Abstract
Sinorhizobium fredii is a dominant rhizobium on alkaline-saline land that can induce nitrogen-fixing symbiotic root nodules in soybean. Two S. fredii strains, CCBAU25509 and CCBAU45436, were used in this study to facilitate in-depth analyses of this species and its interactions with soybean. We have previously completed the full assembly of the genomes and detailed transcriptomic analyses for these two S. fredii strains, CCBAU25509 and CCBAU45436, that exhibit differential compatibility toward some soybean hosts. In this work, we performed high-throughput Orbitrap analyses of the whole proteomes and secretomes of CCBAU25509 and CCBAU45436 at different growth stages. Our proteomic data cover coding sequences in the chromosome, chromid, symbiotic plasmid, and other accessory plasmids. In general, we found higher levels of protein expression by genes in the chromosomal genome, whereas proteins encoded by the symbiotic plasmid were differentially accumulated in bacteroids. We identified secreted proteins from the extracellular medium, including seven and eight Nodulation Outer Proteins (Nops) encoded by the symbiotic plasmid of CCBAU25509 and CCBAU45436, respectively. Differential host restriction of CCBAU25509 and CCBAU45436 is regulated by the allelic type of the soybean Rj2(Rfg1) protein. Using sequencing data from this work and available in public databases, our analysis confirmed that the soybean Rj2(Rfg1) protein has three major allelic types (Rj2/rfg1, rj2/Rfg1, rj2/rfg1) that determine the host restriction of some Bradyrhizobium diazoefficiens and S. fredii strains. A mutant defective in the type 3 protein secretion system (T3SS) in CCBAU25509 allowed this strain to nodulate otherwise-incompatible soybeans carrying the rj2/Rfg1 allelic type, probably by disrupting Nops secretion. The allelic forms of NopP and NopI in S. fredii might be associated with the restriction imposed by Rfg1. By swapping the NopP between CCBAU25509 and CCBAU45436, we found that only the strains carrying NopP from CCBAU45436 could nodulate soybeans carrying the rj2/Rfg1 allelic type. However, no direct interaction between either forms of NopP and Rfg1 could be observed.
Highlights
Rhizobia are Gram-negative bacteria that form symbiotic root nodules in leguminous plants
The peptides obtained from the tryptic digestion of extracted proteomes were analyzed by Orbitrap FusionTM LumosTM TribridTM Mass Spectrometer coupled to a nano-liquid chromatography (LC)
Almost corresponding to 11,000 peptides, or 2,000 proteins in each 600 proteins were detected from bacteroids collected from the nodules of G. max (C08) and G. soja (W05) plants inoculated with S. fredii CCBAU45436 and CCBAU25509, respectively, after 28 days (Figure 1A and Supplementary Tables S3, S4)
Summary
Rhizobia are Gram-negative bacteria that form symbiotic root nodules in leguminous plants. They reduce atmospheric dinitrogen to ammonia which can be efficiently used by host plants for growth and development, in return for carbon and other nutrients provided by their hosts. They constitute an important mechanism for sustainable agriculture by reducing the dependence on synthetic nitrogen fertilizers (Dixon and Kahn, 2004). The genus Sinorhizobium can induce nodule formation on the roots of many host plants from the Leguminosae family. Sinorhizobium fredii, on the other hand, has a very wide range of hosts, representing all three subfamilies of the Leguminosae family, including cultivated (Glycine max) and wild (G. soja) soybeans (Sugawara et al, 2013)
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