An immunodominant membrane protein (Imp) of 'Candidatus Phytoplasma mali' binds to plant actin.

The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (Imp▴Tm) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant Imp▴Tm-His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or Imp▴Tm-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wild-type plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized.

Analysis of the effectiveness of reused primary and secondary antibodies in Western blotting analysis.

Western blot analysis is a common technique for detecting a target protein on a nitrocellulose membrane. The target protein is detected using specific antibodies. The positive signal can be visualized either by chemiluminescence or by a colorimetric reaction. Some specific antibodies can be produced in-house in high amounts and can be used without considering costs. However many antibodies (especially antibodies for detection tag fused proteins) must be purchased from a company. Unfortunately most of them are very expensive. Here we report the effectiveness of different antibodies after reusing them several times and also compare the influence of different storage conditions.

Bottom-up assembly of a bilayer structure of icosahedral viral nanoparticles.

The development of 2D and 3D structures on the nanoscale containing viral nanoparticles (VNPs) as interesting nanobuilding blocks has come into focus for a bottom-up approach as an alternative to the top-down approach in nanobiotechnology. Our research has focused on the plant Tomato Bushy Stunt Virus (TBSV). In a previous study, we reported the impact of the pH value on the 2D assembly of viral monolayers. Here, we extend these studies into the third dimension by using specific interactions between the layers in combination with selective side chains on the viral capsid. The virus bilayer structure is prepared by an alternating deposition of His-tagged TBSV (4D6H-TBSV, first layer), Ni-NTA nanogold (second layer) complexes and 4D6H-TBSV, respectively, and 6D-TBSV (6xaspartic acid TBSV) as the third layer, i.e., the second layer of VNPs. The formed layer structures were imaged by using scanning force and scanning electron microscopy. The data show that a virus bilayer structure was successfully built up by means of the interaction between Ni-NTA nanogold and histidine. By comparing 4D6H- with 6D-TBSV in the third layer, the importance of these specific interactions is shown. This work paves the way for 3D nanodevices based on VNPs.

A RT/PCR-partial restriction enzymatic mapping (PREM) method for the molecular characterisation of the large satellite RNAs of Arabis mosaic virus isolates.

The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from various geographical origins and natural hosts for the presence of large satellite RNAs and analyse their degree of variability, a RT/PCR-partial restriction enzymatic mapping (PREM) method was developed. The method is based on the incorporation of 5-methyl-dCTP in the RT/PCR reaction, and the subsequent digestion of the RT/PCR products by methyl-sensitive restriction enzymes. Satellites RNAs were detected by RT/PCR in eight isolates out of 47, six of them originating from grapevine, one from hop and one from lilac. The partial restriction digestion patterns allowed to distinguish six different types of satellites. Cloning and sequencing of the different satellites confirmed these results, the PREM proving able to discriminate sequences with 96% identity. The sizes of the different satellites varied between 1092 and 1139 nucleotides, their encoded proteins between 338 and 360 amino acids. Conserved domains were found in the amino and carboxy-termini between the sequences of the proteins encoded by the satellites of the different isolates of ArMV.

Complete nucleotide sequences of the RNAs 2 of German isolates of grapevine fanleaf and Arabis mosaic nepoviruses.

The RNAs 2 of an Arabis mosaic virus (ArMV) and a grapevine fanleaf virus (GFLV) isolate, originating from South West of Germany near Neustadt an der Weinstrasse (NW), were sequenced. They are 3820 and 3775 nucleotides long respectively, and both contain one open reading frame encoding a polypeptide of 1110 amino acids. Their 5' non-coding regions contain conserved and repeated sequences, which are able to form stem-loop structures. Nucleotide sequence comparisons between the full-length RNAs 2 revealed homology levels of 84 and 82% between the ArMV-NW and the ArMV-L and -U, respectively, 90% between GFLV-NW and GFLV-F13, and 72% between ArMV-NW and GFLV-NW. Amino acid sequence comparisons showed that the greatest difference was found between the 2A proteins of the different ArMV isolates, the 2A protein of the ArMV-NW showing more similarity to the 2A protein of GFLV-NW than to those of ArMV-L2 or -U2.

Complete nucleotide sequence of an isolate of the nepovirus raspberry ringspot virus from grapevine.

The complete nucleotide sequence of the RNAs 1 and 2 of the nepovirus Raspberry ringspot virus cherry isolate (RpRSV-ch) from grapevine was determined. The RNA 1 is 7935 nucleotides (nt) long excluding the poly(A) tail, and contains one long open reading frame (ORF) encoding a polypeptide of 2367 amino acids. This ORF is preceeded by a 136nt 5' non-coding region, and followed by a 695nt 3' non-coding region. Conserved amino acid motifs, characteristic of the viral protease cofactor, the NTP-binding protein, proteinase and polymerase, were found in the sequence of the RNA 1-encoded polyprotein. The RNA 2 is 3915nt long excluding the poly(A) tail, and contains one long ORF encoding a polypeptide of 1106 amino acids. This ORF is preceeded by a 203nt 5' non-coding region, and followed by a 390nt 3' non-coding region. When compared to the corresponding sequences of other nepoviruses, a maximum level of 34% identity was found between the RNA 1-encoded polypetides of RpRSV-ch and other nepoviruses. For the RNA 2-encoded polypeptide, 88% identity was found between RpRSV-ch and RpRSV-S, a Scottish isolate of RpRSV from raspberry, and a maximum 29% identity between RpRSV-ch and other nepoviruses.

Simultaneous RT/PCR detection and differentiation of arabis mosaic and grapevine fanleaf nepoviruses in grapevines with a single pair of primers.

The movement protein genes from several isolates of ArMV and GFLV of different geographical origins were amplified by RT/PCR using degenerate primers, cloned and sequenced. A single pair of degenerate primers was designed from these sequences to allow the simultaneous amplification of parts of the movement protein genes of ArMV and GFLV. Their use in an immunocapture-RT/PCR for the detection of ArMV or GFLV in infected grapevines proved to be ten times more sensitive than the corresponding ArMV or GFLV ELISA tests. A Sph1 restriction site found in the sequences corresponding to the amplified products from the GFLV isolates, but not in the amplified products from the ArMV isolates, allowed the differentiation between ArMV and GFLV in the infected grapevines by a Sph1 restriction digestion of the amplified products.

An evaluation of antibiotics for the elimination of Agrobacterium tumefaciens from walnut somatic embryos and for the effects on the proliferation of somatic embryos and regeneration of transgenic plants.

Four antibiotics were evaluated for their effects on eliminating the hypervirulent Agrobacterium tumefaciens strain C58C1 ATHV RifR (pEHA101)/p35-gus-intron from walnut somatic embryos and on the production of secondary somatic embryos and the transformed somatic embryos. Exposure to 100-1000 mg l-1 of ampicillin, carbenicillin or cefotaxime respectively for up to 60 days did not eliminate the A. tumefaciens while timentin at 500-1000 mg l-1 eradicated it from somatic embryos. One-hour acidified medium treatments and the addition of 100 mg l-1 kanamycin to 500 mg l-1 ampicillin, carbenicillin, cefotaxime or timentin were of little help in eliminating the Agrobacterium. All four antibiotics reduced somatic embryo production, carbenicillin minimally and cefotaxime maximally, especially at higher concentrations, in comparison with antibiotic-free medium. Putative transformed embryos were selected for continued proliferation on a 100 mg l-1 kanamycin-containing medium. Histochemical assessments indicated that more gus-positive somatic embryos, particularly fully gus-positive embryos, regenerated from timentin-containing medium than from other antibiotic-containing media under equivalent conditions. Transformed embryos have been grown and converted into plants and gus activity was observed in whole plants.

First Report of Cacopsylla picta as a Vector of Apple Proliferation Phytoplasma in Germany.

Since 2000, a serious epidemic of apple proliferation (AP) reappeared in southwestern Germany. Molecular analyses revealed that the AP phytoplasma is associated with this disease. Since no curative treatments or resistant cultivars exist, the only means to reduce spread of the disease is the control of the insect vector. Recently, Frisinghelli et al. (1) identified Cacopsylla costalis as a vector of AP phytoplasma in northern Italy. Following this result, transmission trials with C. picta (synonym C. costalis) were conducted in southwestern Germany at Neustadt (Rheinland-Pfalz) and Dossenheim (Baden-Württemberg) since 2001. Overwintering psyllids were captured from March to May in different orchards. Groups of 5 to 30 C. picta were caged for 2 to 4 weeks on apple seedlings or healthy micropropagated plants. Leaf midribs of test plants were sampled 2 to 3 months after inoculation feeding and tested by polymerase chain reaction (PCR) for AP phytoplasma with specific primers AP5/AP4 (2). In 2001, 1 of 10 test plants, and in 2002, 7 of 40 test plants became AP infected. In 2002, one to four C. picta specimens fed on plants which became infected were tested AP phytoplasma positive by PCR while all psyllids recollected from PCR-negative plants were tested negative. Transmission of the AP phytoplasma was successful at both sites. To our knowledge, this is the first report of C. picta as a vector of the AP phytoplasma in Germany. References: (1) C. Frisinghelli et al. J. Phytopathol. 148:425, 2000. (2) W. Jarausch et al. Appl. Environ. Microbiol. 60:2916, 1994.

Tomato bushy stunt viruses (TBSV) in nanotechnology investigated by scanning force and scanning electron microscopy.

Spherical plant viruses like the tomato bushy stunt virus (TBSV) allow for multiple applications in nanotechnology due to their shape. In this article, different types of the virus were created by extending coat protein (CP) at carboxylic termini with 2 different charged amino acids by point mutation. The obtained CPs carried 6 aspartic acid (negative charge) and 4 histamine (positive charge) residues. The ability of TBSV to form self assembled monolayers with large ordered areas on native and chemically modified mica will be presented. The structural differences between layers formed by the wild type and by the genetically modified types will be discussed in detail.

Complete nucleotide sequence of a putative new cytorhabdovirus infecting lettuce.

The full-length nucleotide sequence of the genomic RNA of a new cytorhabdovirus infecting lettuce was determined. Six open reading frames were found in the antigenomic sequence of the 12,926-nt negative-sense viral RNA genome. The genomic organisation was similar to that of lettuce necrotic yellows virus (LNYV), the type member of the genus Cytorhabdovirus: 3'-N-P-3-M-G-L-5', where N is the capsid protein gene, P the putative phosphoprotein gene, 3 a gene coding for a putative protein of unknown function, M the putative matrix protein gene, G the glycoprotein gene, and L the putative polymerase gene. Amino acid sequence comparison with the corresponding sequences of other rhabdoviruses revealed the closest relationship to LNYV, with identities ranging from 41% for the matrix proteins and 65% for the L polymerase proteins. These results indicate that this virus may be a member of a new cytorhabdovirus species, for which the name Lettuce yellow mottle virus (LYMoV) is proposed.

Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation.

An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.

Nucleotide sequence of a new isolate of ribgrass mosaic tobamovirus infecting Impatiens New Guinea.

The complete nucleotide sequence of a tobamovirus isolated from Impatiens New Guinea was determined. The genome was 6302 nt long, and its genomic organisation was similar to those of other crucufer tobamoviruses. Sequence comparisons with the corresponding sequences of other crucifer tobamoviruses revealed highest levels of identity with the ribgrass mosaic virus (Shanghai isolate). A small open reading frame putatively encoding a 4.5-kDa protein with a low degree of similarity to the ORF6 of tobacco mosaic virus was found nested in the movement protein gene.

Sequence analysis of grapevine isolates of Raspberry ringspot nepovirus.

The nucleotide sequences of RNAs 1 and 2 of a German isolate of Raspberry ringspot virus (RpRSV) infecting grapevine (RpRSV-Grapevine), as well as partial sequences of another grapevine isolate from Switzerland (RAC815) were determined. The sequences of the protease-polymerase region encoded by RNA1, and the movement protein and coat protein genes encoded by RNA 2, of these isolates were compared with those of other isolates available in databases. The coat proteins of the grapevine isolates formed a sister group to all those from other RpRSV isolates, but whether this resulted from divergence or recombination was uncertain.

Analysis of tombusvirus revertants to identify essential amino acid residues within RNA-dependent RNA polymerase motifs.

The RNA-dependent RNA polymerase (RdRp) of Tomato bushy stunt virus (TBSV) contains an arginine- and proline-rich (RPR) motif. This motif functions as an RNA-binding domain and is essential for tombusvirus replication. A mutant carrying three arginine substitutions in this motif rendered the virus unable to replicate in Nicotiana benthamiana plants and protoplasts. When the replicase function was provided in trans, by expressing the TBSV RdRp in N. benthamiana plants, an infectious variant could be isolated. Sequence analysis showed that only the substituted glycine residue (position 216) had reverted to arginine; all other substitutions remained unchanged. This finding suggested that strong selection pressure is active to maintain necessary sequences of the viral RdRp and that the analysis of revertants may help to identify essential viral functions.

Complete nucleotide sequence of the RNA 1 of a grapevine isolate of Arabis mosaic virus.

The complete nucleotide sequence of the genomic RNA 1 of the grapevine isolate NW (Neustadt an der Weinstrasse) of Arabis mosaic virus (ArMV) was determined. It is 7334 nucleotides long excluding the poly(A) tail, and contains one long open reading frame encoding a polypeptide of 2284 amino acids. The 5' and 3' non-coding regions were 227 and 252 nucleotides long respectively, and showed stretches of high identity with the corresponding 5' and 3' non-coding regions of ArMV-NW RNA 2. The analysis of the amino acid sequence of the polyprotein encoded by the RNA 1 of the ArMV-NW showed that the conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cystein protease, and the RdRp core domains, were all present. Amino acid sequence comparisons between the polyproteins encoded by the RNAs 1 of ArMV-NW and other nepoviruses showed 75% identity with the GFLV-F13, and up to 36% with other nepoviruses.

Size and sequence variability of the Arabis mosaic virus protein 2A.

The RNA 2 of the nepovirus Arabis mosaic virus (ArMV) encodes a polyprotein from which protein 2A is released by proteolytic cleavage at the N-terminus. The 2A gene of 19 ArMV isolates from different geographical origin and 9 distinct natural hosts was amplified by RT/PCR and subsequently cloned and sequenced. These 19 isolates and those from databanks were classified into four groups based on the size of the protein 2A which ranged from 233 to 280 amino acids, and sequence identities. Sequence variability was mainly located in the N-terminus of the proteins, whereas the core region and the C-terminus were conserved.

Serological comparison of tospoviruses with polyclonal antibodies produced against the main structural proteins of tomato spotted wilt virus.

A new purification procedure for the tospoviruses of serogroups II and III was developed. SDS-polyacrylamide gel electrophoresis of purified tomato spotted wilt virus (TSWV; serogroup I), groundnut ringspot virus (GRSV: serogroup II), tomato chlorotic spot virus (TCSV; serogroup II) and impatiens necrotic spot virus (INSV; serogroup III) showed that the glycoprotein G2 of serogroup II members differs significantly in size from that of the serogroup I virus. Western immunoblot analysis using polyclonal antisera produced against purified glycoproteins TSWV-G1 and G2 as well as peptides of hydrophilic sequences of TSWV-G1 and G2 expressed in Escherichia coli demonstrated a higher homology amongst G1 of different serogroups than for G2. These results are supported by comparing the glycoprotein gene sequences of different serogroups.

Characterization of petunia flower mottle virus (PetFMV), a new potyvirus infecting Petunia x hybrida.

With the introduction of cutting-grown Petunia x hybrida plants on the European market, a new potyvirus which showed no serological reaction with antisera against any other potyviruses infecting petunias was discovered. Infected leaves contained flexuous rod-shaped virus particles of 750-800 nm in length and inclusion bodies (pinwheel structures) typical for potyviruses in ultrathin leaf sections. The purified coat protein with a Mr of approximately 36 kDa could be detected in Western immunoblots with a specific antibody to the coat protein of the petunia-infecting virus. The 3' end of the viral genome encompassing the 3' non-coding region, the coat protein gene, and part of the NIb gene was amplified from infected leaf material by IC/PCR using degenerate and specific primers. Sequences of PCR-generated cDNA clones were compared to other known sequences of potyviruses. Maximum homology of 56% was found in the 3' non-coding region between the petunia isolate and other potyviruses. A maximum homology of 69% was found between the amino acid sequence of the coat protein of the petunia isolate and corresponding sequences of other potyviruses. These data indicate that the petunia-infecting virus is a previously undescribed potyvirus and the name petunia flower mottle virus (PetFMV) is suggested.