Abstract
Bradyrhizobium elkanii USDA61 is incompatible with mung bean (Vigna radiata cv. KPS1) and soybean (Glycine max cv. BARC2) and unable to nodulate either plant. This incompatibility is due to the presence of a functional type III secretion system (T3SS) that translocates effector protein into host cells. We previously identified five genes in B. elkanii that are responsible for its incompatibility with KPS1 plants. Among them, a novel gene designated as innB exhibited some characteristics associated with the T3SS and was found to be responsible for the restriction of nodulation on KPS1. In the present study, we further characterized innB by analysis of gene expression, protein secretion, and symbiotic phenotypes. The innB gene was found to encode a hypothetical protein that is highly conserved among T3SS-harboring rhizobia. Similar to other rhizobial T3SS-associated genes, the expression of innB was dependent on plant flavonoids and a transcriptional regulator TtsI. The InnB protein was secreted via the T3SS and was not essential for secretion of other nodulation outer proteins. In addition, T3SS-dependent translocation of InnB into nodule cells was confirmed by an adenylate cyclase assay. According to inoculation tests using several Vigna species, InnB promoted nodulation of at least one V. mungo cultivar. These results indicate that innB encodes a novel type III effector controlling symbiosis with Vigna species.
Highlights
Symbiotic relationships between legumes and soil bacteria, collectively called rhizobia, substantially contribute to agricultural production and the nitrogen cycle on the Earth (Broughton et al, 2000; Perret et al, 2000)
Our results reveal that innB encodes a novel type III effector that controls symbiosis with Vigna species
B. elkanii USDA61 and mutant strains were grown at 28◦C on arabinose–gluconate (AG) medium (Sadowsky et al, 1987) or peptone salts yeast extract (PSY) medium (Regensburger and Hennecke, 1983) and Escherichia coli strains were grown at 37◦C on LB medium (Green and Sambrook, 2012)
Summary
Symbiotic relationships between legumes and soil bacteria, collectively called rhizobia, substantially contribute to agricultural production and the nitrogen cycle on the Earth (Broughton et al, 2000; Perret et al, 2000). Rhizobia induce the formation of specialized organs, known as root nodules, and are accommodated within them. Certain flavonoids exuded by host legume roots interact with the rhizobial protein NodD, which binds to specific promoter sequences (called nod boxes) and activates the transcription of nodulation (nod) genes (Cullimore et al, 2001; Radutoiu et al, 2007). Flavonoids from host legumes induce secretion of specific proteins via the bacterial type III protein secretion system (T3SS). Rhizobial type III secreted proteins are designated as nodulation outer proteins (Nops) (Krause et al, 2002; Marie et al, 2004; López-Baena et al, 2008; Wassem et al, 2008; Okazaki et al, 2009). Our results reveal that innB encodes a novel type III effector that controls symbiosis with Vigna species
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