Tetsuro Okuno

Brome mosaic virus defective RNAs generated during infection of barley plants.

Brome mosaic virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By using in vitro transcripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV.

Development of Increased Oxolinic Acid-resistance in Burkholderia glumae

Genomic DNA, partially digested with Sau3AI, of Burkholderia glumae Pg-13, resistant to oxolinic acid (OA), was ligated into pUC118, and Escherichia coli DH5α resistant to 2.5 μg/ml OA was transformed with the plasmid. After incubation on a medium supplemented with 20 μg/ml OA, a clone harboring pB′46 was selected. The nucleotide sequence of the 724-bp insert of pB′46 had no homology to any characterized gene. B. glumae Pg-5, resistant to 10 μg/ ml OA, was transformed with plasmid pUCD3101B′46 containing the insert. An obtained transformant, B25, was resistant to 50 μg/ml OA and had greater resistance to other quinolones than did Pg-5. Transformants of OA-sensitive B. glumae with pUCD3101B′46 had OA sensitivity similar to that of the parental isolates.

Detection of tobamoviruses from soils by non-precoated indirect ELISA.

A method for detecting tobamoviruses from field soils was developed using non-precoated indirect enzyme-linked immunosorbent assay (Id-ELISA). Absorbance values in Id-ELISA were relatively low after directly applying Pepper mild mottle virus (PMMoV)-infested soil extract. However, heat treating the soil extract before application greatly enhanced the absorbance values. The heat treatment was essential for the Id-ELISA detection of tobamoviruses from infested soil, although the efficiency of virus recovery varied depending on the properties of soil. The number of local lesions in the infectivity assay was consistent with the absorbance values in Id-ELISA. Moreover, the absorbance values in Id-ELISA were correlated with the incidence of soil transmission of PMMoV. Thus, Id-ELISA combined with heat treatment is a practical technique for the diagnosis of infestation with Tobamovirus in field soils, Gray Lowland soil and Sand-dune Regosol.

Isolation of the Oxolinic Acid Resistance Related-Gene from Genomic DNA of Burkholderia glumae

Burkholderia glumae is an agent of bacterial seedling rot and bacteria grain rot of rice, devastating diseases in north area and southwest area of Japan, respectively. In growing seedlings, bacterial seedling rot is caused by a rapid increase in the B. glumae population in the plumules (3). After transplanting into paddy fields, the pathogen exists in the upper leaf sheaths containing the flag leaf sheaths, invades into the flowering spikelets and multiplies rapidly, and causes bacterial grain rot (4).

Roles of popA for the Pathogenicity of Ralstonia solanacearum Pathogenic to Tobacco

Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating bacterial plant diseases worldwide (8). R. solanacearum strains including GMI1000, which are nonpathogenic to tobacco and pathogenic to tomato, elicit a hypersensitive response (HR) in tobacco leaves infiltrated with the bacteria (5,9). GMI1000 possesses hrp genes in a megaplasmid which direct their ability to elicit the HR in tobacco leaves or to be pathogenic to tomato (4). Several putative Arp-encoded proteins of GMI1000 share homology with proteins from the animal pathogens such as Yersinia, Salmonella and Shigella, and are assumed to be structural constituents of a secretion machinery, the type III secretion system that is required for the release of extracellular proteins (3).