Functional Analysis of NopM, a Novel E3 Ubiquitin Ligase (NEL) Domain Effector of Rhizobium sp. Strain NGR234
Figure 2
Characterization of the mutant strains NGRΩnopM and NGRnopM(C338A).
(A) Schematic representation of the knock-out mutant NGRΩnopM (with an insertion of a 2-kb spectinomycin Ω interposon (Ωsp) at the constructed BamHI restriction site) and of the point mutant NGRnopM(C338A) (cysteine 338 mutated to alanine; additional Aor51HI restriction site). (B) Immunoblot analysis of NopM of Rhizobium sp. NGR234 and indicated mutant derivatives. Immunoblots were performed with secreted proteins and the anti-NopM antibodies. As a loading control, secreted proteins in the molecular weight range of NopM were visualized on a parallel SDS-PAGE gel by silver staining (C) Symbiotic phenotype of Rhizobium sp. NGR234 (column 1), NGRΩnopM (column 2), NGRnopM(C338A), (column 3), NGRΩnopM carrying pFAJ-nopM (column 4) and NGRnopM(C338A) carrying pFAJ-nopM (column 5) on the host plant L. purpureus (cv. Chaojibiandou). In total, 70 plants (14 plants per strain) were inoculated. The numbers of nodules formed per plant were determined 35 days post inoculation. Data indicate means ± SE. Asterisks indicate significant reduced nodule formation as compared to the parent strain NGR234 (Kruskal-Wallis rank sum test; P<0.01).