Ramesh R. Chavan

A generic method to identify plant viruses by high-resolution tandem mass spectrometry of their coat proteins.

Although a number of protocols have been developed for detection of viruses at the genus or family level, universal approaches to detect and identify unknown viruses are still required. High-resolution tandem mass spectrometry was used to identify accurately peptide masses and their constituent sequences from partially purified plant virus preparations. Analysis of the peptide fragment masses against a virus database using pattern-matching algorithms identified sequences with homology to known virus peptides and also predicted peptides using de novo sequence analysis. This method provided sufficient information to confirm the identity of two known viruses that were included as controls (Cucumber mosaic virus and Tomato spotted wilt virus) and to identify unknown viruses in six viral isolates. The unknown viruses have been identified as four common viruses (Alfalfa mosaic virus, Tobacco streak virus, Citrus leaf blotch virus and Ribgrass mosaic virus), and two novel viruses (a potexvirus and a vitivirus). The identification of viruses from five distinct families by the tandem mass spectrometric determination of their coat protein demonstrates that this is a useful method for initial virus identification. This method, complemented with molecular or immunological procedures, provides a rapid and convenient way to identify both known and novel plant viruses.

Detection and characterisation of two novel vitiviruses infecting Actinidia

Two co-infecting novel vitiviruses from Actinidia chinensis were identified from mechanically inoculated Nicotiana occidentalis. Both virus genomes were sequenced and share 64% nucleotide identity. Their overall structure is typical of vitiviruses, with five open reading frames (ORFs) and a polyadenylated 3′ end. Open reading frame 4 (ORF4) encodes the coat protein, the most conserved gene of the vitiviruses, in which they share 75% amino acid identity, 61-68% with grapevine virus B, 55-59% with grapevine virus A, and 37-42% with grapevine virus E. Based on the molecular criteria for species demarcation in the family Betaflexiviridae, these are two novel viruses, tentatively named Actinidia virus A and Actinidia virus B.

Tracheid cell-wall structures and locations of (1 → 4)-β-D-galactans and (1 → 3)-β-D-glucans in compression woods of radiata pine (Pinus radiata D. Don)

BackgroundCompression wood (CW) forms on the underside of tilted stems of coniferous gymnosperms and opposite wood (OW) on the upperside. The tracheid walls of these wood types differ structurally and chemically. Although much is known about the most severe form of CW, severe CW (SCW), mild CWs (MCWs), also occur, but less is known about them. In this study, tracheid wall structures and compositions of two grades of MCWs (1 and 2) and SCW were investigated and compared with OW in slightly tilted radiata pine (Pinus radiata) stems.ResultsThe four wood types were identified by the distribution of lignin in their tracheid walls. Only the tracheid walls of OW and MCW1 had a S3 layer and this was thin in MCW1. The tracheid walls of only SCW had a S2 layer with helical cavities in the inner region (S2i). Using immunomicroscopy, (1 → 4)-β-D-galactans and (1 → 3)-β-D-glucans were detected in the tracheid walls of all CWs, but in only trace amounts in OW. The (1 → 4)-β-D-galactans were located in the outer region of the S2 layer, whereas the (1 → 3)-β-D-glucans were in the inner S2i region. The areas and intensities of labelling increased with CW severity. The antibody for (1 → 4)-β-D-galactans was also used to identify the locations and relative amounts of these galactans in whole stem cross sections based on the formation of an insoluble dye. Areas containing the four wood types were clearly differentiated depending on colour intensity. The neutral monosaccharide compositions of the non-cellulosic polysaccharides of these wood types were determined on small, well defined discs, and showed the proportion of galactose was higher for CWs and increased with severity.ConclusionThe presence of an S3 wall layer is a marker for very MCW and the presence of helical cavities in the S2 wall layer for SCW. The occurrence and proportions of (1 → 4)-β-D-galactans and (1 → 3)-β-D-glucans can be used as markers for CW and its severity. The proportions of galactose were consistent with the labelling results for (1 → 4)-β-D-galactans.

Quantification of (1→4)-β-D-Galactans in Compression Wood Using an Immuno-Dot Assay

Compression wood is a type of reaction wood formed on the underside of softwood stems when they are tilted from the vertical and on the underside of branches. Its quantification is still a matter of some scientific debate. We developed a new technique that has the potential to do this based on the higher proportions of (1→4)-β-d-galactans that occur in tracheid cell walls of compression wood. Wood was milled, partially delignified, and the non-cellulosic polysaccharides, including the (1→4)-β-d-galactans, extracted with 6 M sodium hydroxide. After neutralizing, the solution was serially diluted, and the (1→4)-β-d-galactans determined by an immuno-dot assay using the monoclonal antibody LM5, which specifically recognizes this polysaccharide. Spots were quantified using a dilution series of a commercially available (1→4)-β-d-galactan from lupin seeds. Using this method, compression and opposite woods from radiata pine (Pinus radiata) were easily distinguished based on the amounts of (1→4)-β-d-galactans extracted. The non-cellulosic polysaccharides in the milled wood samples were also hydrolysed using 2 M trifluoroacetic acid followed by the separation and quantification of the released neutral monosaccharides by high performance anion exchange chromatography. This confirmed that the compression woods contained higher proportions of galactose-containing polysaccharides than the opposite woods.

Characterization of the complete genome of ribgrass mosaic virus isolated from Plantago major L. from New Zealand and Actinidia spp. from China

The complete genomes of tobamovirus isolates from Plantago major L. from New Zealand (NZ-439), Plantago sp. from Germany (Kons 1105), Actinidia chinensis (Actinidia-AC) and A. deliciosa (Actinidia-AD) from China were sequenced and compared to previously published tobamovirus genomes. Their genome organization and phylogenetic analysis of the putative replicase component, replicase readthrough component, movement protein, coat protein and complete genome placed all four isolates in subgroup 3 of the tobamoviruses. The complete genomes differed from each other by <8.5% and from published sequences of turnip vein clearing virus and youcai mosaic virus by about 12-13% and 19-20%, respectively. The aa sequences of the individual ORFs of the Plantago and Actinidia isolates differed from each other by <4% and were most similar to published (partial) sequences of ribgrass mosaic virus (RMV). We propose that these sequences constitute the first complete published sequences for RMV.

First report of Turnip vein clearing virus and Ribgrass mosaic virus from New Zealand

Leaves of Plantago spp. growing in two regions of New Zealand were sampled and tested for the presence of tobamoviruses. Transmission, immunological and molecular studies revealed the presence of both Turnip vein clearing virus (TVCV) and Ribgrass mosaic virus (RMV). The phylogenetic analysis of complete coat protein amino acid sequences grouped these viruses in the RMV and TVCV clusters of sub group 3 tobamoviruses. This is the first confirmed report of these viruses from New Zealand.

Dimensional Changes of Tracheids during Drying of Radiata Pine (Pinus radiata D. Don) Compression Woods: A Study Using Variable-Pressure Scanning Electron Microscopy (VP-SEM)

Variable-pressure scanning electron microscopy was used to investigate the dimensional changes in longitudinal, tangential and radial directions, on wetting and drying, of tracheids of opposite wood (OW) and three grades of compression woods (CWs), including severe CW (SCW) and two grades of mild compression wood (MCW) (MCW1 and MCW2) in corewood of radiata pine (Pinus radiata) saplings. The CW was formed on the underside and OW on the upper side of slightly tilted stems. In the longitudinal direction, the shrinkage of SCW tracheids was ~300% greater than that of OW tracheids, with the shrinkage of the MCW1 and MCW2 tracheids being intermediate. Longitudinal swelling was also investigated and hysteresis was demonstrated for the tracheids of all corewood types, with the extent of hysteresis increasing with CW severity. A statistical association was found between longitudinal shrinkage and the content of lignin and galactosyl residues in the cell-wall matrix. The galactosyl residues are present mostly as (1→4)-β-galactans, which are known to have a high capacity for binding water and swell on hydration. The small proportions of (1→3)-β-glucans in the CWs have similar properties. These polysaccharides may play a functional role in the longitudinal shrinking and swelling of CW tracheids. Tangential shrinkage of tracheids was greater than radial shrinkage but both were greatest for OW and least for SCW, with the MCW1 and MCW2 being intermediate.