Functional Role Of Multidrug Efflux Pump Genes Of Bradyrhizobium Japonicum In Nodulation Competitiveness

Previous studies found that the expression profiles of genomic locus BjG30 in B.j110 is extremely unique in response to the genistein, and has shown that multidrug efflux pump genes bll7019-7021 on BjG30 may play an important role in the early symbiotic interaction.The absence of this genes will result in the change of rhizobium nodulation and nitrogen fixation capability.In the present study, a further study on bll7019-7021 and its neighboring regulator gene blr7023 is carried out to address the function and mechanism of gene blr7023. Firstly, Real Time RT-PCR technology was used to test the expression of nodulation gene in both mutant strains, while the sensitivity of mutant strains against various secondary metabolites of leguminous plant was analyzed. Then, the inoculation experiment on soybean varieties (e.g. Yellow soybeans, Japanese soybeans and Jingdou No.19) was performed to determine the effect of target genes on nodulation and nitrogen fixation capabilities after 60 days. Finally, we monitored the rhizosphere microbial community of mutant strain-inoculated soybean. The results were as follows:1.bll7019-7021 and blr7023 gene mutation had no effect on the expression of ttss and nod genes. However, blr7023 gene is likely involved in the regulation of expression of genes cluster bll7019-7021.2.Analysis of sensitivity of mutant strain against leguminous plants secondary metabolites showed that gene bll7019-7021 mutation would weaken the sensitivity of cells againt leguminous plant flavonoids. But the mutation of gene blr7023 was on the contrary.3. The inoculation experiment demonstrated that both the number of nodules and dry weight of nodules would not be changed for gene bll7019-7022 mutation. On the other side, gene blr7023 mutation would lead to the increasing of nodules but had no effect on dry weight of nodules. However, both gene blr7023 and bll7019-7022 mutation affected the dry weight and total nitrogen content of plant, depending on host plants.4.Community diversity analysis showed that blr7023 and bll7019-7022 gene mutation would lead to obvious changes of the diversity and abundance indigenous bacteria in rhizosphere of soybean 20 days after inoculation. However, blr7023 gene mutation continues its effect up to 60 days after inoculation.Taken together, the multidrug efflux pump encoded by b117019-7021 and blr7023 operon affect competitive nodulation ability in the early stage of symbiotic nitrogen-fixation through the changing of the microbial community diversity in rhizosphere, but not expression of nodulation genes.