Yoshitaka Sano

Nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus, the prototype of the tenuiviruses

The complete nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus (RSV), was determined using two sets of overlapping cDNA clones. RNA segment 1 comprises 8970 nucleotides and on the viral complementary sequence has a single long open reading frame coding for a protein of 2919 amino acids with an estimated M(r) of 336860. Amino acid sequence comparisons of the putative protein indicated strong homology (30% amino acid identity over about 1500 residues) with the L protein of the genus Phlebovirus of the Bunyaviridae, but no detectable similarity with other members of the Bunyaviridae. However, weak similarity was detected with the L protein of Tacaribe arenavirus. The highly homologous sequence domain includes the conserved motifs of the putative RNA-dependent RNA polymerase. The data presented here, along with previous work clearly show significant similarities in genome organization, structure and expression between RSV and members of the genus Phlebovirus of the Bunyaviridae. Taken together, we propose that tenuiviruses should be included in the Bunyaviridae under the genus Tenuivirus.

Isolation and characterization of a temperature-sensitive mutant of Bombyx mori nucleopolyhedrovirus for a putative RNA polymerase gene.

Temperature-sensitive (ts) mutants of Bombyx mori nucleopolyhedrovirus (BmNPV), which exhibited no polyhedra formation at the non-permissive temperature of 33 degrees C, were produced using the base analogue 5-bromodeoxyuridine. A unique ts mutant, designated ts-S1, was characterized in terms of gene mutation and virulence in cultured cells and silkworm larvae. Mutant-infected BmN4 cells at 33 degrees C showed normal viral DNA synthesis but defective budded virus production and polyhedrin synthesis, suggesting the absence of late and very late gene expression. Silkworm larvae were injected with ts-S1 and reared at 33.5 degrees C. At 7 days post-injection, none of the larvae exhibited nucleopolyhedrosis but some possessed viral DNA, detected by PCR using virus-specific primers. Continued rearing of the larvae at a permissive temperature of 25 degrees C showed that, while most developed into normal adults, some developed nucleopolyhedrosis, indicating that the former larvae had aborted the virus infection during the course of rearing at 33.5 degrees C. No viral DNA was detected in the adults. Marker rescue tests to identify the lesion involved in the ts phenotype of ts-S1, and nucleotide sequencing of the identified genome region, showed a single nucleotide mutation of a putative RNA polymerase gene, late expression factor-8 (lef-8). These results indicate that lef-8 is essential for BmNPV replication in vitro and in vivo.

Changes in the Proteome of Xylem Sap in Brassica oleracea in Response to Fusarium oxysporum Stress

Fusarium oxysporum f.sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F. oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change > = 2-fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and 10 of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.