been identified. However, previous results showed that PPV-resistant 'Garrigues' almond (P. dulcis) was able to transfer its resistance to 'GF305' peach through grafting, reducing symptoms and viral load in PPV-infected plants. A recent study tried to identify genes responsible for this effect by studying messenger RNA expression through RNA sequencing in peach and almond plants, before and after grafting and before and after PPV infection. In this work, we used the same peach and almond samples but focused the high-throughput analyses on small RNA (sRNA) expression. We studied massive sequencing data and found an interesting pattern of sRNA overexpression linked to antiviral defense genes that suggested activation of these genes followed by downregulation to basal levels. We also discovered that 'Garrigues' almond plants were infected by different plant viruses that were transferred to peach plants. The large amounts of viral sRNA found in grafted peaches indicated a strong RNA silencing antiviral response and led us to postulate that these plant viruses could be collaborating in the observed "Garrigues effect."

Effect of Gene Silencing of Translation Initiation Factors eIF(iso)4G and eIF(iso)4E on Sour Cherry Rootstock Resistance to Sharka Disease.

Sharka disease, caused by the Plum pox virus (PPV), is one of the most harmful, quarantine viral diseases that affect stone fruit crops. The absence of natural resistance to the virus in stone fruits has become a decisive factor for the use of genetic transformation methods to obtain stable forms. The eIF(iso)4G and eIF(iso)4E genes encode translation initiation factors used in the PPV life cycle. In the presented study, the effect of silencing these genes using the RNA interference method on the resistance of sour cherry rootstock 146-2 plants (Prunus pumila L. x Prunus tomentosa Thunb) to the sharka disease was studied. Two vectors have been created for the genetic transformation of plants, with self-complementary sequences of the eIF(iso)4G and eIF(iso)4E gene fragments. The hairpin expression cassette contains a strong promoter of the peach ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) gene, as well as an intron and terminator of the same gene. We used the pMF1 vector containing recombinase R and a codA-nptII gene which makes it possible to obtain intragenic marker-free plants. A successful genetic transformation was carried out by the AGL0 strain of A. tumefaciens. Whole leaves of shoots cultivated in vitro were used as a source of explants. Eight independent transgenic lines of rootstock 146-2 were obtained in experiments (sixlines with a hairpin to the eIF(iso)4G gene and two lines with a hairpin to the eIF(iso)4E gene). Their status was confirmed by the PCR and Southern blotting. The obtained plants were acclimatized in a greenhouse. The silencing of the eIF(iso)4G and eIF(iso)4E genes in transgenic plants was confirmed by the quantitative PCR. The presence of specific small interfering (si) RNAs was confirmed by the method of Northern blotting. Plants of all transgenic rootstock lines were infected with PPV by the method of grafting with infected buds. Resistance to the PPV infection of the obtained transgenic plants was carried out by using an enzyme immunoassay. The ELISA results showed that silencing the eIF(iso)4G gene did not lead to increased resistance while silencing the eIF(iso)4E factor gene led to increased resistance to the PPV, and the one line's plants showed no signs of infection for two years after infecting. The work demonstrates a (promising) approach in which the creation of stone cultures resistant to the plum pox virus can be achieved by suppressing the genes of translation initiation factors in clonal rootstocks.

Biolistic Inoculation of Fruit Trees with Full-Length Infectious cDNA Clones of RNA Viruses.

Long life cycle and lack of efficient and robust virus inoculation technique are the major technical challenges for studying virus infection in perennial woody plants such as fruit trees. Biolistic technology also called particle bombardment is a physical approach that can directly introduce virions or viral full-length cDNA infectious clones into target cells and tissues by high velocity microcarrier particles. The flexibility and high efficiency of the biolistic inoculation method facilitate research on fruit tree virology and the screening and identification of fruit tree germplasms resistant to viruses. Here, we describe a detailed protocol for the biolistic inoculation of peach with of a cDNA infectious clone of Plum pox virus (PPV) using the Helios gene gun, a biolistic particle delivery system.

Genome sequence analysis of new plum pox virus isolates from Japan.

OBJECTIVE: To find mutations that may have recently occurred in Plum pox virus (PPV), we collected six PPV-infected plum/peach trees from the western part of Japan and one from the eastern part. After sequencing the full-length PPV genomic RNAs, we compared the amino acid sequences with representative isolates of each PPV strain. RESULTS: All new isolates were found to belong to the PPV-D strain: the six isolates collected from western Japan were identified as the West-Japan strain while the one collected from eastern Japan as the East-Japan strain. Amino acid sequence analysis of these seven isolates suggested that the 1407th and 1529th amino acid residues are characteristic of the West-Japan and the East-Japan strains, respectively. Comparing them with the corresponding amino acid residues of the 47 non-Japanese PPV-D isolates revealed that these amino acid residues are undoubtedly unique. A further examination of the relevant amino acid residues of the other 210 PPV-D isolates collected in Japan generated a new hypothesis regarding the invasion route from overseas and the subsequent diffusion route within Japan: a PPV-D strain might have invaded the western part of Japan from overseas and spread throughout Japan.

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.

P1 of Sweet Potato Feathery Mottle Virus Shows Strong Adaptation Capacity, Replacing P1-HCPro in a Chimeric Plum Pox Virus.

Potyviridae is the largest family of plant RNA viruses. Their genomes are expressed through long polyproteins that are usually headed by the leader endopeptidase P1. This protein can be classified as type A or type B based on host proteolytic requirements and RNA silencing suppression (RSS) capacity. The main Potyviridae genus is Potyvirus, and a group of potyviruses infecting sweet potato presents an enlarged P1 protein with a polymerase slippage motif that produces an extra product termed P1N-PISPO. These two proteins display some RSS activity and are expressed followed by HCPro, which appears to be the main RNA silencing suppressor in these viruses. Here, we studied the behavior of the P1 protein of Sweet potato feathery mottle virus (SPFMV) using a viral system based on a canonical potyvirus, Plum pox virus (PPV), and discovered that this protein is able to replace both PPV P1 and HCPro. We also found that P1N-PISPO, produced after polymerase slippage, provides extra RNA silencing suppression capacity to SPFMV P1 in this viral context. In addition, the results showed that presence of two type A P1 proteins was detrimental for viral viability. The ample recombination spectrum that we found in the recovered viruses supports the strong adaptation capacity of P1 proteins and signals the N-terminal part of SPFMV P1 as essential for RSS activity. Further analyses provided data to add extra layers to the evolutionary history of sweet potato-infecting potyvirids. IMPORTANCE Plant viruses represent a major challenge for agriculture worldwide and Potyviridae, being the largest family of plant RNA viruses, is one of the primary players. P1, the leader endopeptidase, is a multifunctional protein that contributes to the successful spread of these viruses over a wide host range. Understanding how P1 proteins work, their dynamic interplay during viral infection, and their evolutionary path is critical for the development of strategic tools to fight the multiple diseases these viruses cause. We focused our efforts on the P1 protein of Sweet potato feathery mottle virus, which is coresponsible for the most devastating disease in sweet potato. The significance of our research is in understanding the capacity of this protein to perform several independent functions, using this knowledge to learn more about P1 proteins in general and the potyvirids infecting this host.

Occurrence, Distribution, and Genomic Characteristics of Plum Pox Virus Isolates from Common Apricot (Prunus armeniaca) and Japanese Apricot (Prunus mume) in China.

Plum pox, or Sharka disease, caused by infection with plum pox virus (PPV), results in enormous economic losses to the stone fruit industry. However, the frequency and distribution of PPV remain unclear in China, the world's largest stone fruit producer. Systemic visual surveys were performed on stone fruit trees in China from 2008 to 2018, and the results suggest that plum pox disease is widely distributed on common apricots (Prunus armeniaca) and Japanese apricots (Prunus mume), with an average symptoms incidence rate >30% in the latter. In samples collected from Beijing, Nanjing, Shanghai, Wuhan, Wuxi, and Yuncheng, PPV was detected in 77% (85 of 110) of collected samples by immunochromatographic (IC) strip tests and reverse transcription PCR, and 96% (67 of 70) of samples showing Sharka symptoms were PPV positive. Transmission electron microscopy revealed filamentous particles of ∼640 × 12.5 nm (n = 19) in size and pinwheel inclusions in symptomatic plants but not in the asymptomatic and PPV-negative plants. Full-length genomes were determined for four isolates (three from Japanese apricot and one from common apricot), and phylogenetic analyses indicated that all four isolates belong to a clade PPV-D, despite slight differences in genome size. These findings not only highlight the widespread occurrence and distribution of PPV in China but also provide detailed information about the genomic characteristics and evolutionary position of PPV isolates in China.

Gene Expression Analysis of Induced Plum pox virus (Sharka) Resistance in Peach (Prunus persica) by Almond (P. dulcis) Grafting.

No natural sources of resistance to Plum pox virus (PPV, sharka disease) have been identified in peach. However, previous studies have demonstrated that grafting a "Garrigues" almond scion onto "GF305" peach rootstock seedlings heavily infected with PPV can progressively reduce disease symptoms and virus accumulation. Furthermore, grafting a "Garrigues" scion onto the "GF305" rootstock has been shown to completely prevent virus infection. This study aims to analyse the rewiring of gene expression associated with this resistance to PPV transmitted by grafting through the phloem using RNA-Seq and RT-qPCR analysis. A total of 18 candidate genes were differentially expressed after grafting "Garrigues" almond onto healthy "GF305" peach. Among the up-regulated genes, a HEN1 homolog stands out, which, together with the differential expression of RDR- and DCL2-homologs, suggests that the RNA silencing machinery is activated by PPV infection and can contribute to the resistance induced by "Garrigues" almond. Glucan endo-1,3-beta D-glucosidase could be also relevant for the "Garrigues"-induced response, since its expression is much higher in "Garrigues" than in "GF305". We also discuss the potential relevance of the following in PPV infection and "Garrigues"-induced resistance: several pathogenesis-related proteins; no apical meristem proteins; the transcription initiation factor, TFIIB; the speckle-type POZ protein; in addition to a number of proteins involved in phytohormone signalling.

Dynamic changes impact the plum pox virus population structure during leaf and bud development.

Plum pox virus (PPV) is a worldwide threat to stone fruit production. Its woody perennial hosts provide a dynamic environment for virus evolution over multiple growing seasons. To investigate the impact seasonal host development plays in PPV population structure, next generation sequencing of ribosome associated viral genomes, termed translatome, was used to assess PPV variants derived from phloem or whole leaf tissues over a range of plum leaf and bud developmental stages. Results show that translatome PPV variants occur at proportionately higher levels in bud and newly developing leaf tissues that have low infection levels while more mature tissues with high infection levels display proportionately lower numbers of viral variants. Additional variant analysis identified distinct groups based on population frequency as well as sets of phloem and whole tissue specific variants. Combined, these results indicate PPV population dynamics are impacted by the tissue type and developmental stage of their host.

Common and Strain-Specific Post-Translational Modifications of the Potyvirus Plum pox virus Coat Protein in Different Hosts.

Phosphorylation and O-GlcNAcylation are widespread post-translational modifications (PTMs), often sharing protein targets. Numerous studies have reported the phosphorylation of plant viral proteins. In plants, research on O-GlcNAcylation lags behind that of other eukaryotes, and information about O-GlcNAcylated plant viral proteins is extremely scarce. The potyvirus Plum pox virus (PPV) causes sharka disease in Prunus trees and also infects a wide range of experimental hosts. Capsid protein (CP) from virions of PPV-R isolate purified from herbaceous plants can be extensively modified by O-GlcNAcylation and phosphorylation. In this study, a combination of proteomics and biochemical approaches was employed to broaden knowledge of PPV CP PTMs. CP proved to be modified regardless of whether or not it was assembled into mature particles. PTMs of CP occurred in the natural host Prunus persica, similarly to what happens in herbaceous plants. Additionally, we observed that O-GlcNAcylation and phosphorylation were general features of different PPV strains, suggesting that these modifications contribute to general strategies deployed during plant-virus interactions. Interestingly, phosphorylation at a casein kinase II motif conserved among potyviral CPs exhibited strain specificity in PPV; however, it did not display the critical role attributed to the same modification in the CP of another potyvirus, Potato virus A.

The global phylogeny of Plum pox virus is emerging.

The 206 complete genomic sequences of Plum pox virus in GenBank (January 2019) were downloaded. Their main open reading frames (ORF)s were compared by phylogenetic and population genetic methods. All fell into the nine previously recognized strain clusters; the PPV-Rec and PPV-T strain ORFs were all recombinants, whereas most of those in the PPV-C, PPV-CR, PPV-CV, PPV-D, PPV-EA, PPV-M and PPV-W strain clusters were not. The strain clusters ranged in size from 2 (PPV-CV and PPV-EA) to 74 (PPV-D). The isolates of eight of the nine strains came solely from Europe and the Levant (with an exception resulting from a quarantine breach), but many PPV-D strain isolates also came from east and south Asia and the Americas. The estimated time to the most recent common ancestor (TMRCA) of all 134 non-recombinant ORFs was 820 (865-775) BCE. Most strain populations were only a few decades old, and had small intra-strain, but large inter-strain, differences; strain PPV-W was the oldest. Eurasia is clearly the 'centre of emergence' of PPV and the several PPV-D strain populations found elsewhere only show evidence of gene flow with Europe, so have come from separate introductions from Europe. All ORFs and their individual genes show evidence of strong negative selection, except the positively selected pipo gene of the recently migrant populations. The possible ancient origins of PPV are discussed.

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.

Multilocation comparison of fruit composition for 'HoneySweet', an RNAi based plum pox virus resistant plum.

'HoneySweet', a transgenic plum (Prunus domestica) resistant to plum pox virus through RNAi, was deregulated in the U.S. in 2011. The compositional study of 'HoneySweet' fruit was expanded to include locations outside of the US as well as utilizing a wide variety of comparators and different collection years to see the variability possible. The results revealed that plums have a wide variation in composition and that variation among locations was greater than variation among cultivars. This was also the case for different years at one location. The results supported the supposition that the transgene and insertion event had no significant effect on the composition of 'HoneySweet' fruit even under virus pressure, and that it fell in the normal range of composition of commercially grown plums. It also suggested that the effect of environment is as great as that of genetics on the fruit composition of plums.

Development of one-tube real-time RT-qPCR for the universal detection and quantification of Plum pox virus (PPV).

In this study a one-tube real-time RT-qPCR assay was developed using the TaqMan chemistry for the universal detection and quantification of PPV, one of the most important pathogens affecting stone fruit trees. In order to design appropriate primers and probe, nucleotide sequences from different PPV isolates originating from all known strains were recovered from the databases. Various genomic regions were screened and finally primers were selected from a conserved region of the 3'- terminal part of the CP gene amplifying a 146 bp DNA fragment while the probe was designed to bind within the amplicon. Ten-fold serial dilutions of in vitro synthesized RNA transcripts were applied for the construction of standard curve. The amplification efficiency of the assay was 93.8% and the linear range of quantification was from 40 up to 4 × 108 RNA copies. The real time RT-PCR was successfully tested with a collection of genetically diverse isolates with different geographical origin belonging to seven PPV strains. The present method is proposed as a useful tool for various basic or applied research studies of PPV as well as for routine testing of plant material during phytosanitary control or in certification schemes of Prunus species.

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.