Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III-secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

Sinorhizobium fredii HH103 is a rhizobial soybean symbiont that exhibits an extremely broad host-range. Flavonoids exuded by legume roots induce the expression of rhizobial symbiotic genes and activate the bacterial protein NodD, which binds to regulatory DNA sequences called nod boxes (NB). NB drive the expression of genes involved in the production of molecular signals (Nod factors) as well as the transcription of ttsI, whose encoded product binds to tts boxes (TB), inducing the secretion of proteins (effectors) through the type 3 secretion system (T3SS). In this work, a S. fredii HH103 global gene expression analysis in the presence of the flavonoid genistein was carried out, revealing a complex regulatory network. Three groups of genes differentially expressed were identified: i) genes controlled by NB, ii) genes regulated by TB, and iii) genes not preceded by a NB or a TB. Interestingly, we have found differentially expressed genes not previously studied in rhizobia, being some of them not related to Nod factors or the T3SS. Future characterization of these putative symbiotic-related genes could shed light on the understanding of the complex molecular dialogue established between rhizobia and legumes.

Summary Sinorhizobium fredii HH103 RifR, a broad‐host‐range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen‐fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 RifR nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen‐fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod‐factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 RifR. Complementation of the nodD2 and nolR mutants provoked a decrease in Nod‐factor production, the incapacity to form nitrogen‐fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 RifR nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod‐factor overproduction, although other bacterial symbiotic signals could also be involved.

Sinorhizobium fredii HH103-Rif, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rif invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rif. A S. fredii HH103-Rif nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rif mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rif fla mutants nodulated L. burttii. None of the S. fredii HH103-Rif surface-polysaccharide mutants gained effective nodulation with L. japonicus.

Summary Sinorhizobium fredii HH103 RifR is a broad host‐range rhizobial strain able to nodulate with soybean and Lotus burttii, but it is ineffective with L. japonicus. Here, we study the role of the HH103 RifR SyrM protein in the regulation of gene expression and its relevance in symbiosis with those three legumes. RNAseq analyses show that HH103 SyrM is an important transcriptional regulator not only in the presence of inducer flavonoids but also in its absence. Lack of SyrM increases Nod factors production and decreases genistein‐mediated repression of exopolysaccharide production in HH103. In symbiosis, mutation of syrM partially impaired interaction with soybean but improves effectiveness with L. burttii and extends the host‐rage to L. japonicus Gifu. In addition, HH103 syrM mutants enter in both Lotus species by infection threads, whereas HH103 uses the more primitive intercellular infection to enter into L. burttii roots These symbiotic phenotypes were previously observed in two other HH103 mutants affected in symbiotic regulators, nodD2 and nolR, revealing that in S. fredii HH103 numerous transcriptional regulators finely modulate symbiotic gene expression.