Scrutiny of NolA and NodD1 Regulatory Roles in Symbiotic Compatibility Unveils New Insights into Bradyrhizobium guangxiense CCBAU53363 Interacting with Peanut (Arachis hypogaea) and Mung Bean (Vigna radiata).

Bradyrhizobium guangxiense CCBAU53363 efficiently nodulates peanut but exhibits incompatible interaction with mung bean. By comparing the common nod region with those of other peanut bradyrhizobia efficiently nodulating these two hosts, distinctive characteristics with a single nodD isoform (nodD1) and a truncated nolA were identified. However, the regulatory roles of NodD1 and NolA and their coordination in legume-bradyrhizobial interactions remain largely unknown in terms of explaining the contrasting symbiotic compatibility. Here, we report that nolA was important for CCBAU53363 symbiosis with peanut but restricted nodulation on mung bean, while nodD1 was dispensable for CCBAU53363 symbiosis with peanut but essential for nodulation on mung bean. Moreover, nolA exerted a cumulative contribution with nodD1 to efficient symbiosis with peanut. Additionally, mutants lacking nolA delayed nodulation on peanut, and both nolA and nodD1 were required for competitive nodule colonization. It is noteworth that most of the nodulation genes and type III secretion system (T3SS)-related genes were significantly downregulated in a strain 53ΔnodD1nolA mutant compared to wild-type strain CCBAU53363, and the downregulated nodulation genes also had a greater impact than T3SS-related genes on the symbiotic defect of 53ΔnodD1nolA on peanut, which was supported by a more severe symbiotic defect induced by 53ΔnodC than that with the 53ΔnodD1nopP, 53ΔnodD1rhcJ, and 53ΔnodD1ttsI mutants. NolA did not regulate nod gene expression but did regulate the T3SS effector gene nopP in an indirect way. Meanwhile, nolA, nodW, and some T3SS-related genes besides nopP were also demonstrated as new "repressors" that seriously impaired CCBAU53363 symbiosis with mung bean. Taken together, the roles and essentiality of nolA and nodD1 in modulating symbiotic compatibility are sophisticated and host dependent. IMPORTANCE The main findings of this study were that we clarified that the roles and essentiality of nodD1 and nolA are host dependent. Importantly, for the first time, NolA was found to positively regulate T3SS effector gene nopP to mediate incompatibility on mung bean. Additionally, NolA does not regulate nod genes, which are activated by NodD1. nolA exerts a cumulative effect with nodD1 on CCBAU53363 symbiosis with peanut. These findings shed new light on our understanding of coordinated regulation of NodD1 and NolA in peanut bradyrhizobia with different hosts.

Coordinated regulation of symbiotic adaptation by NodD proteins and NolA in the type I peanut bradyrhizobial strain Bradyrhizobium zhanjiangense CCBAU51778.

Type I peanut bradyrhizobial strains can establish efficient symbiosis in contrast to symbiotic incompatibility induced by type II strains with mung bean. The notable distinction in the two kinds of key symbiosis-related regulators nolA and nodD close to the nodABCSUIJ operon region between these two types of peanut bradyrhizobia was found. Therefore, we determined whether NolA and NodD proteins regulate the symbiotic adaptations of type I strains to different hosts. We found that NodD1-NolA synergistically regulated the symbiosis between the type I strain Bradyrhizobium zhanjiangense CCBAU51778 and mung bean, and NodD1-NodD2 jointly regulated nodulation ability. In contrast, NodD1-NolA coordinately regulated nodulation ability in the CCBAU51778-peanut symbiosis. Meanwhile, NodD1 and NolA collectively contributes to competitive nodule colonization of CCBAU51778 on both hosts. The Fucosylated Nod factors and intact type 3 secretion system (T3SS), rather than extra nodD2 and full-length nolA, were critical for effective symbiosis with mung bean. Unexpectedly, T3SS-related genes were activated by NodD2 but not NodD1. Compared to NodD1 and NodD2, NolA predominantly inhibits exopolysaccharide production by promoting exoR expression. Importantly, this is the first report that NolA regulates rhizobial T3SS-related genes. The coordinated regulation and integration of different gene networks to fine-tune the expression of symbiosis-related genes and other accessory genes by NodD1-NolA might be required for CCBAU51778 to efficiently nodulate peanut. This study shed new light on our understanding of the regulatory roles of NolA and NodD proteins in symbiotic adaptation, highlighting the sophisticated gene networks dominated by NodD1-NolA.

Multiple Genes of Symbiotic Plasmid and Chromosome in Type II Peanut Bradyrhizobium Strains Corresponding to the Incompatible Symbiosis With Vigna radiata.

Rhizobia are capable of establishing compatible symbiosis with their hosts of origin and plants in the cross-nodulation group that the hosts of origin belonged to. However, different from the normal peanut Bradyrhizobium (Type I strains), the Type II strains showed incompatible symbiosis with Vigna radiata. Here, we employed transposon mutagenesis to identify the genetic loci related to this incompatibility in Type II strain CCBAU 53363. As results, seven Tn5 transposon insertion mutants resulted in an increase in nodule number on V. radiata. By sequencing analysis of the sequence flanking Tn5 insertion, six mutants were located in the chromosome of CCBAU 53363, respectively encoding acyltransferase (L265) and hypothetical protein (L615)-unique to CCBAU 53363, two hypothetical proteins (L4 and L82), tripartite tricarboxylate transporter substrate binding protein (L373), and sulfur oxidation c-type cytochrome SoxA (L646), while one mutant was in symbiotic plasmid encoding alanine dehydrogenase (L147). Significant differences were observed in L147 gene sequences and the deduced protein 3D structures between the Type II (in symbiotic plasmid) and Type I strains (in chromosome). Conversely, strains in both types shared high homologies in the chromosome genes L373 and L646 and in their protein 3D structures. These data indicated that the symbiotic plasmid gene in Type II strains might have directly affected their symbiosis incompatibility, whereas the chromosome genes might be indirectly involved in this process by regulating the plasmid symbiosis genes. The seven genes may initially explain the complication associated with symbiotic incompatibility.