Date of Award

December 2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Gyaneshwar Prasad

Committee Members

Mark J. McBride, Heather A. Owen, Ching H. Yang, Sonia L. Bardy

Keywords

Biofilm, Lipoplysaccharide, Nitrogen, Rhizobium Sp. IRBG74, Rice Endophyte

Abstract

Rhizobium sp. IRBG74 (IRBG74) develops a classical nitrogen-fixing symbiosis with the legume Sesbania cannabina and also promotes the growth of rice (Oryza sativa), but not much is known about the rhizobial determinants important for these interactions. We hypothesize that Rhizobium sp. IRBG74 utilizes similar mechanisms to endophytically colonize both legume and cereal hosts. In this study, we analyzed the colonization of rice and S. cannabina using a strain of IRBG74 marked with β-glucuronidase (GUS) and Green Fluorescent Protein (GFP). IRBG74 infected both of the host plants through crack entry under submerged conditions, but showed root hair mediated infection under aerobic conditions. In rice, IRBG74 was localized within intercellular spaces in the cortex, in the xylem of roots and stems, and intracellularly within epidermal and hypodermal cell layers. We have also shown that the colonization of rice by Rhizobium sp. IRBG74 requires the nod factor. A transposon insertion mutant of IRBG74 in rffB, which encodes dTDP-glucose dehydratase, exhibited significant defects not only in rice colonization but also in nodulation of S. cannabina. IRBG74 was found to synthesize a rhamnose-rich LPS and the rffB mutant produced a truncated version of LPS lacking rhamnose. Colonization of both rice and S. cannabina roots by the rffB mutant was restored by supplementation with purified LPS from wild type IRBG74, but not by LPS from the rffB mutant. Another transposon insertion mutant of IRBG74, thiQ, was identified as a biofilm defective mutant. It exhibited significant defects in rice colonization and showed no nodulation phenotype in S. cannabina. Preliminary results on the thiQ mutant suggest that it is likely involved in flavonoid transport. Taken together, these results indicate that IRBG74 is an effective rice endophyte that utilizes a similar physical mode of entry and a common signaling mechanism to invade rice and its legume host, Sesbania. Understanding this rhizobial-cereal interaction may provide novel avenues for engineering a symbiotic nitrogen-fixing system in these important crops.

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