Tracking the potyviral P1 protein in Nicotiana benthamiana plants during plum pox virus infection.

The P1 protein is derived from the N terminus of potyvirus-coded polyprotein. In addition to the proteolytic activity essential for its maturation, it probably participates in suppression of host defense and/or in virus replication. Clear validation of the P1 in vivo function(s), however, is not yet available. We applied an infectious cDNA clone of plum pox virus (PPV), where the P1 was N-fused with a hexahistidine tag, to trace this protein in Nicotiana benthamiana plants during the PPV infection. Immunoblot analysis with the anti-his antibody showed a diffuse band corresponding to the molecular weight about 70-80 kDa (about twice larger than expected) in the root samples from early stage of infection. This signal culminated on the sixth day post inoculation, later it rapidly disappeared. Sample denaturation by boiling in SDS before centrifugal clarification was essential, indicating strong affinity of P1-his to some plant compound sedimenting with the tissue and cell debris.

Differentially expressed genes in healthy and plum pox virus-infected Nicotiana benthamiana plants.

Viruses use both material and energy sources of their hosts and redirect the production of disposable compounds in order to make viral replication more efficient. Metabolism of infected organisms is modified by these enhanced requirements as well by their own defense response. Resulting complex story consists of many regulation events on various gene expression levels. Elucidating these processes may contribute to the knowledge on virus-host interactions and to evolving new antiviral strategies. In our work we applied a subtractive cloning technique to compare the transcriptomes of healthy and plum pox virus (PPV)-infected Nicotiana benthamiana plants. Several genes were found to be induced or repressed by the PPV infection. The induced genes were mainly related to general stress response or photosynthesis, several repressed genes could be connected with growth defects evoked by the infection. Interestingly, some genes usually up-regulated by fungal or bacterial infection were found repressed in PPV-infected plants. Potential involvement of particular differently expressed genes in the process of PPV infection is discussed.

Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.

PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.

Plum pox virus accumulates mutations in different genome parts during a long-term maintenance in Prunus host plants and passage in Nicotiana benthamiana.

Plum pox virus (PPV) isolates of the strain PPV-M prevalently infect peaches under natural conditions in Middle Europe. Comparison of complete genome sequences obtained from subisolates of a PPV-M isolate maintained experimentally over a 6-year period in different Prunus host species and passaged in Nicotiana benthamiana was performed with the aim to highlight the mutations potentially connected with the virus-host adaptation. The results showed that the lowest number of non-silent mutations was accumulated in PPV-M maintained in peach (original host species), approximately two times higher diversity was recorded in plum, apricot and N. benthamiana, indicating the genetic determination of the PPV host preference. The sequence variability of Prunus subisolates was distributed more or less evenly along the PPV genome and no amino acid motif could be outlined as responsible for the host adaptation. In N. benthamiana the mutations were accumulated notably in the P1 and P3 genes indicating their non-essentiality in the infection of this experimental host plant.

Unfolding the secrets of plum pox virus: from epidemiology to genomics.

Over an approximately 80-year period since the description of the Sharka disease, the plum pox virus (PPV) has been thoroughly studied on various levels of the infection cycle. World-wide distribution of the virus, severity of the disease for the fruit industry with a potential to be further increased and discovery/emergence of new strains make PPV the most epidemiologically important viral pathogen of stone fruit trees. The history of PPV research reflects the development of analytical methods applicable in plant virology. In particular the establishment of molecular biology with reverse genetics and improvement of DNA sequencing technology have further contributed to the increase in knowledge on PPV variability, evolution, replication and interaction with host plants. This review gives a comprehensive summary of PPV data accumulated progressively, from the biological characterization of the disease to recent attempts aimed at using the PPV-based vectors for expression of exogenous proteins in plants.

Cloning of the complete infectious cDNA of the plum pox virus strain PPV-Rec.

UNLABELLED: Plum pox virus (PPV) is the causal agent of Sharka, considered to be the most detrimental viral disease of Prunus spp. worldwide. So far, several PPV strains have been recognized, three of them (PPV-D, PPV-M, and PPV-Rec) having shown serious economic impact in the European area. Infectious cDNA clones of plant RNA viruses are excellent tools for functional studies of viral genomes. Preparation and use of PPV-D and PPV-M infectious clones have been previously reported. Here we describe the construction of an infectious cDNA clone of the strain PPV-Rec (isolate BOR-3) by the strategy involving the subsequent exchanges of homologous BOR-3 genome parts in the backbone of the previously prepared PPV-D infectious construct. The infectivity of each intermediate chimeric cDNA as well as that of the final construct (pIC-PPV-Rec) was confirmed by biolistic transfection of Nicotiana benthamiana plants. Complete sequence of the cloned viral BOR-3 cDNA revealed 0.14% of difference at the nucleotide level compared to original BOR-3 sequence, resulting in four amino acid changes. This slight inequality was related to the population heterogeneity of the initial BOR-3 isolate; no difference in the amino acid sequence resulted from the cloning steps performed. KEYWORDS: inter-strain chimera; biolistics; genome sequence.

Simple and efficient biolistic procedure for the plant transfection with cDNA clones of RNA viruses.

Simple, fast, low-cost, and efficient procedure for DNA delivery to the cell nuclei of whole plants was developed. The procedure was optimized for the Plum pox virus (PPV) and its host Nicotiana benthamiana. It is based on the leaf bombardment with tungsten microparticles with bound DNA using common air gun. The procedure did not require special equipment and source of driving gas. The transfection efficiency obtained by the newly developed procedure was close to 100%, but this level dropped with the age of bombarded plants.

A single amino acid mutation alters the capsid protein electrophoretic double-band phenotype of the Plum pox virus strain PPV-Rec.

Plum pox virus (PPV) isolates differ by their capsid protein (CP) mobility in SDS-PAGE. These electrophoretic phenotypes are likely to result from post-translational modifications of the CP. We demonstrated that the CP mobility was solely determined by the CP N-terminal region. Sequence comparison pinpointed a possible role of mutations at position 66 in determining the CP phenotype of PPV-Rec isolates. Site-directed mutagenesis of a chimeric clone demonstrated that Gly(66) in the CP resulted in the double-band phenotype, while Arg(66) led to a single-band CP pattern, possibly by preventing the phosphorylation of a nearby Ser residue by steric hindrance.

Plum pox virus variability detected by the advanced analytical methods.

Plum pox virus (PPV) infects stone-fruit trees with important economical impact mainly in Europe and Mediterranean region. The data about PPV intra-species variability accumulated markedly in the last two decades. Six PPV strains have been recognized using different approaches including serology, protein analysis, specific amplification, and genome sequencing. Reliable and sensitive diagnostics is the most important requirement for application of early control and safety measures. Therefore, many techniques and their modifications have been adapted to detect PPV and its different forms. Here, we review the improvement of the PPV detection and variability analysis in the context of progress in laboratory methods since the virus discovery till today.

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.

Infectious full-length clones of plant viruses and their use for construction of viral vectors.

In the last two decades, preparation of infectious RNA or DNA clones of plant viruses has transformed to a standard laboratory technique, providing an excellent tool for the research of viral gene functions and virus-host interactions. A full-length clone can be transformed in vitro to a viral vector expressing foreign poly/oligopeptides in soluble form or fused to a viral capsid protein. The use of the plants as producers of antigens, allergens, and other pharmaceuticals is cheaper than the use other eukaryotic bioreactors. Transient expression of proteins using the vector technique has several advantages in comparison with a transgenic approach. Virus-induced gene silencing vectors have been widely adapted for research of plant genes functions and screening of plant genomes for resistance genes. The potential instability of the constructs remains the main problem of virus vector-based techniques leading to a wild-type virus recovery by recombination. A novel approach including "a deconstructed virus-strategy" may overcome these difficulties. A preparative-scale production of numerous biologically active compounds in the various plants using viral vectors are expected in the near future.

Expression of a part of the Potato virus A non-structural protein P3 in Escherichia coli for the purpose of antibody preparation and P3 immunodetection in plant material.

The N-terminal part of the Potato virus A (PVA) P3 protein was cloned into two E. coli fusion expression systems. An overexpression of the P3 fragment fused with thioredoxin was observed between 2 and 21 h after induction. The protein formed insoluble inclusions. Decreasing the cultivation temperature did not enhance its solubility. To obtain antigen for antibody preparation, inclusions were concentrated and purified by sucrose gradient centrifugation, and subjected to SDS-polyacrylamide gel electrophoresis. The band specific for the protein was excised from the gel and used for rabbit immunization. Obtained antibody tested positive with high specificity in immunoblots of expressed PVA P3 fused with either thioredoxin or GST. The antibody was also applied for the detection of P3 protein in plant material by immunoblot. Previous plant sap concentration was essential for most samples. Three concentration methods were tested: simple centrifugal size-exclusion filtration, the same preceded with high-speed centrifugation at 250,000 x g, and differential ammonium sulfate precipitation. The last approach was the most convenient. Plants tested included PVA P3-transgenic tobacco lines as well as PVA-infected wild-type tobacco. In all cases, mature P3 with a molecular mass of 40 kDa was detected.

Detection of transgene copy number by analysis of the T1 generation of tobacco plants with introduced P3 gene of potato virus A.

Real-time PCR, namely the deltadeltaCt method was used to determine the relative copy number of Potato virus A (PVA) P3 gene in the genome of the T1 generation of 18 transgenic tobacco lines. These results were compared with segregation ratios of kanamycin (Km)-resistant phenotype in T1 plants of each line and were found to be, in general, concordant. All the five lines with the Mendelian segregation ratio of 3:1 carried one gene copy. In 12 of 13 lines with uneven segregation more inserted gene copies were detected. Only for one line the real-time PCR and phenotype segregation differed. According to our results the real-time PCR of T1 generation may be used as supplementary method of estimation of the number of transgene copies in the case of nonavailability of the original T0 plants.

Plum Pox Virus Mixed Infection Detected on Apricot in Pakistan.

Sharka, caused by Plum pox virus (PPV), is the most detrimental viral disease of stone fruit trees. First reported from Bulgaria in 1917, the virus is now widespread in Europe, the Mediterranean Basin, and Asia Minor and is sporadically present in North and South America. On the basis of molecular and serological properties, six PPV subgroups are recognized, from which PPV-D, PPV-M, and PPV-Rec are the most common (1,2). Several apricot trees (Prunus armeniaca) showing mild, pale green rings and diffuse chlorotic spots on leaves were found in a small orchard in the Baltistan District in northern Pakistan at approximately 2,400 m above sea level. Dried leaf samples from one symptomatic tree randomly selected from the orchard were positive for PPV using double-antibody sandwich enzyme-linked immunosorbent assay with antisera prepared in the laboratory, immunoblot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid protein (CP) gene using standard procedures (1). To check the subgroup affiliation and evaluate the molecular variability, the 562-bp variable region spanning the C-terminus of NIb and the N-terminus of the CP was amplified, the RT-PCR product was cloned into the pGEM-T Easy vector (Promega, Madison, WI), and positive clones were analyzed by restriction and sequence analyses. Interestingly, sequence analysis of four clones revealed mixed infection, i.e., the presence of two different PPV isolates in the apricot sample. One isolate belonged to PPV-D (GenBank Accession No. DQ422147) and the other belonged to the PPV-Rec subgroup (GenBank Accession No. DQ422148). Multiple alignment of the sequenced genome portion of the Pakistan PPV-D isolate indicated 96 to 99% nt identity with various PPV-D isolates without unique, clear-cut differences. Similarly, the PPV-Rec isolate had 98 to 99% identity with European PPV-Rec isolates and retained the cross-over at nucleotide position 8450 in the 3' terminus of NIb. This sequence had the amino acid signature at the N-terminus of the CP typical of the PPV-Rec subgroup (2). Moreover, no particular clustering of the Pakistan isolates within PPV-D and PPV-Rec could be observed after phylogenetic analysis. The DAG motif, essential for aphid transmission, was present in both sequences. To our knowledge, this is the first indication of PPV occurrence in Pakistan and first identification of the PPV-Rec isolate outside Europe. Together with previous reports on the PPV presence in China and Kazakhstan (3,4), this report indicates the need for more detailed epidemiological studies focusing the PPV spread and its molecular diversity in Asia. References: (1) T. Candresse et al. Phytopathology 88:198, 1998. (2) M. Glasa et al. J. Gen. Virol. 85:2671, 2004. (3) M. Navrátil et al. Plant Dis. 89:338, 2005. (4) S. Spiegel et al. Plant Dis. 88:973, 2004.

A simplified RT-PCR-based detection of recombinant Plum pox virus isolates.

Closely related natural Plum pox virus (PPV) isolates derived from homologous RNA recombination between PPV-D and PPV-M have been recently identified and shown naturally spread in several European countries. As their serological properties were identical with those of conventional PPV-M isolates, they could be detected only by combined analysis of at least two different genome parts. To simplify the detection of such recombinants primers specific to PPV-M and PPV-D sequences with binding sites located on both sides of the recombination crossover situated in the C-terminal part of NIb were designed. They were used for direct differentiation of PPV-M, PPV-D and their recombinants by reverse transcription-polymerase chain reaction (RT-PCR). This method is convenient for identification of a recombinant PPV in single as well as mixed infection with PPV-M or PPV-D.

Molecular analysis of the 3' terminal region of the genome of Beet mosaic virus and its relation with other potyviruses.

A1633 nucleotides long 3'-terminal sequence of genome of the Beet mosaic virus (BtMV) Ren1 isolate from Slovakia, spanning a part of the NIb gene, complete capsid protein (CP) gene and a 3'non-coding region was determined. Computer-assisted translation resulted in a polypeptide encompassing typical conserved potyviral amino acid (aa) motifs. Homology BLAST search and phylogenetic analysis revealed that BtMV was closest to Bean common mosaic virus, Soybean mosaic virus and Zucchini yellow mosaic virus. An unexpectedly high, 99.6% aa sequence identity was found between BtMV-Ren1 and a previously sequenced British BtMV isolate. Restriction analysis and sequencing of the CP gene of other Slovak BtMV isolates revealed their high homology. Developed reverse transcription-polymerase chain reaction protocols enabled sensitive, rapid and reliable detection of the virus.

Plum pox virus capsid protein mobility in SDS-polyacrylamide gel electrophoresis.

Slovak plum pox virus (PPV) isolates BOR-3 and KR-4 behaved atypically in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) of their capsid proteins (CPs). Whereas other tested PPV isolates could be sorted by SDS-PAGE into groups corresponding to the M or D strains, as controlled by restriction fragment length polymorphism (RFLP) analysis, the two above mentioned isolates behaved as the M strain according to RFLP analysis but not SDS-PAGE. Slight mobility differences were observed also among the isolates belonging to the D strain. SDS-PAGE of the CP thus cannot clearly distinguish between these two main PPV strains. BOR-3 isolate has been shown atypical also in its biological properties, and it reacted very weakly with a monoclonal antibody (MAb) recognizing well both M and D strains in immunoblot analysis.

Preparation of diagnostic monoclonal antibodies against two potyviruses.

Diagnostic monoclonal and polyclonal antibodies against bean yellow mosaic virus (BYMV) and plum pox virus (PPV) were prepared, characterized and used for detection of these viruses in infected plants by enzyme-linked immunosorbent assay (ELISA), immunoblot analysis and tissue print immunoblot assay (TPIBA).