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The rhizobial type III secretion system secretes effector proteins into host plant cells, which may either promote or inhibit symbiosis with legumes. We herein demonstrated that the type III secretion system of Bradyrhizobium sp. SUTN9-2 obstructed symbiosis with Lotus japonicus Miyakojima, L. japonicus Gifu, and Lotus burttii. A mutant of SUTN9-2 that is unable to secrete effector proteins showed better nodulation and plant growth promotion than wild-type SUTN9-2 when paired with these Lotus spp. We propose that SUTN9-2 is a useful strain for understanding the mechanisms by which effector proteins obstruct symbiosis between Bradyrhizobium and Lotus spp.

International audience; The Bradyrhizobium sp. DOA9 strain displays the unusual properties to have a symbiotic plasmid and to fix nitrogen during both free-living and symbiotic growth. Sequence genome analysis shows that this strain contains the structural genes of dinitrogenase (nifDK) and the nifA regulatory gene on both the plasmid and chromosome. It was previously shown that both nifDK clusters are differentially expressed depending on growth conditions, suggesting different mechanisms of regulation. In this study, we examined the functional regulatory role of the two nifA genes found on the plasmid (nifAp) and chromosome (nifAc) that encode proteins with a moderate level of identity (55%) and different structural architectures. Using gusA (beta-glucuronidase) reporter strains, we showed that both nifA genes were expressed during both the free-living and symbiotic growth stages. During symbiosis with Aeschynomene americana, mutants in only one nifA gene were not altered in their symbiotic properties, while a double nifA mutant was drastically impaired in nitrogen fixation, indicating that the two NifA proteins are functionally redundant during this culture condition. In contrast, under in vitro conditions, the nifAc mutant was unable to fix nitrogen, and no effect of the nifAp mutation was detected, indicating that NifAc is essential to activate nif genes during free-living growth. In accordance, the nitrogenase fixation deficiency of this mutant could be restored by the introduction of nifAc but not by nifAp or by two chimeric nifA genes encoding hybrid proteins with the N-terminus part of NifAc and the C-terminus of NifAp. Furthermore, transcriptional analysis by RT-qPCR of the WT and two nifA mutant backgrounds showed that NifAc and NifAp activated the expression of both chromosome and plasmid structural nifDK genes during symbiosis, while only NifAc activated the expression of nifDKc during free-living conditions. In summary, this study provides a better overview of the complex mechanisms of regulation of the nitrogenase genes in the DOA9 strain that involve two distinct NifA proteins, which are exchangeable during symbiosis for the activation of nif genes but not during free-living growth where NifAc is essential for the activation of nifDKc.

Plant associations by bradyrhizobia have been detected not only in leguminous plants, but also in non-leguminous species including rice. Bradyrhizobium sp. SUTN9-2 was isolated from Aeschynomene americana L., which is a leguminous weed found in the rice fields of Thailand. This strain promoted the highest total rice (Oryza sativa L. cultivar Pathum Thani 1) dry weight among the endophytic bradyrhizobial strains tested, and was, thus, employed for the further characterization of rice-Bradyrhizobium interactions. Some known bacterial genes involved in bacteria-plant interactions were selected. The expression of the type III secretion component (rhcJ), type IV secretion component (virD4), and pectinesterase (peces) genes of the bacterium were up-regulated when the rice root exudate was added to the culture. When SUTN9-2 was inoculated into rice seedlings, the peces, rhcJ, virD4, and exopolysaccharide production (fliP) genes were strongly expressed in the bacterium 6-24 h after the inoculation. The gene for glutathione-S-transferase (gst) was slightly expressed 12 h after the inoculation. In order to determine whether type III secretion system (T3SS) is involved in bradyrhizobial infections in rice plants, wild-type SUTN9-2 and T3SS mutant strains were inoculated into the original host plant (A. americana) and a rice plant (cultivar Pathum Thani 1). The ability of T3SS mutants to invade rice tissues was weaker than that of the wild-type strain; however, their phenotypes in A. americana were not changed by T3SS mutations. These results suggest that T3SS is one of the important determinants modulating rice infection; however, type IV secretion system and peces may also be responsible for the early steps of rice infection.

Bradyrhizobial inoculants used for soybean seed inoculation to maximize the benefit of N2-fixation should include bradyrhizobial strain with high N2-fixation rates and ability to compete with the indigenous rhizobial populations. In this study, co-inoculation of plant growth promoting rhizobacteria (PGPR) Azospirillum sp. with either of Bradyrhizobium japonicum CB 1809 or USDA 110 increased shoot and root dry weight of soybean over non-inoculated control under pot condition with no indigenous soybean nodulating bradyrhizobia. Moreover, competition for nodulation and the effects on rhizosphere soil eubacterial community structures by using single or co-inoculation of B. japonicum and Azospirillum sp. under rhizobia-established Myanmar and Thailand soils were investigated. By inoculation of gus-marked USDA 110 singly or its co-inoculation gave 93.21 to 94.75% and 74.21 to 100% in nodule occupancy, and 23.50 to 41.95% and 50.37 to 73.24% promotion in biomass dry weight over non-inoculated control in Myanmar and Thailand soil samples, respectively. Each of all the tested inoculum levels, that is 106, 107 and 108 cfu/ml of Azospirillum sp. enhanced nodulation in combination with USDA 110 with a corresponding increase in 73.8, 62.25 and 95.34%; and 51.52, 62.38 and 79.46% over non-inoculated control, respectively in Myanmar and Thailand soil, respectively. In addition, soybean rhizosphere soil eubacterial community structures were not shifted by bacterial inoculation. Therefore, Azospirillum sp. could be used in co-inoculant production with B. japonicum for soybean.Keywords: Bradyrhizobium, plant growth promoting rhizobacteria (PGPR), soybean, co-inoculation, competition, rhizosphere eubacterial community structureAfrican Journal of Biotechnology Vol. 12(20), pp. 2850-2862