NopB, a Type III Secreted Protein of Rhizobium sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages

Abstract

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGR⍀rhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.Rhizobia are gram-negative soil inhabitants that induce the formation of highly specialized, nitrogen-fixing organs called nodules on the roots or stems of leguminous plants. Some rhizobial species provoke nodule formation on a limited number of legume genera and are said to have narrow host ranges, e.g., Rhizobium meliloti, which nodulates only three genera of legumes. Other, broad-host-range rhizobia provoke the formation of nodules on many different legumes; e.g., Rhizobium sp. strain NGR234 (hereafter called NGR234) nodulates more than 112 genera of legumes as well as the nonlegume Parasponia andersonii (32,38). The formation of root nodules is a result of an elaborate developmental program directed by signal exchange between the two partners. These signals include flavonoids, Nod factors, surface polysaccharides, and extracellular proteins (7,31,34). After flavonoid induction, NGR234 secretes a number of extracellular proteins called Nops (nodulation outer proteins) via a type III secretion system (TTSS) (24, 41). TTSSs are virulence determinants shared by many diverse gram-negative bacteria that cause disease in plants and animals. The TTSS machinery is highly conserved and encoded by cluster of hrp (hypersensitive response and pathogenesis) or hrc (hrp conserved) genes in phytopathogens (2, 16). Homologues of the hrc genes were found in NGR234 and renamed rhc (Rhizobium conserved) (13,41

PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum , Are Translocators Belonging to the HrpF/NopX Family

“…In S. fredii, Western blot analysis with antibodies raised against NopX revealed that this protein is associated with the TTSS pili (43). In NGR234, Saad and colleagues (63) showed that the TTSS pili are mainly composed of three proteins: NopA, the major component of the TTSS-pilus, NopB, a protein resembling FlgK, a protein involved in flagellum assembly, and NopX (63). In R. solanacearum, PopF1 is also associated with Hrp pili, as determined by SDS-PAGE analysis of purified pili preparations.…”

“…Moreover, HrpF can form pores in a planar lipid bilayer system (12); this protein may thus act as a translocator. HrpF displays similarity to NolX (recently renamed NopX [63]), a TTSS-secreted protein of S. fredii and Rhizobium sp. strain NGR234 (34,42,51).…”

“…strain NGR234, and Xanthomonas campestris pv. vesicatoria (43,63,76). Mutations in genes encoding Hrp pili prevent secretion of effectors and other accessory proteins in vitro (31).…”

Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103

“…Genes responsible for the biosynthesis of the rhizobial T3SS are organized in the tts region, which also contains genes that encode secreted proteins collectively known as nodulation outer proteins (Nops). To date, eight secreted proteins have been identified: NopA, NopB, NopC, NopD, NopL, NopM, NopP and NopX (Ausmees et al, 2004;Bartsev et al, 2003;Deakin et al, 2005;Krishnan, 2002;Lorio et al, 2004;Rodrigues et al, 2007;Saad et al, 2005;Skorpil et al, 2005). Recently, eight proteins have been found to be type III-secreted in B. japonicum; one of them, GunA2, has been identified as an endoglucanase (Süss et al, 2006).…”

Signals in Soybean's Inoculants

“…This led to the discovery of a complex interplay between the T3SS and surface polysaccharides in the molecular dialogue of the rhizobium-host interaction (Broughton et al, 2006). In Rhizobium species strain NGR234 at least six T3SS-secreted Nops have been identified: NopA, NopB, NopC, NopL, NopP, and NopX (Ausmees et al, 2004;Deakin et al, 2005;Marie et al, 2003;Saad et al, 2005;Viprey et al, 1998). Depending upon the legume host, abolition of Nop secretion by NGR234 can improve or block symbiotic interactions.…”