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Expression Analysis of Rice Pathogenesis-related Proteins Involved in Stress Response and Endophytic Colonization Properties of gfp-tagged Bacillus subtilis CB-R05

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Abstract

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.

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Authors and Affiliations

  1. Department of Molecular Biotechnology, Konkuk University, Seoul, 143-701, Korea

    Sang Hye Ji & Se-Chul Chun

  2. Subtropical Horticulture Research Institute, Faculty of Biotechnology, Jeju National University, Jeju, 690-756, Korea

    Mayank Anand Gururani

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  1. Sang Hye Ji
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  2. Mayank Anand Gururani
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  3. Se-Chul Chun
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Correspondence to Se-Chul Chun.

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Supplementary Figure 1

Growth rates of wild type (CB-R05) and gfp-tagged CB-R05 (GFP + CB-R05-ECE2 strain) bacterial strains in a time period of 0 to 80 h. (JPEG 58 kb)

Supplementary Figure 2

CLSM images showing colonization of gfp-tagged CB-R05 roots of rice variety Ilpum at 24 hours after inoculation with gfp-tagged CB-R05 grown in hydroponic system. After 24 h, the bacteria colonization was similar to the soil culture system, B. subtilis CB-R05 bacterial cells were mostly distributed on the vascular cylinder (elongation zone) (B,F), root apex (division zone) (A,C,E), root cap (D) and root hair zone of roots. The gfp-tagged CB-R05 bacteria were densely distributed in the stele cells of the elongation zone than the cortex cells. These bacteria also colonized in the epidermis cells. (JPEG 182 kb)

Supplementary Figure 3

CLSM images showing colonization of gfp-tagged CB-R05 roots of rice variety Ilpum at 5 days after inoculation with gfp-tagged CB-R05 grown in soil system by seed drenching method. The gfp-tagged CB-R05 bacteria colonized predominantly in elongation zones of the roots (A,C) and root tips (B.D). (JPEG 81 kb)

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Ji, S.H., Gururani, M.A. & Chun, SC. Expression Analysis of Rice Pathogenesis-related Proteins Involved in Stress Response and Endophytic Colonization Properties of gfp-tagged Bacillus subtilis CB-R05. Appl Biochem Biotechnol 174, 231–241 (2014). https://doi.org/10.1007/s12010-014-1047-3

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  • DOI: https://doi.org/10.1007/s12010-014-1047-3

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