Distinct circular single-stranded DNA viruses exist in different soil types.

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

Novel bacteriophages containing a genome of another bacteriophage within their genomes.

A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3' protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a 'fast track' route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri.

Resistance of transgenic tobacco plants incorporating the putative 57-kDa polymerase read-through gene of Tobacco rattle virus against rub-inoculated and nematode-transmitted virus.

Nicotiana tabacum plants were transformed with the 57-kDa read-through domain of the replicase gene of Tobacco rattle virus (TRV) isolate SYM. From a total of six lines containing the viral transgene, four displayed various levels of resistance to TRV infection. Transgenic plants from line 81G were highly resistant to foliar rub-inoculation with the homologous isolate, or with isolates TRV-PpK20 and TRV-PLB, which are almost identical to TRV-SYM in RNA1 sequence. Moreover, 81G plants were moderately resistant to the serologically and genetically distinct, highly pathogenic isolate TRV-GR. Resistance characteristics of line 81G remained stable over six generations. No unambiguous correlation was established between number of transgene insertion loci and level of resistance. Transgene-specific mRNA was clearly detected in plants from susceptible lines but only at an early developmental stage in resistant plants, indicating the operation of a RNA silencing resistance mechanism. Following challenge using viruliferous vector nematodes carrying TRV-PpK20 or by rub inoculation of roots, 81G plants did not show any symptoms and virus was not detected in leaves. However, virus was detected in roots but without apparent effects on plant growth and often at low concentration. When challenged with nematodes carrying TRV-GR, symptoms in aerial parts of 81G plants were less severe and much delayed compared to non-transgenic plants, although younger plants showed less resistance than older ones. No difference was detected in transgene transcript accumulation between leaves and roots of 81G plants. This is the first work reporting a broad level of pathogen derived resistance against two geographically and genetically distinct TRV isolates transmitted directly by their nematode vectors and provides further insight into the expression of transgenic resistance against naturally transmitted soil-borne viruses.

Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants.

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection.

Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells.

BACKGROUND: RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system. RESULTS: Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt) RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein. CONCLUSION: These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.

Functional replacement of the tobacco rattle virus cysteine-rich protein by pathogenicity proteins from unrelated plant viruses.

Mutation of the 16K gene encoded by RNA1 of Tobacco rattle virus (TRV) greatly reduced the levels of viral RNA that accumulated in both infected protoplasts and plants, showing that the 16K cysteine-rich protein (CRP) is required for efficient multiplication of TRV. Overexpression of the 16K protein, either from an additional copy of the gene carried on TRV RNA2 or from a PVX vector, led to an increase in the severity of disease symptoms, suggesting that the protein has a role in the pathogenicity of the virus. Mutation of the 16K gene could be overcome by expression from RNA2 of the Cucumber mosaic virus 2b gene, the Soil-borne wheat mosaic virus 19K gene, or the Barley stripe mosaic virus gammab gene, indicating that the proteins encoded by these diverse genes may have similar functions. One known function of the CMV 2b gene is as a suppressor of posttranscriptional gene silencing, suggesting that the TRV 16K protein may also possess this activity.

A naturally occurring deleted form of RNA 2 of Potato mop-top virus.

A spontaneous deletion in RNA 2 of Potato mop-top virus (PMTV) was identified by RT-PCR. The deletion occurred reproducibly during manual passage of two isolates of PMTV and during fungal inoculation of plants with viruliferous soil. The borders of the deletion were conserved in all instances and sequence analyses showed that a contiguous segment of 2113 nucleotides was deleted internally from the genomic RNA 2, leaving the 5'- and 3'-terminal sequences. RT-PCR experiments also showed that the deletion was present in preparations of PMTV particles.