MucR Is Required for Transcriptional Activation of Conserved Ion Transporters to Support Nitrogen Fixation of Sinorhizobium fredii in Soybean Nodules

Abstract

To achieve effective symbiosis with legume, rhizobia should fine-tune their background regulation network in addition to activating key genes involved in nodulation (nod) and nitrogen fixation (nif). Here, we report that an ancestral zinc finger regulator, MucR1, other than its paralog, MucR2, carrying a frameshift mutation, is essential for supporting nitrogen fixation of Sinorhizobium fredii CCBAU45436 within soybean nodules. In contrast to the chromosomal mucR1, mucR2 is located on symbiosis plasmid, indicating its horizontal transfer potential. A MucR2 homolog lacking the frameshift mutation, such as the one from S. fredii NGR234, can complement phenotypic defects of the mucR1 mutant of CCBAU45436. RNA-seq analysis revealed that the MucR1 regulon of CCBAU45436 within nodules exhibits significant difference compared with that of free-living cells. MucR1 is required for active expression of transporters for phosphate, zinc, and elements essential for nitrogenase activity (iron, molybdenum, and sulfur) in nodules but is dispensable for transcription of key genes (nif/fix) involved in nitrogen fixation. Further reverse genetics suggests that S. fredii uses high-affinity transporters to meet the demand for zinc and phosphate within nodules. These findings, together with the horizontal transfer potential of the mucR homolog, imply an intriguing evolutionary role of this ancestral regulator in supporting nitrogen fixation.

Medicago truncatula Zinc‐Iron Permease6 provides zinc to rhizobia‐infected nodule cells

“…However, pinpointing the specific cause is extremely difficult with our current understanding of the nodule zinc proteome and the techniques to analyse it. The phenotype observed in symbiotic mtzip6 RNAi plants is consistent with that observed in soybean inoculated with S. fredii znuA mutant (Jiao et al, ), lacking a bacterial high‐affinity Zn transporter (Vahling‐Armstrong, Zhou, Benyon, Morgan, & Duan, ), which is mainly expressed in nodule interzone and ZIII (Roux et al, ). This would suggest that Zn requirement for SNF is probably of bacterial origin.…”

Extracellular polysaccharide protects Rhizobium leguminosarum cells against zinc stress in vitro and during symbiosis with clover

“…In nitrogen-fixing bacteria, zinc is a component of transcriptional regulatory proteins from the Ros/MucR family, which play an essential role in EPS synthesis, motility and symbiosis with legumes. These proteins are found in S. meliloti, S. fredii, Rhizobium etli and R. leguminosarum (Keller et al, 1995;Bittinger et al, 1997;Rachwał et al, 2015;Acosta-Jurado et al, 2016;Jiao et al, 2016). Therefore, Zn 21 deficiency seriously disturbs several biochemical and physiological processes in various organisms.…”

The nitrate‐reduction gene cluster components exert lineage‐dependent contributions to optimization of Sinorhizobium symbiosis with soybeans

“…Gene clusters and individual genes were deleted using the cre‐lox system (Marx and Lidstrom, ). Briefly, PCR fragments encompassing the upstream and the downstream regions of the genomic region to be deleted were sequentially amplified and cloned into two multiple cloning sites, respectively, of pCM351 by digestion‐ligation‐transformation manipulation (Jiao et al ., ). The resultant derivatives of pCM351 harbouring correct cloned sequences, as verified by Sanger sequencing, were then conjugated into Sinorhizobium strains using pRK2013 as the helper plasmid (Ditta et al ., ; Jiao et al ., ).…”

“…Briefly, PCR fragments encompassing the upstream and the downstream regions of the genomic region to be deleted were sequentially amplified and cloned into two multiple cloning sites, respectively, of pCM351 by digestion-ligationtransformation manipulation . The resultant derivatives of pCM351 harbouring correct cloned sequences, as verified by Sanger sequencing, were then conjugated into Sinorhizobium strains using pRK2013 as the helper plasmid (Ditta et al, 1980;Jiao et al, 2016). To construct the plasmids expressing hemN homologues, the coding sequences and upstream promoter regions were amplified and cloned into a multiple cloning site of pBBR1MCS-2 (Kovach et al, 1995).…”