Expression analysis of rice pathogenesis-related proteins involved in stress response and endophytic colonization properties of gfp-tagged Bacillus subtilis CB-R05.

Bacillus subtilis CB-R05, possessing antagonistic effects against several fungal pathogens, is a diazotrophic plant growth-promoting bacteria marked with the green fluorescent protein (gfp) gene. To confirm the expression level of the pathogenesis-related (PR) proteins in rice inoculated with CB-R05, the expressions of four pathogenesis-related (PR) proteins (PR2, PR6, PR15, and PR16) were examined in the rice leaves treated with wounding stress over a time period. The PR proteins were generally more strongly expressed in the rice leaves inoculated with CB-R05 compared with the untreated control. The marked gfp-tagged B. subtilis CB-R05 strain was inoculated onto the rice seedlings under axenic conditions. Under the confocal laser scanning microscope (CLSM), the gfp-tagged CB-R05 bacterial cells were observed to penetrate the rhizoplane, especially in the elongation and differentiation zones of the rice roots, and colonize the root intracellularly. The bacteria, 24 h after the gfp-tagged CB-R05 inoculation, were seen to penetrate into the cell wall, cortex, xylem, and concentrate mainly in the vascular bundle. Numerous bacteria were observed within the intercellular spaces, root cortical cells, and xylem vessels. Over time, these bacteria dispersed to the lateral root junctions and propagated slowly from the roots to the stems and leaves. The B. subtilis CB-R05 population in the rice root rhizosphere was also monitored. These results show a very widespread colonization of the B. subtilis CB-R05 in the rice rhizosphere. Further attempts are under way to investigate the competition between the CB-R05 bacteria and the fungal pathogen in vivo.