Reconstruction of putative DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution

BACKGROUND: Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV)-like sequences (ERTBVs) have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response. RESULTS: We have collected ERTBVs from the rice genomes. They contain rearranged structures and no intact ORFs. The identified ERTBV segments were shown to be phylogenetically divided into three clusters. For each phylogenetic cluster, we were able to make a consensus alignment for a circular virus-like structure carrying two complete ORFs. Comparisons of DNA and amino acid sequences suggested the closely relationship between ERTBV and RTBV. The Oryza AA-genome species vary in the ERTBV copy number. The species carrying low-copy-number of ERTBV segments have been reported to be extremely susceptible to RTBV. The DNA methylation state of the ERTBV sequences was correlated with their copy number in the genome. CONCLUSIONS: These ERTBV segments are unlikely to have functional potential as a virus. However, these sequences facilitate to establish putative virus that provided information underlying virus integration and evolutionary relationship with existing virus. Comparison of ERTBV among the Oryza AA-genome species allowed us to speculate a possible role of endogenous virus segments against its related disease

Screening and analysis of genes expressed upon infection of broad bean with Clover yellow vein virus causing lethal necrosis

Clover yellow vein virus (ClYVV) causes lethal systemic necrosis in legumes, including broad bean (Vicia faba) and pea (Pisum sativum). To identify host genes involved in necrotic symptom expression after ClYVV infection, we screened cDNA fragments in which expression was changed in advance of necrotic symptom expression in broad bean (V. faba cv. Wase) using the differential display technique and secondarily with Northern blot analysis. Expression changes were confirmed in 20 genes, and the six that exhibited the most change were analyzed further. These six genes included a gene that encodes a putative nitrate-induced NOI protein (VfNOI), and another was homologous to an Arabidopsis gene that encodes a glycine- and proline-rich protein GPRP (VfGPRP). We recently reported that necrotic symptom development in ClYVV-infected pea is associated with expression of salicylic acid (SA)-dependent pathogenesis-related (PR) proteins and requires SA-dependent host responses. Interestingly, VfNOI and VfGPRP expression was correlated with that of the putative SA-dependent PR proteins in ClYVV-infected broad bean. However, broad bean infected with a recombinant ClYVV expressing the VfGPRP protein showed weaker symptoms and less viral multiplication than that infected with ClYVV expressing the GFP protein. These results imply that VfGPRP plays a role in defense against ClYVV rather than in necrotic symptom expression

Two Resistance Modes to Clover yellow vein virus in Pea Characterized by a Green Fluorescent Protein-Tagged Virus

This study characterized resistance in pea lines PI 347295 and PI 378159 to Clover yellow vein virus (ClYVV). Genetic cross experiments showed that a single recessive gene controls resistance in both lines. Conventional mechanical inoculation did not result in infection; however, particle bombardment with infectious plasmid or mechanical inoculation with concentrated viral inocula did cause infection. When ClYVV No. 30 isolate was tagged with a green fluorescent protein (GFP) and used to monitor infection, viral cell-to-cell movement differed in the two pea lines. In PI 347595, ClYVV replicated at a single-cell level, but did not move to neighboring cells, indicating that resistance operated at a cell-to-cell step. In PI 378159, the virus moved to cells around the infection site and reached the leaf veins, but viral movement was slower than that in the susceptible line. The viruses observed around the infection sites and in the veins were then recovered and inoculated again by a conventional mechanical inoculation method onto PI 378159 demonstrating that ClYVV probably had mutated and newly emerged mutant viruses can move to neighboring cells and systemically infect the plants. Tagging the virus with GFP was an efficient tool for characterizing resistance modes. Implications of the two resistance modes are discussed

Hypothesis on particle structure and assembly of rice dwarf phytoreovirus: interactions among multiple structural proteins

To study the morphogenesis and packaging of rice dwarf phytoreovirus (RDV), the interactions among multiple structural proteins were analysed using both the yeast two-hybrid system and far-Western blotting analysis. The following protein-protein interactions were observed. P3 (major core protein) bound to itself as well as to P7 (nucleic acid-binding protein) and P8 (major outer capsid protein). P7 bound to P1 (RNA-dependent RNA polymerase) and P8, in addition to P3. Based on these findings, we hypothesize that the core shell structure is based on P3-P3 interactions and that P7 has the ability to bind to multiple structural proteins as well as to genomic RNAs during viral particle assembly. Based on the observed protein-protein interactions and on computer-aided analysis of the numbers of structural proteins per particle, possible RDV assembly events are proposed

White clover mosaic virus-induced gene silencing in pea

Double-stranded RNAs formed in secondary structures and replicative intermediates of viral genomes are thought to strongly elicit RNA silencing. This phenomenon is known as virus-induced gene silencing (VIGS). VIGS is a powerful tool for modifying gene expression in host plants. We constructed a virus vector based on White clover mosaic virus (WClMV) and demonstrated VIGS of phytoene desaturase (PDS) in pea. Photobleaching of tissues, caused by VIGS of PDS, was observed in restricted areas of upper leaves and stems. We confirmed that the PDS mRNA and subgenomic RNAs of WClMV were reduced in the photobleached tissues