Characterization of NopP, a type III secreted effector of Rhizobium sp. strain NGR234

Abstract

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.

FIG. 1.

Comparison of flavonoid-induced extracellular proteins secreted by Rhizobium sp. strain NGR234, R. fredii USDA257, and various mutant strains. (A) Silver-stained SDS—15% PAGE gel of secreted proteins of NGR234 (lanes 1 and 2) and USDA257 (lanes 3 and 4) collected after 40 h of induction with apigenin (lanes 1 and 3) or genistein (lanes 2 and 4). (B) Western blot of flavonoid-induced secreted proteins of NGR234 (lanes 1 and 2), USDA257 (lanes 3 and 4), NGRΔnopX (lane 5), NGRΩnopL (lane 6), and the double mutant NGRΔnopXΩnopL (lane 7) separated on an SDS—12% PAGE gel and immunostained with SR3 antibodies. Cells were induced with either apigenin (lanes 1, 3, 5, 6, and 7) or genistein (lanes 2 and 4). The positions of NopX, NopL, and SR3 are indicated by arrows. Estimated molecular masses (in kilodaltons) are indicated on the left.

FIG. 2.

NopP cross-reacts with SR3 antibodies, and its secretion requires a functional TTSS. (A) Silver staining of an SDS—12% PAGE gel containing extracellular proteins of apigenin-induced strains NGR234 (lane 1), NGRΔnopP (lane 2), and NGRΩrhcN (lane 3). (B, C, and D) Western blots of identical gels immunostained with SR3, NopP, and SRT antibodies, respectively. The positions of NopP (P), NopL (L), and NopX (X) are indicated by arrows. Estimated molecular masses (in kilodaltons) are indicated.

FIG. 3.

ClustalV alignment of NopP homologues. NopP of NGR234 (NopP), ORF3 of R. etli strain CNPAF512 (ORF3), y4yP of R. fredii USDA257 (y4yP), HipA of R. fredii USDA201 (HipA), and ID84 of Bradyrhizobium japonicum USDA110 (ID84) were aligned by using ClustalV. Note that the predicted N terminus of HipA was extended to meet the same start codon as that in NopP. Amino acids common to all NopP homologues are indicated by red type, and the peptide encoded by the SR3c13 clone is indicated by green type. Putative proteins, such as ID185, ID186, and ID322, that correspond to either the N-terminal half of NopP (ID186) or the C-terminal half of NopP (ID185 and ID322) were not included in the alignment.