Presentation Title

Role of nodC and nodD genes in Paraburkholderia tuberum

Faculty Mentor

Michelle Lum

Start Date

23-11-2019 8:00 AM

End Date

23-11-2019 8:45 AM

Location

73

Session

poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Rhizobia are soil bacteria that are present in the root nodules of plants, where they have the ability to form nitrogen fixing symbioses with members of the legume family. Paraburkholderia tuberum is a β-proteobacteria rhizobia that nodulates legumes such as Phaseolus vulgaris (bean). Additionally, P. tuberum has been recognized to contain the nodulation genes, nodABC, as well as two copies of nodD, which in α-rhizobia are specifically responsible for the synthesis of Nod factor, a lipochitin oligiosaccharide (LCO), which is required for root nodule morphogenesis in plants. The primary objective of this study was to determine whether nodC and nodD are critical for β-rhizobia symbiosis as it is in α -rhizobia, through generating deletions in nodC and nodD genes of P. tuberum.

Primers were designed to amplify the border regions of nodC, nodD1, and nodD2. These were cloned into the pk18mobsacB vector to generate each deletion. The upstream/downstream regions were fused and digested with BamHI and HindIII, and ligated into the pK18mobsacB vector, which carries kanamycin resistance and sucrose sensitivity. The ligation mix was transformed into E. coli S17, mated with the deletion construct and wild type P. tuberum. A double crossover event was selected for using kanamycin resistance and sucrose sensitivity. Black bean was inoculated with the selected nodC, nodD1, nodD2, and nodD1D2 mutants and were quantified for the number of nodules observed.

The black bean plants inoculated with nodC and nodD1D2 mutants were not able to form nodules, while those with nodD1 and nodD2 mutants formed nodules. Furthermore, the nodD2 mutants were found to have an increased number of observed nodules when compared to the nodD1 mutants. Overall, this indicates that both nodC and nodD genes are essential for both α- and β-rhizobia symbiosis and that the conservation of genes are required in the pathway towards nodulation.

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Nov 23rd, 8:00 AM Nov 23rd, 8:45 AM

Role of nodC and nodD genes in Paraburkholderia tuberum

73

Rhizobia are soil bacteria that are present in the root nodules of plants, where they have the ability to form nitrogen fixing symbioses with members of the legume family. Paraburkholderia tuberum is a β-proteobacteria rhizobia that nodulates legumes such as Phaseolus vulgaris (bean). Additionally, P. tuberum has been recognized to contain the nodulation genes, nodABC, as well as two copies of nodD, which in α-rhizobia are specifically responsible for the synthesis of Nod factor, a lipochitin oligiosaccharide (LCO), which is required for root nodule morphogenesis in plants. The primary objective of this study was to determine whether nodC and nodD are critical for β-rhizobia symbiosis as it is in α -rhizobia, through generating deletions in nodC and nodD genes of P. tuberum.

Primers were designed to amplify the border regions of nodC, nodD1, and nodD2. These were cloned into the pk18mobsacB vector to generate each deletion. The upstream/downstream regions were fused and digested with BamHI and HindIII, and ligated into the pK18mobsacB vector, which carries kanamycin resistance and sucrose sensitivity. The ligation mix was transformed into E. coli S17, mated with the deletion construct and wild type P. tuberum. A double crossover event was selected for using kanamycin resistance and sucrose sensitivity. Black bean was inoculated with the selected nodC, nodD1, nodD2, and nodD1D2 mutants and were quantified for the number of nodules observed.

The black bean plants inoculated with nodC and nodD1D2 mutants were not able to form nodules, while those with nodD1 and nodD2 mutants formed nodules. Furthermore, the nodD2 mutants were found to have an increased number of observed nodules when compared to the nodD1 mutants. Overall, this indicates that both nodC and nodD genes are essential for both α- and β-rhizobia symbiosis and that the conservation of genes are required in the pathway towards nodulation.