Symbiosis-Promoting and Deleterious Effects of NopT, a Novel Type 3 Effector of Rhizobium sp. Strain NGR234

Abstract

Establishment of symbiosis between certain host plants and nitrogen-fixing bacteria ("rhizobia") depends on type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS). Here, we report that the open reading frame y4zC of strain NGR234 encodes a novel rhizobial type 3 effector, termed NopT (for nodulation outer protein T). Analysis of secreted proteins from NGR234 and T3SS mutants revealed that NopT is secreted via the T3SS. NopT possessed autoproteolytic activity when expressed in Escherichia coli or human HEK 293T cells. The processed NopT exposed a glycine (G50) to the N terminus, which is predicted to be myristoylated in eukaryotic cells. NopT with a point mutation at position C93, H205, or D220 (catalytic triad) showed strongly reduced autoproteolytic activity, indicating that NopT is a functional protease of the YopT-AvrPphB effector family. When transiently expressed in tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. Arabidopsis plants transformed with nopT showed chlorotic and necrotic symptoms, indicating a cytotoxic effect. Inoculation experiments with mutant derivatives of NGR234 indicated that NopT affected nodulation either positively (Phaseolus vulgaris cv. Yudou No. 1; Tephrosia vogelii) or negatively (Crotalaria juncea). We suggest that NopT-related polymorphism may be involved in evolutionary adaptation of NGR234 to particular host legumes.In root nodules of legumes, symbiotic bacteria ("rhizobia" belonging to the Rhizobiaceae family) reduce N 2 gas into ammonia by the process termed nitrogen fixation. Nodule formation requires specific bacterial signals and determinants (12). Rhizobial nodulation factors (Nod factors) trigger various host responses, including root hair deformation, expression of symbiosis-related host genes, and cortical cell divisions resulting in nodule formation (31, 32). Host-specific nodulation depends also on surface carbohydrates, such as oligosaccharides released from exopolysaccharides, lipopolysaccharides, cyclic ␤-glucans, and K antigens (also named capsular polysaccharides). In Rhizobium sp. strain NGR234, for example, mutants that do not produce exo-oligosaccharides or flavonoid-inducible lipopolysaccharide are unable to establish symbiosis with various host plants (36,48). In addition to these symbiotic determinants, host-specific nodulation depends on proteins secreted via a pilus-like secretory apparatus, the type 3 secretion system (T3SS) of NGR234. Recent data from various laboratories provide evidence that T3SSs of certain rhizobial strains modulate establishment of symbiosis and the efficiency of nitrogen fixation (14,23,25,56).T3SSs from pathogenic bacteria deliver effector proteins (type 3 effectors) into eukaryotic cells to manipulate the host metabolism, e.g., to suppress defense responses. Many reports indicate that type 3 effectors are virulence factors that play a key role in the pathogenesis of humans, animals, and plants (9,20). On the other hand, eukaryotic host cells developed strategies ...

Y4lO of Rhizobium sp. Strain NGR234 Is a Symbiotic Determinant Required for Symbiosome Differentiation

“…When expressed in tobacco or Lotus japonicus plants, NopL suppressed the expression of pathogen-related defense genes (6). NopT, another T3 effector of NGR234, elicited a hypersensitive reaction when expressed in tobacco cells (16).…”

Functional Analysis of the Type 3 Effector Nodulation Outer Protein L (NopL) from Rhizobium sp. NGR234

“…Thus, specific type 3 effectors can function as avirulence factors on certain host plants. Similarly, rhizobial type 3 effectors, such as NopT of NGR234, possess either a positive or negative role in the symbiosis with legume host plants (27,28), and R-proteins control host-specific nodulation in the interaction between soybean and specific rhizobial strains (37). In this study, the nopL mutant NGR⍀nopL was compared with the parent strain NGR234 in nodulation tests with the host plant P. vulgaris (cv.…”

“…strain NGR234 (20) possesses a functional type 3 secretion system (21), which can affect nodulation of host plants either positively or negatively. Up to now, four nodulation outer proteins (Nops) of NGR234 have been identified as type 3 effectors, namely NopL (21-24), NopP (25,26), NopT (27,28), and NopM (28). Their functions remain unclear, however.…”

“…and AvrPphB of Pseudomonas syringae pv. phaseolicola (27,28); NopM displays sequence similarities with YopM of Yersinia spp. and contains ubiquitin ligase motifs that are present in the E3 ubiquitin ligase IpaH 9.8 of Shigella spp.…”

Immunosuppression duringRhizobium-legume symbiosis

“…NGR234 secrets a few effectors including NopJ, NopL, NopM, NopP and NopT into host plant cells, where most of them interact with immune signaling pathways to suppress host defense responses. [21][22][23][24] For example, NopL mimics a MAP substrate to impair the MAPK signaling, while NopM is a ubiqutin ligase to reduce the ROS production induced by flg22. 20,21 Therefore, we propose that rhizobial T3SS effectors could trigger ETS to promote Rhizobium infection and survival in host cells.…”